in-cell western™ assay development handbook

Published August 2020. The most recent version ofthis document is posted at licor.com/bio/support.

Application GuideIn-Cell Western™ Assay DevelopmentHandbook

http://www.licor.com/bio/support

Table of Contents

Page

I. What Is an In-Cell Western Assay? 7

What Advantages Does an In-Cell Western Assay Have Over Western Blotting? 7

What Are the Main Differences Between an In-Cell Western Assay and a Western Blot? 7

Difference 1: Context in Which Proteins Are Presented As Target Antigens 7

Difference 2: Lack of Protein Separation 7

Who Can Use an In-Cell Western Assay? 8

What Can You Use An In-Cell Western Assay For? 8

Introduction References 9

II. How Do I Develop an In-Cell Western Assay? 11

Step 1. Define Your Experimental System 11

Step 2. Determine the Cell Number to Seed 11

Step 3. Validate Antibodies 12

Step 4. Choose a Blocking Buffer 12

Step 5. Choose Fixation and Permeabilization Conditions 12

Step 6. Determine Primary Antibody Concentration 12

III. Example Protocol Overview 13

IV. Cell Type 14

Cell Culture 14

Primary Cells 14

Immortalized Cell Lines 14

Cell Culture Systems 15

Common Conditions 15

Growth Culture Medium 15

Experimental Design 16

Choosing a Cell Type 16

Choosing a Plate Type 17

Considerations for Primary Cells 17

Considerations for Cell-Based Assays 17

Obtaining Cells 18

Confirm Cell Line Identity After Subculturing in the Lab 18

- In-Cell Western™ Assay Development Handbook

Other Factors to Consider 18

Plate Treatments 18

Cell Characteristics 19

Cell Type References 19

Other Resources 19

V. Cell Seeding 20

Cell Seeding Factors 20

Plates 20

Cell Seeding Density 20

Confluence 21

Generalized Protocol for Seeding Cells 21

VI. Cell Stain Linearity 22

Determining Cell Stain Linearity in Empiria Studio® Software 22

Creating a Plate Template 22

Analyzing Cell Stain Linearity Experiment to Determine Linear Range 22

VII. Antibody Selection 24

Concept References 24

Definitions 24

Distinguishing Specificity and Selectivity 25

Identify Assay Requirements 25

Get to Know Your Target Antigen 25

Identify Cells and Tissues 27

Establish Experimental Prerequisites 28

Determine Antibody Requirements 29

Acquire Acceptable Antibodies 30

Identify Specific Antibodies 30

Review Literature 30

Locate Available Antibodies 30

Obtain Antigen Information 31

Select Desired Host Species 31

Research Verified Applications 31

Compare Antibody Data to Existing Biological Information 32

Research the Supplier 32

Notes About Custom Antibodies 32

Validate Antibody 32

Gather Antibody Validation Information 32

Validate Antibody Experimentally 34

In-Cell Western™ Assay Development Handbook -

Document and Report Antibody Details and Validation Procedures 38

Antibody Selection References 39

Additional Resources 39

Single Antibody Titration Experiment 39

Antibody Resources 40

VIII. Blocking Buffer Evaluation 43

Buffer System 43

Blocking Agents 44

Bovine Serum Albumin 44

Synthetic Blocking Buffers 44

Normal Serum 44

Blocking Buffer Optimization 45

IX. Fixation and Permeabilization 46

Fixation Methods 46

Aldehyde Fixatives 46

Organic Solvents 47

Evaluating Fixation 48

Evaluating Fixation for In-Cell Western Assays 48

Optimizing Fixation for On-Cell Western Assays 48

Permeabilization 49

Detergents 49

Organic Solvents 50

Optimize Permeabilization 50

Choosing Fixation and Permeabilization Options 51

On-Cell Westerns 53

Recommended Fixation and Permeabilization Methods 54

Method 1: Formaldehyde/Triton™ X-100 54

Method 2: Methanol 55

Method Comparison Example 55

Alternative Methods 56

80% Acetone in Water (100% acetone not compatible with plastic plates) 56

Formaldehyde-Methanol 57

Ethanol-Acetic Acid 57

Methanol-Acetone (100% acetone not compatible with plastic plates) 57

Paraformaldehyde 57

Paraformaldehyde-Methanol 57

Fixation and Permeabilization References 58


Example Fixation and Permeabilization Optimization Plate Layout 59

X. Replicates 60

Technical and Biological Replicates: Which Do You Need to Include? 61

Technical Replicates 61

Biological Replicates 61

Technical and Biological Replicates: How Many Replicates Do You Need to Include? 61

Technical Replicates 61

Biological Replicates 62

Replicates References 62

XI. Normalization 64

Why Normalization is Necessary 64

Ideal World 64

Real World 65

Normalization Basics 65

Normalization Concepts 65

Normalization Cannot Account for Everything 65

Normalization Strategies 66

Post-Translational Modification Normalization Strategy 66

Cell Stain Strategies 67

Cell Stain Selection Guide 70

Normalization References 71

XII. Experimental Controls 72

Control Types and Well Types 72

XIII. Z’-Factor Determination 74

Experimental Overview 74

Guidelines for the Z'-Factor Experiment 75

Z'-Factor Calculation Overview 75

Z'-Factor Equation 75

Z'-Factor Interpretation 76

Z'-Factor Summary 77

XIV. Frequently Asked Questions 78

General 78

Assay Development 79

Cell Seeding 80

Fixation and Permeabilization 80


Primary Antibody 80

Normalization 81

Wash Step in the Protocol 81

Plates 82

Imaging Microwell Plates 83

Miscellaneous 84

XV. Plate Loading Guides 85

Plate Layout for Loading Antibody Dilutions 85

Blank Plate Loading Guides 86

XVI. Empiria Studio® Software Multiwell Plate Calculations Reference 87

Explanation of Analysis Table Data on the Quantify Wells Page 87

Well Information 87

Replicate Information 88

Set Up Chart 89

In-Cell Western 89

Z’-Factor Determination Experiment 90

Required Wells 90

In-Cell Western 90

Analysis Table Well Tab Calculation Examples 90

Total (800 nm Channel) 91

Background (800 nm Channel) 91

Signal (800 nm Channel) 91

Normalized Signal 92

Set Up Chart Page Calculation Examples 92

Well Sets vs Well Groups 92

Normalization 93

Fold Change 93

Percent Change 95

XVII. Glossary 96


I. What Is an In-Cell Western Assay?

An In-Cell Western is a quantitative immunofluorescence (IF) assay performed in microwell plates (optimized for 96- or 384-well formats). Data analysis is based on whole-well fluorescence quantification. The In-Cell Western Assay offers a convenient alternative to Western blotting and is a powerful platform for meaningful in situ analyses.

What Advantages Does an In-Cell Western Assay Have Over Western Blotting?

In-Cell Western Assays combine the specificity of Western blotting with the replicability and throughput of ELISA. These assays are a higher throughput way to detect and quantify multiple targets at the same time in their cellular context. Using these assays, you can rapidly collect data from many replicates under various experimental conditions. Additionally, fewer steps in the workflow mean this cell-based assay has improved consistency over traditional Western blotting.

What Are the Main Differences Between an In-Cell Western Assay and a Western Blot?

These are two of the main differences between Western blots and In-Cell Western Assays.

Difference 1: Context in Which Proteins Are Presented As Target Antigens

In-Cell Western Assays are commonly performed on cultured cells that have been fixed to the bottom of a microplate. Antigens in an In-Cell Western often have different conformational structures than those that have been removed from the cell and processed by SDS-PAGE. Consequently, some primary antibodies that perform well for Western blotting may exhibit poor binding characteristics for fixed protein epitopes, resulting in low or absent signal during the detection step. On the other hand, an antibody might work better for a fixed antigen.

Difference 2: Lack of Protein Separation

In an In-Cell Western Assay, there is no electrophoresis step to separate individual proteins by their molecular weight. Whole cells are attached to the bottom of the multiwell plate for analysis, consequently proteins are probed within the context of the entire cell. Since nonspecific antibody interactions are indistinguishable in the microplate well, antibody specificity must be confirmed prior to performing the In-Cell Western Assay. For more information about choosing the correct antibody, see "Antibody Selection" on page 24.


Who Can Use an In-Cell Western Assay?

Researchers in sectors from academia to industry can make use of In-Cell Western Assays for basic and translational research.

What Can You Use An In-Cell Western Assay For?

These are just some of the ways an In-Cell Western Assay can be used.

l Virus research including quantification of viral load (1 - 8)

l Protein phosphorylation and signaling (9 - 11)

l Off-target effects of drugs on signaling pathways (12)

l Timing and kinetics of signaling events (13, 14)

l Genotoxicity assays (15, 16)

l Chemical library screening (17 - 19)

l Glycoprotein analysis (20, 21)

l Screening of monoclonal antibody clones (22)

l Bacterial-induced epithelial signaling (23)

l Cell proliferation and apoptosis assays (24)


Introduction References

1. Counihan, N. A., Daniel, L. M., Chojnacki, J., and Anderson, D. A. (2006). Infrared fluorescent immunofocus assay (IR-FIFA) for the quantitation of non-cytopathic and minimally cytopathic viruses. J Virol Methods, 133, 62-69. https://doi.org/10.1016/j.jviromet.2005.10.023

2. Lin, Y. C., Li, J., Irwin, C. R., Jenkins, H., DeLange, L., and Evans, D. H. (2008). Vaccinia virus DNA ligase recruits cellular topoisomerase II to sites of viral replication and assembly. J Virol, 82, 5922-5932. https://doi.org/10.1128%2FJVI.02723-07

3. Weldon, S. K., Mischnick, S. L., Urlacher, T. M., and Ambroz, K. L. (2010). Quantitation of virus using laser-based scanning of near-infrared fluorophores replaces manual plate reading in a virus titration assay. J Virol Methods, 168, 57-62. https://doi.org/10.1016/j.jviromet.2010.04.016

4. Lopez, T., Silva-Ayala, D., Lopez, S., and Arias, C. F. (2012). Methods suitable for high-throughput screening of siRNAs and other chemical compounds with the potential to inhibit rotavirus replication. J Virol Methods, 179, 242-249. https://doi.org/10.1016/j.jviromet.2011.11.010

5. Wan, Y., Zhou, Z., Yang, Y., Wang, J., and Hung, T. (2010). Application of an In-Cell Western assay for measurement of influenza A virus replication. J Virol Methods, 169, 359- 364. https://doi.org/10.1016/j.jviromet.2010.08.005

6. Fabiani, M., Limongi, D., Palamara, A. T., De Chiara, G., and Marcocci, M. E. (2017). A novel method to titrate Herpes simplex virus-1 (HSV-1) using laser-based scanning of near-infrared fluorophores conjugated antibodies. Frontiers in Microbiology, 8, 1–8. https://doi.org/10.3389/fmicb.2017.01085

7. DuShane, J. K., Wilczek, M. P., Crocker, M. A., and Maginnis, M. S. (2019). High-throughput characterization of viral and cellular protein expression patterns during JC polyomavirus infection. Frontiers in Microbiology, 10, 1–11. https://doi.org/10.3389/fmicb.2019.00783

8. Ma, H. W., Ye, W., Chen, H. S., Nie, T. J., Cheng, L. F., Zhang, L., Zhang, F. L., et al. (2017). In-cell western assays to evaluate Hantaan virus replication as a novel approach to screen antiviral molecules and detect neutralizing antibody titers. Frontiers in Cellular and Infection Microbiology, 7 https://doi.org/10.3389/fcimb.2017.00269

9. Chen, H., Kovar, J., Sissons, S., Cox, K., Matter, W., Chadwell, F., Luan, P., Vlahos, C. J., Schutz-Geschwender, A., and Olive, D. M. (2005). A cell-based immunocytochemical assay for monitoring kinase signaling pathways and drug efficacy. Analytical biochemistry, 338, 136- 142. https://doi.org/10.1016/j.ab.2004.11.015

10. Aguilar, H. N., Zielnik, B., Tracey, C. N., and Mitchell, B. F. (2010). Quantification of rapid Myosin regulatory light chain phosphorylation using high-throughput in-cell Western assays: comparison to Western immunoblots. PLoS One, 5, e9965. https://doi.org/10.1371/journal.pone.0009965

11. Wong, S. K. (2004). A 384-well cell-based phospho-ERK assay for dopamine D2 and D3 receptors. Analytical biochemistry, 333, 265-272. https://doi.org/10.1016/j.ab.2004.05.011

12. Kumar, N., Afeyan, R., Kim, H. D., and Lauffenburger, D. A. (2008). Multipathway model enables prediction of kinase inhibitor cross-talk effects on migration of Her2-overexpressing mammary epithelial cells. Mol Pharmacol, 73, 1668-1678. https://doi.org/10.1124/mol.107.043794

13. Hannoush, R. N. (2008). Kinetics of Wnt-driven beta-catenin stabilization revealed by quantitative and temporal imaging. PLoS One, 3, e3498. https://doi.org/10.1371/journal.pone.0003498


https://doi.org/10.1016/j.jviromet.2005.10.023

https://doi.org/10.1128%2FJVI.02723-07




https://doi.org/10.3389/fmicb.2017.01085

https://doi.org/10.3389/fmicb.2019.00783

https://doi.org/10.3389/fcimb.2017.00269

https://doi.org/10.1016/j.ab.2004.11.015

https://doi.org/10.1371/journal.pone.0009965

https://doi.org/10.1016/j.ab.2004.05.011

https://doi.org/10.1124/mol.107.043794


14. Chen, W. W., Schoeberl, B., Jasper, P. J., Niepel, M., Nielsen, U. B., Lauffenburger, D. A., and Sorger, P. K. (2009). Input-output behavior of ErbB signaling pathways as revealed by a mass action model trained against dynamic data. Mol Syst Biol, 5, 239. https://doi.org/10.1038/msb.2008.74

15. Jamin, E. L., Riu, A., Douki, T., Debrauwer, L., Cravedi, J. P., Zalko, D., and Audebert, M. (2013). Combined genotoxic effects of a polycyclic aromatic hydrocarbon (B(a)P) and an heterocyclic amine (PhIP) in relation to colorectal carcinogenesis. PLoS One, 8, e58591. https://doi.org/10.1371/journal.pone.0058591

16. Khoury, L., Zalko, D., and Audebert, M. (2013). Validation of high-throughput genotoxicity assay screening using gammaH2AX in-cell western assay on HepG2 cells. Environ Mol Mutagen, 54, 737-746. https://doi.org/10.1002/em.21817

17. Guo, K., Shelat, A. A., Guy, R. K., and Kastan, M. B. (2014). Development of a cell-based, high-throughput screening assay for ATM kinase inhibitors. J Biomol Screen, 19, 538-546. https://doi.org/10.1177/1087057113520325

18. Hoffman, G. R., Moerke, N. J., Hsia, M., Shamu, C. E., and Blenis, J. (2010). A high- throughput, cell-based screening method for siRNA and small molecule inhibitors of mTORC1 signaling using the In Cell Western technique. Assay Drug Dev Technol, 8, 186-199. https://doi.org/10.1089/adt.2009.0213

19. Schnaiter, S., Furst, B., Neu, J., Waczek, F., Orfi, L., Keri, G., Huber, L. A., and Wunderlich, W. (2014). Screening for MAPK modulators using an in-cell western assay. Methods Mol Biol, 1120, 121-129. https://doi.org/10.1007/978-1-62703-791-4_8

20. Urlacher, T., Xing, K., Cheung, L. et al. (2013). Glycoprotein applications using near-infrared detection. Poster presentation, Experimental Biology. https://doi.org/10.1096/fasebj.27.1_supplement.592.6

21. McInerney, M. P., Pan, Y., Short, J. L., and Nicolazzo, J. A. (2017). Development and Validation of an In-Cell Western for Quantifying P-Glycoprotein Expression in Human Brain Microvascular Endothelial (hCMEC/D3) Cells. J Pharm Sci, 106, 2614-2624. https://doi.org/10.1016/j.xphs.2016.12.017

22. Daftarian, M. P., Vosoughi, A., and Lemmon, V. (2014). Gene-based vaccination and screening methods to develop monoclonal antibodies. Methods Mol Biol. 1121, 337-346. https://doi.org/10.1007/978-1-4614-9632-8_30

23. Du, Y., Danjo, K., Robinson, P. A., and Crabtree, J. E. (2007). In-Cell Western analysis of Helicobacter pylori-induced phosphorylation of extracellular-signal related kinase via the transactivation of the epidermal growth factor receptor. Microbes Infect, 9, 838-846. https://doi.org/10.1016/j.micinf.2007.03.004

24. Yunn, N-O.,Koh, A., Han, S., Lim, J.H., Park, S., Lee, J., et al. (2015). Agonistic aptamer to the insulin receptor leads to biased signaling and functional selectivity through allosteric modulation. Nucleic Acids Res, 43, 7688–7701. https://doi.org/10.1093/nar/gkv767


https://doi.org/10.1038/msb.2008.74


https://doi.org/10.1002/em.21817

https://doi.org/10.1177/1087057113520325

https://doi.org/10.1089/adt.2009.0213

https://doi.org/10.1007/978-1-62703-791-4_8

https://doi.org/10.1096/fasebj.27.1_supplement.592.6

https://doi.org/10.1096/fasebj.27.1_supplement.592.6

https://doi.org/10.1016/j.xphs.2016.12.017

https://doi.org/10.1007/978-1-4614-9632-8_30

https://doi.org/10.1016/j.micinf.2007.03.004

https://doi.org/10.1093/nar/gkv767

II. How Do I Develop an In-Cell Western Assay?

The development process will vary between research projects and must be determined by the researcher. Generally, the stages of development can follow the steps in the protocol (see Example Protocol Overview on page 13). The following is a brief overview of an assay development process and the objectives of each stage of the process.

Note: At any point during the assay development process, you can use a Z'-Factor assay to evaluate the robustness of the assay under development.

Preset Plate Templates (see the [Preset] Z-Factor Determination Plate Templates) are provided in Empiria Studio® Software to help you get started.

Step 1. Define Your Experimental System

Although your project will have specific questions, here are some common and important questions to consider.

l What target do you want to detect?

l What antibodies are available to detect your target?

l Is the target on the cell surface or intracellular?

l What cell line(s) should you use?

l What controls should you use?

l Which imager will you be using for detection?

Step 2. Determine the Cell Number to Seed

Determine the cell number that results in cell stain signal that falls within the linear range of detection.

The Plate Analysis Template [Preset] Cell Stain Linearity is provided in Empiria Studio to help you analyze or design your experiment for determining cell stain linearity in your assay.


Step 3. Validate Antibodies

Research which antibodies are available to detect your target. The Antibody Selection chapter (page 24) has recommendations for researching antibodies to use in your assay. A Western blot, In-Cell Western, or immunofluorescence assay could be used to validate antibodies.

In a Western blot validation experiment, ensure there are no unexpected bands. Empiria Studio® Software provides workflows to help with antibody validation for Western blots and antibody titration for In-Cell Western assays (see Step 6 for more information).

Step 4. Choose a Blocking Buffer

Choose a variety of blocking buffers to evaluate, and run an In-Cell Western to determine the best blocking buffer for your assay.

You can analyze In-Cell Western blocking buffer experiment results using these Plate Templates provided in Empiria Studio: [Preset] Blocker Evaluation (Single Antibody) and [Preset] Blocker Evaluation (Two Antibodies).

Step 5. Choose Fixation and Permeabilization Conditions

Start with vendor recommendations, if available. A Fixation and Permeabilization Evaluation can be performed to increase confidence that you'll be using the best conditions for your assay.

The [Preset] Fixation and Permeabilization Evaluation Plate Template can be used to determine the optimal fixation and permeabilization conditions for an experiment.

Step 6. Determine Primary Antibody Concentration

Run an Antibody Titration experiment to determine the antibody concentration that provides optimal signal and lowest background.

Try starting with the dilution given on the pack insert for IF and include a dilution that is two-fold above and one that is two-fold below. Follow dilution recommendations on the pack insert for IRDye® Secondary Antibodies.

Two Plate Templates are provided in Empiria Studio to help you design and analyze Antibody Titration experiments. The two Plate Templates are [Preset] Antibody Titration (Single Antibody) and [Preset] Antibody Titration (Two Antibodies).


III. Example Protocol Overview

The following steps provide an overview of an example In-Cell Western Assay.

More Info: More detailed protocols and preparation instructions for solutions are provided in the pack inserts for CellTag™ 700 Stain Kits I and II and CellTag 520 Stain Kits III an IV.

1. Seed cells - See Cell Type on the next page and Cell Seeding on page 20 for more information.

2. Cell treatment (optional)

3. Fix cells - See Fixation and Permeabilization on page 46 for more information.

4. Permeabilize cells - See Fixation and Permeabilization on page 46 for more information.

5. Block cells - See Blocking Buffer Evaluation on page 43 for more information.

6. Prepare primary antibody solution

7. Incubate primary antibody solution

8. Wash plate

9. Prepare secondary antibody solution and cell stain

10. Incubate secondary antibody solution and cell stain

11. Wash plate

12. Measure signal - See licor.com/AcquisitionQuickStart for more information about using LI-COR® Acquisition Software and an Odyssey® Imager to acquire multiwell plate images. For Image Studio™ Software, see your imager's User Manual for more information.


https://www.licor.com/documents/lbzoh3dlfw9j887vqh60iud3h4azrlcf

https://www.licor.com/documents/daa33rlr2p0gp9nhimoyb9w42vq3wf2q

https://www.licor.com/documents/daa33rlr2p0gp9nhimoyb9w42vq3wf2q

https://www.licor.com/AcquisitionQuickStart

IV. Cell Type

This section covers differences between cell types, technical considerations for using various cell types, experimental design considerations for various cell types, and cell culture.

Cell Culture

Cell culture refers to the growth of cells in a controlled, artificial environment. Isolated cells come from various biological sources, such as animals, plants, and fungi. Cell culture is typically divided into two groups: primary cell culture and immortalized cell lines. Cell lines are cells isolated from tissue and grown primarily in culture dishes or flasks until they reach confluency and must be subcultured. To answer today’s research questions, scientists will use both primary cells and established cell lines in assays such as the In-Cell Western.

Primary Cells

Primary cells are isolated directly from the tissue of interest and grown for a finite time in culture. These cells exhibit many of the same features as the tissue, such as morphology and expression profiles. The similarity between the tissue and the primary cells make primary cells useful for the study of various human diseases.

Immortalized Cell Lines

To maintain cell lines in culture, cells must be immortalized. Cells can be immortalized in a few ways.

l The first is through spontaneous genetic changes, which give cells the ability to avoid senescence. This occurs in the case of cancer cells.

l A second way cells can become immortalized is by introducing a viral gene, which confers the ability of cells to override the cell cycle. Genes for the large and small T antigens from Simian Virus 40 are frequently used for this purpose.

l A third method to confer immortality to cells is the expression of genes such as Telomerase (hTERT), which abate senescence by extending the lengths of telomeres and allowing the cells to divide indefinitely.

Once the immortalized cell line is established, it can be grown using specific growth conditions and maintained for long periods of time. Some cell lines have been grown for decades, resulting in phenotypic and genotypic distinctions from the tissue they were originally isolated from. Even with these differences, scientists have relied on cell lines to answer important scientific questions for decades.


Cell Culture Systems

There are two systems for growing cells in culture: adherent cells and suspension cells.

l Adherent: Cells are attached to a surface in a single layer (monolayer)

l Suspension: Cells float freely in a medium

Cell culture conditions vary greatly depending on the cell type being grown, but there are some common conditions.

Common Conditions

There are some common considerations for all cells grown in culture, such as media, temperature, osmotic pressure, and pH (CO2). Whether you are growing cells in adherent or suspension culture, the optimal pH, temperature, and CO2 recommendations are identical for mammalian cells.

Table 1. The recommendations in this table are for mammalian cell culture, both adherent culture (cell lines and primary cells) and suspension culture.

Condition Recommendation

Media +/- Serum

pH 7.4

Temperature 36-37 °C

CO2 4-10%

Growth Culture Medium

The composition of the growth culture medium can vary depending on cell type. The primary components in a cell culture medium are:

l Buffer base

l Amino acids

l Vitamins

l Inorganic salts

l Polysaccharide

l Serum

The primary function of the medium components is to support and promote cell growth. Most components are required for all cell growth, independent of cell type. One factor which differs for different cell types is the presence of serum. High concentrations of serum can cause primary cells to change their cell type, a process known as differentiation. It can also promote the growth of certain cell types like fibroblasts more than others.


Primary cells also often require additional nutrients from traditional cell culture mediums, so each cell type may have a specialized media that is formulated which is low in serum or serum-free for their growth. Cell lines require less specialized mediums, but the presence and amount of serum can still vary depending on the type of cell line. Synthetic sera, which can have reduced variation between batches, are commercially available.

More Info: For more detailed recommendations for cell culture medium and culturing techniques for each cell line, you can refer to www.atcc.org.

Table 2. This table shows the components of growth culture medium and the purpose of those components.

Component Purpose

Buffer Maintain pH, Source of sodium, potassium, carbon, nitrogen, hydrogen

Amino Acids Promote cell growth

Vitamins Promote cell growth

Inorganic Salts Promote cell growth

Polysaccharide (e.g., Glucose) Energy source

Serum (e.g., Fetal Bovine Serum)

Provides hormones, growth and adhesion factors, lipids, etc. for cell growth

Experimental Design

Choosing a Cell Type

The cell type chosen for an In-Cell Western Assay is critical for the success and relevance of the results. Both primary cells and immortalized cell lines can be used in an In-Cell Western Assay, and there are specific considerations for each. If the appropriate cell type is chosen and antibodies are validated, the In-Cell Western can provide accurate and relevant results to scientific questions.

Primary cells can be hard to grow and hard to transfect. For both primary cells and cell lines, different phenotypic and genotypic characteristics can directly affect the results of an assay. For example, if performing a cytotoxicity assay, cells that normally exhibit a high cell death rate may contribute background to the assay. Primary cells have a higher spontaneous cell death rate than cell lines, and therefore may not be optimal for cytotoxic assays. For cell viability assays, cell lines tend to have a higher metabolic rate.


http://www.atcc.org/

Choosing a Plate Type

Different plates are recommended for adherent vs suspension cells for plate-based assays.

l For adherent cells, LI-COR recommends a 96-well plate with a clear, flat bottom and black wells, such as the Greiner Bio-One CELLSTAR® Black μClear® Microplate, LI-COR PN 926-19156 (8 pack) or 926-19157 (32 pack).

l For suspension cells, LI-COR recommends growing cells in a 96-well U-bottom plate and transferring cells to Greiner Bio-One CELLSTAR® Black μClear® Microplate, LI-COR PN 926-19156 (8 pack) or 926-19157 (32 pack), for imaging.

Considerations for Primary Cells

Primary cells can be difficult to culture in vitro. These are some general points to consider for culturing primary cells in vitro.

l Prevent contamination: Upon initial isolation from tissue, cells should be treated to prevent contamination from bacteria or fungi.

l Maintain less than 100% confluency: Cells should be grown in the appropriate medium with supplements until the desired confluency is attained, so that the primary cells can be subcultured. It is important not to allow primary cells to reach 100% confluency, because they can enter senescence (a process which inhibits cell division). Growing the primary cell culture past confluency may also lead to differentiation, where the cellular profile will be permanently altered and may ultimately affect how the cells will perform in an assay.

l Limit length of time cells are exposed to trypsin: Primary cells are sensitive to trypsin, so adherent cells should not be exposed to trypsin for prolonged periods of time. Exposing primary cells to trypsin for prolonged periods of time can lead to cell death.

l Maintain other growth conditions: Maintenance of the pH, temperature, and CO2 concentration is also very important, as primary cells are more sensitive to changes in these growth conditions than cell lines.

Considerations for Cell-Based Assays

Many factors will influence the outcome of a cell-based assay. The list below has important information to know regarding the cell type you choose for your experiments.


Obtaining Cells

Ideally, research would be conducted with authenticated, quality-controlled cell lines with a low passage number from a dependable biological resource center. Depending on the needs of your research and where you were able to obtain your cells, you may need to perform verification tests on your cell line to ensure the cells can be used for reliable and publishable research.

ATCC recommends “basic benchmark verification tests that can be employed by any lab and included in publication” (25), specifically covering the following subject areas.

l Low passage number

l Morphology check by microscope

l Growth curve analysis

l Species confirmation by isoenzyme analysis

l Identity verification with STR profiling

l Mycoplasma detection

Confirm Cell Line Identity After Subculturing in the Lab

Detailed discussion about confirming cell line identify is beyond the scope of this document, but ATCC provides useful articles on this topic. You can read more about how STR (short tandem repeat) profiling can contribute to quality and integrity of research using human cell lines in their article Searchable STR Database For Human Cell Lines (26). You can learn more about identifying mycoplasma contamination in cell cultures on their Mycoplasma Testing Service page (27).

Other Factors to Consider

Plate Treatments

l TC-treated

l Poly-L or Poly-D-Lysine

l Gelatin


Cell Characteristics

l Cell doubling time

l Treatment conditions (such as serum starvation)

Cell Type References

25. ATCC. Cell Line Authentication - Why It Matters, (Acc. August 27, 2020).

26. ATCC. Searchable STR Database For Human Cell Lines, (Acc. August 27, 2020).

27. ATCC. Mycoplasma Testing Service, (Acc. August 27, 2020).

Other Resources

1. ATCC. Animal Cell Culture Guide, (Acc. August 27, 2020).

2. Zhanqiu, Y., Xiong, H. (2012). Culture Conditions and Types of Growth Media for Mammalian Cells. IntechOpen. https://doi.org/10.5772/52301


https://www.atcc.org/en/Services/Testing_Services/Cell_Authentication_Testing_Service/Cell_Line_Authentication_Test_Recommendations.aspx

https://www.atcc.org/en/Products/Cells_and_Microorganisms/Testing_and_Characterization/STR_Profiling_Analysis.aspx

https://www.atcc.org/Services/Testing_Services/Mycoplasma_Testing.aspx

https://www.atcc.org/resources/culture-guides/animal-cell-culture-guide

https://doi.org/10.5772/52301

V. Cell Seeding

Cell seeding is the process of pipetting a specific number of cells into each well of a microplate. This procedure has a marked effect on the assessed biological result of a treatment or condition. Multiple cell seeding concentrations should be tested to determine a concentration that will result in a level of confluency that generates significant well fluorescence at the time of imaging. Standardization of a cell seeding protocol for a particular treatment is important for reproducibility of the experiment and assay output.

Cell Seeding Factors

The following are important factors to consider when seeding adherent cells.

Plates

For cells that adhere strongly to the wells (e.g., A431, HEK293, CHO), we recommend regular tissue culture microplates with low auto-fluorescence, particularly those with black sides and clear well bottoms (e.g., Greiner CELLSTAR® 96 well plates, flat bottom black polystyrene wells). For adherent cells that could detach from wells during In-Cell Western Assay wash steps (e.g., NIH3T3), we recommend Poly-D-lysine coated 96-well microplates.



Cell Seeding Density

Typically, 5,000 to 40,000 cells are seeded per well (96-well plate). One to three days are required for cells to reach the appropriate confluency, depending on growth rate and cell size. The growth rate can be different at different cell densities. It is best to attempt to operate within the exponential growth phase for your cell line.

Seeding with lower cell numbers is recommended if you plan to culture for several days before use. Plates seeded with higher cell numbers will be ready to use earlier.


Confluence

In general, cells should be imaged at about 80 – 85% confluency while cells are in the log phase of growth and in a monolayer on the plate. However, the appropriate level of confluency depends on the cell line. Experimental conditions must be optimized for the cell type to determine the appropriate level of confluency required to achieve significant well fluorescence.

Generalized Protocol for Seeding Cells

1. Allow cells to grow in a T75 flask using standard tissue culture procedures until ~80% confluency is achieved.

2. Remove growth media and wash cells with sterile 1X PBS (RT).

3. Add 5 mL Trypsin-EDTA (Sigma) and incubate for 3 - 5 minutes at 37 °C and 5% CO2 to displace cells.

4. Neutralize trypsin by adding serum-containing growth media and pellet by centrifugation.

5. Remove supernatant and disrupt the cell pellet manually by hand-tapping the collection tube.

Note: To maintain cell integrity, do not pipet or vortex during pellet disruption.

6. Dilute cells in complete media so that cells are at a concentration that is 5-fold the desired amount per well.

7. Manually mix the cell suspension thoroughly.

8. Under sterile conditions, dispense 200 μL of the cell suspension per well in a 96-well plate.

9. Incubate cells at 37 °C and monitor cell density until desired confluency is achieved.


VI. Cell Stain Linearity

During the development of an In-Cell Western Assay, it is important to determine the relationship between cell number and signal intensity to ensure that signals are detected within the linear range of the assay.

Determining Cell Stain Linearity in Empiria Studio® Software

Empiria Studio provides unique functionality for determining linear range.

Creating a Plate Template

To determine the cell stain linearity for an In-Cell Western Assay, first design a Cell Stain Linearity experiment, and set up a Plate Template (with the Cell Stain Linearity category) in the Empiria Studio Experiment Designer. The Plate Template serves dual roles: It is a visual way to plan your experiment, and it is also used for analysis.

Figure 1. This screenshot shows the experimental design for the [Preset] Cell Stain Linearity Plate Template provided in Empiria Studio. The abbreviation "spl" indicates "sample" and "bkg" indicates "back-ground". The cell number for each sample well is indicated on the well. You can open the Plate Template in Empiria Studio for more information.

Analyzing Cell Stain Linearity Experiment to Determine Linear Range

In a multiwell plate analysis workflow in Empiria Studio, you choose a multiwell plate image and a Plate Template. The wells designated in the Plate Template are quantified and analyzed.

In the Cell Stain Linearity workflow, wells are quantified and the relationship between cell number and signal intensity is graphed on the Find Linear Range page. A color bar above the


graph indicates where the relationship between cell number and signal intensity has excellent linearity, good linearity, or no linearity.

l Green sections on the bar correspond to regions on the graph where there is excellent linearity between cell number and signal intensity.

l Yellow sections on the bar correspond to regions on the graph where there is good linearity.

l Red sections on the bar correspond to regions on the graph where the relationship between cell number and signal intensity is not linear.

Drag the slider bars above the graph so that they bracket a region with linearity. The median cell number within this region is indicated above the graph. In general, this is a good cell number to use in your assay to stay within the linear range; however, it is the responsibility of the researcher to choose the appropriate cell number to use.

Figure 2. This screenshot shows an example Find Linear Range page in Empiria Studio® Software. Sliders above the Signal vs Cell Number graph have been dragged to bracket a region of excellent linearity. The median cell number in the bracketed region is 10,000. This number may be an appropriate cell number to use in the assay to stay within the linear range of the assay.


VII. Antibody Selection

Selection and validation of primary and secondary antibodies are vital to achieving reliable results from an In-Cell Western Assay.

In this guide, we present antibody validation strategies, examples, and guidelines to ensure accurate and reproducible data. The approach is focused on achieving scientific rigor and lab economics for the In-Cell Western Assay, but the underlying best practices are applicable to other cell-based assays. Guidelines presented in this document have been written to align with the thorough guidelines provided by the European Antibody Network (euromabnet.com/guidelines).

For a summary of useful resources, see "Antibody Resources" on page 40.

Concept References

To begin, this section explains critical concepts and defines critical terms that will be referenced throughout this section.

Definitions

A detailed glossary of key terms is provided at the end of this document (see page 96), but it is worth emphasizing some important definitions that will be used in this section. In particular, it is worth clarifying how "specificity" and "selectivity" will be used.

This section relies on the following definitions from the Workshop Report from the Antibody Validation: Standards, Policies, and Practices Workshop hosted by the Global Biological Sciences Institute (29).

l Affinity: Binding strength between the antibody’s binding site and its epitope.

l Antigen: A substance stimulates the production of and binds to antibodies.

l Epitope: A part of the antigen to which the antibody directly binds.

l Selectivity: Ability to preferably bind the target antigen in the presence of other antigens.

l Specificity: Ability to recognize the target antigen.


https://www.euromabnet.com/guidelines/

Distinguishing Specificity and Selectivity

The antibody binds

a single epitope but

multiple targets.

The antibody binds

a single epitope and

a single target.

The antibody binds

multiple epitopes and

multiple targets.

The antibody binds

multiple epitopes

within a single target.

Selectivity

Sp

ecifi

city

Figure 3. A diagram illustrating specificity and selectivity and how to distinguish them.

Identify Assay Requirements

When beginning a new In-Cell Western Assay, identify these basic experimental requirements before acquiring antibodies and completing preliminary method development.

Get to Know Your Target Antigen

To identify an antibody that is both specific and selective for your target, you will want to acquire a thorough knowledge of your target antigen. In addition to a comprehensive literature review, online databases can be useful resources when searching for information regarding your target antigen. A list of helpful resources is included on page 40.

Know your antigen by name(s)

Start your research by identifying the various nomenclature for your antigen to help identify all the relevant information and reagents. The HUGO Gene Nomenclature Committee site (genenames.org) enables you to search human gene nomenclature by symbol, keyword, and ID.

Know your antigen’s sequence(s)

Locate the protein sequence of the antigen, including any known variants (e.g., splicing isoforms, proteolytic products, and post-translational modifications). The UniProt protein database (uniprot.org) is a useful tool for determining your antigen's sequence. Knowing the protein sequence is helpful for making some key decisions.


https://www.genenames.org/

https://www.uniprot.org/

l Is it important to detect all variants of the target antigen or only one in particular?

l Is it critical to distinguish domains with different subcellular localizations (e.g., labeling the extracellular versus intracellular portions of membrane protein)?

When variants of a target antigen are present in a sample, you may observe unexpected cellular localization or tissue localization using IF or unexpected bands on a Western blot. Knowing that variants of the target antigen are present may prevent misidentifying non-specificity of the antibody.

Know your antigen’s relatives

It is also valuable to investigate any sequence homology between your target antigen and any related and unrelated proteins. A Basic Local Alignment Search Tool (BLAST) search is useful for identifying these regions of similarity by comparing your target sequence against a large database (blast.ncbi.nlm.nih.gov/Blast.cgi). This search can help you identify proteins that share sequence identity with your target antigen and could potentially bind your antibody. A BLAST search can also identify sequence conservation across species, if you want to utilize a monoclonal antibody against a common epitope found in multiple species.

From this information you can begin to define and confirm optimal epitopes for your antibody. This information is critical for antibody production and helpful when selecting commercial antibodies.

Know your antigen's biological characteristics

Understanding the biological relevance of your target, such as when, where, and which variant of your target is expressed, is valuable information when designing your experiment. This information can help identify potential non-specific interactions when validating your antibody.

For Example

A protein involved in the electron transport chain is likely to have a mitochondrial localization. Therefore, a nuclear staining pattern would be questionable. Databases (such as Uniprot, Genecards, AceView, Protein Atlas, or Expression Atlas) are particularly useful sites for gathering this type of information. See "Additional Resources" on page 39 for a list of helpful resources.

Note: Be cautious when evaluating resources based on mRNA expression, because protein and mRNA expression patterns do not always correlate.


http://blast.ncbi.nlm.nih.gov/Blast.cgi

Identify Cells and Tissues

A critical component of demonstrating antibody specificity and selectivity is the correct use of controls. Determining which samples will be used for control materials is critical for the validation process.

l Positive controls should confirm expression of your target antigen in the relevant cells and tissues.

l Negative controls should consist of samples where the target is known to be absent, expressed at low levels, knocked down, or knocked out.

Positive and negative controls

Potential positive and negative controls can often be identified through online and literature searches. When selecting control samples, the expression of the target antigen should ideally be verified using more than one assay format, e.g., confirming expression by Western blot before use as an In-Cell Western Assay control.

It is best if one of these assays is a non-antibody method, such as genomic analysis. This increases confidence in the expression profile of the control samples, thereby enhancing the validation process. In addition to using tissues or cell lines that endogenously over- and under-express your target, both genetic and chemical approaches can be used to obtain valuable biological controls.

Genetic approaches for controls

Cultured cell lines are particularly beneficial when the target antigen expression can be altered by genetic modifications.

CRISPR/Cas9

This system offers an exacting method of gene editing. It can be used for complete genetic insertions and knockouts, as well as the insertion or correction of genetic mutations.

RNAi

Thoughtfully planned RNAi experiments, resulting in the absence or decrease of the specific target protein, are a useful tool for antibody validation. However, due to possible effects of off-target toxicity (28), care should be taken in how RNAi-based experiments are interpreted.

Chemical approaches for controls

In addition to genetic manipulation, chemical treatments with a well characterized substance (e.g., drug, growth hormone, or mineral) can be used to precisely alter protein expression in a controlled manner to obtain positive and negative controls.


For example

Samples treated with and without a growth hormone, that stimulates the phosphorylation of a signaling protein, can be used as the positive and negative controls for an antibody targeting the phosphorylated antigen.

Positive controls with variable expression

Finally, selecting a few positive controls expressing differing amounts of the target antigen (e.g., high versus moderate or weak) can increase confidence in antibody specificity. These controls are helpful for selecting antibodies with the highest binding affinity and highest specificity for the antigen.

Establish Experimental Prerequisites

Establish some preliminary assay parameters for the In-Cell Western Assay prior to antibody selection to maximize the likelihood of antibody success.

The success of an antibody in one application does not predict its success in another application. Some antibodies will specifically recognize a fully denatured protein in a Western blot but will not recognize the same protein in its natural conformation in an immunoprecipitation experiment. Some of the assay parameters that may influence antibody performance and deserve consideration before antibody selection include:

l Antibody/reagent compatibility

l Live versus fixed cells

l Fixation conditions

l Permeabilization conditions

l Blocking buffer constraints

l Preferred normalization method


Determine Antibody Requirements

Examples of antibody requirements to consider include:

l Polyclonal versus monoclonal versus recombinant antibodies

l Direct versus indirect detection

l Quantity of antibody for the project

l Isotype

l Host species

Polyclonal vs Monoclonal

Choosing between polyclonal and monoclonal antibodies can depend on antibody availability. If both options are available, you must decide if the technical advantages of a polyclonal antibody outweigh the disadvantages.

Advantages Disadvantages

Monoclonal l Monoclonal antibodies are often preferrable because of their specificity for a single epitope.

l Large quantities of identical monoclonal antibody can be produced with high batch-to-batch consistency.

l If your goal is to detect a target antigen and its orthologues in a different species, then a monoclonal antibody may be too specific. This consideration may be relevant for research that involves studying corresponding animal models (e.g., mouse/monkey) or research involving evolutionary genetics.

l Monoclonal antibodies are more sensitive to the loss of a single epitope during chemical treatment or other processing that occurs during the experiment.

l Monoclonal antibodies are time consuming to produce and can be more expensive.

Polyclonal l Polyclonal antibodies recognize multiple epitopes on the same antigen, which may result in a stronger signal and success in a broader selection of techniques.

l Increased chance of cross-reactivity

l Batch-to-batch variability


Isotype

Selecting primary antibodies from different species or different isotypes is important when detecting multiple antigens in the same samples using multiple antibodies. These antibodies can subsequently be detected with individually labeled isotype-specific or species–specific secondary antibodies. Also, to avoid cross-reactivity of the secondary antibody, the species of the primary antibody should vary from that of your sample and blocking buffer.

Acquire Acceptable Antibodies

Once you have identified the fundamental requirements for the antibody, the next step is to source and compare available antibodies to find one that will meet the needs of your research. This section details the information that you should gather before purchasing antibodies to validate.

Identify Specific Antibodies

When reviewing antibodies, it is important to note the clone name or product number. This information is helpful when surveying scientific literature for successful antibody applications. Since there are often multiple suppliers of the same antibody, it can also prevent purchasing the same antibody from different suppliers. In addition to clone names and product codes, Research Resource Identifiers (RRID) are unique identifiers to label biological resources like antibodies. These identifiers can be obtained through the RRID Portal website (scicrunch.org) or by contacting the suppliers directly.

Review Literature

Antibody data sheets and scientific literature are typically the primary sources of information when researching antibodies. Peer-reviewed scientific literature is an excellent place to start for locating which antibodies have previously been used to study a target protein. Research articles can be found on PubMed, CiteAb, BenchSci, and commonly used search engines.

For some antigens, there is a clear choice of antibody based on supporting validation data. For other antibodies, there may be few antibody options and little supporting data available. Extensive validation may be required after purchase to ensure an antibody will work for you under your specific experimental conditions.

Locate Available Antibodies

An important consideration when prioritizing antibodies is whether or not they are easily available (not all are commercially available). Occasionally, research laboratories will choose not to share their reagents and supply of some polyclonal antibodies may be limited or no longer available.


https://scicrunch.org/

In addition to searching individual company catalogs, there are several web-based search engines useful for identifying and comparing antibodies from commercial suppliers. These websites include Biocompare, The Antibody Resource, Antibodypedia, Linscott’s Directory, and the CPTAC Antibody Portal.

When planning a potentially lengthy research project, ensure you can acquire enough antibody to complete the project. This is particularly important with polyclonal antibodies. Keep in mind that researchers from other labs may have difficulty reproducing research conducted with polyclonal antibodies if antibodies from the same lot are no longer available. It may be worth validating the reagent and then purchasing multiple vials from the same lot to reduce difficulties with assay reproducibility.

Do not assume that antibody performance will be the same between lots. Each time you purchase antibodies from a new lot, validate and qualify antibodies from the new lot.

Obtain Antigen Information

The antigen used as an immunogen affects the number of possible antibody epitopes and impacts the antibody’s selectivity for the target protein. If a company does not readily supply this information, they may be willing to confirm if specific protein regions are contained within their immunogen without disclosing the actual immunogen.

If no information is available, assume the antibody may recognize any portion of the full-length protein.

Select Desired Host Species

Host species information is important for In-Cell Western Assay experiments that require multiple antibodies.

l Antibodies raised in one species may recognize orthologous antigens in other species. Should you choose to use primary antibodies from the same species, you will need to find a way to avoid the problem of secondary antibodies binding to both primary antibodies.

l Using primary antibodies raised in different species will avoid problems caused by secondary antibodies binding to both primary antibodies. It is also best to use affinity-purified secondary antibodies.

If you have questions about host species reactivity that is not listed on the antibody data sheet, contact the supplier.

Research Verified Applications

Antibody data sheets often include a list of validated techniques, suggested working dilutions, and additional technical information. Images from representative experiments are also often included to help you evaluate the data and draw your own conclusions. If an application is not


listed, it may mean that the antibody has not been tested in that application. Some antibody suppliers will state when an antibody is not suited for a specific application, if evidence exists that shows the antibody does not work in that application.

For In-Cell Western Assays, antibodies validated for immunofluorescence, immunocytochemistry, and immunohistochemistry are sometimes a good place to start. It is the researcher’s responsibility to verify.

Compare Antibody Data to Existing Biological Information

As discussed previously, the biological information of the target is vital. Use this information to guide your selection and testing of an antibody. Compare the data found in the data sheet with known biological information (molecular weight, subcellular localization, tissue distribution, etc.).

Example

If immunofluorescent staining revealed nuclear localization for a membrane receptor protein expected to be expressed at the plasma membrane, this would indicate that the antibody is non-specific.

If the data generated with one antibody differ from another antibody, further investigation is required to determine which antibody is selective for the target antigen.

Research the Supplier

When shopping for an antibody, there is less risk when you purchase from a supplier with a good reputation for customer satisfaction.

Notes About Custom Antibodies

Specific guidelines for producing and validating custom antibodies are beyond the scope of this guide, besides a brief mention. When producing custom antibodies, the type and quality of immunogen, host animal species, purification method, and pre-adsorption process should all be carefully considered.

Validate Antibody

Whether producing your own antibody or selecting from commercial offerings, it is important to validate antibody specificity and ensure that there is sufficient sensitivity across the entire dynamic range of samples to be tested in the In-Cell Western Assay.

Gather Antibody Validation Information

The antibody information in this section can likely be gleaned from published sources.


Reactivity to the immunogen

The following assays are often used to assess antibody reactivity against the immunogen:

l Enzyme-linked immunosorbent assay (ELISA)

l Flow cytometry

l Western blotting

Note: Antibody validation limited to an ELISA assay using the immunizing peptide alone is insufficient and does not guarantee reactivity with the endogenous protein.

Detection of a target antigen

Antibody reactivity with purified recombinant proteins or cells overexpressing the target protein are commonly used to demonstrate binding to the target antigen. Tagged expression constructs or lysates for most proteins are now commercially available to facilitate antibody validation. However, this still does not ensure antibody reactivity with the endogenous antigen.

Recognition of endogenous protein

Positive and negative controls are used to assess endogenous antibody binding. For thorough validation, multiple positive and negative controls should be used in the same experiment. Proper controls are essential for verification of endogenous protein detection and antibody specificity.

Antibody specificity and selectivity

Detection of the target antigen does not exclude the possibility that the antibody is cross-reactive with another protein(s). In addition to proteins sharing sequence similarity, antibodies can also recognize proteins with similar conformational epitopes, which cannot be predicted from amino acid sequence comparison. Antibody cross-reactivity with related proteins should be tested experimentally. A common approach is to screen transiently transfected cells engineered to express protein members of the same gene family.

Expression pattern

Experimentally confirm that the antibody detects the appropriate tissue and subcellular localization of the antigen.

Corroboration of multiple antibodies

Evaluating several antibodies in parallel and comparing their patterns of reactivity can significantly increase confidence in the antibodies. This is especially important when new or


unexpected results are obtained.

Reagents

Some reagents work in a wide number of techniques whereas others are very limited in their application. Select an antibody that has been validated, when possible, in the same tissue type and species. If this information is absent, then additional experimental testing is advised.

Evidence for application suitability

An antibody might be validated in only a few techniques, but this does not suggest it is unsuitable for other applications. For example, an antibody recommended for IF may also work in the In-Cell Western Assay. Furthermore, many antibodies are validated for Western blotting, where a denatured antigen is usually recognized, and antibodies that selectively recognize the native conformation of the antigen may not work in this technique.

Validate Antibody Experimentally

For an In-Cell Western Assay, antibody specificity should be evaluated by both Western blot and immunocytochemistry (ICC). Western blotting can yield useful information about antibody specificity, even though antigen presentation is different than in a cell-based assay. An antibody that generates any non-specific bands on a Western blot may indicate that the antibody is not suitable for In-Cell Western Assay experiments or that the blocking and antibody incubation conditions require further optimization.

Western blot analysis is a reliable way to determine if the desired cellular response has been triggered in your experiment. You may need to optimize cell treatment conditions or timing to capture the cellular response at its peak. For example, some phosphorylation events increase and decrease rapidly, so sampling at the wrong time may cause you to over or underestimate the response.

Determine necessary validation and optimization

If there is insufficient data to indicate that the antibody is suitably validated for your experimental context, then it is incumbent on the researcher to perform the additional required validation using appropriate controls.

Even if there is sufficient data to indicate that the antibody is suitably validated for your experimental context, then optimization is probably still required to achieve optimal performance of the antibody.


Western Blot Validation

Prepare your samples and perform the Western blot procedure

Follow LI-COR Protocols (licor.com/icw) for blocking, primary antibody incubation, secondary antibody incubation, wash steps, and image acquisition. Use LI-COR IRDye® Secondary Antibodies appropriate to the primary antibody you are evaluating.

l Estimate total protein concentration, preferably by BCA, Bradford, or Lowry spectrophotometric assays, and load equal amounts of total protein onto the SDS-PAGE gel for each of the prepared lysate samples.

l Whole cell lysates should be prepared from a representative cell line or cell type of interest. Recombinant proteins can also be used as a control.

l Lysates should be prepared from cells treated under representative In-Cell Western Assay conditions.

l Be sure to include a negative control lysate to detect any non-specific binding.

l Include a high-quality protein molecular weight marker (licor.com/bio/reagents/protein-ladders) to verify target size. Follow standard protocols for gel electrophoresis and Western blot transfer.

l Stain membrane with Revert™ 700 Total Protein Stain (licor.com/revert) for total protein detection and normalization.

Verify Molecular Weight and Specificity

l The position of the band(s) of interest on the Western blot should be close (e.g., ± 10% if using MW markers to size proteins) to the expected molecular weight of the target protein. Empiria Studio® Software (licor.com/empiria) provides analysis workflows to help with antibody validation.

l Only one band should be visible in the lane of interest (Figure 5). If multiple bands are present – especially strong ones – the primary antibody is possibly cross-reactive and not suitable for use in the In-Cell Western Assay (Figure 6).

l For activation-state-specific antibodies (e.g., phospho-specific antibodies), ensure that basal signal in the negative control lysate is not too high.


https://www.licor.com/icw

https://www.licor.com/bio/reagents/protein-ladders

https://www.licor.com/bio/reagents/protein-ladders

https://www.licor.com/revert

https://www.licor.com/empiria

Comparing Antibody Selectivity and Specificity by Western Blot

Specific

Selective

Target

Specific

Selective

Specific

Selective

Figure 4. A specific and selective antibody will produce a single band in a Western blot lane (leftmost lane illustration). The presence of additional bands in the lane indicates problems with specificity (middle lane illustration) or specificity and selectivity (rightmost lane illustration).

Example Western Blots

Examples of primary antibodies with good Western blot specificity are shown in Figure 5. An example of a primary antibody with poor Western blot specificity is shown in Figure 6.

Figure 5. Serial dilution of EGF-treated A431 cell lysate; phospho-EGFR (700 nm) and total ERK (800 nm) detec-tion. Rabbit anti-phospho-EGFR was obtained from Cell Signaling Technology (2237), and mouse anti- ERK from Santa Cruz (sc-1647). The presence of a single prominent band in each lane indicates good antibody specificity for the target. Research conducted at LI-COR Biosciences.


Non-specific

Target

band

Figure 6. Serial dilution of EGF-treated A431 lysate; phospho-ERK (800 nm) detection. The anti-phospho-ERK primary antibody is recognizing a spurious, strong high molecular weight band. Mouse anti-phospho-ERK was obtained from Santa Cruz (sc-7383). Research conducted at LI-COR Biosciences.

Immunocytochemistry Validation

Antibody specificity and selectivity should also be evaluated in ICC to ensure optimal assay performance and interpretation. Signal can be assessed using microplate assays on an Odyssey® M, Odyssey DLx, or Odyssey CLx Imager, or by near-infrared fluorescence microscopy. General methods for specificity evaluation are described below. The primary antibody supplier may have additional suggestions or information about antibody specificity and optimization.

Primary antibody titration

Primary antibodies should be titrated to determine the concentration that provides optimal signal and lowest background. Careful titration may reduce background staining.

The multiwell format of the In-Cell Western Assay makes it easy to quickly screen a range of antibody concentrations. Fluorescence microscopy should be used to confirm the protein localization at whichever primary antibody concentration is determined to be optimal.

Technical experimental conditions

If target signal is low, consider whether alternate experimental conditions (e.g., fixation, permeabilization, and blocking conditions) might improve performance.


Validation of primary antibodies in general

For validation of primary antibodies in general, create conditions under which the target signal increases or decreases (in each case, confirm the change in target signal by Western blotting or In-Cell Western). Suggestions include:

l Stain cells that express the target protein (endogenously and/or overexpressing), and cells that do not (endogenously or by knockout/knockdown). This is the most conclusive test. If this is not practical, look for cell lines that are high or low expressors of your target protein, or transfect the target into a cell line that does not endogenously express it.

l Expose the cells to conditions, ligands, or inhibitors that affect target abundance.

o Run antibody isotype controls (with Ig concentration and isotype that match your primary antibody) to assess non-specific binding.

o Examine stained cells with a fluorescence microscope, to determine if target signal is localized to the proper cell compartment.

Validation of phospho-antibodies specifically

For validation of phospho-antibodies used to monitor cell signaling:

l Treat fixed cells with phosphatase to reduce target phosphorylation. Signal should be reduced in treated cells.

Note: This test cannot determine if the antibody cross-reacts with another phospho-epitope.

l Treat cells with a target-specific ligand or inhibitor that modulates only the pathway of interest, to determine if signal responds accordingly. Confirm results by Western blotting.

Document and Report Antibody Details and Validation Procedures

Be sure to document your antibody details and validation procedure to ensure that all experimental details will be available for publication and to share with other researchers. Provide enough technical information to enable other researchers to acquire the same antibody and to get the same results from the same validation procedures and experiments. Empiria Studio® Software (licor.com/empiria) provides data fields in the antibody validation workflows that allow you to record this information. The Empiria Studio Antibody Validation Library provides a central location where you can find your antibody validation information. If allowed by your institution, share details about your antibody validation on online antibody databases (see Additional Resources on the facing page).


https://www.licor.com/empiria

Antibody Selection References

28. Lin, A., Giuliano, C.J., Palladino, A., John, K.M., Abramowicz, C., Yuan, M.L., et al. (2019). Off-target toxicity is a common mechanism of action of cancer drugs undergoing clinical trials. Science Translational Medicine, 11(509). https://doi.org/10.1126/scitranslmed.aaw8412

29. Global Biological Standards Institute. Antibody Validation Standards, Policies, and Practices Workshop Report. 2016.

Additional Resources

Single Antibody Titration Experiment

The following plate layout shows a template experiment that can be adapted for the specifics in your research to determine the best antibody dilution to use in your experiment. Plate layouts may also be created and printed from Empiria Studio® Software.

Note: The section "Plate Loading Guides" on page 85 has loading guides designed to fit under a plate to help keep track of which samples should be loaded in which wells.

blnkblnk blnkblnk blnkblnk blnkblnk blnkblnk blnkblnk blnkblnk blnkblnk bkgbkg blnkblnk blnkblnk blnkblnk

blnkblnk blnkblnk blnkblnk blnkblnk blnkblnk blnkblnk blnkblnk blnkblnk blnkblnk blnkblnk blnkblnk blnkblnk

blnkblnk ++ -- bkgbkg ++ -- bkgbkg ++ -- bkgbkg blnkblnk blnkblnk






Primary Antibody Dilutions

A

Dilution 1 Dilution 2 Dilution 3

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

Figure 7. This is the same design used in the [Preset] Antibody Titration (Single Antibody) Plate Template provided in Empiria Studio Software. Read the Plate Layout Legend below or open the Plate Template in Empiria Studio for more information.


https://doi.org/10.1126/scitranslmed.aaw8412

Plate Layout Legend

Symbol Definition

blnk Blank wells contain only TBS or PBS buffer and are used to mitigate the potential impact of plate well edge effects.

bkg Background wells that contain secondary antibody without primary antibody or normalization reagent.

+ Positive control wells contain primary and secondary antibody. Depending on your experiment, pos-itive controls could be an over-expressed cell line, a treatment known to stimulate target expression, etc.

- Negative control wells contain primary and secondary antibody. Depending on your experiment, negative controls could be a CRISPR knockdown, a treatment known to inhibit target expression, etc.

Antibody Resources

European Monoclonal Antibodies Networkeuromabnet.com

"EuroMAbNet represents the first European network of laboratories linked to academic institutions, each having an internationally recognized reputation in the production and use of monoclonal antibodies."

CiteAbciteab.com

Data provider for life sciences information.

The Antibody Registryantibodyregistry.org

"The Antibody Registry gives researchers a way to universally identify antibodies used in their research." You can find a list of LI-COR antibodies on The Antibody Registry by searching for "LI-COR".

The Biocompare Antibody Search Toolbiocompare.com/Antibodies

The Biocompare Antibody Search Tool lets researchers search over 3 million antibodies from hundreds of antibody suppliers according to criteria such as target antigen, species reactivity, host, conjugate, isotype, and application.

Antibodypediaantibodypedia.com

"Antibodypedia scores antibodies to guide researchers to choose an appropriate antibody for a particular application."


https://www.euromabnet.com/

https://www.citeab.com/

https://antibodyregistry.org/

https://antibodyregistry.org/search?q=LI-COR

https://www.biocompare.com/Antibodies/

https://www.antibodypedia.com/

RRID Portalscicrunch.org/resources

The Research Resource Identifier (RRID) Portal provides shared identifiers for citing research resources in biomedical literature. You can add a resource and get a unique identifier if you notice that a resource you are using is missing.

Clustal Omegaclustal.org/omega

Clustal Omega is a software tool that enables alignment of hundreds of thousands of sequences in a few hours.

Linscott’s Directorylinscottsdirectory.com

Search engine for finding suppliers for antibodies, ELISA assay kits, cytokines, enzymes, recombinant proteins, siRNAs, tissues, organs, antibody services, etc.

Expression Atlasebi.ac.uk/gxa/home

The mission of the Expression Atlas is to provide the scientific community with freely available information on the abundance and localization of RNA and proteins for different species, biological conditions, tissues, cell types, developmental stages, disease state, and others.

AbMinerdiscover.nci.nih.gov/abminer/home.do

"AbMiner is a tool that allows users to search for appropriate, commercially available antibodies for research purposes, and to match each antibody to its respective genomic identifiers."

HUGO Gene Nomenclature Committee Search

genenames.org

This site enables you to search human gene nomenclature by symbol, keyword, and ID. The HGNC approves unique symbols and names for human loci.

National Center for Biotechnology Informationncbi.nlm.nih.gov

"The National Center for Biotechnology Information advances science and health by providing access to biomedical and genomic information."

UniProtuniprot.org


https://scicrunch.org/resources

http://www.clustal.org/omega/

https://www.linscottsdirectory.com/

https://www.ebi.ac.uk/gxa/home

https://discover.nci.nih.gov/abminer/home.do

https://www.genenames.org/

https://www.ncbi.nlm.nih.gov/

https://www.uniprot.org/

"The mission of UniProt is to provide the scientific community with a comprehensive, high-quality and freely accessible resource of protein sequence and functional information."

BenchScibenchsci.com

BenchSci provides reagent selection and experimental design aided by artificial intelligence.

GeneCardsgenecards.org

"GeneCards is a searchable, integrative database that provides comprehensive, user-friendly information on all annotated and predicted human genes."

Center for Strategic Scientific Initiatives Antibody Portalproteomics.cancer.gov/antibody-portal

The CPTAC Antibody Portal serves as a National Cancer Institute (NCI) community resource that provides access to a large number of standardized renewable affinity reagents (to cancer-associated targets) and accompanying characterization data.

The Human Protein Atlasproteinatlas.org

The Human Protein Atlas is a program with the goal to map all the human proteins in cells, tissues, and organs using various omics approaches.

The Antibody Resourceantibodyresource.com

Source for Antibody and ELISA products from major suppliers.

AceViewncbi.nlm.nih.gov/IEB/Research/Acembly/

"AceView provides a curated, comprehensive and non-redundant sequence representation of all public mRNA sequences..."


https://www.benchsci.com/

https://www.genecards.org/

https://proteomics.cancer.gov/antibody-portal

https://www.proteinatlas.org/

https://www.antibodyresource.com/

https://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/

VIII. Blocking Buffer Evaluation

Blocking is an important step in cell-based assay protocols. Once cells in an In-Cell Western Assay have been fixed and permeabilized, blocking buffer is added to bind spaces in the well not occupied by cells and to bind potential nonspecific binding sites in the cell. Blocking buffers minimize non-specific binding of primary and secondary antibodies to wells and to non-target sites in the cell.

Non-specific antibody binding can mask signal from the target protein, reducing specificity and sensitivity. Blocking allows detection of specific antibody binding to the target. Ultimately, blocking improves the signal-to-noise ratio by reducing background. High background can be a problem with immunoassays, so blocking is an especially important step that should always be performed.

No single blocking buffer is ideal for every target of interest. A blocking buffer used for one application may not necessarily be optimal for other applications. This section provides essential knowledge for choosing and optimizing a blocking buffer system for immunoassays and shows an experimental design that can be used to help identify the optimal buffer for an immunoassay.

Buffer System

Immunoassay protocols require solutions for blocking, antibody dilution, and washing.

PBS (Phosphate-buffered saline) and TBS (Tris-buffered saline) are commonly used buffers during the various stages in immunoassays, and it is useful to know when to use each of them. The buffer you choose will depend on your proteins of interest and the antibodies used. It is recommended that you compare PBS and TBS buffers to see which gives you the highest signal and lowest background for your specific targets.

PBS and TBS are often interchangeable as dilution buffers for blocking. However, not all blocking buffer formulations are suitable for every situation or antibody, and there are instances where PBS cannot be used.

TBS blocking buffers are typically preferred when detecting post-translational modifications such as phosphorylation. In some cases, the primary antibody may not only bind to the phosphate on the protein of interest, but also to phosphate in a PBS-based buffer, significantly reducing your target signal. However, this is not the case for all phospho-specific antibodies. For example, some PBS-based buffers can reduce the weaker non-specific interactions of a phospho-specific antibody while leaving the stronger antibody-antigen interactions intact. Therefore, both buffers need to be tested.


Blocking Agents

The most common blocking agents for cell-based assays include concentrated protein solutions and pre-formulated commercial solutions.

No single blocking buffer is ideal for every target of interest. It is important to optimize the blocking conditions for your cell line and protein target and determine which are most appropriate for the context and goals of your experiment. A good place to start is to look at published IF/ICC experiments where the same cell type and/or protein of interest has been examined and see what blocking conditions were used. This can give you some confidence as to which condition may work for your experiments.

Bovine Serum Albumin

Bovine serum albumin (BSA), derived from cow serum, can be used when detecting post-translational modifications. Because it is made of a single purified protein, BSA is less likely to interfere with antibody binding. Gelatin is also a purified protein, hydrolyzed collagen, that can come from different sources. Fish gelatin does not react with mammalian proteins and can decrease background in immunoassays, but may mask some antigens and should be carefully tested prior to use in immunoassays.

Synthetic Blocking Buffers

Synthetic and/or non-animal blocking agents were formulated to prevent any cross reactivity with the primary and secondary antibodies produced in animals. Pre-formulated commercial solutions include those produced by LI-COR. LI-COR offers several blocking buffers for use when performing an immunoassay.

Intercept® Blocking Buffer products are optimized for use with IRDye® products and other near-infrared fluorophores. They provide excellent performance with low variability and background for immunoassays. Intercept Blocking Buffer products are non-mammalian and provide a high signal-to-noise ratio in a 1X ready-to-use formula. Intercept Protein-Free Blocking Buffers, available in both PBS and TBS formulations, are essential for applications where animal-based products are prohibited, or protein-based blocking buffers are contributing to cross-reactivity.

Normal Serum

Normal serum is a common blocking agent that helps reduce the nonspecific binding of the primary and secondary antibodies to the cellular proteins by binding to cross-reactive sites. Serum is rich in antibodies, albumin, and other proteins that readily bind to nonspecific protein-binding sites which can improve the signal-to-noise of the assay. The most important factor to remember is the serum should be from the same host species as the secondary antibody. For example, if the primary antibody is a rabbit anti-target and the secondary is a labeled goat anti-rabbit, then the blocking serum would be normal goat serum (NGS).


Blocking Buffer Optimization









Blocking Buffer Optimization

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

Blocking Buffer 1 Blocking Buffer 2 Blocking Buffer 3

Figure 8. This plate layout shows an experimental design that will allow you to test up to three different blocking buffers on the same plate. This plate layout shows the same design that is used in the [Preset] Blocker Evaluation (Single Antibody) Plate Template provided in Empiria Studio® Software. Open the Plate Template in Empiria Stu-dio for more information.


IX. Fixation and Permeabilization

Fixation and permeabilization are tools that safeguard cell structure and allow entry of the antibody through the cellular and nuclear membranes. This section reviews common fixation and permeabilization methods, addresses experimental and technical considerations, and includes the two most common fixation/permeabilization protocols for In-Cell Western Assays.

Fixation Methods

Fixatives preserve biological specimens by limiting enzymatic degradation, preventing microbial contamination, and fixing (cross-linking) cells to the plate. This is especially important for samples that have long lag times from collection to analysis. Stabilizing cellular architecture is another important benefit of fixation when using conditions too harsh for live cells or employing immunostaining techniques that have multiple solution changes.

There are a variety of strategies to fix cells, but the priority must be for preservation of antigenicity and epitope accessibility. The two groups of reagents most often used for fixation in cell biology, immunology, and medicine are aldehydes and organic solvents.

Recommendation: A 3.7% formaldehyde solution is the standard fixative when optimal fixation conditions are not known.

Aldehyde Fixatives

Aldehyde-based fixatives include formaldehyde, paraformaldehyde, and glutaraldehyde.

Formaldehyde and Paraformaldehyde

Formaldehyde (the saturated form in water at 37 - 40% is called formalin) is the most commonly used fixative that works for a wide range of cells and tissues, preserving the cellular structure without substantial cellular shrinkage (30 - 31). Formaldehyde is purchased as a saturated solution containing 10 - 15% methanol as a stabilizer to prevent repolymerization into paraformaldehyde or oxidation to formic acid. When diluted to an often-used concentration of 3.7 - 4%, the methanol concentration is 1 - 1.5%. This means that methanol is likely a factor in the permeabilization process and will be discussed further.

Paraformaldehyde is polymerized formaldehyde that is dissolved in heated (60 °C) water or PBS, and the pH is adjusted to 6.9. Paraformaldehyde does not contain added methanol. Formaldehyde and paraformaldehyde act by cross-linking functional groups of proteins, glycoproteins, nucleic acids. and polysaccharides.


Figure 9. Formaldehyde molecular structure (Wikimedia, Public Domain). Red = oxygen, black = carbon, and white = hydrogen.

Glutaraldehyde

The bifunctional glutaraldehyde molecule has aldehyde groups at either end providing the advantage of greater cross-linking with the drawback of potential reduction in the immunogenicity of proteins for immunostaining (32).

Figure 10. Glutaraldehyde molecular structure (Wikimedia, CC0 1.0). Red = oxygen, black = carbon, and white = hydrogen.

Combinations

Sometimes combinations of these reagents, such as formaldehyde and glutaraldehyde, are used to balance out the advantages and disadvantages of each one. Inclusion of glutaraldehyde, at a concentration between 0.05 – 0.1%, along with formaldehyde or paraformaldehyde may enhance the cross-linking action and thereby provide stronger cell fixation. The extent and nature of the crosslinking of any aldehyde can be controlled by the concentration, time, and temperature (see Choosing Fixation and Permeabilization Options on page 51).

Organic Solvents

Organic solvents adhere cells to surfaces by dehydration and precipitation of proteins. Methanol, acetone, and ethanol are commonly used and are very effective for nucleic acid preservation.


https://commons.wikimedia.org/wiki/File:Formaldehyde-3D-balls-A.png

https://commons.wikimedia.org/wiki/File:Glutaraldehyde_3D_ball.png

Figure 11. Molecular structures of methanol (Wikimedia, Public Domain), acetone (Wikimedia, Public Domain), and ethanol (Wikimedia, Public Domain). Red = oxygen, black = carbon, and white = hydrogen.

With the exception of methanol, other organic solvents can be significantly damaging to cell structure, cell organelles, microtubules, and nuclear content (33 - 34). Methanol can maintain protein cytoskeletal structure, although small molecules can be lost if not precipitated. This solvent often gives very good results for immunostaining with low background (35). Methanol fixation is frequently combined with an acetone rinse to improve permeabilization and overall results.

Evaluating Fixation

Optimizing fixation requires that you first decide if you will permeabilize the cells or not. The In-Cell Western Assay protocol calls for cell permeabilization. The On-Cell Western assay protocol is similar to the In-Cell Western Assay protocol, but cells are not permeabilized, enabling quantitative monitoring of cell surface protein expression.

Evaluating Fixation for In-Cell Western Assays

If you plan to permeabilize cells as you would in an In-Cell Western Assay, then try an aldehyde fixative followed with a permeabilization method or use an alcohol-based fixative at varying concentrations (Figure 12) on a single sample type to determine which method gives the best results.

Optimizing Fixation for On-Cell Western Assays

Because cells are not permeabilized in an On-Cell Western assay, you will need to choose paraformaldehyde or glutaraldehyde (Figure 12) for fixing cells. Other fixation methods will result in some degree of permeabilization. Evaluate various concentrations on a single sample type to determine if paraformaldehyde or glutaraldehyde is best for your experimental system.


https://commons.wikimedia.org/wiki/File:Methanol-3D-balls.png

https://commons.wikimedia.org/wiki/File:Acetone-3D-balls.png

https://commons.wikimedia.org/wiki/File:Ethanol-3D-balls.png

Fixation and

Permeabilization

Formaldehyde

Paraformalehede

Glutaraldehyde

Methanol

Acetone

Ethanol

Paraformaldehyde

Glutaraldehyde

Triton X-100

Saponin

Acetone Rinse

After Methanol

Fixation without

PermeabilizationNo Permeabilization

Solution

Figure 12. Example fixation and permeabilization systems.

Permeabilization

Fixation is commonly followed by a permeabilization step so that antibodies can gain access to the intracellular components. Permeabilization can be achieved with either detergents or solvents.

Detergents

l Saponin is a relatively gentle detergent that removes cholesterol from the membrane, leaving holes to permit entry of antibodies (36).

l Triton™ X-100 is a nonionic surfactant that is widely used, and its effects are non-specific and dependent on the amount and duration of exposure to the cells (37).

Recommendation: A 0.1% Triton X-100 solution is the standard permeabilization agent when optimal permeabilization conditions are not known.

l Nonidet™ P-40 - Similar to Triton X-100, Nonidet P-40 is now available as IGEPAL CA-630. Nonidet P-40 is a non-specific, non-denaturing detergent that can have harsh effects on the nuclear and plasma membranes of cells if left for too long (36).

l Tween® 20 - One of the gentlest nonionic detergents, Tween 20, can create pores without affecting the membrane integrity and is suitable for detection of cytoplasmic and nucleic acid targets in the cytoplasm (38).


Organic Solvents

Organic solvents can be used for permeabilization or both fixation and permeabilization combined. Two solvents frequently used together are methanol and acetone. Although they can be less effective and cause cell shrinkage, solvents do work well for nuclear targets.

Keep in mind that most 96- and 384-well plates are plastic and are not compatible with 100% acetone or chloroform fixatives. Only use glass bottom plates for those fixation solvents. For compatibility information, consult information provided by the plate manufacturer or ask the plate manufacturer for more information.

Optimize Permeabilization

Optimization of permeabilization requires that you decide on a single fixative protocol then vary the agents used to permeabilize the cell. The choice is dependent on the goal of your experiment. For example, cytoplasmic and nuclear targets will require gentler detergents (Tween® 20, saponin) and careful timing. Small molecule targets may not be retained if methanol is used.

No single fixation/permeabilization method will perform best for every cell line or condition. Optimal conditions need to be empirically determined. Past experience with an antigen may be helpful, as are established conditions for this antigen for immunofluorescent microscopy or other cell-based assays. Primary antibody manufacturers will often provide fixation and permeabilization conditions for immunofluorescent staining of a particular antigen. Choosing Fixation and Permeabilization Options on the facing page gives general concentration ranges, times, and temperatures to try for both fixative and permeabilization solutions. Optimization is the key to a quality, robust experiment.


Choosing Fixation and Permeabilization Options

In-Cell Western Pro Con

Formaldehyde Low level of cell shrinkage

Good preservation of cell structure

Wide range of cells and tissues

Always get a degree of permeabilization due to presence of methanol

Paraformaldehyde Low level of cell shrinkage

Good preservation of cell structure

Wide range of cells and tissues

Glutaraldehyde Good for high MW DNA preservation

Methanol Preserves nucleic acid content

High-level immunostaining with low background

Acetone Can only use 80% or lower on plastic plates

Triton™ X-100 Good at removing adducts

Non-selective

Tween® 20 Gentle on cell lipids

Saponin Specifically interacts and removes cholesterol

No significant membrane alteration

NP-40 Non-selective


Fixative Permeabilization Concentration Range

Formaldehyde* X X 2 - 3.7% in PBS

Paraformaldehyde** X - 4% in PBS

Glutaraldehyde X - 2.5% in PBS

Methanol X X 50 - 100% cold

Acetone X X 70 - 100% cold

Triton™ X-100 - X 0.1 - 0.2%

Tween® 20 - X 0.1 - 0.5%

Saponin - X 0.1 - 0.5%

NP-40 - X 0.1 - 0.2%

*37% (wt/vol) in H2O, contains 10 - 15% Methanol as stabilizer to prevent polymerization** Paraformaldehyde is a methanol-free, stabilizer-free formaldehyde solution

On-Cell Western Paraformaldehyde Paraformaldehyde

Concentration 4% 0.25%

Incubation time 20 minutes 1 hour

Citation Miller, J.W. Tracking G protein-coupled receptor trafficking using Odyssey® imaging.

Hjerten S, Johansson KE: Selective solubilization with Tween 20 of membrane proteins from Acholeplasrna laidlawii. Bioch Bioph Acta 288:312-325, 1972.


https://www.licor.com/documents/k5r52vpo5vx1y6ddapsewpzrrpfjdlit



On-Cell Westerns

If you are interested in cellular membrane targets rather than intracellular targets, then permeabilization is a step that you would either skip or shift to a later stage of the protocol. Your choice of washing buffer would not include detergents but would include buffers as close to physiological pH as possible, such as PBS or cell culture media.

If you intend to normalize cell number using a cell impermeant stain, such as CellTag™ 700 Stain, then prior to the addition of the secondary antibody step, you would need to add a permeabilization step to allow entry of CellTag 700 Stain. The stain would be mixed with the secondary antibody and added at the same time. The secondary antibody will only recognize the primary antibody that has already bound to the cell membrane proteins. Below is an example of a generic On-Cell Western workflow.

1. Fix cells with paraformaldehyde or glutaraldehyde.

2. Wash with PBS or cell culture media.

3. Block cells.

4. Add primary antibody.

5. Wash with Triton™ X-100 / PBS.

6. Add secondary antibody and cell normalization stain.

7. Wash with Triton X-100 / PBS.

8. Image


Recommended Fixation and Permeabilization Methods

Method 1: Formaldehyde/Triton™ X-100

The following conditions are a good place to begin optimization if you have little or no experience with your system. In this method, formaldehyde is used as the cross-linking reagent, and Triton X-100 is used for permeabilization.

1. Just prior to use, prepare Fixation Solution:

o Prepare 20 mL per plate.

o Mix 2 mL 37% Formaldehyde and 18 mL 1X PBS (final formaldehyde concentration = 3.7%)

Important: Formaldehyde is hazardous and should be handled and discarded according to the manufacturer’s MSDS.

2. Carefully remove growth medium from the microwell plate by manual aspiration.

3. Immediately fix cells. Using a multichannel pipette, carefully add 150 µL of Fixation Solution down the sides of each well to avoid detaching cells.

4. Cover the plate and allow incubation on the bench top for 20 minutes at ambient temperature without agitation.

5. During the fixing step, prepare Permeabilization Solution by mixing 200µL of 10% Triton X-100 and 19.8 mL of 1X PBS (final Triton X-100 concentration = 0.1%).

6. Remove Fixation Solution from the microwell plate by manual aspiration to an appropriate hazardous waste container (refer to manufacturer’s SDS).

7. Using a multichannel pipette, carefully add 150 µL of Permeabilization Solution down the sides of each well to avoid detaching cells.

8. Gently agitate plate for 20 minutes at room temperature.

9. Remove Permeabilization Solution carefully from the wells using the multichannel pipette without removing any cells.

10. After removing the permeabilization wash by manual aspiration, proceed immediately to blocking and the remaining In-Cell Western incubation steps.


Method 2: Methanol

The second method uses methanol for both fixation and permeabilization of cells. Methanol fixation/permeabilization may be a better option than formaldehyde in some cases (for example, when using some phospho-Stat3 antibodies or antibodies against some major structural proteins or nuclear matrix proteins).

1. Use 100% methanol that has already been cooled to -20 ˚C.

Important: Methanol is flammable and should only be placed in a freezer that is appropriately rated for flammable liquids.

2. Carefully remove growth medium from the microplate by manual aspiration.

3. Immediately fix and permeabilize cells by adding 150 µL cold methanol to each well.

Note: Be sure to carefully add the methanol down the sides of the wells to avoid detaching the cells from the well bottom.

4. Allow the plate to incubate at -20 ˚C for 10 minutes without shaking.

5. Carefully remove the methanol by manual aspiration into an appropriate waste container.

6. Gently rinse wells three times with 100 µL of 1X PBS. Be sure to add and remove the PBS carefully by manual aspiration to avoid detaching the cells.

7. After removing the last 1X PBS rinse, proceed immediately to blocking and remaining In-Cell Western incubation steps.

Method Comparison Example

The graph in Figure 13 shows the Z’-factor results when comparing permeabilization Method 1 and Method 2 for detection of Stat3 phosphorylation in EGF-treated and untreated A431 cells. In this case, methanol produced consistently more desirable results than formaldehyde and Triton™ X-100 (Z’-factor values > 0.5 are desirable).


Z-Factor Analysis of Two Independent Fixation and Permeabilization TechniquesFormaldehye

+ Triton X-100

Methanol

Plate 1

1.0

Plate 2 Plate 3

0.8

0.6

0.4

0.2

0.0

-0.2

Z’-

Facto

r

-0.04

0.61

0.19

0.54

-0.10

0.72

Figure 13. Using methanol for fixation and permeabilization in this assay consistently produces a Z’-factor above 0.5, demonstrating that methanol is a better choice than formaldehyde fixation with Tri-ton™ X-100 permeabilization for this particular assay. Z’-factor analysis of two independent In-Cell Western experiments for detection of phospho Stat3 in EGF-treated and un-treated A-431 cells. In the first exper-iment, cells in three separate plates were fixed with formaldehyde and permeabilized with Triton X-100 (Method 1; blue bars). In the second experiment, cells in three separate plates were fixed and permeabilized using meth-anol (Method 2; green bars).

Alternative Methods

The following reagents/methods are alternatives to the aforementioned fixation/permeabilization methods. Because these methods have not been specifically tested for the In-Cell Western Assay, they are not supported by LI-COR. However, they are relatively common to other immunocytochemical assays, and may be viable options when optimizing your assay.

The steps in these methods are not rigid, and optimization of the various methods is recommended. As a general rule, aldehydes must be followed by a permeabilization step, but alcohols and organic solvents do not require a separate permeabilization step; however, this is not always true. In each case, proceed with In-Cell Western blocking and incubation steps after the fixation/permeabilization procedure.

80% Acetone in Water (100% acetone not compatible with plastic plates)

Consult your manufacturer’s documentation for specifics.

1. Fix cells with ice-cold (-20 ˚C) 80% acetone for 10 minutes.

2. Rinse with 1X PBS.


Formaldehyde-Methanol

1. Fix cells with room temperature 3.7% formaldehyde for 20 minutes.

2. Incubate cells with cold 100% methanol for 10 min.


Ethanol-Acetic Acid

1. Fix cells with refrigerated (4 ˚C) 95% ethanol + 5% glacial acetic acid for 10 minutes.


Methanol-Acetone (100% acetone not compatible with plastic plates)

1. Fix cells with methanol for 10 minutes at -20 ˚C.

2. Remove methanol and add acetone (-20 ˚C) for 1 minute.


Paraformaldehyde

1. Fix cells in 4% paraformaldehyde for 20 minutes at room temperature.


3. Wash 1 time for 20 minutes with 0.1% Triton™ X-100 (diluted in 1X PBS).

Paraformaldehyde-Methanol

1. Fix cells in 4% paraformaldehyde for 20 minutes at room temperature.


3. Add methanol (-20 ˚C) for 5 minutes.



Fixation and Permeabilization References

30. Thavarajah, R., Mudimbaimannar, V.K., Elizabeth, J., Rao, U.K., and Rangathan, K. (2012). Chemical and physical basics of routine formaldehyde fixation. J Oral Maxillofac Pathol, 16(2), 400-405. https://doi.org/10.4103/0973-029x.102496

31. Srinivasan, M., Sedmak, D., and Jewell, S. (2002). Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol, 161(6), 1961-1971. https://doi.org/10.1016/s0002-9440(10)64472-0

32. Suthipintawong, C., Leong, A.S., and Vinyuvat, S. (1996). Immunostaining of cell preparations: a comparative evaluation of common fixatives and protocols. Diagn Cytopathol, 15(2), 167-174. https://doi.org/10.1002/(sici)1097-0339(199608)15:2%3C167::aid-dc17%3E3.0.co;2-f

33. Hoetelmans, R.W., Prins, F.A., Cornelese-ten Velde, I., van der Meer, J., Van de Velde, C.J., and van Dierendonck, J.H. (2001). Effects of acetone, methanol, or paraformaldehyde on cellular structure, visualized by reflection contrast microscopy and transmission and scanning electron microscopy. Appl Immunohistochem Mol Morphol, 9(4), 346-351. https://doi.org/10.1097/00129039-200112000-00010

34. Vielkind, U. and Swierenga, S.H. (1989). A simple fixation procedure for immunofluorescent detection of different cytoskeletal components within the same cell. Histochemistry, 91(1), 81-88. https://doi.org/10.1007/bf00501916

35. Levitt, D. and King, M. (1987). Methanol fixation permits flow cytometric analysis of immunofluorescent stained intracellular antigens. J Immunol Methods, 96(2), 233-237. https://doi.org/10.1016/0022-1759(87)90319-x

36. Amidzadeh, Z., Behbahani, A.B., Erfani, N., Sharifzadeh, S., Ranjbaran, R., Moezi, L., et al. (2014). Assessment of different permeabilization methods of minimizing damage to the adherent cells for detection of intracellular RNA by flow cytometry. Avicenna J Med Biotechnol, 6(1), 38-46. http://www.ncbi.nlm.nih.gov/pmc/articles/pmc3895578/

37. Oliver, C. and Jamur, M.C. (2010). Immunocytochemical Methods and Protocols. Methods Mol Biol, 588, 63-66. https://doi.org/10.1007/978-1-59745-324-0

38. le Maire, M., Champeil, P., and Moller, J.V. (2000). Interaction of membrane proteins and lipids with solubilizing detergents. Biochim Biophys Acta, 1508(1-2), 86-111. https://doi.org/10.1016/s0304-4157(00)00010-1


https://doi.org/10.4103/0973-029x.102496

https://doi.org/10.1016/s0002-9440(10)64472-0

https://doi.org/10.1016/s0002-9440(10)64472-0

https://doi.org/10.1002/(sici)1097-0339(199608)15:2<167::aid-dc17>3.0.co;2-f

https://doi.org/10.1097/00129039-200112000-00010

https://doi.org/10.1007/bf00501916

https://doi.org/10.1016/0022-1759(87)90319-x

http://www.ncbi.nlm.nih.gov/pmc/articles/pmc3895578/

https://doi.org/10.1007/978-1-59745-324-0

https://doi.org/10.1016/s0304-4157(00)00010-1

https://doi.org/10.1016/s0304-4157(00)00010-1

Example Fixation and Permeabilization Optimization Plate Layout









Fixation and Permeabilization Verification

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

Fix/Perm 1 Fix/Perm 2 Fix/Perm 3

Figure 14. This plate layout shows an experimental design that will allow you to test 1 fixative and 3 per-meabilization agents on the same plate. Signal in this experimental design is normalized, for example, to a cell stain. To test more fixatives or permeabilization agents, repeat the experiment. This plate layout shows the same design that is used in the [Preset] Fixation and Permeabilization Evaluation Plate Template provided in Empiria Studio® Software. Open the Plate Template in Empiria Studio for more information.


X. Replicates

The In-Cell Western Assay is a higher throughput assay than a Western blot. Technical replicates and biological replicates can be performed in a single plate, which eliminates the inter-membrane variability associated with Western blots.

Replicates improve the reproducibility and accuracy of experimental findings. They are important because they confirm the validity of observed changes in a biological sample, such as protein levels. Without replication, it is impossible to know if an effect is real or simply an artifact of experimental noise or variation, which can directly affect conclusions made about experimental findings.

There are two types of replicates: biological and technical. Each type addresses different questions (39 - 41). Peer-reviewed journals, such as the Journal of Biological Chemistry and Nature, have specific guidelines regarding replicates.

Clearly define replicates. How many technical and biological replicates were performed during how many independent experiments?

- The Journal of Biological Chemistry, Collecting and Presenting Data (44)

The number of sampled units, N, upon which each reported statistic is based must be stated.

- Nature, Instructions for Authors of Research Articles (Initial Submission) (45)


Technical and Biological Replicates: Which Do You Need to Include?

Technical Replicates

Technical replicates are repeated measurements used to establish the variability of a protocol or assay and determine if an experimental effect is large enough to be reliably distinguished from the assay noise (39). Examples may include loading multiple wells with each sample on the same plate, running multiple plates in parallel, or repeating the plate with the same samples on different days.

Technical replicates evaluate the precision and reproducibility of an assay, which determine if the observed effect can be reliably measured using the assay. When technical replicates are highly variable, it is more difficult to separate the observed effect from the assay variation. You may need to identify and reduce sources of error in your protocol to increase the precision of your assay. Even though technical replicates evaluate the precision and reproducibility of an assay, they do not address the biological relevance of the results.

Biological Replicates

Biological replicates are parallel measurements of biologically distinct and independently generated samples, used to control for biological variation and determine if the experimental effect is biologically relevant. The effect should be reproducibly observed in independent biological samples. Demonstration of a similar effect in another biological context or system can provide further confirmation. Examples include analysis of samples from multiple batches of independently cultured and treated cells.

To demonstrate the same effect in a different experimental context, the experiment might be repeated in multiple cell lines, in related cell types or tissues, or with other biological systems.

An appropriate replication strategy should be developed for each experimental context. Several recent papers discuss considerations for choosing technical and biological replicates (39 - 41).

Technical and Biological Replicates: How Many Replicates Do You Need to Include?

Technical Replicates

Technical replicates are the measurement of the same sample within an assay multiple times. Increasing or decreasing the number of technical replicates can affect the experimental outcome of a research study. A general rule of thumb for plate-based assays is ‘the more replicates the better.’ LI-COR recommends 4 - 6 technical replicates for a 96-well plate. The coefficient of variation increases as well size and working volume decrease. Therefore, for a 384-well plate, a minimum of 4-8 replicates is suggested.

There are several advantages to using many technical replicates within an assay:


1. Variation can be measured between replicates and statistical tests applied to evaluate differences between the samples.

2. Smaller changes can be discerned between samples when the average across replicates is calculated.

3. A larger number of replicates makes identification of outlier samples easier to detect.

Standard Deviation versus Standard Error of the Mean

Standard Deviation

When performing three or more technical replicates, the most important statistical calculation is standard deviation. Standard Deviation (SD) is a measure of the variability of data points with respect to the mean. SD indicates the variation of data within individual samples.

Standard Error of the Mean

The Standard Error of the Mean (SEM) is also sometimes used, although caution should be employed when using the SEM because it is easy to misrepresent variability. SEM compares the means of populations and gives information about the variation in two samples. This information can be valuable to researchers in some circumstances, such as when trying to compare two different experimental groups. For example, a cell line treated with an inhibitor versus an untreated cell line.

Biological Replicates

Biological replicates are biologically distinct samples measured in parallel that capture random biological variation. For statistically significant measurements, a minimum of three biological replicates is recommend for plate-based assays in either a 96-well or 384-well format.

Replicates References

39. Naegle, K., Gough, N.R., and Yaffe, M.B. (2015). Criteria for biological reproducibility: what does “n” mean? Sci Signal, 8 (371), fs7. https://doi.org/10.1126/scisignal.aab1125

40. Blainey, P., Krzywinski, M., and Altman, N. (2014). Replication: Quality is often more important than quantity. Nat Meth, 11(9), 879-880. https://www.nature.com/articles/nmeth.3091.pdf

41. Vaux, D.L., Fidler, F., and Cumming, G. (2012). Replicates and repeats – what is the difference and is it significant? EMBO reports, 13(4) 291-296. https://dx.doi.org/10.1038%2Fembor.2012.36

42. Nagele, P. (2003). Misuse of standard error of the mean (SEM) when reporting variability of a sample. A critical evaluation of four anaesthesia journals. Br J Anaesth, 90(4), 514–516. https://doi.org/10.1093/bja/aeg087

43. Lee, D.K., In, J., and Lee, S. (2015). Standard deviation and standard error of the mean. Korean J Anesthesiol, 68(3), 220-223. https://doi.org/10.4097/kjae.2015.68.3.220

44. Journal of Biological Chemistry. Collecting and Presenting Data, (Acc. August 27, 2020).


https://doi.org/10.1126/scisignal.aab1125

https://www.nature.com/articles/nmeth.3091.pdf

https://dx.doi.org/10.1038%2Fembor.2012.36

https://doi.org/10.1093/bja/aeg087

https://doi.org/10.4097/kjae.2015.68.3.220

http://jbcresources.asbmb.org/collecting-and-presenting-data

45. Nature. Instructions for Authors of Research Articles (Initial Submission), (Acc. August 27, 2020).


https://www.sciencemag.org/authors/instructions-authors-research-articles-initial-submission

XI. Normalization

In the context of an In-Cell Western Assay, normalization refers to a method of correcting for small differences in the number of cells between wells. Because some variation in cell number is unavoidable, normalization is a critical part of attaining reproducible In-Cell Western data.

This section explains why normalization is necessary, which normalization methods are available, and how to use these methods to get accurate data from your In-Cell Western Assay.

Why Normalization is Necessary

Ideal World

Imagine an In-Cell Western Assay where each well in the plate contained exactly the same number of cells. Normalization would not be necessary for this imaginary experiment.

If you were to add a cell stain to the wells that was known to produce a signal proportional to the number of cells in the well, then the cell stain signal measured for each replicate well would be the same.

The signal measured for a target protein might be the same in each well, or it might differ based on your experimental treatments or conditions.

Either way, you would be sure that the variation in target signal was due to changes in target levels and not the number of cells.

Sig

nal

Sample 1 Sample 2 Sample 3

Cell Stain

Target

Figure 15. This graph illustrates how signal from a plate in an imaginary experiment would look if each well con-tained an equal number of cells. Although the target protein expression may vary, the number of cells would remain the same.


Real World

However, in the real world, the total number of cells will vary, and the target protein levels can also vary. Therefore, normalization is necessary to account for the different cell numbers and to obtain accurate In-Cell Western results.

Sig

nal

Cell Stain

Target

Sample 1 Sample 2 Sample 3Figure 16. In this illustration of signal detected from sample wells on an In-Cell Western plate, both the total number of cells vary and target protein levels vary.

Normalization Basics

Normalization Concepts

Normalization refers to the use of an internal control to mathematically correct for small, unavoidable differences in the number of cells between wells.

An internal control (IC) refers to endogenous reference proteins that are present in all samples at a stable level and are unaffected by experimental conditions. Signal from detection of a reliable IC should be directly proportional to the number of cells in a well.

The term linear range refers to the range of cell seeding densities that produce a linear, proportional relationship between signal intensity and the number of cells in the well.

Normalization Cannot Account for Everything

Normalization is necessary to account for unavoidable cell number differences. Excessive cell number differences must be minimized, because normalization cannot correct for all types and degrees of variation.


Sources of Variability That Normalization Can Correct

Pipetting inconsistency and edge effects can introduce variation from well to well. Normalization can account for small differences. For normalization to be effective, it is important to quantify cell number before seeding to seed cells as accurately as possible.

Sources of Variability That Normalization Cannot Correct

Normalization cannot account for the following possible sources of error.

l Fixation/Permeabilization: Normalization cannot compensate for variability introduced during the fixation/permeabilization process.

l Inconsistent or inaccurate technique: Care must be taken to ensure accuracy and consistency in lab technique. Normalization can only account for small variations.

Normalization Strategies

There are a couple of primary normalization methods that can be used for In-Cell Western Assays. The different methods either rely on the detection of a constitutively expressed protein or a cell stain. The following methods may work better in some In-Cell Western experiments than in others. It is up to the researcher to determine which strategy is appropriate for given experimental conditions.

Post-Translational Modification Normalization Strategy

The post-translational modification strategy (PTM) is used to monitor changes in post-translational modification of proteins by normalizing a specific modification of a target protein against all target protein, regardless of modification. The target protein is used as its own internal control.

When using the PTM normalization strategy, you are able to answer the question, “How much does the expression of a specific protein modification change compared to the total amount of the protein, regardless of modification?”

PTM normalization can be used for normalization in the study of post-translational modification, where suitable antibodies are available.

PTM normalization employs two primary antibodies raised in different hosts:

l Modification-specific antibody: An antibody against a specific modification of the target.

l Pan-specific antibody: An antibody that recognizes all target regardless of modification.


Spectrally distinct fluorescent secondary antibodies are used to detect the modification-specific and pan-specific antibodies at the same time in the same well (multiplex fluorescent detection).

Validating a PTM Normalization Method

Consider If Epitope Interference Could Be a Problem

Epitope interference may occur if antibodies against different epitopes on the same target interfere with each other's ability to bind the target. If the two antibodies interfere with each other's ability to bind, normalization will not be accurate. Check antibody datasheets to ensure that the two primary antibodies recognize different regions of the protein. The Antibody Publication Database can help you find antibody pairs that work for your experiment (licor.com/antibodyrequest).

Polyclonal pan-specific antibodies are less likely to experience epitope interference. Even if one epitope is blocked, the polyclonal antibody may be able to bind other epitopes.

Depending on the antibodies you are using, you may wish to verify empirically that epitope interference is not occurring. For example, in 2015, Bakkenist et al. (46) demonstrated a Western blot method that can be used to evaluate binding interference between primary antibody pairs. Identical blots were incubated with modification and pan-specific antibodies separately (singleplex), or with both antibodies simultaneously (multiplex). In that experiment, relatively little variation in signal intensity was measured between pan-specific antibody from multiplex and singleplex blots.

Document Validation Procedure and Results

Document your normalization procedure and results to ensure that all experimental details will be available for publication or reproduction of your results. Include lot numbers of all reagents. New lots of the same catalog number should be verified.

In-Cell Western Protocol with Post-Translational Modification Normalization

After ensuring that your normalization procedure will produce accurate results for your particular experiment, follow the standard protocol for an In-Cell Western Assay to detect the modification-specific and pan-specific proteins in the same well.

Cell Stain Strategies

A cell stain is a reagent that binds to cells so extensively that signal detected from the cell stain proportionately represents the number of cells in the well. A cell stain can be used as a tool for normalization. Because cell stains target different classes of biomolecules within a cell (e.g., proteins and/or nucleic acids), error and variability are minimized. In general, LI-COR recommends using cell stain normalization.


http://www.licor.com/antibodyrequest

CellTag™ Stain

LI-COR offers a one-step solution for In-Cell Western Assay normalization using the CellTag Stain (i.e., CellTag 520 Stain and CellTag 700 Stain). CellTag stains accumulate in both the nucleus and cytoplasm of permeabilized cells, and provide a linear fluorescent signal across a wide range of cell types and cell numbers.

CellTag 700 Stain is detected in the 700 nm channel of Odyssey® Imagers and CellTag 520 Stain is detected in the 520 nm channel (available on the Odyssey M), allowing accurate quantification of target protein levels when combined with an IRDye® 800CW Secondary Antibody. See licor.com/celltag for more product information.

CellTag Stain for the In-Cell Western Assays

CellTag Stain requires permeabilization, so it cannot be used for normalization in live cells. CellTag Stain is applied to the cells during the IRDye Secondary Antibody incubation step, enabling accurate quantification of target protein levels in an assay with much higher throughput than Western blotting.

CellTag Stain for On-Cell Western Assays

On-Cell Western assays are similar to In-Cell Western Assays, with the notable difference that the target is detected on the surface of the cell. In some cases, researchers have detected a target on the surface of a cell, then permeabilized the cells and used CellTag Stain for normalization.


https://www.licor.com/celltag

Example Data

0

25

12.5

50

100

200

400

EG

F n

g/m

L

800 ChannelPhospho-EGFR

700 ChannelCellTag 700 Stain

Two-Color

2° Ab 2° Ab

+

CellTag

CellTag 2° Ab CellTag 2° Ab CellTag2° Ab

+

CellTag

2° Ab

+

CellTag

Figure 17. In-Cell Western™ Assay with CellTag™ 700 Stain in EGF-stimulated A431 cells. EGF-stimulated A431 cells were fixed, permeabilized, and blocked with Intercept® Blocking Buffer (LI-COR, P/N: 927-70001). Phos-phorylated EGFR was measured using rabbit anti-phospho-EGFR primary antibody followed by detection with IRDye® 800CW Goat anti-Rabbit IgG (LI-COR, P/N: 926-32211). CellTag 700 Stain (LI-COR, P/N: 926-41090) was used for normalization to cell number. The data demonstrate that phosphorylated levels of EGFR increase with EGF treatment. The plate was scanned on an Odyssey® CLx Imaging System (Resolution: 169 µm; Quality: low-est; Focus offset: 3.5 mm; Intensity: Auto for both channels).

Sapphire700 and DRAQ5

Sapphire700™ Stain and DRAQ5™ Stain are cell staining agents that can be used in combination for accurate normalization across the same range of cell densities as CellTag 700 Stain. Simultaneous staining of cells with Sapphire700 and DRAQ5 expands the linear range, allowing more accurate normalization of cell number across both low-and high-cell densities. Similar to CellTag 700, these stains are detected in the 700 nm channel of Odyssey® Imagers, allowing accurate measurement of target protein levels in the 800 nm channel when combined with an IRDye® 800CW Secondary Antibody.

l Sapphire700 Stain is a non-specific cell stain that accumulates in both the nucleus and cytoplasm of fixed or dead cells, but not live cells. When used to stain serial dilutions of A431 cells in 96-well plates, Sapphire700 displays linearity of fluorescent signal for higher cell densities, from ~50,000 to ~250,000 cells/well.

l DRAQ5 Stain is a cell-permeable DNA-interactive agent which can be used for stoichiometric staining of DNA in live or fixed cells. For more information about DRAQ5 Stain, please visit the Biostatus Limited web site (biostatus.com). When serial dilutions of A431 human epithelial carcinoma cells are plated in 96-well plates, DRAQ5 Stain alone demonstrates linearity of fluorescent signal for lower cell densities, from ~3,000 to ~50,000 cells/well in the 700 nm channel for normalization.


http://www.biostatus.com/

Cell Stain Selection Guide

There are many things to consider when selecting the right stain for your application. Below is a list of some questions to ask when choosing the correct stain for your application.

l What is the excitation and emission spectra for the stain?

l Does your stain work for your organism and/or cell type of interest?

l What is the cellular target and/or mechanism of action?

l Is the stain compatible with your fixation and permeabilization methods?

l Is the stain cell permeable?

l Does the dye stain live cells? Dead cells? Or both?

l Is the detection range compatible with your experimental design?


Selection Guide for LI-COR Supported Dyes

StainEx/Em (nm)

Detection Range (cells/well) *

Cell Permeable

Localization Comment

CellTag™ 700 Stain

675/697 200 – 100,000

No Nucleus/Cytoplasm Wide linear range

Cost-effective

Easy to use

Sapphire700™ Stain/DRAQ5™

675/697 3,000 – 250,000

No Nucleus/Cytoplasm Requires two stains

Easy to use

IRDye® NHS Esters

689/700

773/792

200 – 200,000

No Nucleus/Cytoplasm

If cells are not permeabilized, the cell surface will be stained.

If cells are permeabilized, the nucleus and cytoplasm will be stained.

Use for On-Cell Westerns and In-Cell Westerns

Provides flexibility for which channel you use for normalization

Requires two additional steps

Cost-effective

Wide linear range

High sensitivity at lower cell numbers

* The number of cells you use in your experiment will depend on your experimental conditions (experimental conditions can change size/morphology of cells) and the cell line that you are using.

Normalization References

46. Bakkenist, C.J., Czambel, R.K., Hershberger, P.A., Tawbi, H., Beumer, J.H., and Schmitz J.C. (2015). A quasiquantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX Phosphorylation in human peripheral blood mononuclear cells. Oncoscience, 2(5), 542–554. https://doi.org/10.18632%2Foncoscience.162

47. Hoffman, G., et al. (2008). A functional siRNA screen for novel regulators of mTORC1 signaling. Poster presentation, American Society for Cell Biology Annual Meeting.


https://doi.org/10.18632%2Foncoscience.162

XII. Experimental Controls

The breadth and power of the In-Cell Western Assay is highly dependent on the chosen antibodies, optimization of each step, and careful consideration and selection of controls. Accurate experimental analysis relies on the framework of data established by the controls.

Control Types and Well Types

Well Type Primary

Antibody

Treatment Positive

Sample*

Negative

Sample**

Cells Normalization

Reagent

PBS

Buffer***

Blank

Control

Secondary

Antibody

Background

Cell Only

Background

Normalization

Control

Positive

Control

Negative

Control

Secondary

Antibody

Sample Sometimes

l Blank control wells contain only PBS buffer and are used to mitigate the potential impact edge effects can have on wells located near the outside of plate.

l Secondary antibody background control wells contain secondary antibody without primary antibody or normalization reagent. This control accounts for signal caused by binding of a secondary antibody to material other than the target. By eliminating the primary antibody, any binding of secondary antibody caused by cross-reactivity to cellular antigens or to charged groups created during the fixation process is exposed. If you are using more than one primary antibody, this control will also differentiate secondary antibody binding to its appropriate primary versus cross-reactivity between the multiple secondary antibodies. This control should be included on every plate.


l Positive control wells contain primary and secondary antibody. Depending on your experiment, positive controls could be an over-expressed cell line, a treatment known to stimulate target expression, etc.

l Negative control wells contain primary and secondary antibody. Depending on your experiment, negative controls could be a CRISPR knockdown, a treatment known to inhibit target expression, etc.


XIII. Z’-Factor Determination

During In-Cell Western Assay development, it is important to assess the overall quality and reliability of the assay. The Z’-factor statistic provides a way to evaluate whether or not assay conditions (reagents, protocols, instrumentation, kinetics, and other conditions not directly related to the test compounds) are optimized. Z’-factor, introduced by Zhang et al., is a dimensionless value that represents both the variability and the dynamic range between two sets of sample control data.

Z’-factor experiments are performed on one or more In-Cell Western Assay plates containing replicate wells designated for background subtraction, negative control samples, and positive control samples. A Z’-factor assay can be performed at any point during the assay development process.

Experimental Overview

As with any statistical parameter, the accuracy of the Z’-factor value improves with larger data sets used for the calculation (see Replicates on page 60). For a quality assessment, 3 - 4 plates should be run on different days. Wells can be arranged as desired, but a typical arrangement may look like the one shown in Figure 18.

++ -- bkgbkg ++ -- bkgbkg ++ -- bkgbkg ++ -- bkgbkg








Z’-Factor Determination Plate Template

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

Figure 18. This typical Z'-Factor Plate Layout is the same design that is used in the [Preset] Z-Factor Determin-ation 1 Plate Template provided in the Empiria Studio® Software Template Library. Open the Plate Template in Empiria Studio for more information.


Guidelines for the Z'-Factor Experiment

l All reagents (apart from test compounds and reagents), antibodies, materials, and assay conditions should be the same as those that you intend to use for the final In-Cell Western Assay.

l Background wells must be included on the assay plate to account for nonspecific secondary antibody binding (see the Experimental Controls section on page 72).

l Be sure to correct for well-to-well variation by normalizing (see the Normalization section on page 64).

l When choosing treatment compounds to be used for Z’-factor analysis (i.e., stimulants, inhibitors, drugs, etc.), select well-characterized agents which give the best known response in an appropriate cell line.

Z'-Factor Calculation Overview

The Z'-factor value can be calculated manually from normalized signal intensity values, or you can use the Z'-factor calculation in Empiria Studio® Software or Image Studio™ Software.

Z'-Factor Equation

This is the equation for calculating the Z'-factor and a list of the terms in the equation.

Where:

= Standard deviation of positive controls

= Standard deviation of negative controls

= Mean of positive controls

= Mean of negative controls


Z'-Factor Interpretation

The Z'-factor equation can also be written as follows.

Where:

represents the assay dynamic range

is the separation band between positive and negative control signals.

Z'-factor values are always 1 or less as described.

Data Variability Band

Data Variability Band

Separation Band

Sample Number0 96

Sig

nal

3σc-

3σc+

Figure 19.

Z’ = 1 Indicates an ideal assay. As standard deviations become very small or the difference between signals for positive and negative controls approaches infinity, Z’-factor approaches 1.

1 > Z’ ≥ 0.5 Indicates a high-quality assay exhibiting a wide separation between signal and background, and low data variability.

0.5 > Z’ > 0 Indicates a poor-quality assay with marginal distinction between signal and background, and higher data variability.

Z’ ≤ 0 Indicates unreliable data. Assay conditions are not optimized or the assay is not capable of generating meaningful data.


Z’ = -1 There is no distinguishable difference between background signal and sample signal. If the lower limit of detection (LLD) is expressed as the mean of the background signal plus three SD of the background signal, then the measured signal is equal to the LLD of the instrument.

Z'-Factor Summary

The Z’-factor experimental guidelines described above can be utilized to evaluate In-Cell Western Assay quality at any time throughout assay development, or near completion of assay optimization. Z’-factor measurement is also useful for monitoring assay quality over time, from user to user, or when conditions or reagents within the assay must be changed.


XIV. Frequently Asked Questions

General

Question Answer

What is the In-Cell Western Assay?

The In-Cell Western is a quantitative IF assay performed in microwell plates (optimized for 96- or 384-well formats). Data analysis is based on whole-well fluor-escence quantification. The In-Cell Western Assay offers a convenient alternative to Western blotting and is a powerful platform for meaningful in situ analyses.

What is the difference with the antigen conformation between In-Cell Western and Western blot?

In a Western blot, the antigen is in its denatured state, and the proteins are sep-arated by size. In an In-Cell Western, the antigen is in a more native conformation and the proteins are not separated by size.

What are the advantages of the In-Cell Western vs IF?

An In-Cell Western is a higher-throughput assay that allows for more samples to be quantified quickly in a plate based format.

What are the advantages of the In-Cell Western vs ELISA?

The ELISA can only screen for 1 target, depends on the enzymatic reaction for chemiluminescence detection, requires a standard curve for quantification.

Why should I use the In-Cell Western over flow cytometry?

The protocol and data analysis for the In-Cell Western is more streamlined than the flow cytometry protocol. The In-Cell Western lacks information on cell size and granularity.

What is the difference between In-Cell Western and On-Cell Western?

In the In-Cell Western, the cell is permeabilized to allow targeting proteins that are present inside of the cell. In an On-Cell Western, the target protein is present on the outer membrane.

What are some common applic-ations for this assay?

Some common examples of the use of the In-Cell Western are: studies of signaling pathways, detection of post-translational modifications such as protein phos-phorylation, timing/kinetics of signal transduction, IC50 determination, GPCR activ-ation, protein accumulation and inhibition, viral titer/load, apoptosis, cell surface proteins and receptor internalization, RNAi efficacy, cell proliferation assays, and targeted therapeutic development.

What specific reagents do I need to get started?

Black walled, flat bottom plates of 96 or 384 wells, fixation and permeabilization reagents (typically PFA and Triton™ X-100), blocking buffer (Intercept® Blocking Buffer), primary antibody, secondary antibody (IRDye® 800CW), and normalizing stain (CellTag™ 700 Stain or CellTag 520 Stain).


Assay Development

Question Answer

What time point should I use for this assay?

The time point for treatment will depend on previous experimental data that indic-ates the time point that produces the desired result.

Can I perform In-Cell Western on organoids?

Yes, the In-Cell Western is compatiable with organoids as well as differentiated cells.

What steps and reagents need to be optimized?

Every step of any assay can be optimized, the steps and reagents that are com-monly optimized for the In-Cell Western are the cell seeding, blocking buffer eval-uation, fixation/permeabilization, and primary antibody concentration.

In what order should the steps be optimized?

The preferred order in which steps will be optimized will be dependent on the par-ticular assay but as a rule of thumb, we typically develop the protocol in the order each step takes place.

What are some common chal-lenges with technique?

The most common challenges with the technique for the In-Cell Western is pipet-ting too far into the well and removing the cells, shaking the plate too fast, and not optimizing reagents/conditions.

Can the In-Cell Western be per-formed with bacteria and yeast?

The In-Cell Western can be performed on bacteria and yeast, however, there are a few additional modifications to the protocol to account for the presence of a cell wall.

Can I use the In-Cell Western for secreted proteins?

No, the In-Cell Western cannot be used for secreted proteins as they are washed away during the wash steps.

What type of controls do I need? When optimizing the reagent conditions, include a secondary antibody back-ground control (no primary antibody or CellTag™), a negative control, and a pos-itive control.

What should I do if I don't see a difference between my positive and negative controls?

Confirm that there are different expression levels between the positive and neg-ative control in another assay, such as a Western blot. Proceed with further pro-tocol development.

How do I prevent my cells from clumping in the center of the well?

Homogenize the cells carefully before seeding and incubate for 1 hour at room temperature before placing in the incubator. Prevent cell over growth.

Can I strip a plate and reprobe for other targets?

Stripping and reprobing of plates has not been validated by LI-COR.


Cell Seeding

Question Answer

How do you handle suspension cells?

Grow and treat suspension cells in the appropriate growth conditions, and add them in the flat bottom plate at the time of fixation. In some cases, the treatment can be performed with the cells already seeded in the flat bottom plate.

What number of cells should my plate have?

The number of cells to load in each well varies based on the cell line. Typically, 5,000 to 40,000 cells are seeded per well. One to three days are required for cells to reach the appropriate confluency, depending on growth rate and cell size. Seed-ing with lower cell numbers is recommended if you plan to culture for several days before use. Choose a cell seeding amount where the signal is in the linear range.

Can I use the In-Cell Western for primary/differentiated cell cul-tures?

Yes, when working with primary cell lines prevent contamination, maintain less than 100% confluency, limit the length of times the cells are exposed to trypsin, and maintain growth conditions (such as pH, temperature, etc.).

How many cells do I need to have per well to see signal?

The number of cells is only one contributing factor to the signal in the well. Cell line, antibody specificity, protein expression level, and other factors will contribute to the signal. Therefore, those conditions need to be determined empirically.

Fixation and Permeabilization

Question Answer

What are the most common fix-ation and permeabilization reagents?

The most common fixation and permeabilization reagents are 3.7% formaldehyde and 0.1% Triton™ X-100, respectively.

When optimizing permeabilization and blocking: which concentration of primary antibody do I have to use?

Use the recommended primary antibody concentration as indicated on the data sheet from the manufacturer for IF.

Primary Antibody

Question Answer

How do I select which primary antibody to use?

Choose a primary antibody that is validated for IF/ICC/IHC. You can reach out to LI-COR for suggestions of primary antibodies based on published data, and val-idate the antibodies using Western blot and IHC.

How do I validate my primary anti-body?

Confirm antibody specificity through Western blot looking for the size of your tar-get protein, absence of non-specific bands, and appropriate expression levels of tar-get protein in positive and negative controls. Use IHC/IF to confirm that the protein localizes to the appropriate cellular location and to determine the concentration of the primary antibody.


Normalization

Question Answer

What are the options for nor-malization?

The signal for the target protein can be normalized against a cell stain, such as CellTag™ 520 Stain or CellTag 700 Stain. Post translational modifications can be analyzed with the In-Cell Western. The phopshorylated target is normalized against the pan or total protein signal.

How should I normalize in an On-Cell Western Assay?

The target signal can be normalized against a cell stain (such as CellTag 520 Stain or CellTag 700 Stain) after the plate has been imaged for the primary target of interest, because the cells will need to be permeabilized at this point.

Can I use Revert for In-Cell Western normalization?

No, Revert cannot be used for an In-Cell Western normalization. We recommend using a total cell stain, such as CellTag 520 Stain or CellTag 700 Stain.

Wash Step in the Protocol

Question Answer

How do I wash the cells? The cells can be washed manually by gently pipetting the wash solution down the inner wall of the plate or using an automated plate washer.

Can I use an automated plate washer?

Yes, when using an automated plate washer ensure that the pressure is low and does not wash away the cell from the well.

Is it better to remove solution from the well by pipetting or by vacuum aspiration?

Both methods have their advantages and disadvantages and should be chosen based on your research needs.


Plates

Question Answer

What plates should I use? Use black walled, clear flat bottom plates.



Do I have to use the recom-mended Greiner plates?

No, you do not have to use the Greiner plates. Plates with any number of wells can be used. If you choose to use another plate, be sure that the plate has a clear bot-tom and has black walls. The focus offset must be optimized by scanning the plates at several focus offset settings and choosing the focus offset that yields the highest signal-to-noise ratio.

Can I use treated/coated plates? For adherent cells that could detach from wells during In-Cell Western Assay wash steps (e.g., NIH3T3), we recommend Poly-D-lysine coated microwell plates.


Imaging Microwell Plates

Question Answer

How do I determine the correct focus offset for my plate?

The Multiwell Plate imaging workflows in LI-COR Acquisition Software are setup with recommended imaging parameters for LI-COR imagers. For more information, see the LI-COR Acquisition Software Help (licor.com/AcquisitionQuickStart) or the Operator's Manual for your imager. Be sure to determine the correct focus offset if you are not using the recommended plates (see "Plates" on the previous page) with your Odyssey Imager.

To manually determine a focus offset, scan the plate at various focus offsets and choose the focus offset that yields the highest signal for the target protein. Most plates have a focus offset between 3.5 - 4.0 mm.

How would I know from looking at an image that the focus offset is incorrect?

If the focus offset is incorrect, the image will appear fuzzy and there will be a ring of signal on the outer edges of the wells.

Why is there a halo on the edge of the wells in my image?

There could be several causes for this effect. The focus offset may not be correct and the plate is being scanned out of focus. The cells may be accumulating at the edges of the well. If white or transparent walled plates are used, then the plate is exhibiting autofluorescence.

What settings should I use to acquire images?

The Multiwell Plate imaging workflows in LI-COR Acquisition Software are setup with recommended imaging parameters for LI-COR imagers and the recom-mended plates (see "Plates" on the previous page). For more information, see the LI-COR Acquisition Software Help (licor.com/AcquisitionQuickStart) or the Operator's Manual for your imager.

I see cells under a microscope but no signal on the plate what does that mean?

Confirm that the plate is imaged using the correct focus offset. If the focus offset is correct, optimize reagents and conditions. Include CellTag™ 700 Stain as a positive control.

How much liquid should be in the plate when I image?

Plates should be inverted and blotted dry and then sealed prior to imaging and stor-age.

Why is my dilution series (i.e., cell seeding density, drug treatment, etc.) not giving a linear signal?

The protocol likely needs to be further developed to ensure you are operating within the linear range of the assay. See Cell Stain Linearity on page 22 for more information about cell stain linearity. Empiria Studio® has a Cell Stain Linearity work-flow and a Preset Plate Template for analyzing a Cell Stain Linearity experiment. See the Empiria Studio Software Help (licor.com/AnalysisQuickStart) for more inform-ation about using Plate Templates for analylsis.




https://www.licor.com/AnalysisQuickStart

Miscellaneous

Question Answer

How long can I store the plates after completion of the In-Cell Western experiment?

Store at 4 °C and sealed in a light tight container for several weeks.

How can I know if my assay is properly developed?

The Z'-factor can confirm robustness of the assay.

See Z’-Factor Determination on page 74 for more information about the Z'-factor. Empiria Studio has a Z'-Factor Determination workflow and a Preset Plate Template for analyzing a Z'-factor experiment. See the Empiria Studio Software Help (licor.com/AnalysisQuickStart) for more information about using Plate Templates for analylsis.

How do I analyze the data? See the Empiria Studio Software Help (licor.com/AnalysisQuickStart) for more inform-ation about using Plate Templates for analylsis.

Has In-Cell Western data been published?

Yes, there are >250 publications that use the In-Cell Western Assay.

What could lead to speckling on the plate?

Lint or dust contamination can come from the working surface or scan area and can lead to speckling.

What could lead to speckling in the wells?

Speckling could be caused by antibody aggregation. Spin down the antibody tube and sterile filter the primary and secondary antibody solution.

What is a good stopping point in the In-Cell Western protocol?

The fixation step may work as a stopping point for your protocol.

Can the plates be heated to dry-ness or taken from the fridge immediately before imaging?

Temperature can affect fluorescence and reading at inconsistent temperatures can cause results to vary.

Which free dye should be used to quantify total cell number in the 700 nm channel?

IRDye® 680RD and IRDye 680LT work but, IRDye 680LT is very bright compared to 680RD. Dilution optimization is necessary.




XV. Plate Loading Guides

To use the following plate loading guides, cut out the layout, then place the layout underneath the plate to make it easier to load samples in the correct wells. Plate layouts may also be created and printed from Empiria Studio® Software.

Note: More Plate Loading guides are available at licor.com/plate-loading-guides.

Plate Layout for Loading Antibody Dilutions

The following plate layout can be used as a guide for loading antibody dilutions according to the Plate Layout shown in "Single Antibody Titration Experiment" on page 39.

A

Dilution 1 Dilution 2 Dilution 3

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

bkg

bkg

bkg

bkg

-

-

-

-

+

+

+

+

+

+

+

+

-

-

-

-

bkg

bkg

bkg

bkg

+

+

+

+

-

-

-

-

bkg

bkg

bkg

bkg

Symbol Cells Primary Antibody Secondary Antibody CellTag™ 700 Stain

bkg

+

-


https://www.licor.com/plate-loading-guides

Blank Plate Loading Guides

Label the following blank Loading Guides as needed for your In-Cell Western Assay.

Note: More Plate Loading guides are available at licor.com/plate-loading-guides.

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12


https://www.licor.com/plate-loading-guides

XVI. Empiria Studio® Software Multiwell Plate Calculations Reference

Explanation of Analysis Table Data on the Quantify Wells Page

You can view Well Quantification data by clicking Analysis Table on the Quantify Wells page.

Information in the Analysis Table is divided into two main categories: Well information and Replicate information.

Well Information

Well information is information for specific wells in the microwell plate image. The Wells tab contains a thorough list of this information. The Total tab, Background tab, Signal tab, and Normalized Signal tab contain focused subsets of well information to help you find the information you need at a quick glance. Click to export all the Plate Analysis table data. If you export to the Excel format, each tab for each channel will be in different sheets in the exported Excel workbook.

Well information can be divided into Well Metadata and Well Calculations.

Well Metadata

l Well ID – A label for the well that shows which row and column the well is in. The letter indicates which row the well is in and the number indicates which column the well is in.

l Well Type – Sample, background, or control from the Template.

l Well Value – This is the value for a Well entered in the Template Editor.

l Well Name – This is the name text for a Well Type entered in the Template Editor.


l Well Description – This is the description text for a Well Type entered in the Template Editor.

l Group Attribute - This is the attribute text for the Well Group that the well is part of. The Group Attribute is entered in the Template Editor.

l Group Name - This is the name text for the Well Group that the well is part of. The Group Name is entered in the Template Editor.

Well Calculations

l Total – Sum of the pixel intensity values in a well.

l Signal – Sum of the individual pixel intensity values (Total) for a well minus the Background value.

l Normalized Signal – Signal in a target channel divided by the internal loading control Signal detected in the normalization channel.

l Background – The Background value shown for a well is the Average of the Total values of all Background wells in the well’s Well Group.

Replicate Information

The Replicate tab in the Analysis Table has information for Well Replicates (also called Well Sets).

Figure 20. Replicate tab in the Analysis Table.

l Avg. Total – Average of the Total values for all the wells in a Well Set.

l Background – See the definition in Well Calculations above.


l Avg. Signal – Average of the Signal values for all wells in the Well Set.

l Signal Std. Dev. – Standard deviation of signal values for all wells in the Well Set.

l Signal % CV – Percent coefficient of variation for all the well Signal values in the Well Set.

l Avg. Norm. Signal - Average of the Normalized Signal values for all the wells in the Well Set.

l Norm. Signal % CV – Coefficient of variation for all the well Normalized Signal values in the Well Set.

l Norm. Signal Std. Dev. – Standard deviation of the Normalized Signal values for all wells in the Well Set.

l SNR – Signal-to-noise ratio for a Well Set.

Set Up Chart

In-Cell Western

Target Analysis, Antibody Titration, Blocker Evaluation, Fixation and Permeabilization, and Target Analysis

Normalization

The Normalization values for the Normalization chart type are the average of the Normalized Signal values for replicate wells in a Well Set.

Fold Change

The fold change is the ratio of the average Normalized Signals from a treatment Well Set to the average Normalized Signals in a control Well Set. Select the control for a Fold Change chart type on the Set Up Charts page.

Percent Change

The Percent change is the fold change in percent format.


Z’-Factor Determination Experiment

Follow steps on the Z’-Factor Experiment workflow to the Set Up chart page to view the Z’-factor value and chart.

Z’-Factor Calculation

The Z'-factor value is calculated from the standard deviation and the mean of normalized signal values from positive and negative control wells using the following equation. For the Z’-Factor Templates, all wells are in a single Well Group.

This is the equation for calculating the Z'-factor and a list of the terms in the equation.

Where:

= Standard deviation of positive controls

= Standard deviation of negative controls

= Mean of positive controls

= Mean of negative controls

Required Wells

In-Cell Western

All calculations, except Total and Avg. Total, require at least two background wells to be defined in the Well Group in the Plate Template and included in the analysis.

Analysis Table Well Tab Calculation Examples

The example calculations in this section are based on the following simple Plate Template. This template does not represent a recommended experimental design. The following example is simplified to one Well Group for illustrative purposes.


Total (800 nm Channel)

6 7 8C 300 200 100D 310 210 110E 320 220 120F 330 230 130

Background (800 nm Channel)

This is the background value for the Well Group that will be used as the background value for calculating signal in for all the wells in the group.

Note: You can set up a Plate Template so that one background value can be used for multiple Well Groups. To do this, select Include all backgrounds on the Setup Well Groups page of the Template, then create groups. The average Total value from all background wells will be used as the background value for Well Groups created when the Include all backgrounds option is selected.

Signal (800 nm Channel)

An example signal calculation in shown in C7. The other Positive and Negative wells are also calculated by subtracting the Total value for the well minus the Average Background value.


6 7

C 185200 – 115 =

85D 195 95E 205 105F 215 115

Normalized Signal

The Normalized Signal for a target channel is calculated by dividing the well Signal in the given target channel from the well Signal in the normalization channel. In this example, the 700 channel is the normalization channel.

Signal (700 nm Channel)

Signal for the normalization channel (in this example, 700 nm channel) is calculated the same way as Signal is calculated for the target channel (in this example, 800 nm channel). The Signal for the 700 nm channel is shown here for reference, but calculations are omitted for brevity.

6 7C 400 300D 410 310E 420 320F 430 330

Normalized Signal (800 nm Channel)

6 7

C 0.46385 ÷ 300 =

0.283D 0.476 0.306E 0.488 0.328F 0.500 0.348

Set Up Chart Page Calculation Examples

Examples in this section are based on the Normalized Signal values shown in the previous section.

Well Sets vs Well Groups

To understand how values displayed on the Set Up Charts page are calculated, it is helpful to distinguish the terms “Well Set” and “Well Group”.


l Well Set – Replicate Well Types within a Well Group.

o For example, all negative control wells inside a Well Group comprise a Well Set.

l Well Group – Experimental group on a microwell plate that includes well types linked for analysis. It may help to think about Well Groups as individual experiments.

o For example, positive control, negative control, and background wells that receive the same secondary antibody dilution in an antibody titration experiment constitute a Well Group.

Normalization

Fold Change

To demonstrate fold change calculations, we will switch examples. The antibody titration example with one well group is not sufficient for this section.


Plate Template

Example Data and Calculation

Control Normalized Signal

C 10

D 20

E 30

F 40

Average 25

Treatment Normalized Signal

C 20

D 40

E 60

F 80

Average 50


Percent Change


XVII. Glossary

A

Adherent cellsSingle layer of cells attached to a surface.

AffinityBinding strength between the antibody’s binding site and its epitope.

AntigenA substance stimulates the production of and binds to antibodies.

B

Biological replicateA test performed on biologically distinct samples representing an identical time point or treat-ment, used to control for biological variation and to determine if the experimental effect is bio-logically relevant.

Blocking bufferA solution (often consisting of proteins) that reduces nonspecific antibody binding by binding to areas in a well that are not occupied by cells and by binding to nonspecific interaction sites in/on cells.

C

Cell confluencyPercentage of a container surface covered by adherent cell.

Cell cultureGrowing cells in a controlled artificial environment.

Cell seedingProcess of pipetting a specific number of cells into each well of a microwell plate.

Cell stainA cell stain (or whole cell stain) is a label that binds to components of a cell to provide a signal readout that is proportional to the number of cells in a sample for normalization.

D

DMSODimethyl Sulfoxide. DMSO is an organic solvent. It is also used as an excipient for many drugs and other active compounds (including IRDye NHS Esters).


E

EpitopeA part of the antigen to which the antibody directly binds.

Experimental groupA set of samples in an experiment (e.g., controls, replicates, treatments) that serve the same pur-pose in the experimental design.

F

FixationChemically preserving biological specimens to limit degradation, prevent microbial con-tamination, and to adhere cell monolayers to a glass or plastic surface.

FixativeChemicals that preserve biological specimens by limiting enzymatic degradation and preventing microbial contamination.

I

Internal loading controlEndogenous reference protein(s) that are present in all samples at a stable level and are unaf-fected by experimental conditions. Signal from detection of a reliable ILC should be directly pro-portional to the number of cells in a well.

L

Linear rangeThe range of detection that produces a linear, proportional relationship between signal intensity and target quantity (e.g., protein concentration or cell number).

N

NormalizationUse of an internal loading control to mathematically correct for small, unavoidable differences in the number of cells between wells.

Normalized signalThe normalized signal for each well is calculated by dividing the target signal for each well by the normalization factor for that well.

P

PermeabilizationUsing solvents or detergents to create openings in the cell membrane so that antibodies and stains can gain access to intracellular targets.


S

SelectivityAbility to preferably bind the target antigen in the presence of other antigens.

SignalSum of the individual pixel intensity values (Total) for a quantification shape minus the back-ground value.

SpecificityAbility to recognize the target antigen.

Suspension cellsCells free-floating in a medium.

T

TargetThe protein you are interested in studying.

Technical replicateA test performed on the same sample multiple times to establish the variability of a protocol or assay and to determine if an experimental effect is large enough to be distinguished from noise.


LI-COR Biosciences Regional Offices

4647 Superior StreetLincoln, NE 68504Phone: +1-402-467-0700Toll free: [email protected]

licor.com/bio

LI-COR Biosciences GmbH

Siemensstraße 25A61352 Bad HomburgGermanyPhone: +49 (0) 6172 17 17 [email protected]

LI-COR Biosciences UK Ltd.

St. John’s Innovation CentreCowley Road • CambridgeCB4 0WS • United KingdomPhone: +44 (0) 1223 [email protected]

© 2021 LI-COR, Inc. LI-COR, In-Cell Western, Odyssey, CellTag, Intercept, IRDye, Revert, Sapphire700, Empiria Studio, and Image Studio are trademarks or registered trademarks of LI-COR, Inc. in the United States and other countries. All other trademarks belong to their respective owners.

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in-cell western™ assay development handbook

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