ISOLATE MONONUCLEAR CELLS DIRECTLY FROM WHOLE BLOODWith EasySep™ Direct

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Obtaining peripheral blood mononuclear cells (PBMCs) from blood samples with traditional methods, such as density gradient centrifugation, can be time consuming and difficult to automate. In addition, isolating cells from older blood samples can result in red blood cell (RBC) and granulocyte contamination, requiring additional RBC lysis and centrifugations.

Why Use EasySep™ Direct Human PBMC Isolation Kit?

FAST. Isolate human PBMCs in as little as 20 minutes without the need for lysis or centrifugation.

EASY. Avoid laborious and time consuming methods such as density gradient centrifugation and RBC lysis.

HIGH PURITY. Obtain highly purified mononuclear cells, even on older blood samples.

CONVENIENT. Automate blood sample processing with RoboSep™ instruments to increase laboratory throughput.

FLEXIBLE. Obtain cells from whole blood, cord blood, bone marrow, buffy coats, leukapheresis products, and leukocyte reduction system (LRS) cones.

How Does It Work?

Pour off sample into a new tube, add RapidSpheres™ and mix.

Add EasySep™ Direct Human PBMC Isolation Cocktail and mix well.

Add recommended medium to top up sample. Add RapidSpheres™ to sample and mix.

Place the tube into the magnet and incubate for 5 minutes*.

Incubate for 5 minutes

*Times will vary depending on the EasySep™ magnet platform that is used.

Place the tube into the magnet and incubate for 5 minutes*.

Pour off sample into a new tube.

Repeat steps 5 and 6. Cells are now ready for downstream applications.

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Sample Protocol Video: www.stemcell.com/easysepdirectvideo

Using EasySep™ Direct Human PBMC Isolation Kit (Catalog #19654), you can immunomagnetically obtain PBMCs without lysis or centrifugation in as little as 20 minutes. The resulting cells are highly purified with no RBC contamination, even on older samples (Figure 1 and 2).

To meet all of your laboratory needs, the EasySep™ Direct Human PBMC Isolation Kit can be used on:

• Whole blood• Cord blood• Bone marrow• Buffy coats• Leukapheresis products• Leukocyte reduction system (LRS) cones

Increase sample processing throughput by automating your PBMC isolation with RoboSep™ instruments. Automation can minimize sample handling and free up valuable hands-on time (Table 1, page 3).

2 STEMCELL Technologies Inc.

ISOLATE MONONUCLEAR CELLS DIRECTLY FROM WHOLE BLOOD

PBMC Isolation with EasySep™ DirectSkip centrifugation and isolate PBMC immunomagnetically directly from whole blood with EasySep™ Direct Human PBMC Isolation Kit. With immunomagnetic cell isolation PBMCs are separated based on cell surface markers rather than cell density, resulting in highly purified PBMCs and fewer contaminating cells, even when processing older blood samples (Figure 2C).

Figure 1. Typical EasySep™ Direct Human PBMC Isolation Profile

(A) Starting with human whole blood from normal healthy donors, the typical mononuclear cell content of the non-lysed final isolated fraction is 98.3 ± 2.8% (gated on CD45). In the above example, the mononuclear cell content of the whole blood start sample (lysed by ammonium chloride) and non-lysed final isolated fraction is 27.0% and 98.6% (not gated on CD45), respectively. (B and C) Mononuclear cells were isolated from whole blood samples using either density gradient centrifugation or EasySep™ Direct Human PBMC Isolation Kit. (B) Representative FSC vs. SSC flow cytometry plots. (C) Representative t-SNE plots of PBMCs stained with 19 markers and analyzed with mass cytometry (CyTOF). Cells are clustered and colored based on the combination of markers they express. Both density gradient centrifugation and EasySep™ Direct Human PBMC Isolation Kit resulted in comparable cell populations with the exception of the contaminating granulocyte population (CD16+ CD15+ CD11b+ CD66b+ CD45low).

Figure 2. EasySep™ Direct Human PBMC Isolation Kit Results in Fewer Contaminating Cells Compared to Density Gradient Centrifugation

Mononuclear cells were isolated from whole blood samples using either density gradient centrifugation or EasySep™ Direct Human PBMC Isolation Kit. Cells were counted and analyzed by flow cytometry. (A) Both density gradient centrifugation and EasySep™ Direct Human PBMC Isolation Kit resulted in an equivalent total number of nucleated cells recovered from 24-hour-old blood samples (mean ± SD; n=14). (B) Using EasySep™ Direct Human PBMC Isolation Kit to obtain mononuclear cells from 24-hour-blood samples resulted in lower numbers of residual platelets (CD41+), red blood cells (Glycophorin A+/CD45−), and granulocytes (CD66b+) compared to density gradient centrifugation (mean ± SD; n=15). (C) Cell isolation from 24-, 48-, 72-, and 96-hour-old blood samples using EasySep™ Direct Human PBMC Isolation Kit resulted in lower numbers of residual granulocytes compared to cell isolation using density gradient centrifugation (mean ± SD; n=3).

Figure 3. PBMCs Isolated with EasySep™ Direct Human PBMC Isolation Kit Proliferate and Maintain High RNA Integrity

Mononuclear cells were isolated from whole blood samples using either density gradient centrifugation or EasySep™ Direct Human PBMC Isolation Kit. (A) Isolated mononuclear cells were used for downstream RNA isolation. There was no significant difference in RNA integrity as measured with the Agilent RNA Bioanalyzer (mean ± SEM, n=3). (B) Isolated mononuclear cells were labeled with Proliferation Dye eFluor 450 and stimulated with ImmunoCult™ Human CD3/CD28 T Cell Activator and 0.5 ng/ml IL-2. After 4 days in culture, cells were analyzed for proliferation by flow cytometry. Representative histogram showing dividing cells (eFluor 450low).

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Functional Data – Whole BloodT Cell Proliferation Data

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NK cells - CD45+ CD16+ CD56+ CD8+ CD11blow

B cells - CD45+ CD19+ HLA-DR+

CD8 T cells - CD45+ CD3+ CD8+ CD25low

Antigen presenting cells - CD45+ CD19+

HLA-DR+ CD16+ CD123low CD4low

T cell - CD45+ CD3+

CD4 T cells - CD45+ CD3+ CD4+

Hematopoietic progenitor subtype - CD45+ CD38+ CD123+ CD294+

Density Gradient Centrifugation

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Functional Data – Whole BloodT Cell Proliferation Data

Frann AntignanoMelina Messing

Hematopoietic progenitor subtype - CD34+

CD117+ CD294+

Granulocytes - CD16+ CD15+ CD11b+

CD66b+ CD45low

Monocytes - CD45+ CD11c+ CD14+ CD11b+

HLA-DR+ CD38 CD4low

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Automating PBMC Isolation with RoboSep™

Minimize sample handling and free up valuable hands-on time by automating PBMC isolation with the EasySep™ Direct Human PBMC Isolation Kit and the RoboSep™-S instrument. By automating all sample labeling and magnetic separation steps you can perform simultaneous cell isolations from up to 4 samples and increase sample throughput.

Select desired protocol and load samples, EasySep™ reagents, buffer, and tips.

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Return in 30 minutes to collect your separated cells.3

Press “Run”.2

How Does It Work?

Step Duration (minutes)

Manual Density Gradient

Centrifugation

ManualEasySep™ Direct

Automated EasySep™ Directon RoboSep™-S

Preparation of tubes with density gradient medium and layering sample

7 - -

Loading samples and reagentsonto the RoboSep™-S instrument

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Cell isolation 45 20 30

Cell collection 10 1 1

Optional: Wash step toremove platelets

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Optional: Red blood cell lysis 15 - -

Final centrifugation step 15 Optional Optional

Total time for cell separation ≥ 77 Minutes ~ 21 Minutes ~ 36 Minutes

Total hands-on time ≥ 32 Minutes ~ 21 Minutes ~ 6 Minutes

Table 1. Automated PBMC Isolation Using RoboSep™-S Reduces the Overall Cell Separation Time Compared to Density Gradient Centrifugation

Why Automate PBMC Isolation with RoboSep™?

EFFICIENT. Minimize hands-on time and increase sample throughput.

HIGH PURITY. Obtain highly purified mononuclear cells with improved RBC, granulocyte, and platelet clearance compared to density gradient centrifugation.

CONVENIENT. Load your samples and reagents, press “Run”, and come back to your isolated cells.

FLEXIBLE. Obtain cells from cord blood, bone marrow, buffy coats, leukapheresis products, and leukocyte reduction system (LRS) cones.

Steps and approximate times to isolate monononuclear cells from whole blood samples when following using density gradient centrifugation or EasySep™ Direct Human PBMC Isolation Kit (manual separation or automated on the RoboSep™-S). For density gradient centrifugation, additional washing steps and red blood cell lysis might be required.

ISOLATE MONONUCLEAR CELLS DIRECTLY FROM WHOLE BLOOD

STEMCELL TECHNOLOGIES INC.’S QUALITY MANAGEMENT SYSTEM IS CERTIFIED TO ISO 13485. PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED.

TOLL FREE PHONE 1 800 667 0322 • PHONE +1 604 877 0713 • INFO@STEMCELL.COM • TECHSUPPORT@STEMCELL.COM

FOR GLOBAL CONTACT DETAILS VISIT WWW.STEMCELL.COM DOCUMENT #27156 VERSION 2.0.0 MARCH 2020

To see RoboSep™ instruments in action, visit www.RoboSep.com.

Copyright © 2020 by STEMCELL Technologies Inc. All rights reserved including graphics and images. STEMCELL Technologies & Design, STEMCELL Shield Design, Scientists Helping Scientists, EasySep, and RoboSep

are trademarks of STEMCELL Technologies Canada Inc. Lymphoprep is a registered trademark of Alere Technologies. All other trademarks are the property of their respective holders. While STEMCELL has made all

reasonable efforts to ensure that the information provided by STEMCELL and its suppliers is correct, it makes no warranties or representations as to the accuracy or completeness of such information.

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Figure 4. PBMC Isolation from a Full-Size Leukapheresis Pack Using the EasySep™ Direct Human PBMC Isolation Kit Automated with RoboSep™-S Results in Fewer Contaminating Cells Compared to Density Gradient Centrifugation

Mononuclear cells were isolated from single concentrated leukapheresis packs using either density gradient centrifugation with Lymphoprep™ density gradient medium (Density Gradient Centrifugation) or EasySep™ Direct Human PBMC Isolation Kit automated on the RoboSep™-S instrument (EasySep™ Direct on RoboSep™-S). Cells were counted and analyzed by flow cytometry. Compared to density gradient centrifugation, EasySep™ Direct Human PBMC Isolation Kit automated on the RoboSep™-S instrument resulted in (A) equivalent numbers of total mononuclear cells, (B) equivalent numbers of total monocytes and lymphocytes and (C) lower numbers of residual platelets (CD41+), red blood cells (Glycophorin A+ /CD45− ), and granulocytes (CD66b+ ) (mean ± SD n=6).

ProductBlood Volume

ProcessedCatalog #

EasySep™ Direct Human PBMC Isolation Kit

100 mL 19654

EasySep™ Direct Human PBMC Isolation Kit for RoboSep™ 100 mL 19654RF

RoboSep™ Instruments for Automated Cell Isolation

RoboSep™-S and Robosep™-16 fully automate all cell labeling and separation steps of the EasySep™ procedure, minimizing sample handling and freeing up technician time. Setup is simple: just load your samples and reagents and walk away. Return later to separated cells, which are immediately ready for downstream applications.

RoboSep™-S

Catalog #21000

RoboSep™-16

Catalog #23000

NEED FRESH WHOLE BLOOD PRODUCTS?Get fresh whole peripheral blood and leukapheresis packs when you need it and specify your donor requirements.*

Learn more at www.stemcell.com/PrimaryCells

Special Application

Automate PBMC Isolation from a Full-Size Leukapheresis Pack

Obtain PBMCs from a full-size leukapheresis pack in a single run with EasySep™ Direct Human PBMC Isolation Kit and RoboSep™-S. This automated cell isolation system results in fewer contaminating cells compared to density gradient centrifugation (Figure 4). First, concentrate the leukapheresis pack (see below). Then, load the tubes with the resuspended cells, as well as the EasySep™ reagents, buffers, and tips onto the instrument. Start the instrument’s isolation protocol and come back in 30 minutes to collect your isolated cells.

Below is an example of how to concentrate an entire leukapheresis pack for downstream PBMC isolation using RoboSep™-S. For more information please consult the product information sheet for your specific product or contact our Product & Scientific Support team at techsupport@stemcell.com.

1. Transfer the entire contents of the leukapheresis bag into tubes

and centrifuge at 300 x g for 10 minutes with the brake on.

2. Carefully remove the supernatant and resuspend the cells in EasySep™ Buffer (Catalog #20144) to obtain a final volume of 20 mL and approximate concentration of 4x108 cells/mL.

3. Add EDTA at 6 mM final concentration and aliquot the 20 mL into four 14 mL polystyrene round-bottom tubes (e.g. Catalog #38008). Each tube will now contain 5 mL of resuspended cells.

4. Select the appropriate protocol on the screen.

5. Follow the onscreen prompts and load the 4 x 14 mL tubes, reagents, tips and buffer onto the RoboSep™-S carousel.

6. Press “Run”.

7. Come back in 30 minutes after the run is complete and collect the isolated cells.

*Certain products only available in select territories.