FLUORESCENT ACTIVATED CELL

SORTING Present by,

Muhammad Ashraf Bin MazlanCalvin Lim Lai Hock

Soon Tsuey NingChia Kay Veng

Liew Lee Bing

BASIC CONCEPTS OF FACS

Measurement of cells in a flow system - delivers the cell singly past a point of measurement- light is focused

FACS enables sorting of a population of cells into subpopulations.- based on fluorescent labeling- sorting involves mechanisms in flow cytometer

Antibodies bond with specific protein are coated with a fluorescent dye.

Very effective

Principles of Fluorescent Activated Cell Sorting

History

- In 1980s, immunophenotyping rise as CD4 T cells was killed.

- Antibodies for CD3, CD4 and CD8 are among the first characterised monoclonal antibodies

- AIDS First major clinical application Flow cytometry

Applications of FACS in Diagnosis of HIV

infection

Diagnosis of Immunodeficiency disease

● Separation of T-lymphocytes subsets

● Fluorochrome + monoclonal antibodies + antigen → fluorescence

● Measurement of CD4+ (helper) and CD8+ (cytotoxic) T cells ratio.

● AIDS development CD4+ T cells (~200 cells/μL)

● Normal CD4+ T cells level = 1000 cells/μL

Monoclonal antibody cloning using FACS

Immunotherapy for HIV infection● Broad neutralizing antibodies.

● Protect against SIVs that express HIV-1 envelope glycoproteins

SimilaritiesFLOW CYTOMETRY FACS

- Fluorescent labels, cellular propertiesDifferences

Commonly found in research labs, user-friendly

More personalized, designed for researcher

Cannot sort cells Sort heterogeneous mixture of cells into different populations

Intuitive software interfaces

Automated features

Current Development

Derived of smaller and lower cost equipments

PanLeucogating technologyAnchor marker

Validation of small instrumentsCell count errorOutline of the procedures

Challenges

Conclusion➔ Fluorescent activated cell sorting

(FACS) is a specialized type of flow cytometry, It allows multiparametric analysis of heterogeneous cells populations based on their cellular characteristics

➔ Diagnosis of HIV Infection.

◆ separation of subsets of T-lymphocytes

◆ Fluorescent marker ---> identify cell type ---> individual antigenic marker

◆ Measurement of CD4+ (helper) and CD8+ (cytotoxic) T cells ratio.

➔ Monoclonal antibody cloning.

◆ immunotherapy for HIV infection.

➔ Current development

◆ Producing compact and cheap equipments.

◆ PanLeucogating technology

◆ Anchor marker

➔ Challenges

◆ cell count error

◆ outline of the procedures

◆ validation of small instrumets.

➔ FACS can be utilize in analysis and treating HIV infection.

ReferencesAbcam, 2011, Fluorescence activated cell sorting of live cells. Available from:

http://www.abcam.com/protocols/fluorescence-activated-cell-sorting-of-live-cells [23 September 2016]Flowbook.denovosoftware.com, 2016, Chapter 1: Introduction | Flow Cytometry - A Basic Introduction.

Available at: http://flowbook.denovosoftware.com/chapter-1-introduction-0 [26 September 2016]Mukherjee A, 2011, Fluorescent-Activated Cell Sorting. Available from:

http://www.biotecharticles.com/Applications-Article/Fluorescence-Activated-Cell-Sorting-570.html [29 September 2016]

Parks D & Perez O, 2002, The History and Future of the Fluorescence Activated Cell Sorter and Flow Cytometry: A View from Stanford. Available from: http://clinchem.aaccjnls.org/content/48/10/1819 [29 September 2016]

Robinson R, Pellenz S, 2013, What is flow cytometry (FACS analysis)?, Available from: http://www.antibodies-online.com/resources/17/1247/what-is-flow-cytometry-facs-analysis. [24 September 2016]

VenturiOne, 2012, Flow Cytometry. Available from: http://www.appliedcytometry.com/flow_cytometry.php [29 September 2016]