What is Fluorescence-activated cell sorting (FACS)?

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Fluorescence-activated cell sorting (FACS) is a specialized type of lymphocyte flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument, as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. BD FACSCalibur or FACSCanto are the most use sorters.

What is a fluorescence-activated cell sorter (FACS) ?

It is a sorting method based on an antibody specific for a particular cell surface protein. That cluster of differentiation or anti CD monoclonal antibody is associated to a fluorescent molecule and then added to a mixture of cells. For fluorescence when the specific cells pass through a laser beam, they are monitored. Droplets containing single cells are given a positive or negative charge, based on whether the cell has limited the fluorescently-tagged antibody or not. Droplets containing a single cell are then detected by an electric field into collection tubes according to their charge. There exist also Miltenyi Biotech magnetic cell sorters called Macs.

How many Cluster of differentiation Flow Cytometry Antibody are there ?

Strictly validated monoclonal anti CD antibodies are the premise of good results obtained in Flow Cytometry or FACS. So far, Gentaur sells about 1000 fully validated antibodies types used for Flow Cytometry assays. All of them are of high specificity, high sensitivity, precise tissue or subcellular localization, and especially, avoiding false positive signal induced by Fc receptors on the surface of certain cell types.

Why mouse monoclonal antibodies are the most used for Flow ?

Mice are the injected with cluster differentiation antigens CDs to clone anti CD antibodies from their lymphocytes. Monoclonal antibodies have certain advantages over their polyclonal counter parts. These advantages include high homogeneity, high specificity, ease of characterization, and no batch-to-batch or lot-to-lot variability in affinity.

How are Cytometry Antibodies Stained ?

Direct FITC or PE monoclonal antibodies are used as primary antibody in a one-step staining. It is more convenient for multiplex targets staining.

Indirect antibody staining for flow cytometry is a kind of indirect immunoassay, which the secondary antibodies were needed as detector antibodies. Secondary antibodies are the “anti-antibodies” against the primary antibody you are using. And when your primary is a mouse monoclonal, you will require an anti-mouse secondary. The secondary antibody was conjugated with a variety of fluorochromes or chromogens.

Djoumana Ounas

Pharmacist Assistant Professor in Analytical Chemisty. Consultance Expertise and Reglatory Biotech Support.