Flow cytometry (FACS) is an immunophenotyping technique that analyze the expression of cell surface and
intracellular molecules. Flow cytometry rapidly measures the specific characteristics of a large number of
individual cells. Living cells in suspension are stained with specific, fluorescently labeled antibodies and then
analyzed with a flow cytometer. The intensity of fluorescence detected by cytometer is directly proportional to the
antigen density or the characteristics of the cell being measured.
Instruments and Consumables
Fluorochrome-labeled Secondary Antibody
Harvest, wash the cells and then determine the total cell number.
Resuspend the cells to approximately 1-5 x 106 cells/mL in PBS.
Add 100 μl of cell suspension to each tube.
Add 0.1-10 μg/mL of the primary antibody binding cells antigen. Dilutions, if necessary, should be made in
Incubate for 60 min at 4°C.
Wash the cells 3 times by centrifugation at 400g for 5 min and resuspend them in ice cold PBS.
Dilute the fluorochrome-labeled secondary antibody in 3% BSA/PBS at the optimal dilution and add to cell
Incubate for 60 min at 4°C in dark.
Wash the cells 3 times by centrifugation at 400g for 5 min and resuspend them in PBS.
Analyze the cells on the flow cytometer. It is recommended that analysis is performed on the same