FLOW CYTOMETRY PROTOCOL

PRINCIPLE

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.

Material

Reagents	Instruments and Consumables
Washing Buffer	Eppendoff
Primary Antibody	Fluorochrome-labeled Secondary Antibody
Centrifuge	Flow Cytometer

Procedure

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 PBS.
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 suspension.
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 day.

HOT TARGETS

PDCD1 HIV1 gp120 CD33 IAV HA PRNP TNF MS4A1 CD274 APP EGFR CD40 ERBB3 TFPI ERBB2 SARS-CoV S PCSK9 MET BSG HAVCR2 IL6 CD3 LAG3 ROR1 TNFRSF4 IL33 CD19 CD38 JAG1 IL17A IFNG CD47 HCV E2 Protein IL1A / IL1B HBsAg IGF1R CD4 AXL ANGPT2 IL23A NGF MUC1 TNFRSF18 TNFRSF9 MSTN IL1B HRSV F Protein KDR SORT1 C5 VEGF CXCR4 TNFRSF17 Tau TFRC CTLA4 IL13 IAV H5N1 HA TSLP MSLN SOST

For research use only, not directly for clinical use.