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Flow Cytometry (FC) is a powerful tool that can be applied in multiple disciplines such as immunology, virology, molecular biology, and cancer biology. It quantitatively analyzes and sorts individual cells or biological particles by detecting fluorescent signals labeled on single cells or other biological particles in suspension, and analyzing the visible light scattering and fluorescence parameters of each particle.

Experimental Applications

I. Related Reagent Formulations

Name	Formula
1×PBS	10 mM PBS, room temperature, valid for 7 days.
Fixative	4% PFA or 100% methanol or 80% methanol, stored at -20°C, valid for 12 months.
Cell Storage Buffer	1×PBS containing 0.1% Proclin^TM 300, stored at 2~8°C, valid for 3 months.
Permeabilization Buffer	1×Permeabilization Buffer (eBioscience) or 90% methanol, prepared fresh before use.
Blocking Buffer	1×PBS containing 1% BSA, 2.26% glycine, 10% negative goat serum, 0.1% Tween-20 and 0.05% Proclin^TM 300, stored at 2~8°C, valid for 1 month.

II. Cell Fixation

1) Obtain cells from the cell room, in a 96-well plate, the number of cells per well needs to reach 5x10⁵~1x10⁶ cells. Centrifuge the obtained cells at 1500 rpm/min for 5 min, discard the supernatant.

2) Add fixative 4% PFA, fix at room temperature for 15 min. Or add ice-cold methanol, fix for 15 min.

3) Remove the fixative, wash twice with 1×PBS at room temperature, 5 min each time.

4) Remove 1×PBS, add cell storage buffer, store at 2~8°C.

III. Permeabilization and Blocking

When organic solvent fixatives such as methanol are used in the fixation step, there is no need to perform the permeabilization step. Note that excessive permeabilization will destroy cell structure and cause signal loss, so the concentration and time must be strictly controlled.

1) Add permeabilization buffer, incubate at room temperature for 10 min.

2) Remove the permeabilization buffer, add sufficient blocking buffer, incubate at room temperature for 15 min.

IV. Antibody Incubation

1) Dilute primary antibody with 1×PBS according to the instructions.

2) Mix the blocked cells, transfer to a 96-well V-shaped plate, centrifuge at 1500 rpm/min for 5 min, discard the supernatant, add diluted primary antibody, mix well, incubate at room temperature for 15 min.

3) Remove the primary antibody, add 1×PBS, wash once at room temperature.

4) Remove 1×PBS, add fluorescent secondary antibody, mix well, incubate at room temperature in the dark for 15 min.

5) Remove the secondary antibody, add 1×PBS and wash twice at room temperature, 5 min each time.

6) Add 1×PBS to resuspend the cells, and detect on a flow cytometer.