Staining Intracellular Targets for Flow Cytometry

1、Add 100 μl of fluorescently labeled surface antibodies and/or corresponding isotype controls, according to the antibody instructions, to a 5 ml flow cytometry intracellular staining tube containing 1 × 10^6 cells in suspension. Incubate at room temperature.
2、Add 100 μl of MEDIUM A and incubate at room temperature for 15 minutes.
3、Add 3 ml of Flow Cytometry Staining Buffer (Catalog No. S1001) or PBS containing 5% FBS to the tube. Centrifuge at 300-350 × g for 5 minutes, discard the supernatant, vortex, and resuspend the cell pellet in the remaining liquid.
4、Add 100 μl of MEDIUM B and the recommended volume of intracellular antibodies and/or corresponding isotype controls.
5、Vortex for 1-2 seconds and incubate according to the antibody instructions.
6、Add 3 ml of Flow Cytometry Staining Buffer or PBS containing 5% FBS to the tube. Centrifuge at 300-350 × g for 5 minutes, discard the supernatant.
7、Add 0.5 ml of Flow Cytometry Staining Buffer or PBS containing 5% FBS for flow cytometry analysis. If analysis cannot be performed on the same day, add 0.5 ml of 0.1% paraformaldehyde, store at 2-8℃ avoiding light, and analyze within 24 hours.