Flow Cytometry Protocol for Indirect Intracellular Staining of Cultured Cells Grown in Suspension.

Reagents Required:

• 16% paraformaldehyde (PFA) (Electron Microsopy Sciences RT 15710)
• 90% Methanol (ice cold)
• PBS (Phosphate Buffered Saline pH 7.2)
• ICSB (IntraCellular Staining Buffer) – 1% FBS, 0.05% Sodium Azide in PBS
• FACS tubes

Cell Fixation and Permeabilization:

1. For cells grown in suspension, add fresh 16% PFA to achieve a final concentration of 1.54% directly to the cells in media (1-2 x 10⁶ cell/ml).
2. Fix cells at room temperature (RT) for 10 minutes.
3. Incubate and fix on ice for an additional 30 minutes.
4. Centrifuge fixed cells at 250 x g for 5 minutes at 20^oC.
5. Pour off media + PFA into dedicated PFA waste containier.
6. Permeabilize cells by resuspending in ice cold 90% methanol to a final cell concentration of 1 X 10⁶ cells/ml.
7. Cells may be stored at -70^o to -20^oC for up to a month.

Intracellular Staining:

1. Aliquot 1 ml (1 x 10⁶ cells) of cells in methanol for each tube/sample.
2. Centrifuge at 250 x g for 5 minutes at room temperature and remove supernatant (perform all subsequent spins at these conditions)
3. Resuspend cells in 1 ml ICSB
4. Centrifuge cells and remove supernatant
5. Resuspend the cell pellet in 100µl of ICSB containing diluted primary antibody per Bethyl’s recommendation
6. Incubate on ice for 1 hour
7. Rinse the cells 2X in ICSB by centrifugation
8. Resuspend in 100µL of conjugated secondary antibody per Bethyl’s recommendation
9. Incubate on ice for 30 minutes in the dark
10. Rinse the cells 2X in ICSB by centrifugation. Continue to minimize exposure of cells to the light
11. Resuspend in 300µl ICSB in the dark
12. Filter cells through a strainer to remove clumps
13. Analyze with a flow cytometer