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)
  11.6 g sodium chloride
  10 ml 1 M phosphate buffer
  0.26 ml 4 M sodium hydroxide
  DI H₂O to 1 liter
ICSB (IntraCellular Staining Buffer)
  495 ml PBS
  5 ml FBS (fetal bovine serum)
  0.36 ml 9% sodium azide
12 x 75 FACS tubes (BD Falcon #352054)

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. Spin 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. Spin at 250 x g for 5 minutes at 20^oC, brake set to slow (perform all subsequent spins at these conditions)
3. Pour off methanol.
4. Resuspend cells in 1 ml ICSB for wash.
5. Pour off wash and blot tube on paper towels.
6. Resuspend cells in 50 mcl of primary antibody at desired dilution.
7. Incubate at RT for 1 hour.
8. Add 1 ml of ICSB.
9. Spin.
10. Pour off and blot tube.
11. Resuspend in 100 mcl of conjugated secondary antibody at desired dilution.
12. Incubate 30 minutes at RT in dark.
13. Add 1 ml ICSB for 2^nd wash.
14. Spin, pour off, and blot tube.
15. Resuspend in 100 mcl ICSB.
16. Analyze on a flow cytometer.