Flow Cytometry Protocol for Indirect Intracellular Staining of Cultured Cells Grown in Suspension.
16% parafolmaldehyde (PFA) (Electron Microsopy Sciences RT 15710)
90% Methanol (ice cold)
PBS (Phosphate Buffered Saline pH 7.2)
11.6 g sodium chloride
100 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 (feteal bovine serum)
0.36 ml 9% soldiuim 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 20oC.
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 -70o to -20oC for up to a month.
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 20oC, break 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.
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 2nd wash.
14. Spin, pour off, and blot tube.
15. Resuspend in 100 mcl ICSB.
16. Analyze on a flow cytometer.
This is a recommended protocol, and additional optimization may be required for any individual experiment.