Flow Cytometry Protocol for Indirect Intracellular Staining of Cultured Cells Grown in Suspension.
- 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:
- 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).
- Fix cells at room temperature (RT) for 10 minutes.
- Incubate and fix on ice for an additional 30 minutes.
- Centrifuge fixed cells at 250 x g for 5 minutes at 20oC.
- Pour off media + PFA into dedicated PFA waste containier.
- Permeabilize cells by resuspending in ice cold 90% methanol to a final cell concentration of 1 X 106 cells/ml.
- Cells may be stored at -70o to -20oC for up to a month.
- Aliquot 1 ml (1 x 106 cells) of cells in methanol for each tube/sample.
- Centrifuge at 250 x g for 5 minutes at room temperature and remove supernatant (perform all subsequent spins at these conditions)
- Resuspend cells in 1 ml ICSB
- Centrifuge cells and remove supernatant
- Resuspend the cell pellet in 100µl of ICSB containing diluted primary antibody per Bethyl’s recommendation
- Incubate on ice for 1 hour
- Rinse the cells 2X in ICSB by centrifugation
- Resuspend in 100µL of conjugated secondary antibody per Bethyl’s recommendation
- Incubate on ice for 30 minutes in the dark
- Rinse the cells 2X in ICSB by centrifugation. Continue to minimize exposure of cells to the light
- Resuspend in 300µl ICSB in the dark
- Filter cells through a strainer to remove clumps
- Analyze with a flow cytometer