Flow Cytometry for Intracellular Staining
Solutions and Reagents
- 1X Phosphate Buffered Saline (PBS): Dissolve 8g NaCl, 0.2g KCl, 1.15g Na2HPO4 and 0.2g KH2PO4 in 800mL distilled water (dH2O). Adjust the pH to 7.4 with HCl and the volume to 1 liter. Store at room temperature.
- Fixation buffer: 2% paraformaldehyde in 1xPBS
- Permeabilization buffer : 0.1% Triton X-100 in 1xPBS
- FACS buffer: 0.5% BSA , 0.05% Azide in 1xPBS
- Fluorescent dye conjugated secondary antibody.
- Collect cells by centrifugation and aspirate supernatant.
- Fix the cell by 125μl cold fixation buffer, vortex briefly.
- Incubate at room temperature for at least 30 min or for 1hr 40C.
- Centrifuge for 5min at 300g,remove the supernatant.
- Add 1ml permeabilization buffer to each tube.
- Centrifuge briefly, and aspirate supernatant.
- Resuspend cells in 125μl of permeabilization buffer and incubate at room temperature for 5min.
- Aliquot 1-2x106 cells into each tube.
- Add 1 ml FACS buffer to each tube, centrifuge to pellet the cells.
- Resuspend cell pellet with 125μl FACS buffer containing diluted primary antibody, vortex and incubate on ice for 30min.
- Rinse as before in FACS buffer by centrifugation.
- Resuspend cells in fluorescent dye conjugated secondary antibody, diluted in FACS buffer per manufacturer’s recommendations.
- Incubate for 30 minutes on ice.
- Rinse the cells as before in FACS Buffer by centrifugation.
- Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.