FACS Protocols

Flow Cytometry for Intracellular Staining

1. Solutions and Reagents
   1. 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.
   2. Fixation buffer: 2% paraformaldehyde in 1xPBS
   3. Permeabilization buffer : 0.1% Triton X-100 in 1xPBS
   4. FACS buffer: 0.5% BSA , 0.05% Azide in 1xPBS
   5. Fluorescent dye conjugated secondary antibody.
2. Fixation
   1. Collect cells by centrifugation and aspirate supernatant.
   2. Fix the cell by 125μl cold fixation buffer, vortex briefly.
   3. Incubate at room temperature for at least 30 min or for 1hr 40C.
   4. Centrifuge for 5min at 300g,remove the supernatant.
3. Permeabilization
   1. Add 1ml permeabilization buffer to each tube.
   2. Centrifuge briefly, and aspirate supernatant.
   3. Resuspend cells in 125μl of permeabilization buffer and incubate at room temperature for 5min.
4. Staining
   1. Aliquot 1-2x106 cells into each tube.
   2. Add 1 ml FACS buffer to each tube, centrifuge to pellet the cells.
   3. Resuspend cell pellet with 125μl FACS buffer containing diluted primary antibody, vortex and incubate on ice for 30min.
   4. Rinse as before in FACS buffer by centrifugation.
   5. Resuspend cells in fluorescent dye conjugated secondary antibody, diluted in FACS buffer per manufacturer’s recommendations.
   6. Incubate for 30 minutes on ice.
   7. Rinse the cells as before in FACS Buffer by centrifugation.
   8. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer