Dual Staining with FITC and PI
Contributors: University of Florida School of Dental Medicine Flow Cytometry Facility
E-mail: SDMFACS@path.dental.upenn.edu
URL: http://biochem.dental.upenn.edu/~sdmfacs/
This protocol calls for the following controls:
  1. Completely unstained cells
  2. FITC only positive control (cells stained with a FITC-Ab that is known to bind
  3. Cells stained with PI alone

Proceudre:
  1. Stain cells with FITC-Ab as usual. You should probably do a titration to determine the minimal working Ab concentration. This will make the PI/FITC compensation easier.
  2. Wash twice with PBS or media + 0.1% NaN3.
  3. Resuspend cells in 1 ml cold 1% paraformaldehyde in PBS.
  4. Votex immediately. Incubate 60 minutes on ice in the dark.
  5. Wash cells once for 5 minutes @ 1800 RPM, 4C.
  6. Pour off supernatant and add 1 ml cold 70% EtOH dropwise while vortexing. This will minimize clumping. The cells can be kept for several days in the dark at 4C.
  7. Pour off EtOH and resuspend in 1 ml PBS or 50 mM citrate with 0.1 mg/ml RNase A. Incubate 5 minutes at room temperature.
  8. Add PI stock (1 mg/ml), mix and incubate 10 minutes in the dark at room temperature. 10 or 20 ul should do the trick, for a final concentration of 10 to 20 ug/ml. As in step 1, use the lowest effective concentration for best compensation.
  9. Analyze by flow cytometry within a day.