PIP3 Staining Protocol

1.   Resuspend 5-10 million splenic cells in 100-200 µl PBS.  Place cells at 37°C to equilibrate for 5 minutes.  Prepare 2x concentration of stimulatory antibody or antigen (e.g. 20 µg/ml of anti-IgM antibody for a final concentration of 10 µg/ml).  Place stimulant at 37°C to equilibrate for 5 minutes.

2.
   Place an equal volume of stimulant to cells and mix.  Stimulate at 37°C for 5 minutes.

3.  
Add 16% paraformaldehyde to cells to a final concentration of 1.5% to stop stimulation and fix cells.  Mix and fix cells at RT for 10 minutes.

4. 
  Spin cells in a tabletop microcentrifuge at 3000 rpm for 3 min.

5.
   Wash with cold PBS.  Spin.

6.  
Resuspend cells in 500 µl PBS/1% BSA/0.2% saponin to permeabilize.  Incubate on ice for 10 minutes.

7.
   Prepare 1:100 or 1:200 dilution of biotinylated anti-PIP3 IgM antibody (Echelon Biosciences Catalog #Z-B345) or biotinylated IgM isotype in PBS/BSA/saponin.   Stain cells on ice for 30 minutes.

8.  
Wash cells 2x with PBS/BSA/saponin

9.
   Resuspend cells in PBS/BSA/saponin containing 1:100 Strepavidin-FITC + 1:100 B220-APC Cy7.  Incubate in the dark on ice for 20-30 minutes.

10.  
  Wash cells 2x with FACS buffer (PBS/1%FBS/0.01% Azide).  Resuspend cells in FACS buffer.

11.  
  Analyze cells by flow cytometry.