1. Isolate cells
2. Stimulated with 20 nM PMA and 1 µM ionomycin at cell conc of 1-2 x 10^6/mL in cR10 in 6 well plate.
3. After four hours, add media with Brefeldin A to final conc of 5 µg/mL.
4. Let stimulate for add’l 2 hours.
5. Pipet off plate with P1000 and put into tubes on ice with cold PBS.
6. Spin down, bring them up in FACS buffer with surface stains.
7. Wash twice.
8. Take pellet, put on vortex (set on 4) and add 4% PFA dropwise at RT. Use 100 µL per 10^6 cells, lower limit of 400 µL. Fix for 8 minutes at RT.
9. Add cold PBS to fill. Spin down. Takes a bit longer to spin down cells once they are fixed. 1400 rpm 6 minutes in tubes or 2200 rpm for 2-3 minutes in plates.
10. Aspirate, wash 1X in cold PBS. Transfer to 96 well plate, spin down. Wash one more time.
11. Flatch out PBS, then beat plate to disperse pellet prior to adding wash buffer.  Wash with perm buffer (PBS/0.5% saponin/1% BSA/0.1% NaAz), 200 µL per sample. Make perm buffer FRESH.
12. Spin down. Add 20 µL of Fc block (1:100) for five minutes. Add 20 µL diluted intracellular stain on top. Let incubate 20-30 minutes at RT in dark. (IFN-g 1:50, IL-4 1:20, IL-2 1:50, IL-10 1:20, IL-5 1:20)
13. Wash 3X with perm buffer.
14. Wash 2X with PBS.
15. FACS.

Extra notes KMA 5/08

Here are a few things that probably matter:

Saponin is "Saponin from Quillaja bark" from Sigma

4%PFA in PBS should be made fresh, or be a freshly thawed aliquot (I make 5-10mL aliquots and store at -20 deg C). Old PFA will perform poorly.

PFA must be added slowly while the cells are being mixed.

PFA fixation should not be allowed to go on too long. If I have many samples, I keep track of when I began and how long it takes me to do each tube (~30 seconds each, usually). Then I add a bunch of cold PBS in the same staggered time course so that they all get about the same exposure time. It is critical to stop before they get overfixed -- this is the most likely reason for getting poor IL4 staining.

All antibodies are Ebioscience or BD/Pharmingen. IL-4 is the usual clone 11B11 (PE or APC).

Antibody dilutions are for the 20 microliters that you put on top of 20 microliters of FcBlock (that's anti-CD16/32 Fc receptors)... so the final dilution of these antibodies is really 1:40 or 1:100 on the cells.