Reagents:

• Diluted recombinant cytokine: 20 µl per well (concentration varies)
• Peripheral Whole Blood (100 µl/well) BD™ Phosflow Lyse/Fix: (558049), 400-500 µl per well
• BD™ Phosflow Perm/Wash Buffer I (557885), Perm Buffer II (558052) or Perm Buffer III (558050) 500 µl per well
• BD Pharmingen™ Stain Buffer (FBS) (554656)
• Dulbecco's PBS (DPBS) 1X , sterile

Equipment:

• Deep-Well Titer Plate Polypropylene, Sterile (267007, Beckman Instruments, Inc.)
• Assay Plate, 96 Well U-Bottom (BD Falcon Cat #353910)
• Aspirator Adaptor: 12 channel manifold for deep well plates with female luer, 19 gauge needles, 35 mm long, 9 mm center to center spacing, (VP 187A, V & P Scientific, Inc.)
• Tabletop centrifuge and plate holder compatible with deep well plates: Beckman Coulter, Model Allegra 6R; 125 x g (800 RPM); 150 x g (900 RPM)
• Ice/bucket
• 37°C Water bath *Note: activation conditions are more consistent with the water bath

Procedure:

1. Collect whole blood in the presence of heparin or EDTA. **See BD™ Phosflow FAQ for information on anticoagulants.
2. Dilute 5 X BD Phosflow™ Lyse/Fix Buffer to 1 X with distilled water.
3. Pre-warm the 1 X BD Phosflow™ Lyse/Fix Buffer in a 37°C water bath for 5-10 minutes before use.
4. Considering that the total volume per well will be 120 µl, dilute anti-CD3 (1µg/100µl) and Goat anti-Mouse Ig cat# 553998 (0.5µg/100µl) to a final working concentration. A recommended minimum volume of stimulant to add is 20 µl/well.
5. Designate wells as "Treated" and "Untreated".
6. Pre-chill deep well plate and 1 X DPBS on ice.
7. Add peripheral whole blood cells (100 µl/well) to both sets of wells, Treated and Untreated.
8. Add diluted anti-CD3 Ab clone UCHT1 to blood with a final working concentration of 1.0 µg/100 µl of blood to wells designated as Treated.
9. Mix thoroughly via pipetting up and down 3 times, gently vortex, and incubate on ice for 15 min.
10. Add 400 µl COLD 1 X DPBS. Mix by pipetting up and down 3 times. Centrifuge at 4°C, 125 x g, 5 min using centrifuge plate adaptor. NOTE: **Excessive speed may result in red blood cells clumping thus causing poor red blood cell lysis and fixation of white blood cells. (Step 15)
11. Remove supernatant, leaving behind 50 µl to 100 µl volume, with minimal disturbance to red blood cells. Do not dab the plate.
12. Add Goat/Anti-Ms Ig (553998) with a final working concentration of 0.5 µg/100 µl of blood to wells designated as Treated.
13. Mix thoroughly via pipetting up and down 3 times, gently vortex, and incubate on ice, 15 min.
14. Transfer the Treated and Untreated plates to 37°C water bath and incubate for 5-7 minutes.
15. Fix both sets of cells immediately in order to maintain their phosphorylation state by adding 1 BD Phosflow™ Lyse/Fix Buffer (400 µl/well). Mix thoroughly via pipetting the entire volume up and down 3 times. NOTES: **Poor mixing may result in poor lysis and fixation. **Caution must be taken to prevent spillover from liquid displacement. **After this step, process Treated and Untreated similarly.
16. Incubate plate with cells in 37°C water bath for 10-15 minutes.
17. Pellet the cells by centrifugation (125 x g) for 5 minutes. Remove supernatant via aspiration. Dab plate onto paper towel to remove all residual supernatant.
18. Repeat lyse/fix by adding 500 µl Phosflow™ Lyse/Fix Buffer. Incubate at 37°C water bath for 10-15 minutes. Mix thoroughly via pipetting the entire volume up and down 3 times. **Ensure that all RBC clumps are broken up by pipetting them up and down.
19. Pellet cells by centrifugation (125 x g) for 5 minutes. Remove supernatant and dab the plate. Cover plate and vortex to loosen cell pellets. Wash with 500 µl 1 X DPBS, cover, and centrifuge (125 x g) 5 min.
20. Completely remove supernatant via aspiration. Dab plate onto paper towel to remove residual supernatant. NOTES: **If excessive RBCs remain, this is mainly due to poor mixing or clumping of RBCs. A third lyse/fix step (followed by another wash) may be necessary in this case. Ensure that all RBC clumps are broken up by pipetting them up and down.
21. Permeabilize the cells by adding appropriate BD Phosflow™ Perm buffer (as per your antibody of choice) at 500 µl/well. Thoroughly mix cells by pipetting them up and down 3 times. Cover plate and gently vortex.
22. For Perm Buffer II and III, incubate on ice (2-4°C) for 30 minutes. NOTES: **Longer incubation times in BD™ Phosflow Perm Buffer II and III may decrease the signal intensity of surface marker staining.
    For Perm/Wash Buffer I, incubate cells at RT and use BD™ Phosflow Perm/Wash Buffer I for all subsequent incubations and washes.
23. Wash by adding 500 µl of BD Pharmingen™ Stain Buffer (FBS). Mixing is not necessary at this point. Centrifuge at 125 x g for 10 minutes. Remove the supernatant and dab the plate.
24. Vortex plate and wash again by adding 500 µl of BD Pharmingen™ Stain Buffer (FBS). Mix thoroughly by pipetting up and down 3 times followed by gentle vortexing. Centrifuge at 125 x g for 10 minutes. Remove the supernatant and dab the plate.
25. Resuspend the cells after the second wash by adding 50 µl of BD Pharmingen™ Stain Buffer (FBS) containing 15 µg/50 µl of Normal Mouse Ig (CALTAG Cat. 10400). Mix thoroughly by pipetting up and down 3 times followed by gentle vortexing. Incubate at RT for 15 min. **Blocking with Normal Mouse Ig is necessary to block unbound Goat anti-Mouse Ig.
26. Aliquot optimal concentrations of fluorescent antibodies to each well, Treated and Untreated, and mix thoroughly by pipetting up and down 3 times.
27. Incubate plate with cells at room temperature for 20 minutes protected from light.
28. Wash the cells once by adding BD Pharmingen™ Stain Buffer (FBS) (500 µl/well), centrifuging at 150 x g for 5-10 minutes. Remove supernatant, dab plate, and repeat wash.
29. Resuspend cells in BD Pharmingen™ Stain Buffer (FBS) (200 µl/well).
30. Transfer samples to a 96 Well U-Bottom plate (BD Falcon Cat #353910) or tubes prior to flow cytometric analysis.