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™ PhosflowFAQ for information on anticoagulants.
2. Dilute 5X BD Phosflow™ Lyse/Fix Buffer to 1X with distilled water.
3. Pre-warm the 1X BD Phosflow™ Lyse/Fix Buffer in a 37°C water bath for 5-10 minutes before use.
4. Considering that the total volume added per well will be 120µl, dilute cytokine (or polyclonal activator like phorbol myristate acetate/PMA) 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. Add peripheral whole blood cells (100 µl/well) to both sets of wells, Treated and Untreated.
7. Add 20 µl aliquots of cytokine to wells designated as Treated.
8. Mix thoroughly via pipetting up and down 3 times, gently vortex, and incubate at 37°C for the optimal/peak phosphorylation time. NOTE: *Methods of activation vary and optimal treatments should be determined by the researcher.
9. 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 will result in poor lysing and fixation. **Caution must be taken to prevent spill over from liquid displacement **After this step, process Treated and Untreated similarly.
10. Incubate plate in 37°C water bath for 10-15 minutes.
11. Pellet plate with cells by centrifugation (125 x g) for 5-10 minutes using centrifuge plate adaptor.
12. Completely remove supernatant via aspiration. Dab plate onto paper towel to remove residual supernatant from the wells.
13. Repeat lysing by adding 500 µl 1x BD™ 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 up and down.
14. Cover and vortex plate(s) to loosen the cell pellets. Wash with 500 µl 1 PBS, cover, and centrifuge (125 g) for 5 minutes. NOTES: **If excessive RBCs remain, this is mainly due to poor mixing. A third lyse and fix step(followed by another wash) may be necessary in this case. **Ensure that all RBC clumps are broken up by pipetting up and down.
15. Completely remove supernatant via aspiration. Dab plate onto paper to remove residual supernatant from the wells.
16. Vortex plates and permeabilize cells by adding appropriate BD™ Phosflow Perm Buffer (as per your antibody of choice) at 500 µl/well. Thoroughly mix by pipetting up and down 3 times.
17. 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.
18. Add 500 µl BD Pharmingen™ Stain Buffer (FBS). Pellet cells by centrifugation (125 x g) for 10 minutes and remove the supernatant via aspiration and dabbing the plate.
19. Cover plate and vortex cells. Wash the cells by adding 500 µl with BD Stain Buffer. Centrifuge at 125 x g for 5 minutes. Remove the supernatant from the wells and repeat.
20. Resuspend the cells after the second wash by adding 50 µl of BD Pharmingen™ Stain Buffer (FBS). Vortex
21. Aliquot optimal concentrations of fluorescent antibodies to each well, Treated and Untreated and mix thoroughly by pipetting up and down 2 times.
22. Incubate the cells at room temperature for 20-30 minutes protected from light.
23. Wash the cells 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 from the wells and dab the plate.
24. Cover plate and vortex. 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 from the wells and dab the plate.
25. Resuspend the cells after the second wash by adding 50 µl of BD Pharmingen™ Stain Buffer (FBS). Mix thoroughly by pipetting up and down 3 times followed by a gentle vortexing.
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 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 125 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 U-bottom 96-well plate (BD Falcon Cat # 353910) or tubes prior to flow cytometric analysis.