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Cytokine Stimulation and 96 Well BD Phosflow Protocol
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:
- Collect whole blood in the presence of heparin or EDTA. **See BD™ PhosflowFAQ for information on anticoagulants.
- Dilute 5X BD Phosflow™ Lyse/Fix Buffer to 1X with distilled water.
- Pre-warm the 1X BD Phosflow™ Lyse/Fix Buffer in a 37°C water bath for 5-10 minutes before use.
- 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.
- Designate wells as "Treated" and "Untreated".
- Add peripheral whole blood cells (100 µl/well) to both sets of wells, Treated and Untreated.
- Add 20 µl aliquots of cytokine to wells designated as Treated.
- 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.
- 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.
- Incubate plate in 37°C water bath for 10-15 minutes.
- Pellet plate with cells by centrifugation (125 x g) for 5-10 minutes using centrifuge plate adaptor.
- Completely remove supernatant via aspiration. Dab plate onto paper towel to remove residual supernatant from the wells.
- 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.
- 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.
- Completely remove supernatant via aspiration. Dab plate onto paper to remove residual supernatant from the wells.
- 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.
- 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. - 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.
- 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.
- Resuspend the cells after the second wash by adding 50 µl of BD Pharmingen™ Stain Buffer (FBS). Vortex
- Aliquot optimal concentrations of fluorescent antibodies to each well, Treated and Untreated and mix thoroughly by pipetting up and down 2 times.
- Incubate the cells at room temperature for 20-30 minutes protected from light.
- 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.
- 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.
- 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.
- Aliquot optimal concentrations of fluorescent antibodies to each well, Treated and Untreated and mix thoroughly by pipetting up and down 3 times.
- Incubate cells at room temperature for 20 minutes protected from light.
- 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.
- Resuspend cells in BD Pharmingen™ Stain Buffer (FBS) (200 µl/well).
- Transfer samples to a U-bottom 96-well plate (BD Falcon Cat # 353910) or tubes prior to flow cytometric analysis.
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