TCR Stimulation and 96 Well BD™ Phosflow Protocol
- 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
- 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)
- 37°C Water bath *Note: activation conditions are more consistent with the water bath
- Collect whole blood in the presence of heparin or EDTA. **See BD™ Phosflow FAQ for information on anticoagulants.
- Dilute 5 X BD Phosflow™ Lyse/Fix Buffer to 1 X with distilled water.
- Pre-warm the 1 X BD Phosflow™ Lyse/Fix Buffer in a 37°C water bath for 5-10 minutes before use.
- 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.
- Designate wells as "Treated" and "Untreated".
- Pre-chill deep well plate and 1 X DPBS on ice.
- Add peripheral whole blood cells (100 µl/well) to both sets of wells, Treated and Untreated.
- 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.
- Mix thoroughly via pipetting up and down 3 times, gently vortex, and incubate on ice for 15 min.
- 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)
- Remove supernatant, leaving behind 50 µl to 100 µl volume, with minimal disturbance to red blood cells. Do not dab the plate.
- Add Goat/Anti-Ms Ig (553998) with a final working concentration of 0.5 µg/100 µl of blood to wells designated as Treated.
- Mix thoroughly via pipetting up and down 3 times, gently vortex, and incubate on ice, 15 min.
- Transfer the Treated and Untreated plates to 37°C water bath and incubate for 5-7 minutes.
- 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.
- Incubate plate with cells in 37°C water bath for 10-15 minutes.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Aliquot optimal concentrations of fluorescent antibodies to each well, Treated and Untreated, and mix thoroughly by pipetting up and down 3 times.
- Incubate plate with 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 150 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 96 Well U-Bottom plate (BD Falcon Cat #353910) or tubes prior to flow cytometric analysis.