A. Solutions and Reagents

NOTE: Prepare solutions with RODI (reverse osmosis deionized) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. Formaldehyde (methanol free).
  3. 2% Formaldehyde (stock formaldehyde diluted in PBS; make fresh on day of experiment).
  4. Triton™ X-100.
  5. Ficoll-Paque™.
  6. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) (#9988) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

NOTE: Surface staining of CD4 and CD25 antibodies should be performed on live cells prior to fixation/permeabilization, as per manufacturer’s requirements.

  1. Isolate PBMCs from whole blood by Ficoll density centrifugation as per manufacturer’s instructions.
  2. Wash 2x by centrifugation using Incubation Buffer.
  3. Resuspend cells in Incubation Buffer and aliquot at a concentration of 1 x 106 cells/100 µl/sample tube.
  4. Add CD4 and CD25 antibodies to assay tubes as per manufacturer’s recommended volume or concentration and incubate for 30 min. on ice.
  5. Add 2 ml of Incubation Buffer and wash by centrifugation.
  6. Aspirate supernatant and resuspend cells in 500 µl of 2% formaldehyde.
  7. Fix for 30 min at room temperature.
  8. Wash 2X by centrifugation in Incubation Buffer.

C. Permeabilization

  1. Resuspend cells in 1 ml of 0.1% Triton™ X-100 (v/v in PBS).
  2. Let stand for 30 min at room temperature.
  3. Wash 2X by centrifugation in Incubation Buffer.

D. Immunostaining

  1. Resuspend cell pellets in 100 µl of FoxP3 (D6O8C) XP® Rabbit mAb #12632 working solution (stock antibody diluted 1:200 in Incubation Buffer).
  2. Incubate for 1 hr at room temperature.
  3. Wash 2X by centrifugation in Incubation Buffer.
  4. If using a fluorochrome-conjugated primary antibody, resuspend cells in 500 µl of Incubation Buffer and analyze on flow cytometer; for unconjugated or biotinylated primary antibodies, proceed to Step 5.
  5. Resuspend cells in fluorochrome-conjugated secondary antibody or fluorochrome-conjugated avidin, diluted in Incubation Buffer at the recommended dilution.
  6. Incubate for 30 min at room temperature.
  7. Wash 2X by centrifugation in Incubation Buffer.
  8. Resuspend cells in 500 µl of Incubation Buffer and analyze on flow cytometer.
  9. FoxP3 can be plotted against CD25 on a bivariate scattergram gated on CD4+ T lymphocytes.

posted July 2013

revised October 2013

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