Specific For:
A. Solutions and Reagents
NOTE: Prepare solutions with RODI (reverse osmosis deionized) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
- Formaldehyde (methanol free).
- 2% Formaldehyde (stock formaldehyde diluted in PBS; make fresh on day of experiment).
- Triton™ X-100.
- Ficoll-Paque™.
- Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) (#9998) 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.
- Isolate PBMCs from whole blood by Ficoll density centrifugation as per manufacturer’s instructions.
- Wash 2x by centrifugation using Incubation Buffer.
- Resuspend cells in Incubation Buffer and aliquot at a concentration of 1 x 106 cells/100 µl/sample tube.
- Add CD4 and CD25 antibodies to assay tubes as per manufacturer’s recommended volume or concentration and incubate for 30 min. on ice.
- Add 2 ml of Incubation Buffer and wash by centrifugation.
- Aspirate supernatant and resuspend cells in 500 µl of 2% formaldehyde.
- Fix for 30 min at room temperature.
- Wash 2X by centrifugation in Incubation Buffer.
C. Permeabilization
- Resuspend cells in 1 ml of 0.1% Triton™ X-100 (v/v in PBS).
- Let stand for 30 min at room temperature.
- Wash 2X by centrifugation in Incubation Buffer.
D. Immunostaining
- Resuspend cell pellets in 100 µl of FoxP3 (D6O8C) XP® Rabbit mAb #12632 working solution (stock antibody diluted 1:200 in Incubation Buffer).
- Incubate for 1 hr at room temperature.
- Wash 2X by centrifugation in Incubation Buffer.
- 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.
- Resuspend cells in fluorochrome-conjugated secondary antibody or fluorochrome-conjugated avidin, diluted in Incubation Buffer at the recommended dilution.
- Incubate for 30 min at room temperature.
- Wash 2X by centrifugation in Incubation Buffer.
- Resuspend cells in 500 µl of Incubation Buffer and analyze on flow cytometer.
- FoxP3 can be plotted against CD25 on a bivariate scattergram gated on CD4+ T lymphocytes.
posted July 2013
revised October 2013