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.
  • 1X ACK Lysis Buffer: Dissolve 8.29 g NH4Cl, 1 g KHCO3, 37.2 mg EDTA in a total volume of 1L; adjust pH to 7.2–7.4. Store at 4°C.
  • 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. Disaggregate spleens using a 100 µm nylon mesh cell strainer and collect in cold PBS.
  2. Collect cells by centrifugation and resuspend in ACK Lysis Buffer for 5 mins. (Red Blood Cell lysis).
  3. Wash by centrifugation in Incubation Buffer.
  4. Resuspend freshly isolated murine spleen cells at 5–10 x 105 cells in 100 µl Incubation Buffer per assay tube.
  5. Collect cells by centrifugation and aspirate supernatant.
  6. Resuspend cells in 500 µl of 2% formaldehyde.
  7. Fix for 15 min at room temperature.
  8. Wash 2X by centrifugation in Incubation Buffer.

C. Permeabilization

  1. Add 1ml of 0.3% Triton™ X-100 (v/v in PBS) to the cell pellet.
  2. Vortex and 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 (D6O8R) XP® Rabbit mAb #12653 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 350 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 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 350 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 June 2013

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