Regulatory T Cell (Treg) Flow Cytometry Panel

Immunophenotyping of Regulatory T cells (Tregs)

Regulatory T cells (Tregs) are a critical subset of CD4+ T cells that regulate the immune response and prevent autoimmunity. They are capable of inducing apoptosis, increasing cellular adenosine, and inhibiting DC maturation.

 

 

Flow Cytometry Panel for Immunophenotyping of Regulatory T Cells

Marker Clone Fluorochrome Catalog #
CD3* UCHT1 Alexa Fluor® 405 FAB100V
CD4 11830 PerCP FAB3791C
CD25/IL-2R 24212 PE FAB1020A
CD127/IL-7R 40131 Alexa Fluor® 700 FAB306N
GITR/TNFRSF18 110416 FITC FAB689F
FoxP3 376209 Alexa Fluor® 647 IC8970P

*Designate clones independently validated by HLDA.

This multicolor flow cytometry panel was validated on human peripheral blood mononuclear cells (PBMCs).

 

 

Flow Cytometry Gating Strategy for Regulatory T Cell Panel

Pseudocolor flow cytometry plot showing gating strategy for Treg markers CD3, CD4, CD25, CD127, and FoxP3.

Immunophenotyping of expanded human Tregs. Human T regulatory cells were expanded from PBMCs using Anti-Human CD3 Monoclonal Antibody (10 μg/mL, Catalog # MAB100) and Anti-human CD28 Monoclonal Antibody (5 μg/mL, Catalog # AF-342-PB) plus rhIL-2 (20 ng/mL, Catalog # 202-IL), rhTGF-β (10 ng/mL, Catalog # 77454-BH) for 2 days. Cells were assessed for expression of human CD3, CD4, CD25, CD127, FoxP3, and GITR.

 

 

 

Staining Protocol for Treg Phenotype Panel

Other Supplies Required

Surface Stain

  1. Wash human PBMCs (1 x 106 cells per sample) with 2 mL of Staining Buffer (1X) (R&D Systems®, Catalog # FC001) or other BSA-containing buffers, by spinning at 300 x g for 5 minutes, using 5 mL flow cytometry tubes. Decant/aspirate supernatant.
  2. Fc-block cells with blocking IgG (1 μg IgG/106 cells) for 10 minutes at room temperature.
  3. Add 5 μL (or previously titrated amount) of each surface marker). Vortex tubes.
  4. (Optional) To a separate tube, add 5 μL of each of the isotype control antibodies. Vortex tubes.
  5. Incubate the mixtures for 30-45 minutes at room temperature in the dark.
  6. At the end of the incubation, wash with 2 mL of Staining Buffer (1X), by spinning at 300 x g for 5 minutes. Decant/aspirate supernatant.

Intracellular Stain (with detergent permeabilization)

  1. Add 0.5 mL of cold Flow Cytometry Fixation Buffer (R&D Systems® Catalog # FC004) and vortex. Incubate at room temperature for 10 minutes. Vortex cells intermittently to maintain a single cell suspension.
  2. Centrifuge cells 350-500 x g for 5 minutes. Decant the Fixation Buffer.
  3. Wash cells PBS (or HBSS) by adding 2 mL of PBS (or HBSS), centrifuge at 350-500 x g for 5 minutes, and decant buffer from pelleted cells. Repeat (2 total washes).
  4. Resuspend the cell pellet in 0.1-0.2 mL of Flow Cytometry Permeabilization Buffer/Wash Buffer I (R&D Systems® Catalog # FC005)

Note: Saponin-mediated cell permeabilization is a reversible process, it is important to keep the cells in the presence of Permeabilization Buffer I during intracellular staining.
Note: Depending on the specific antibody and cell sample being used, the fixation and permeabilization steps can be performed simultaneously using Flow Cytometry Fixation/Permeabilization Buffer I (R&D Systems® Catalog # FC007).

  1. Add 5-10 µL/106 cells (or a previously titrated amount) of conjugated antibody to intracellular activation markers). Vortex tubes.
  2. Incubate cells for 30 minutes at room temperature in the dark.
  3. Wash cells 2 times with Flow Cytometry Permeabilization Buffer/Wash Buffer I (Catalog # FC005) as described in step 3.
  4. Resuspend the cells in 0.2-0.5 mL PBS (or HBSS) buffer and acquire on a Flow Cytometer.