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. Use this validated flow cytometry panel to identify and phenotype CD4+ T regulatory cells.



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
FoxP3 376209 Alexa Fluor® 647 IC8970P

*Designate clones independently validated by HLDA.
Alexa Fluor® is registered trademark of Molecular Probes, Inc.

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.

Multicolor flow cytometry panel to identify human T Regulatory Cells (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 stained with CD3 Alexa Fluor® 405, CD4 PerCP, CD25 PE, CD127 Alexa Fluor® 700, FoxP3 Alexa Fluor® 647, and GITR FITC. Cells were previously gated on lymphocytes and live cells.




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 previously titrated amount of each surface marker. Vortex tubes.

    Treg Antibody Concentration Chart  
    Marker Fluorochrome Treg Antibody Concentration Chart
    CD3 Alexa Fluor® 405 5 μL/106 cells
    CD4 PerCP 10 μL/106 cells
    CD25/IL-2R PE 10 μL/106 cells
    CD127/IL-7R Alexa Fluor® 700 5 μL/106 cells
    GITR/TNFRSF18 FITC 10 μL/106 cells
  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 previously titrated amount of conjugated antibody to intracellular activation markers. Vortex tubes.

    Recommended Antibody Concentrations  
    Marker Fluorochrome Recommended Concentration
    FoxP3 Alexa Fluor® 647 10 μL/106 cells
  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.