Regulatory T Cell (Treg) Flow Cytometry Panel

Gating Strategy | Staining Protocol | Additional Flow Cytometry Resources

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

*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

Other Supplies Required

• PBS
• Flow Cytometry Staining Buffer (Catalog # FC001)
• Flow Cytometry Fixation Buffer (Catalog # FC004)
• Flow Cytometry Permeabilization Buffer/Wash Buffer I (Catalog # FC005)
• Fc-block (blocking IgG)
• (Optional) Isotype Control Antibodies
• 5 mL Flow cytometry tubes

Staining Protocol for Treg Phenotype Panel

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.
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.
    

Treg Antibody Concentration Chart  

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

Recommended Antibody Concentrations  

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