Regulatory T Cell (Treg) Flow Cytometry Panel

*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).

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

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).

5. 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

    
6. Incubate cells for 30 minutes at room temperature in the dark.
7. Wash cells 2 times with Flow Cytometry Permeabilization Buffer/Wash Buffer I (Catalog # FC005) as described in step 3.
8. Resuspend the cells in 0.2-0.5 mL PBS (or HBSS) buffer and acquire on a Flow Cytometer.