A Reliable Way To Visualize Mouse Regulatory T Cells

Protocol Overview

Prepare a single-cell suspension. Stain cells with a Fixable Viability Dye and Fc block in PBS for 20 minutes covered and on ice. Wash cells in FACS buffer, then stain cell surface markers in FACS buffer for 20 minutes covered and on ice. Wash in FACS buffer, then aspirate the supernatant. Add 1 mL of Foxp3 Fixation/Permeabilization working solution to each tube and vortex, then incubate cells for 30-60 minutes covered and at room temperature in the dark or overnight at 4°C in the dark. Add 2 mL of 1X Permeabilization Buffer to each tube and centrifuge at 400-600 x g for 5 minutes at room temperature, then discard the supernatant. Repeat wash step. Add the appropriate amount of intracellular protein-directed antibody (typically 1:100 dilution in Permeabilization Buffer, 100 uL per million cells)to cells and incubate for at least 30 minutes covered at room temperature or overnight at 4°C in the dark. Add 2 mL of 1X Permeabilization Buffer to each tube and centrifuge samples at 400-600 x g for 5 minutes at room temperature, then discard the supernatant. Repeat wash step. Resuspend cells in 100-250 uL PBS containing 1.5% paraformaldehyde.