The FOXP3 Fix/Perm buffer(4X) must be diluted to (1X) working solution with PBS (10 mM sodium phosphate, 150 mM sodium chloride, pH 7.2) prior to staining procedures. e.g. Dilute one (1) part FOXP3 Fix/Perm buffer(4X) to three (3) parts PBS.

FOXP3 Intracellular Staining Procedures:
1. Perform cell surface staining as described in BioLegend's Cell Surface Immunofluorescence Staining Protocol.
2. Add 1 ml of 1X BioLegend's FOXP3 Fix/Perm solution to each tube, vortex and incubate at room temperature in the dark for 20 minutes, then spin down the cells and remove the supernatant.
3. Wash once with cell staining buffer (Cat. No. 420201). Spin at 250Xg for 5 minutes and remove the supernatant.
4. Wash once with 1ml 1X BioLegend's FOXP3 Perm buffer.
5. Re-suspend cells in 1ml 1X BioLegend's FOXP3 Perm buffer, incubate at room temperature in the dark for 15 minutes, spin down cells and discard the supernatant, then resuspend the pellet in 100 ul of 1X BioLegend's FOXP3 Perm buffer.
6. Add appropriate amount of flurochrome conjugated anti-FOXP3 antibody and incubate at room temperature in the dark for 30 minutes.
7. Wash twice with cell staining buffer, and resuspend in 0.5ml cell staining buffer then analyze with flow cytometer with appropriate instrument setting.