Each lot of this FOXP3 antibody is quality control tested by immunofluorescent staining with flow cytometric analysis. For immunofluorescent staining, the suggested use of this reagent is 20µl per test. It is recommended that the reagent be titrated for optimal performance for each application.

Materials Provided:
1. Helios FITC/FOXP3 Alexa Fluor^® 647/CD4 PerCP/Cy5.5, 20uL/test, 25 tests
2. FOXP3 Fix/Perm Buffer Set, 100 tests (Cat. No. 421403)

Materials Not Included:
1. Cell staining buffer (Cat. No. 420201) 
2. FITC Armenian Hamster IgG Isotype (400905)
3. Alexa Fluor® 647 Rat IgG2b, κ Isotype Ctrl (400626)
4. PerCP/Cy5.5 Rat IgG2b, κ Isotype Ctrl (400631)

Immunofluorescence Staining Procedures:

Centrifugation steps: perform at 250Xg for 5min
Incubation steps: perform at room temperature.

1. Prepare 1X working solutions of FOXP3 Fix/Perm buffer and FOXP3 Perm buffer prior to staining. The FOXP3 Fix/Perm buffer (4X) must be freshly diluted by diluting one (1) part FOPX3 Fix/Perm buffer (4X) with three (3) parts PBS. The FOXP3 Perm buffer (10X) should be diluted by diluting one (1) part FOXP3 Perm buffer (10X) with nine (9) parts of PBS.

NOTE: The FOXP3 Perm buffer (10X) may have crystallization or precipitation observed when it is stored at 2-8°C, however, it is normal and does not affect buffer performance. If there is a heavy precipitation observed after diluted to 1X working solution, it can be filtered to clarify the solution.
Caution: The FOXP3 Fix/Perm buffer contains paraformaldehyde, which is toxic and mutagenic. Please wear necessary protection to avoid direct body contacts.

2. Suspend the cells of interest at the concentration of 1X10⁷/ml with BioLegend's cell staining buffer (Cat. No. 420201).
3. Distribute 100 μl of the cell suspension into FACS tubes.
4. Add 1 ml of 1X BioLegend's FOXP3 Fix/Perm solution to each tube, vortex and incubate for 20 minutes; centrifuge, then discard the supernatant.
5. Wash: resuspend with 2ml/tube cell staining buffer; centrifuge, then discard the supernatant.
6. Wash: resuspend with 1ml 1X BioLegend's FOXP3 Perm buffer.
7. Re-suspend cells in 1ml 1X BioLegend's FOXP3 Perm buffer, incubate for 15 minutes; centrifuge, then discard the supernatant.
8. Add 20 μl Mouse Helios Treg Flow^TM Kit or isotype control antibodies into appropriate tube and mix by gently vortexing.
9. Incubate at in the dark for 20 minutes.
10. Wash twice with cell staining buffer (see step 5) and resuspend in 0.5 ml cell staining buffer then analyze using a flow cytometer with appropriate instrument settings.