FOXP3 Intracellular Staining Procedure

Protocol

1. Perform cell surface staining as described in Cell Surface Flow Cytometry Staining Protocol.
2. Prepare a 1x working solution of FOXP3 Fix/Perm Buffer by diluting 1 part FOXP3 Fix/Perm Buffer (4x) (Cat. No. 421401) in 3 parts PBS (10 mM sodium phosphate, 150 mM sodium chloride, pH 7.2).
3. Add 1 mL of 1x FOXP3 Fix/Perm solution to each sample. Vortex, then incubate at room temperature in the dark for 20 minutes. Spin at 250 x g for 5 minutes and discard the supernatant.
4. Add 1 mL of Cell Staining Buffer (Cat. No. 420201) to wash cells. Spin at 250 x g for 5 minutes and discard the supernatant.
5. Prepare a 1x working solution of FOXP3 Perm Buffer by diluting 1 part FOXP3 Perm Buffer (10x) (Cat. No. 421402) to 9 parts PBS.

   Note:

   FOXP3 Perm Buffer (10x) may exhibit crystallization or precipitation when stored at 2-8 °C. This is normal and does not affect the buffer's performance. Heavy precipitation observed after dilution to a 1x solution can be cleared by filtration.
6. Wash once with 1 mL of 1x FOXP3 Perm Buffer.
7. Resuspend cells in 1 mL of 1x FOXP3 Perm Buffer and incubate at room temperature in the dark for 15 minutes. Spin down cells and discard the supernatant. Then resuspend the pellet in 100 μL of 1x FOXP3 Perm Buffer.
8. Add an appropriate amount of fluorochrome-conjugated antibody and incubate at room temperature in the dark for 30 minutes.
9. Wash twice with Cell Staining Buffer, and resuspend in 0.5 mL of Cell Staining Buffer. Then analyze with a flow cytometer.