Cell Preparation and Staining Procedures for Fluorochrome-Conjugated Anti-Human FoxP3 Antibody
1. Bring the buffers to room temperature (RT) before use. Prepare working solutions of the BD Pharmingen Human FoxP3 Buffer Set
Cat. No. 560098 (For the buffer preparation, please see the Technical Data Sheet of Cat. No. 560098 buffer instructions for details).
2. Prepare human PBMC. Suspend the cells with BD Pharmingen™ Stain Buffer (FBS)* to ten million cells/ml.
3. Pipette appropriate amount of surface staining reagent to the bottom of each 12 x 75 mm tube.
4. Add 100 µl of cells per tube, vortex, incubate for 20 minutes at RT protected from light.
5. Add 2 ml of wash buffer. Centrifuge 250 x g for 10 minutes to pellet the cells and remove wash buffer.
6. To fix the cells, gently resuspend the cell pellet in residual volume of wash buffer and then add 2 ml of 1x Human FoxP3 Buffer A.
Vortex. Incubate for 10 minutes at RT in the dark.
7. Centrifuge 500 x g for 5 minutes, and remove fixative. Caution: Be aware the pellet is buoyant.
8. To wash cells, resuspend each cell pellet in 2 ml of BD Pharmingen Stain Buffer (FBS)*, and centrifuge 500 x g for 5 minutes. Remove
9. To permeabilize the cells, gently resuspend pellet in residual volume of wash buffer and then add 0.5 ml of 1x working solution
Human FoxP3 Buffer C to each tube. Vortex. Incubate for 30 minutes at RT protected from light.
10. To wash cells, add 2 ml of BD Pharmingen Stain Buffer (FBS)* to each tube, centrifuge 500 x g for 5 minutes at RT. Remove buffer
and repeat wash step. Remove buffer.
11. Add conjugated FoxP3 antibody at appropriate concentrations to resuspend the pellet. Gently shake or vortex.
12. Incubate for 30 minutes in the dark at RT.
13. Repeat wash step #10.
14. Resuspend in wash buffer and analyze immediately.
Optional Add 300 µl of 1% formaldehyde in 1x PBS and store at 4°C. Analyze cells within 24 hours.
* We recommend using the BD Pharmingen™ Stain Buffer (FBS; Cat No. 554656) for all wash steps and covering tubes during incubation steps with caps or parafilm. We also recommend optimizing forward scatter and side scatter voltages to visualize lymphocytes as separate from debris, red cell ghosts and/or platelets before acquisition.