Description
Cell Preparation and Staining Procedures for 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 TDS Cat. No. 560098 buffer instructions for details). 2. Prepare a suspension of human peripheral blood mononuclear cells. Suspend the cells with BD Pharmingen Stain Buffer (FBS) (Cat. No. 554656)* to ten million cells/ml. 3. Pipet appropriate amount of surface staining reagent to 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 250g for 10 minutes, 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 500g for 5 minutes and remove fixative. Caution: Be aware the cell pellet is buoyant. 8. To wash cells, resuspend each cell pellet in 2 ml of BD Pharmingen Stain Buffer (FBS)*, and centrifuge 500g for 5 minutes. Remove wash buffer. 9. To permeabilize the cells, gently resuspend the cell 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 500g for 5 minutes at RT. Remove buffer and repeat wash step. Remove buffer. 11. Add conjugated FoxP3 antibody at appropriate concentrations to resuspend the cell 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 2 hours. * 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. ** Acquire at least 15,000 to 25,000 gated CD4 positive lymphocyte events.
1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
2. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
