BD Pharmingen™ Alexa Fluor® 647 Rat anti-Mouse Foxp3

Cell Preparation and Staining Procedures for Conjugated Anti-Mouse Foxp3 Antibody

        1.   Prepare working solutions of the BD Pharmingen™ Mouse Foxp3 Buffer Set Cat. No. 560409 (for the buffer preparation, please see TDS

              Cat. No. 560409 buffer instructions for details).

        2.   Pre-warm Permeabilization buffer to 37°C before use. Keep Fixation buffer on ice.

        3.   Prepare a single-cell suspension from the peripheral lymphoid tissue of interest. Remove clumps of cells and/or debris by passing the

              suspension through a 70-µm nylon cell strainer. Use 1 × BD PharmLyse™ Lysing Buffer (Cat. No. 555899) to lyse red blood cells

              if necessary. Dilute the cells with BD Pharmingen™ Stain Buffer (FBS, Cat. No. 554656) to ten million cells/ml.

        4.   Pipette appropriate amount of CD4 or other surface staining reagent(s) to bottom of each 12 x 75 mm tube.

        5.   Add 100 µl of cells per tube, mix well. Incubate for 20 minutes at RT in the dark.

        6.   To wash cells, add 2 ml of BD Pharmingen™ Stain Buffer (FBS) to each tube. Centrifuge 250 x g for 10 minutes, and remove buffer.

        7.   To fix the cells, gently re-suspend pellet in residual volume of staining buffer and then add 2 ml of freshly prepared cold 1 x BD

              Pharmingen™ Mouse Foxp3 Fixation Buffer. Mix well. Incubate for 30 minutes at 4°C in the dark.

        8.   Centrifuge 500 x g for 5 minutes, and remove fixative.

        9.   To wash cells, re-suspend each pellet in 2 ml of freshly prepared pre-warmed 1 × BD Pharmingen™ Mouse Foxp3 Permeabilization

               buffer,  and centrifuge 500 x g for 5 minutes. Remove permeabilization buffer.

        10. To permeabilize the cells, gently re-suspend pellet in another 2 ml of freshly prepared pre-warmed 1 x BD Pharmingen™ Mouse Foxp3

              Permeabilization buffer. Incubate for 30 minutes at 37°C in the dark.

        11. Centrifuge 500 x g for 5 minutes, and remove buffer.

        12. To wash cells, add 2 ml of BD Pharmingen™ Stain Buffer (FBS) to each tube, centrifuge 500 x g for 5 minutes. Remove buffer.

        13. Add 20 µl of conjugated Foxp3 antibody diluted with BD Pharmingen™ Stain Buffer (FBS) at appropriate concentrations (check the  

              figure legend from each format for the concentration) to re-suspend the pellet. Gently shake or vortex briefly.

        14. Incubate for 20 minutes at RT in the dark.

        15. Repeat wash step #12 two times.

        16. Resuspend the cells in 0.5 ml BD Pharmingen™ Stain Buffer (FBS) and analyze immediately.*

Note: We recommend using the BD Pharmingen™ Stain Buffer 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 cells, etc. before acquisition.

* Acquire at least 15,000 to 25,000 CD4 positive lymphocytes.