BD, 560408, BD Pharmingen™ PE Rat anti-Mouse Foxp3

Alternative Name: Forkhead box P3; Forkhead box protein P3; JM2; Scurfin; Scurfy; Sf
Reactivity: Mouse (QC Testing)
Isotype: Rat IgG2b, κ
Immunogen: Mouse FoxP3 Recombinant Protein
Application: Intracellular staining (flow cytometry) (Routinely Tested)
Concentration: 0.2 mg/ml
RRID: AB_1645251
Storage Buffer: Aqueous buffered solution containing ≤0.09% sodium azide.
Regulatory Status: RUO
Preparation And Storage: Preparation And Storage The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The antibody was conjugated to the dye under optimum conditions and unconjugated antibody and free dye were removed.
Recommended Assay Procedures: Recommended Assay Procedures 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. BD® CompBeads can be used as surrogates to assess fluorescence spillover (compensation).  When fluorochrome conjugated antibodies are bound to BD® CompBeads, they have spectral properties very similar to cells.   However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls.  It is strongly recommended that when using a reagent for the first time, users compare the spillover on cell and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.