Description
The 259D/C7 monoclonal antibody specifically recognizes human Forkhead box protein P3 (FoxP3) that is also known as Scurfin. FoxP3 is encoded by FOXP3 (Forkhead box P3), likewise known as IPEX (Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-Linked) and JM2, that belongs to the forkhead/winged-helix family of transcriptional regulators. FoxP3 is expressed in CD4+ regulatory T cells (Treg) and represents a specific marker for these cells. Flow cytometric analyses have shown that FoxP3 is expressed by the majority of CD4+CD25+high T cells in peripheral blood while less than half of the CD4+CD25+intermediate cell population are FoxP3 positive. Approximately 5-10% of peripheral CD4+ cells are CD4+CD25+ T regulatory cells. T regulatory cells are thought to play a critical role in the regulation of T cell-mediated immunity and to protect against autoimmunity by suppressing the proliferation and cytokine production of other T cells. In support of this hypothesis, it has been found that Foxp3 is mutated in Scurfy (sf) mice that have defective T cell tolerance leading to an X-linked lymphoproliferative disease. The 259D/C7 antibody recognizes all currently-identified isoforms of human FoxP3 and is crossreactive with FoxP3 from Cynomolgus, Rhesus and Baboon primates.
Cell Preparation and Staining Procedures for Conjugated Anti-Human FoxP3 Antibody 1. Bring the buffers to 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 human PBMC. Dilute the cells with BD Pharmingen Stain Buffer (FBS)* to ten million cells/ml. 3. Pipette appropriate amount of surface staining reagent to bottom of each 12 x 75 mm tube. 4. Add 100µl 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, and remove wash buffer. 6. To fix the cells, gently re-suspend pellet in residual volume of wash buffer and then add 2ml 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, re-suspend each pellet in 2ml of BD Pharmingen Stain Buffer (FBS)*, and centrifuge 500 x g for 5 minutes. Remove wash buffer. 9. To permeabilize the cells, gently re-suspend 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 re-suspend 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 300µl 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. ** Acquire at least 15,000 to 25,000 CD4 positive lymphocytes.
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.
1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
2. An isotype control should be used at the same concentration as the antibody of interest.
3. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
4. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
6. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
7. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
8. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
9. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
10. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
