Alexa Fluor® 488 Rat anti-Mouse Foxp3
Clone MF23 (RUO)
- Brand BD Pharmingen™
- Alternative Name Forkhead box p3; Forkhead box protein p3; JM2; Scurfin; Scurfy; Sf
- Concentration 0.2 mg/ml
- Isotype Rat IgG2b
- Reactivity Mouse (QC Testing)
Intracellular staining (flow cytometry) (Routinely Tested)
- Immunogen Mouse Foxp3 Recombinant Protein
- Storage Buffer Aqueous buffered solution containing ≤0.09% sodium azide.
- Regulatory Status RUO
Regulatory Status Legend
The MF23 monoclonal antibody specifically binds to mouse Foxp3. Foxp3 is a 50-55 kDa protein also known as Forkhead box p3, JM2, or IPEX. It is a member of the forkhead or winged helix family of transcription factors and is specifically expressed by T regulatory (Treg) cells. Foxp3 has been reported to be a key regulatory protein for Treg cell development and function. Ectopic expression of Foxp3 in conventional T cells is sufficient to induce suppressive activity, repress the production of cytokines such as IL2 and IFN-γ, and upregulate Treg cell-associated molecules such as CD25, CTLA4 and GITR. It has been found that the mutation of Foxp3 is responsible for "scurfy" mice. When overexpressed, Foxp3 leads to poor T cell proliferation and activation. Immunoblotting with MF23 antibody has confirmed it recognizes an epitope between 1-87 amino acids in the N-terminal domain.
Alexa Fluor® conjugates are highly photostable and remain fluorescent over a broad pH range. The excitation and emission maxima are nearly identical to those of FITC. However, Alexa Fluor® 488 tends to be brighter and more optimal for intracellular applications. Due to nearly identical excitation and emission properties but different spillover characteristics, FITC and Alexa Fluor® 488 cannot be used simultaneously. Alexa Fluor® 488 exhibits extraordinary photostability, which makes it highly suitable for fluorescence microscopy.
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Preparation and Storage
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
The antibody was conjugated to Alexa Fluor® 488 under optimum conditions, and unreacted Alexa Fluor® 488 was removed.
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Alexa Fluor® 488 fluorochrome emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC).
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- 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.
- 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.
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.