Contents: Phycoerythrin (PE) anti-mouse Foxp3
Catalog Number: 12-4771
Formulation: Phosphate buffer pH 7.2,
150 mM NaCl, 0.09% NaN3
Storage Conditions: Store at 4°C.
DO NOT FREEZE.
LIGHT-SENSITIVE MATERIAL.
Clone: NRRF-30
Isotype: Rat IgG2a, κ
|
|
 
Mouse (BALB/c) splenocytes were surface-stained with CD4-FITC (clone RM4-5) (cat. 11-0042) and CD25-APC (clone PC61.5) (cat. 17-0251), and subsequently with 0.25μg/million cells PE anti-mouse Foxp3 (NRRF-30) (cat. 12-4771) or PE Rat IgG2a Iso Cntrl (cat. 12-4321) using the Foxp3 Staining Buffer Set (cat. 00-5523). The dot plot on the left demonstrates co-staining of CD4 and NRRF-30, while the right plot demonstrates co-staining of CD25 and NRRF-30 . Cells in the lymphocyte gate were used for analysis.
|
| Available Formats of This Product |
| Cat. No. |
Format |
Excite
(nm) |
Emit
(nm) |
Reported Applications |
| 14-4771 |
Affinity Purified anti-mouse Foxp3 |
N/A |
N/A |
IH/F
WB
|
| 12-4771 |
Phycoerythrin (PE) anti-mouse Foxp3 |
488 |
575 |
IC Flow
|
Flow Cytometry Product Notes:
Test Sizes: To accommodate multicolor flow cytometry, eBioscience is in the process of reducing test size volumes from 20 µl to 5 µl. Please check your antibody vial for the recommended test size.
Fluorochrome Replacements: eBioscience is in the process of replacing all Pacific Blue® and APC-Alexa Fluor® 750 conjugated products with eFluor™ 450 and APC-eFluor™ 780 conjugated products, respectively. |
Questions? Please consult our answers to frequently asked questions at
http://www.ebioscience.com/faq.
Description
The NRRF-30 antibody reacts with mouse Foxp3 also known as FORKHEAD BOX P3, SCURFIN, and JM2; cross reactivity of this antibody to other proteins has not been determined. Foxp3, a 49-55 kDa protein, is a member of the forkhead/winged-helix family of transcriptional regulators, and was identified as the gene defective in ‘scurfy’ (sf) mice. Constitutive high expression of foxP3 mRNA has been shown in CD4+/CD25+ regulatory T cells (Treg cells), and ectopic expression of foxp3 in CD4+/CD25- cells imparts a Treg phenotype in these cells.
Immunoblotting with NRRF-30 antibody has mapped the epitope to amino acids 1-75 of the mouse Foxp3 protein.
Intracellular staining of mouse splenocytes with fluorochrome-conjugated NRRF-30 using the eBioscience Foxp3 Staining Buffers (cat.
00-5523) and corresponding staining protocol reveals approximately 3% of total cells in the C57Bl/6 strain and approximately 5% in the BALB/c mouse strain. Multicolor flow cytometric analysis demonstrates approximately 90% of the CD4+/CD25+ cells and 4% of the CD4+/CD25- cells staining with NRRF-30. Co-staining with FJK-16s (anti-mouse/rat Foxp3 cat.
71-5775), which has been mapped to amino acids 71-125, and NRRF-30 shows 100% correlation, indicating that the same cells are stained with both anti-mouse Foxp3 antibodies.
Please see the following link for FAQ regarding the usage of eBioscience Foxp3 reagents:
http://www.ebioscience.com/ebioscience/Foxp3FAQs.htm
Applications Reported
For research use only, not for diagnostic or therapeutic use. This NRRF-30 antibody has been reported for use in intracellular staining followed by flow cytometric analysis.
Applications Tested
This NRRF-30 antibody has been tested by intracellular flow cytometric analysis of mouse splenocytes using the Foxp3 Staining Buffer Set (cat.
00-5523) and protocol for Foxp3. This can be used at less than or equal to 0.25 μg per million cells in a 100 μl total staining volume. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest.
Related Products
Cat. 12-4321 PE Rat IgG2a Isotype ControlCat. 12-5773 PE anti-mouse/rat Foxp3 (clone FJK-16s)Cat. 14-5773 Affinity Purified anti-mouse/rat Foxp3 (clone FJK-16s)
Protocol for IC Staining
It is critical to use the Foxp3 Staining Buffer Set (cat. 00-5523). The buffer set is included with all Foxp3 Staining Sets.
Prior to staining, dilute the Fixation/Permeabilization Concentrate (1 part) into the Fixation/Permeabilization Diluent (3 parts) to the desired volume of Fixation/Permeabilization working solution. This buffer should not be stored for more than 1 day. For example: For 12 samples, use 3 ml Fixation/Permeabilization Concentrate and 9 ml Fixation/Permeabilization Diluent.
-
Add 100 µl of prepared cells (1x106) to each tube.
-
Stain surface molecules such as CD4, CD8, CD25, etc. following the Surface Staining Protocol (http://www.ebioscience.com/ebioscience/appls/FCS.htm).
-
Wash in cold Flow Cytometry Staining Buffer (or cold PBS).
-
Resuspend cell pellet with pulse vortex and add 1 ml of freshly prepared Fixation/Permeabilization working solution to each sample. Pulse vortex again.
-
Incubate at 4°C between 30 minutes and 18 hours in the dark. (Comparable results, using the same donor, are obtained when incubated in the Fixation/Permeabilization Solution for varying times between 30 minutes and 18 hours.)
-
Wash once by adding 2 ml 1X Permeabilization Buffer (made from 10X Permeabilization Buffer) followed by centrifugation and decanting of supernatant.
-
Wash cells with 2 ml 1X Permeabilization Buffer. Centrifuge and decant supernatant.
-
[OPTIONAL] Block with Fc block in 1X Permeabilization Buffer, in approximately 100 µl volume, at 4°C for 15 minutes.
-
Without washing after blocking step, add fluorochrome conjugated anti-Foxp3 antibody or isotype control in 1X Permeabilization Buffer and incubate at 4°C for at least 30 minutes in the dark. Please perform further titration for optimal staining in your own assay system.
-
Wash cells with 2 ml 1X Permeabilization Buffer. Centrifuge and decant supernatant.
-
Repeat step 10.
-
Resuspend in appropriate volume Flow Cytometry Staining Buffer and analyze on cytometer. Please note that due to the fixation and permeabilization procedure, the FSC/SSC distribution of the cell population will be different than live cells. Therefore the gate and voltages will need to be modified.
Mouse Cellular Antigens Chart