53-4774-42

antibody from Invitrogen Antibodies

Targeting: FOXP3 AIID, DIETER, IPEX, JM2, PIDX, SCURFIN, XPID

Flow cytometry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

53-4774-42

Provider

Invitrogen Antibodies

Product name

FOXP3 Monoclonal Antibody (150D/E4), Alexa Fluor™ 488, eBioscience™

Antibody type

Monoclonal

Antigen

Other

Description

Description: eBioscience offers a panel of monoclonal antibodies to different epitopes of human/primate and mouse/rat Foxp3 protein, providing useful tools for investigating the complete expression pattern of Foxp3 at the protein level, and discerning the precise subsets of Foxp3+ cells. Other antibodies to Foxp3 available from eBioscience, which have been used significantly in scientific literature, include the anti-human Foxp3 PCH101 (Product # 72-5776) and ebio7979 (Product # 12-7979), and the anti-mouse/rat Foxp3 FJK-16s (Product # 72-5775). The 150D/E4 has been mapped to the splice region in Exon 2 found in human cells. The splicing of this region has not been shown to occur in mouse. Characterization of this antibody to the splice variant has been limited to epitope mapping. It should be noted that other antibodies such as PCH101 and 236A/E7 will recognize both the spliced and full length forms of the Foxp3 protein and stain more intensely than 150D/E4 and FJK-16s in human cells. The 150D/E4 antibody reacts with mouse/rat/human 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. Applications Reported: This 150D/E4 antibody has been reported for use in intracellular staining followed by flow cytometric analysis. Applications Tested: This 150D/E4 antibody has been pre-titrated and tested by intracellular staining and flow cytometric analysis of mouse splenocytes using the Foxp3/Transcription Factor Staining Buffer Set (Product # 00-5523-00) and protocol. Please refer to Best Protocols: Protocol B: One-step protocol for intracellular (nuclear) proteins. This antibody can be used at 5 µL (0.5 µg) per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. The staining protocol has been optimized with freshly Ficoll-prepared PBMC or mouse splenocytes. The use of lysed whole blood is not recommended. It is critical that this antibody be used in conjunction with the Foxp3/Transcription Factor Staining Buffer Set (Product # 00-5523-00) for flow cytometric analysis. Excitation: 488 nm; Emission: 519 nm; Laser: Blue Laser. Filtration: 0.2 µm post-manufacturing filtered.

Reactivity

Human, Mouse, Rat

Host

Mouse

Conjugate

Green dye

Isotype

IgG

Antibody clone number

150D/E4

Vial size

100 Tests

Concentration

5 µL/Test

Storage

4° C, store in dark, DO NOT FREEZE!

References

Submitted references

In mouse chronic pancreatitis CD25(+)FOXP3(+) regulatory T cells control pancreatic fibrosis by suppression of the type 2 immune response.

Glaubitz J, Wilden A, Golchert J, Homuth G, Völker U, Bröker BM, Thiele T, Lerch MM, Mayerle J, Aghdassi AA, Weiss FU, Sendler M
Nature communications 2022 Aug 3;13(1):4502

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Differential expression of tissue-restricted antigens among mTEC is associated with distinct autoreactive T cell fates.

Lebel MÈ, Coutelier M, Galipeau M, Kleinman CL, Moon JJ, Melichar HJ
Nature communications 2020 Jul 24;11(1):3734

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Regulatory T Cells License Macrophage Pro-Resolving Functions During Atherosclerosis Regression.

Sharma M, Schlegel MP, Afonso MS, Brown EJ, Rahman K, Weinstock A, Sansbury BE, Corr EM, van Solingen C, Koelwyn GJ, Shanley LC, Beckett L, Peled D, Lafaille JJ, Spite M, Loke P, Fisher EA, Moore KJ
Circulation research 2020 Jul 17;127(3):335-353

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Mitochondrial transfer from MSCs to T cells induces Treg differentiation and restricts inflammatory response.

Court AC, Le-Gatt A, Luz-Crawford P, Parra E, Aliaga-Tobar V, Bátiz LF, Contreras RA, Ortúzar MI, Kurte M, Elizondo-Vega R, Maracaja-Coutinho V, Pino-Lagos K, Figueroa FE, Khoury M
EMBO reports 2020 Feb 5;21(2):e48052

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Microbial, metabolomic, and immunologic dynamics in a relapsing genetic mouse model of colitis induced by T-synthase deficiency.

Jacobs JP, Lin L, Goudarzi M, Ruegger P, McGovern DP, Fornace AJ Jr, Borneman J, Xia L, Braun J
Gut microbes 2017 Jan 2;8(1):1-16

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Histone/protein deacetylase 11 targeting promotes Foxp3+ Treg function.

Huang J, Wang L, Dahiya S, Beier UH, Han R, Samanta A, Bergman J, Sotomayor EM, Seto E, Kozikowski AP, Hancock WW
Scientific reports 2017 Aug 17;7(1):8626

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Characteristics of CD4(+)CD25(+)Foxp3(+) regulatory T cells in patients with multiple organ dysfunction syndrome.

Zang X, Jiang Y, Li X, DU Y, Niu J
Experimental and therapeutic medicine 2016 May;11(5):1908-1912

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Glycolysis controls the induction of human regulatory T cells by modulating the expression of FOXP3 exon 2 splicing variants.

De Rosa V, Galgani M, Porcellini A, Colamatteo A, Santopaolo M, Zuchegna C, Romano A, De Simone S, Procaccini C, La Rocca C, Carrieri PB, Maniscalco GT, Salvetti M, Buscarinu MC, Franzese A, Mozzillo E, La Cava A, Matarese G
Nature immunology 2015 Nov;16(11):1174-84

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CD40 deficiency in mice exacerbates obesity-induced adipose tissue inflammation, hepatic steatosis, and insulin resistance.

Guo CA, Kogan S, Amano SU, Wang M, Dagdeviren S, Friedline RH, Aouadi M, Kim JK, Czech MP
American journal of physiology. Endocrinology and metabolism 2013 May 1;304(9):E951-63

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Regulatory T cell lineage specification by the forkhead transcription factor foxp3.

Fontenot JD, Rasmussen JP, Williams LM, Dooley JL, Farr AG, Rudensky AY
Immunity 2005 Mar;22(3):329-41

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Control of regulatory T cell development by the transcription factor Foxp3.

Hori S, Nomura T, Sakaguchi S
Science (New York, N.Y.) 2003 Feb 14;299(5609):1057-61

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Staining of C57BL/6 splenocytes with Anti-Mouse CD25 APC (Product # 17-0251-82) and Anti-Mouse CD4 PE (Product # 12-0041-82) followed by fixation and permeabilization using Foxp3 Staining Buffers (Product # 00-5523-00) and subsequently stained with Mouse IgG1 kappa Isotype Control Alexa Fluor® 488 (Product # 53-4714-42) (left) or Anti-Foxp3 Alexa Fluor® 488 (right). CD4+ cells in the lymphocyte gate were used for analysis.

Conjugate

Green dye

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Conjugate

Green dye

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Conjugate

Green dye

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Conjugate

Green dye

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Conjugate

Green dye

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Conjugate

Green dye

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Conjugate

Green dye

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Conjugate

Green dye

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Conjugate

Green dye

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 2. Regulatory T cell (Treg) depletion abrogates atherosclerosis regression. A , Experimental design: Atherosclerosis was established in C57BL6&solJ mice by injection of adeno-associated virus (AAV)-PCSK (proprotein convertase subtilisin&solkexin type)-9 followed by Western diet feeding for 20 wk (Baseline). Mice were then switched to chow diet and atherosclerosis regression was initiated by apoB (apolipoprotein B)-antisense oligonucleotide (ASO)-mediated lipid lowering in conjunction with anti-CD25 or IgG isotype control antibody treatment for 3 wk. B , Plasma total cholesterol, HDL (high-density lipoprotein) and non-HDL cholesterol, and triglyceride levels at baseline and after regression &plus IgG anti-CD25 treatment (n=11 mice&solgroup). C , Representative flow cytometric analyses of CD4 &plus CD25 &plus cells in the circulation of mice treated with IgG or anti-CD25 during atherosclerosis regression. D , Quantification of FoxP3 &plus CD25 &plus cells in peripheral blood (n=5&solgroup), thymus (n=5&solgroup), and spleen (n=9&solgroup) of mice by flow cytometry. Data are mean+-SEM and are representative of 2 independent experiments. P values were determined by a Student t test. E , Representative immunofluorescence staining for FoxP3 in aortic root plaques from mice at baseline and after regression &plus IgG or anti-CD25 treatment (n=6 mice&solgroup). Quantification shown at right. Scale bars=100 &mgrm. F and G , Representative images and quantification of atheroscler

Conjugate

Green dye

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 3 Conditional HDAC11 deletion promotes chromatin remodeling at the Foxp3 locus and Treg gene expression. ( A ) ChIP studies of the Foxp3 promoter and 3 enhancer regions in WT vs. conditional HDAC11-/- Treg cells, showing increased acetyl-H3 binding at all 4 sites in HDAC11-/- vs. WT Treg cells. ( B ) Luciferase reporter assays showing the impact of overexpression, and also inhibition, of HDAC11 in 293 T cells that were transiently transfected with murine Foxp3-promoter-Luc vector plus expression vectors for p65, HDAC11, Foxp3, and p300, as specified, or empty vector (EV) along with 50 ng of pRL-TK Renilla luciferase internal control vector; EV was added to ensure equal amounts of DNA per reaction. After 48 h, cells were treated with PMA (6 ng/ml) and ionomycin (1 muM) for 5 to 6 h, and 1 muM HDAC11i where specified, and cell lysates were harvested for luciferase assays. Firefly luminescence data was normalized to Renilla luminescence and analyzed by ANOVA (*p < 0.05, **p < 0.005); data are representative of the results of 3 or more independent experiments. ( C ) Heat-map of selected genes that were upregulated (red) or downregulated (blue) in conditional HDAC11-/- vs. WT Treg cells. ( D ) qPCR evaluation of WT vs. conditional HDAC11-/- Treg cells, showing increased expression of Jun, Junb, Fos and Fosb in HDAC11-/- Tregs; normalized data are shown as mean +- SD, 4 mice/group, and *p < 0.05, **p < 0.01.

Conjugate

Green dye

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Fig. 4 mTEC hi -restricted expression of USA preferentially induces T reg differentiation. Littermate control (LMC), CRP, and INS2 Tg mice (CD45.2, Thy1.2) were irradiated and reconstituted with 1% OT-II (CD45.2, Thy1.1) and 99% WT (CD45.1, Thy1.2) bone marrow. The hematopoietic chimeras were analyzed 6-8 weeks post-irradiation. a Proportion of mature TCRbeta hi CD4 + SP OT-II thymocytes among total live cells and absolute number of live CD4 + SP OT-II thymocytes. b Proportion of OT-II cells within the CD4 + T cell population in the peripheral lymph nodes. c Representative flow plots of CD25 and FoxP3 expression on OT-II T cells. d Proportion and absolute cell number of CD25 + Foxp3 + OT-II T cells in the thymus. e Proportion of CD25 + Foxp3 + OT-II T cells in the lymph nodes. (mean +- SEM, n = 17 LMC, 8 CRP, 10 INS2, from three independent experiments). All p -values indicated are calculated by Kruskal-Wallis test followed by Dunn''s post-hoc comparisons to LMC, two-sided. ** p < 0.01 relative to LMC. Source data are provided as a Source Data file.

Conjugate

Green dye

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Fig. 5 TRA expression by distinct mTEC compartments impacts the mode of tolerance induction in the polyclonal T cell repertoire in naive mice. a Representative flow plots of H-2K b -OVA tetramer on pooled peripheral lymph nodes and spleen cells from naive mice after enrichment, gated on live TCRalphabeta + CD8 + T cells. Relative number of H-2K b -OVA-specific TCRalphabeta + CD8 + T cells in the peripheral secondary lymphoid organs (mean +- SEM, n = 12 LMC, 7 CRP, 7 INS2) ( b ) and intraepithelial lymphocytes (IEL) (mean +- SEM, n = 17 LMC, 11 CRP, 8 INS2) ( c ) from CRP and INS2 Tg mice as compared to their respective littermate controls (LMC). d Representative flow plot of CD8alpha and CD8beta and e relative proportion of TCRalphabeta + CD8alphaalpha within the H-2K b -OVA tetramer double positive TCRalphabeta IEL of CRP and INS2 Tg mice as compared to their respective LMC (mean +- SEM, n = 17 LMC, 11 CRP, 8 INS2). f Representative flow plots of I-A b -gp66 tetramer on pooled peripheral lymph nodes and spleen cells from naive mice, gated on live TCRalphabeta + CD4 + T cells. g Relative number of I-A b -gp66 tetramer DP CD4 + T cells in the peripheral lymph nodes and the spleen of naive mice. h Representative flow plots of CD25 and FoxP3, gated on live I-A b -gp66 tetramer + CD4 + T cells. i Relative proportion of CD25 + FoxP3 + T reg of gp66-specific CD4 + T cells and relative number of gp66-specific CD4 + CD25 + FoxP3 + T reg from CRP and INS2 peripheral lymph nodes and sp

Conjugate

Green dye

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Fig. 6 TRA expression by distinct mTEC compartments impacts the mode of tolerance induction in the polyclonal T cell repertoire in LCMV infected mice. Mice were infected with LCMV-OVA and the antigen-specific response was measured in the spleen 8 days post-infection (NI, not infected mice) a, c Representative CD44 and H-2K b -OVA or H-2D b -gp33 tetramer staining on CD8 + T cells from littermate controls (LMC), CRP, and INS2 Tg mice. Relative response and absolute number of OVA- ( b ) and gp33- ( d ) specific CD8 + T cell response in USA expressing mice as compared to LMC. e Representative CD44 and I-A b -gp66 tetramer staining on CD4 + T cells from LMC, CRP, and INS2 Tg mice. f Relative and absolute gp66-specific response in USA expressing mice. g Representative flow plot of CD25 and FoxP3, gated on live I-A b -gp66-specific CD4 + T cells. h Relative proportion of CD25 + FoxP3 + T reg among gp66-specific CD4 + T cells and absolute number of gp66-specific CD4 + CD25 + FoxP3 + T reg (mean +- SEM, b left panel n = 22 LMC, 7 CRP, 5 CRP lo , 9 INS2, 3 INS2 lo ; b right panel n = 3 NI, 8 CRP LMC, 10 CRP Tg, 9 CRP lo LMC, 9 CRP lo Tg, 10 INS2 LMC, 9 INS2 Tg, 3 INS2 lo LMC, 3 INS2 lo Tg; d and f left panel n = 32 LMC, 12 CRP, 12 CRP lo , 14 INS2, 8 INS2 lo , d right panel n = 6 NI, 11 CRP LMC, 12 CRP Tg, 11 CRP lo LMC, 12 CRP lo Tg, 13 INS2 LMC, 11 INS2 Tg, 6 INS2 lo LMC, 6 INS2 lo Tg, f right panel n = 6 NI, 11 CRP LMC, 13 CRP Tg, 12 CRP lo LMC, 13 CRP lo Tg, 14 INS2 LMC, 12 INS2 T

Conjugate

Green dye