Anti foxp3 fjk 16s

Manufactured by Thermo Fisher Scientific

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Anti-Foxp3 (FJK-16s) is a monoclonal antibody used for the detection and analysis of Foxp3-expressing cells, which are a subset of regulatory T cells (Tregs). The antibody recognizes the Foxp3 (forkhead box P3) transcription factor, which is a key regulator of Treg development and function. This antibody can be used in various applications, such as flow cytometry and immunohistochemistry, to identify and quantify Foxp3-positive cells.

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63 protocols using anti foxp3 fjk 16s

Comprehensive Immunophenotyping of Murine Lymphoid Tissues

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Mice were sacrificed after 60 days and spleens, inguinal and mesenteric lymph nodes were harvested and homogenized into single cell suspensions using HBSS + 2% FBS (Gibco; Gaithersburg, MD). Spleen cells were further treated with ammonium chloride lysing solution to remove erythrocytes. The following anti-mouse antibodies were used for immunophenotyping: anti-CD1d (1D1) (BD Pharmingen; San Diego, CA), anti-CD3 (145-2C11) (BD Pharmingen), anti-CD4 (GK1.5) (eBioscience, San Diego, CA), anti-CD5 (53–7.3) (BD Pharmingen), anti-CD8 (53–6.7) (eBioscience), anti-CD11b (M1/70) (eBioscience), anti-CD19 (6D5) (Caltag Laboratories; Burlingame, CA), anti-CD25 (PC61) (BD Pharmingen), anti-Ter119 (TER-119) (eBioscience), anti-B220 (RA3-6B5) (BD Pharmingen), anti-DX5 (DX5) (eBioscience), anti-Gr-1 (RB6-8C5) (eBioscience), and anti-FoxP3 (FJK-16s) (eBioscience). Staining for FoxP3 was performed according to the manufacturer’s protocol (eBioscience). All samples were acquired on a FACSCalibur flow cytometer (BD Biosciences; San Jose, CA) and analyzed by FlowJo software (Ashland, OR).

BitMansour A., Pop L.M, & Vitetta E.S. (2016). The Role of Regulatory B Cell-Like Malignant Cells and Treg Cells in the Mouse Model of BCL1 Tumor Dormancy. PLoS ONE, 11(12), e0167618.

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Antibodies and Metabolic Inhibitor Assay

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The following antibodies for flow cytometry were from BD Biosciences: anti-CD4 (RM4-5), anti-CD8 (Ly-3), anti-IFN-γ (XMG1.2), and anti-Thy1.1 (OX-7). Anti-Foxp3 (FJK-16s) was from eBioscience. Class I OVA peptide and class II OVA peptide were obtained from AnaSpec. PCC peptide 81–104 was synthesized at Johns Hopkins University. Rabbit polyclonal antibody against mouse OCT1 (SLC22A1, ab55916) was obtained from Abcam. Rabbit polyclonal antibody to pS6 Ser 235/236 was obtained from Cell Signaling. Alexa Fluor 488-conjugated donkey anti-rabbit-IgG F(Ab′)2 was from Life Technologies. 3H-acetate was obtained from Perkin Elmer.
Rapamycin was purchased from LC laboratories and PP242 was from Chemdea. 2-DG was purchased from Carbosynth. Metformin and DON were purchased from Sigma-Aldrich. For all in vivo experiments, individual metabolic inhibitors were dissolved in PBS and administrated intraperitoneally.

Lee C.F., Lo Y.C., Cheng C.H., Furtmüller G.J., Oh B., Andrade-Oliveira V., Thomas A.G., Bowman C.E., Slusher B.S., Wolfgang M.J., Brandacher G, & Powell J.D. (2015). Preventing allograft rejection by targeting immune metabolism. Cell reports, 13(4), 760-770.

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Naive T cell differentiation conditions

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Naive CD4+ T cells were purified from dissociated spleens and lymph nodes of C56BL/6N mice by fluorescence-associated cell sorting (FACS) (CD4+CD62L^hiCD44^lo). Purified cells were cultured under T_H0 condition stimulated with anti-CD3e (5 μg/mL, clone 145– 2C11, eBioscience), anti-CD28 (5 μg/mL, clone 37.51, eBioscience), and human IL-2 (100 U/mL, Peprotech). Additional TGFβ (0.25 ng/mL, Peprotech) was added for T_reg polarization conditions. Bile acids, retinoic acid (1 nM, Sigma), or mitoParaquat (5 μM, Sigma) were added on day 0. IsoalloLCA dissolved in DMSO was resuspended in culture media and sonicated before being added to the culture. Cells were harvested either at 48 hours for RNA-Seq, ATAC-Seq, ChIP analysis, or at 72 hours followed by staining with anti-CD4 (RM4–5, eBioscience) or anti-Foxp3 (FJK-16s, eBioscience) antibodies, and analyzed with LSR II flow cytometer (BD). For mitoROS detection, cells cultured for 48 hours were incubated with 5 μM mitoSOX (ThermoFisher) for 30 min and assayed by flow cytometry. Flow cytometry data were acquired on an LSR II flow cytometer or Symphony flow cytometer (both BD) and data were analyzed with FlowJo software (TreeStar).

Li W., Hang S., Fang Y., Bae S., Zhang Y., Zhang M., Wang G., McCurry M.D., Bae M., Paik D., Franzosa E.A., Rastinejad F., Huttenhower C., Yao L., Devlin A.S, & Huh J.R. (2021). A Bacterial Bile Acid Metabolite Modulates Treg Activity through the Nuclear Hormone Receptor NR4A1. Cell host & microbe, 29(9), 1366-1377.e9.

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Isolation and Activation of Murine T Cells

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Zebularine was purchased from Tocris Bioscience (UK). The recombinant mouse interleukin-6 (rmIL-6), rmIL-12, recombinant human transforming growth factor beta 1 (rhTGF-β1) were purchased from R&D Systems (USA). Functional antibodies anti-CD3 (145-2C11) and anti-CD28 (37.51), FACS antibodies anti-IL-17A (eBio17B7), anti-IFN-γ (XMG1.2) and anti-Foxp3 (FJK-16s) were from eBioscience (USA). CD4 (L3T4) and naïve CD4⁺ T cell Magnetic-activated cell sorting (MACS) isolation kits were purchased from Miltenyi (Germany). The complete Freund's adjuvant and pertussis toxin were purchased from Sigma-Aldrich (USA).

Zou Y., Hu X., Schewitz-Bowers L.P., Stimpson M., Miao L., Ge X., Yang L., Li Y., Bible P.W., Wen X., Li J.J., Liu Y., Lee R.W, & Wei L. (2019). The DNA Methylation Inhibitor Zebularine Controls CD4+ T Cell Mediated Intraocular Inflammation. Frontiers in Immunology, 10, 1950.

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Flow Cytometry for T Cell Characterization

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Anti-CD3 (145-2C11) for in vitro functional studies was obtained from BioXCell. Conjugated anti-CD4 (RM4-5), anti-CD8β (YTS156.7.7), TCR-β (H57-597), Thy1.1 (OX-7), Thy1.2 (30-H12), anti-CD62L (MEL-14), anti-CD44 (IM7), anti-CTLA-4 (UC10-4B9), anti-PD-1 (RMP1-30), anti-PD-L1(10F.9G2), anti-GITR (DTA-1), anti-ICOS (C98.4A), anti-TIGIT (1G9), anti-LAP (TW7-16B4), anti-IL-10 (JES5-16E3), anti-IL-17A (TC11-18H10.1), and anti-IFN-γ (XMG1.2) were purchased from BioLegend. Anti-CD8β (H35-17.2), anti-Ki67 (B56), and anti-PTEN (A2B1) were from BD Biosciences. Anti-FoxP3 (FJK-16s) was purchased from eBioscience. pAKT (D9E), pS6 (D57.2.2E), and pFOXO1/O3A (9464) antibodies were purchased from Cell Signaling Technologies.

Tan C.L., Kuchroo J.R., Sage P.T., Liang D., Francisco L.M., Buck J., Thaker Y.R., Zhang Q., McArdel S.L., Juneja V.R., Lee S.J., Lovitch S.B., Lian C., Murphy G.F., Blazar B.R., Vignali D.A., Freeman G.J, & Sharpe A.H. (2020). PD-1 restraint of regulatory T cell suppressive activity is critical for immune tolerance. The Journal of Experimental Medicine, 218(1), e20182232.

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Murine T Cell Phenotyping and Activation

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The following monoclonal antibodies and reagents were used: for surface staining, anti-CD4 (GK1.5), anti-CD8a (53-6.7), anti-CD25 (PC61), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-CD45.1 (A20), anti-CD45.2 (104), and anti–TCR-β chain (H57-597; all from BioLegend); for intracellular (cytoplasmic and nuclear) staining, anti–IL-17A (TC11-18H10.1), anti–IL-4 (11B11), anti–IFN-γ (XMG1.2; from BioLegend), anti-Foxp3 (FJK-16s), and anti-RORγt (B2D; from eBioscience); and for T cell stimulation, anti-CD3 (145-2c11) and anti-CD28 (37.51; from BioLegend). Recombinant mouse TGF-β1, IL-6, IL-4, IL-12, IL-1β, and IL-23 were from R&D Systems and recombinant mouse IL-2 was from PeproTech. Neutralizing anti–IFN-γ (XMG1.2), anti–IL-4 (11B11), and anti–IL-12 (C17.8) were from BioLegend. CFSE was from Invitrogen.

Fu G., Xu Q., Qiu Y., Jin X., Xu T., Dong S., Wang J., Ke Y., Hu H., Cao X., Wang D., Cantor H., Gao X, & Lu L. (2017). Suppression of Th17 cell differentiation by misshapen/NIK-related kinase MINK1. The Journal of Experimental Medicine, 214(5), 1453-1469.

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Isolation and Analysis of Immune Cells

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Single cell suspensions were performed from spleens, mLN and iLN by mechanical disruption and passage through pre-separation filter. Colonic lamina propria mononuclear cells (LPMCs) were isolated as previously described [61 (link)]. Subsequently, cells were stained with anti-CD4 (eBioscience), fixed and permeabilazed in 3% saponine buffer before intracellular staining. Foxp3- and Helios-expressing cells were routinely detected by using the Foxp3 staining kit (eBioscience), anti-Foxp3 (FJK-16s, eBioscience) and anti-Helios (22F6, eBioscience) Abs. Intracellular detection of IL-17A (eBio17B7) and IFN-γ (XMG1.2) was performed after restimulation of cells with PMA (50 mg/ml)/ionomycin (750 ng/ml) in the presence of brefeldin A (10 mg/ml, all substances were obtained from Sigma-Aldrich) for 4 h. Both Abs were obtained from eBioscience. The stained cells were analyzed by flow cytometry using the FlowJo software (Tree Star).

Luu M., Jenike E., Vachharajani N, & Visekruna A. (2017). Transcription factor c-Rel is indispensable for generation of thymic but not of peripheral Foxp3+ regulatory T cells. Oncotarget, 8(32), 52678-52689.

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Immunological Profiling with Flow Cytometry

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The following antibodies for flow cytometry were from BD Biosciences: anti-B220 (RA3-6B2), anti-CD3 (2C11), anti-CD4 (RM4–5), anti-CD8 (Ly-3), anti-CD25 (PC61), anti-CD44 (IM7), anti-CD124 (mIL4R-M1), anti-IFN-γ (XMG1.2), anti-IL-17a (TC11-18H10), and anti-human CD8 (RPA-T8). Anti-CD62L (MEL-14), anti-IL-13 (eBio13A), anti-IL-5 (TRFK5), anti-T-bet (eBio4B10) and anti-Foxp3 (FJK-16s) were from eBioscience.
The following antibodies for immunoblotting were from Cell Signaling Technologies: Antibody to Rictor (2140), NDRG1 phosphorylated at Thr346 (5482), total NDRG1 (9408), antibody to Akt phosphorylated at S473 (4060), pan-Akt (4685), NEDD4-2 phosphorylated at Ser342 (4080), total NEDD4-2 (4013), TCF-1 (2206), antibody to β-catenin (9562), antibody to phosphorylated β-catenin (9561), antibody to phosphorylated STAT6 (9361). Anti-T-bet (4B10) and anti-GATA-3 (TWAJ) were from eBioscience. Anti-JunB (210) was from Santa Cruz Biotechnology. Anti-ubiquitin was and actin were from Sigma. MG132 was from Sigma, PP242 was from Calbiochem, and CFSE was from Invitrogen.

Heikamp E.B., Patel C.H., Collins S., Waickman A., Oh M.H., Sun I.H., Illei P., Sharma A., Naray-Fejes-Toth A., Fejes-Toth G., Sen J., Horton M.R, & Powell J.D. (2014). The AGC kinase serum- and glucocorticoid-regulated kinase 1 (SGK1) regulates TH1 and TH2 differentiation downstream of mTORC2. Nature immunology, 15(5), 457-464.

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Isolation and Activation of Murine Naive CD4+ T Cells

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Naive CD4+ T cells were purified from the dissociated spleens and lymph nodes of C57BL/6N mice by fluorescence-associated cell sorting (FACS) (CD4+CD62LhiCD44lo 572). Purified cells were cultured under T_H0 condition stimulated with anti-CD3e (5 μg/mL, clone 145– 2C11, eBioscience), anti-CD28 (5 μg/mL, clone 37.51, eBioscience), and human IL-2 (100 U/mL, Peprotech). Bile acids were added on day 0. IsoalloLCA dissolved in DMSO was resuspended in culture media and sonicated before being added to the culture. Cells were harvested at 72 hours followed by staining with anti-CD4 (RM4–5, eBioscience) or anti-Foxp3 (FJK-16s, 580 eBioscience) antibodies, and analyzed with LSR II flow cytometer (BD).

Helgason S., Petursson G., Gudmundsson S, & Sigurdsson J.A. (2000). Prevalence of postherpetic neuralgia after a first episode of herpes zoster: prospective study with long term follow up. BMJ : British Medical Journal, 321(7264), 794.

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Multiparameter Immune Cell Analysis

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Anti-CXCR5 (2G8), GL7 (GL7), and anti-IL-4 (11B11) Abs were from BD Biosciences. Fixable viability dye, anti-CD38 (90), anti-TIGIT (GIGD7), anti-IL-21 (mhalx21), and anti-Foxp3 (FJK-16s) Abs were from eBioscience. Anti-CD4 (GK1.5), anti-B220 (RA3-6B2), anti-PD1 (29F.1A12), anti-CD25 (PC61), anti-IL-10 (JES5-16E3), Annexin V and anti-IFN-γ (XMG1.2) were from Biolegend.

Wu H., Chen Y., Liu H., Xu L.L., Teuscher P., Wang S., Lu S, & Dent A.L. (2016). Follicular regulatory T cells repress cytokine production by follicular helper T cells and optimize IgG responses in mice. European journal of immunology, 46(5), 1152-1161.

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