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Anti rorγt

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Anti-RORγt is a laboratory reagent that binds to and inhibits the activity of the RORγt (Retinoic Acid-related Orphan Receptor gamma t) transcription factor. RORγt is a key regulator of T helper 17 (Th17) cell differentiation and function.

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Lab products found in correlation

Anti t bet, by BD (3 mentions) Anti cd3, by BD (2 mentions) Anti cd45, by BD (2 mentions) Anti il 17a, by BioLegend (2 mentions) Anti foxp3, by BioLegend (2 mentions) Anti cd4, by BioLegend (2 mentions) Cytofix cytoperm kit, by BD (2 mentions) Facscanto flow cytometer, by BD (1 mentions) Anti il 22, by BD (1 mentions) Anti foxp3, by BD (1 mentions) Anti il 17a, by BD (1 mentions) Facsdiva software, by BD (1 mentions) Anti cd4, by BD (1 mentions) Anti h3k4me3, by Cell Signaling Technology (1 mentions) Anti h3k36me3, by Abcam (1 mentions) Anti h3k27ac, by Cell Signaling Technology (1 mentions) Anti tcrβ, by BioLegend (1 mentions) Anti h3k9me3, by Cell Signaling Technology (1 mentions) Anti gata3, by BioLegend (1 mentions) Foxp3 permeabilisation kit, by Thermo Fisher Scientific (1 mentions)

10 protocols using anti rorγt

1

T Cell Differentiation Assay

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Naïve CD4+CD25- T cells were differentiated to the different effector subsets as detailed in Supplemental Experimental Procedures. For proliferation assays, cells were labeled Cell Violet (1 μM) and activated with anti-CD3/CD28 or differentiated as indicated. For intracellular staining of transcription factors, cells were differentiated during 3 days, collected with 5 mM EDTA in PBS, fixed, permeabilized and stained with anti-RORγT and anti-T-bet (BD biosciences). For intracellular cytokine staining, cells were restimulated for 4 hr with PMA and ionomycin in the presence of brefeldin, permeabilized with 0.5% saponin and stained with anti-CD4, anti-IFN-γ, anti-IL-17 and anti-Foxp3. Data were acquired on a FACSCanto flow cytometer (BD Bioscience) and analyzed using the FLOWJO software (Tree Star).
Baixauli F., Acín-Pérez R., Villarroya-Beltrí C., Mazzeo C., Nuñez-Andrade N., Gabandé-Rodriguez E., Dolores Ledesma M., Blázquez A., Martin M.A., Falcón-Pérez J.M., Redondo J.M., Enríquez J.A, & Mittelbrunn M. (2015). Mitochondrial respiration controls lysosomal function during inflammatory T cell responses. Cell metabolism, 22(3), 485-498.
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2

Isolation and Analysis of Intestinal Immune Cells

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To isolate lamina propria immune cells, IEC and intraepithelial lymphocyte layers were first stripped by shaking sections of large intestine in 5 mM EDTA/1 mM DTT. Remaining tissue was digested with collagenase (0.5 mg/ml) to obtain single cell suspensions. For flow cytometry, cells were stained with a combination of the following fluorescence-conjugated monoclonal antibodies, anti-CD3 (clone: 500A2), anti-CD4 (clone: RM4-5), anti-Foxp3 (clone: FJK-16s), anti-CD45 (clone: 30-F11), anti- RORγt (clone: Q21-559), anti-IL-17A (clone: TC11-18H10), and anti-IL-22 (clone: 1H8PWSR) (BD-Biosciences or Therma-Fisher). Samples were acquired on an LSR II (BD Biosciences) and were analyzed with FACSDiva software (BD Biosciences).
Sun D., Wang W., Guo F., Pitter M.R., Du W., Wei S., Grove S., Vatan L., Chen Y., Kryczek I., Fearon E.R., Fang J.Y, & Zou W. (2022). DOT1L affects colorectal carcinogenesis via altering T cell subsets and oncogenic pathway. Oncoimmunology, 11(1), 2052640.
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3

Multiparametric Flow Cytometry for Immune Profiling

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Flow cytometric staining was performed as previously described (Chang et al., 2015 (link)). To assess cytokine staining in vitro and ex vivo, cells were re-stimulated with 50 ng/mL PMA and 1 μg/mL ionomycin in the presence of brefeldin A (Biolegend) for 5 hours. Intracellular cytokine staining was performed using BD CytoFix/CytoPerm kit (BD Biosciences) and nuclear staining of transcription factors using the FoxP3 Permeabilisation kit (eBioscience). Cells were stained with Live/Dead viability dye (Thermo) prior to antibody staining. Cells were collected on LSR II and Fortessa flow cytometers (BD Biosciences) and analyzed using FlowJo (TreeStar) software. The following antibodies were used: anti-CD4, anti-TCRβ, anti-CD45, anti-IL17A, anti-IL-17B, anti-IFN-γ, anti-T-bet, anti-GATA-3, anti-FoxP3 (all Biolegend), anti-RORγt (BD Bioscience), anti-ODC, anti-spermine synthase (both Abcam). For analysis of chromatin marks, cells were gated on Ki-67+ cells and diploid cells with ‘single’ DNA content based on FxCycle (dapi, Thermo) staining in the live cell gate. Primary antibodies were stained for 90 minutes in permeabilization buffer at room temperature, followed by staining with the relevant secondary antibody for 30 minutes. The following antibodies were used: anti-H3k4Me3, anti-H3k9Ac, anti-H3k27Ac, anti-H3k9Me3 (all Cell Signaling), and anti-H3k36Me3 (Abcam).
Puleston D.J., Baixauli F., Sanin D.E., Edwards-Hicks J., Villa M., Kabat A.M., Kamiński M.M., Stanckzak M., Weiss H.J., Grzes K.M., Piletic K., Field C.S., Corrado M., Haessler F., Wang C., Musa Y., Schimmelpfennig L., Flachsmann L., Mittler G., Yosef N., Kuchroo V.K., Buescher J.M., Balabanov S., Pearce E.J., Green D.R, & Pearce E.L. (2021). Polyamine metabolism is a central determinant of helper T cell lineage fidelity. Cell, 184(16), 4186-4202.e20.
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4

Multiparametric Profiling of T Cell Subsets

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eFluor660-anti–IL-21, Alexa Fluor 488–anti–IL-10, PerCP-Cy5.5-anti–IFN-γ, FITC-anti-CD45RA, PE-anti-ICOS, and anti-Tbet, and biotin–PD-1 were from eBioscience. Alexa Fluor 488–anti-GATA3, Alexa Fluor 647–anti-CXCR5, anti-pSTAT4, anti-pSTAT5, anti-pSTAT6, APC-anti-CD10, APC-Cy7-anti-CD4, BV605-anti-IgG, BV421-CD40L, BV711-anti–IL-2, PE-anti-pSTAT1, anti-RORγt, and Bcl-6, PE-Cy7-anti-CD25, and anti-CD27, PerCP-Cy5.5-anti-CD127, anti-pSTAT3, and anti-Tbet, SA-PerCpCy5.5, and IFN-γ were obtained from BD. Pacific Blue–anti-CD20 and SA-BV605 were purchased from BioLegend. Recombinant human IL-12 was purchased from R&D Systems. TGF-β, IL-1β, IL-6, IL-21, and IL-23 were obtained from PeproTech. PGE2 was purchased from Sigma-Aldrich. Human IL-4 and IL-10 were provided by R. de Waal Malefyt (DNAX Research Institute, Palo Alto, CA).
Ma C.S., Wong N., Rao G., Nguyen A., Avery D.T., Payne K., Torpy J., O’Young P., Deenick E., Bustamante J., Puel A., Okada S., Kobayashi M., Martinez-Barricarte R., Elliott M., Sebnem Kilic S., El Baghdadi J., Minegishi Y., Bousfiha A., Robertson N., Hambleton S., Arkwright P.D., French M., Blincoe A.K., Hsu P., Campbell D.E., Stormon M.O., Wong M., Adelstein S., Fulcher D.A., Cook M.C., Stepensky P., Boztug K., Beier R., Ikincioğullari A., Ziegler J.B., Gray P., Picard C., Boisson-Dupuis S., Phan T.G., Grimbacher B., Warnatz K., Holland S.M., Uzel G., Casanova J.L, & Tangye S.G. (2016). Unique and shared signaling pathways cooperate to regulate the differentiation of human CD4+ T cells into distinct effector subsets. The Journal of Experimental Medicine, 213(8), 1589-1608.
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5

Antibody-based Transcription Factor Analysis

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Antibodies used in these studies were anti-SUMO1 (1:500; #FL-101, Santa Cruz), anti–ROR-γt (1:800; BD Bioscience), anti-Ubc9 (1:1000, CST), anti-HIF-1α (1:200; BD Biosciences), mouse IgG (Santa Cruz Biotechnology), PE-CD45Rb (Biolegend), APC-CD4 (Biolegend), FITC-CD25 (Tonbo biosciences) and Clean-Blot™ IP detection reagent (HRP) (1:50; Pierce Biotechnology). Dulbecco’s modified Eagle’s medium (Gibco), fetal bovine serum (Gibco), QIAmp DNA mini kit (Qiagen, USA), EpiTect bisulfite kit (Qiagen, USA), EpiTaq HS polymerase (TaKaRa), ChIP assay kit (Millipore).
Kumar R., Singh A.K., Starokadomskyy P., Luo W., Theiss A.L., Burstein E, & Venuprasad K. (2021). Hypoxia Induced Ubc9 promoter hyper-methylation regulates IL-17 expression in ulcerative colitis. Journal of immunology (Baltimore, Md. : 1950), 206(5), 936-940.
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6

Berberine Modulates Immune and Apoptotic Pathways

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Berberine (purity:≥98%) was purchased from Chem Faces (#CFN98049). Malondialdehyde (MDA) assay kit (#A003‐1) and Superoxide Dismutase (SOD) assay kit (#A001‐3) were purchased from NanJing Jiancheng Bioengineering Institute (Nanjing, China). Antibodies against Wnt5a (#sc‐365370) were purchased from SantaCruz and were dissolved in primary antibody dilution buffer (#P0023A, Beyotime, Wuhan, China). Antibodies against β‐catenin (#51067‐2‐AP) were provided by the Proteintech Group (Wuhan, China). The annexin V apoptosis detection kit I (#559763) was obtained from BD Biosciences (Franklin Lakes, America). Antibodies used for flow cytometry include anti‐CD4 (#470041‐32), anti‐CD8 (#48–0081‐82), and anti‐Foxp3 (#35–5773‐82), which were purchased from eBiosciences (Carlsbad, America). Antibodies including anti‐ROR‐γt (#562007), anti‐Ly6C (#560592), anti‐Ly6G (#560600), anti‐CD3 (#533066), anti‐CD19 (#550992), anti‐CD11b (#562950), anti‐CD11c (#558079), anti‐F4/80 (#565411), and anti‐CD45 (#103108) were purchased from BD Biosciences. Human/Mouse/Rat TGF‐β1 ELISA Kit (#EK981‐96) and Mouse IL‐18 ELISA Kit (#EK218‐96) were purchased from Multi Sciences (Hangzhou, China). The Mouse CBA Kits of cytokines were obtained from Biolegend (California, America). TUNEL staining kit (#556547) was purchased from BD Biosciences.
Tian C., Li M., Shuai X., Jiang F., Dong Y., Gui Y., Zhang Z., Qin R., Kang Z., Lin L., Sarapultsev A., Wu B., Luo S, & Hu D. (2022). Berberine plays a cardioprotective role by inhibiting macrophage Wnt5a/β‐catenin pathway in the myocardium of mice after myocardial infarction. Phytotherapy Research, 37(1), 50-61.
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7

T Helper 17 Cell Differentiation

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Purified CD4+CD45RA+ naïve T cells (2 ×105 to 5 ×105 per well) were cultured for 6 or 7 days under TH17 differentiation condition with plate-bound anti-human CD3 (OKT-3, BioXcell), soluble antihuman CD28 mAb (CD28.2, eBioscience), human TGF-β (5 ng/ml), human IL-6 (20 ng/ml), human IL-23 (10 ng/ml), human IL-1β (10 ng/ml) (PeproTech), anti–IFN-γ (5 μg/ml) (NIB42, eBioscience), and anti–IL-4 (MP4-25D2, eBioscience). In some experiments, MDSCs (at a 1:1 ratio to the naïve CD4 T cells) and nor-NOHA (at 300 μM) were added to determine the role of MDSCs and Arg-1 in TH17 cell differentiation. The cells were further stimulated with PMA (300 ng/ml) plus ionomycin (1 μg/ml) (Sigma) for the last 5 hours in the presence of brefeldin A (BD Pharmingen) and then stained intracellularly with anti–IL-17A, anti–IL-17F, anti-mTOR (eBioscience), anti-RORγt (BD Pharmingen), anti-GCN2 (ab134053, Abcam), anti-EIF2S1 (eIF2α-P; Abcam), or anti-AHR (eBioscience). IL-17A and IL-17F concentrations in the culture supernatants were measured in duplicate by human IL-17A (BioLegend) and IL-17F (eBioscience) ELISA kits, respectively.
Wu H., Zhen Y., Ma Z., Li H., Yu J., Xu Z.G., Wang X.Y., Yi H, & Yang Y.G. (2016). Arginase-1–dependent promotion of TH17 differentiation and disease progression by MDSCs in systemic lupus erythematosus. Science translational medicine, 8(331), 331ra40.
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8

Immunohistochemical Analysis of Murine Lung Tissue

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Lung tissues were perfused and then incubated in a fixation and permeabilization solution (BD Bioscience, 554722) overnight followed by dehydration in 30% sucrose before embedding in OCT compound (Sakura Finetek). 18-μm sections were cut on a CM3050S cryostat (Leica) and adhered to Superfrost Plus slides (VWR). Frozen sections were permeabilized and blocked in PBS containing 0.3% Triton X-100 (Sigma-Aldrich) and Fc-blocker (CD16/CD32 Monoclonal Antibody, eBioscience) followed by staining with antibodies diluted in the blocking buffer. The following antibodies were used for staining: anti-CD3 (17A2; Biolegend), anti-CD4 (GK 1.5, Biolegend), anti-CD8 (53–6.7, BD), anti-RORγt (Q31–378, BD) and anti-TCRγδ (GL3, Biolegend). After staining, slides were mounted with Fluormount G (Southern Biotech), and examined on a Leica TCS SP8 confocal microscope. Images were analyzed with Imaris software (Bitplane).
Jin C., Lagoudas G., Zhao C., Bullman S., Bhutkar A., Hu B., Ameh S., Sandel D., Liang X.S., Mazzilli S., Whary M.T., Meyerson M., Germain R., Blainey P.C., Fox J.G, & Jacks T. (2019). Commensal Microbiota Promote Lung Cancer Development via γδ T Cells. Cell, 176(5), 998-1013.e16.
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9

Cytokine and Transcription Factor Analysis in Immune Cells

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For measurement of intracellular cytokine expression, cells were isolated ex vivo and stimulated with 50 ng/ml PMA and 500 ng/ml ionomycin, in the presence of GolgiStop (BD Biosciences) for 3 h. Dead cells were excluded by Fixable Viability Dye eFluor 450 (eBioscience). Cells were stained with antibodies to surface antigens, and then fixed and permeabilized with the Cytofix/Cytoperm kit (BD Biosciences), followed by staining with anti-IL-5 (Biolegend) and anti-IL-13 (eBioscience).
For analysis of transcription factor expression, cells were stained with antibodies to surface antigens, then fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s instructions, and stained with anti-GATA3 (eBioscience), anti-RORγt, anti-T-bet (BD Biosciences), or anti-Ki-67 (eBioscience).
For measurement of intracellular HIF1α, Lin− bone marrow cells were cultured in complete media with 10 ng/ml IL-7 (Peprotech) and 10 ng/ml IL-33 (R&D Systems) for 4 days. Cells were collected and stained with antibodies to surface antigens, fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s instructions, and then stained with anti-HIF1α (R&D Systems).
Li Q., Li D., Zhang X., Wan Q., Zhang W., Zheng M., Zou L., Elly C., Lee J.H, & Liu Y.C. (2018). The E3 ligase VHL promotes group 2 innate lymphoid cell maturation and function via inhibition of glycolysis and induction of interleukin 33 receptor. Immunity, 48(2), 258-270.e5.
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10

Comprehensive Murine Regulatory T Cell Analysis

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For flow cytometry, the cell surface was stained with the following antibodies specific for mouse proteins: anti-CD4, anti-CCR6, anti-CD25, anti-CTLA-4, anti-GITR, and anti-ICOS (BioLegend). Mouse Regulatory T Cell Staining Kit (eBioscience; San Diego, CA) was used to stain the transcription factors with anti-Foxp3 (Biolegend) and anti-RORγt antibodies (BD), anti-Blimp-1 (Biolegend) and anti-Helios (Biolegend), or intracellular cytokine staining with anti-IL-17A and anti-IL-10 (BioLegend). Data were acquired using the FACSVerseFlow Cytometer (Becton Dickinson; Mountain View, CA) or the SH800 Cell Sorter (Sony, Tokyo, Japan) and analyzed with FlowJo software (Tree Star; Ashland, OR).
Furuyama K., Kondo Y., Shimizu M., Yokosawa M., Segawa S., Iizuka A., Tanimura R., Tsuboi H., Matsumoto I., & Sumida T. (2021). RORγt+Foxp3+ regulatory T cells in the regulation of autoimmune arthritis.
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