Anti rorγt

Manufactured by Thermo Fisher Scientific

Sourced in United States

Anti-RORγt is a laboratory reagent used to detect and quantify the expression of the RORγt protein, which is a key transcription factor involved in the differentiation and function of Th17 cells. This reagent can be used in various immunological and cell biology applications, such as flow cytometry, Western blotting, and immunohistochemistry, to investigate the role of RORγt in immune responses and disease processes.

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26 protocols using anti rorγt

Profiling Lung Tissue Protein Expression

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The proteins of the lung tissues in mice were extracted for Western blot analysis as described previously.20 Rat monoclonal antibodies of anti‐T‐bet (eBioscience) (1:250 dilution), anti‐GATA‐3 (Cell Signaling Technology) (1:1000 dilution), anti‐RORγ(t) (eBioscience) (1:100 dilution) and GAPDH (Cell Signaling Technology) (1:1000 dilution) were used as the primary antibodies. HRP‐conjugated anti‐rat IgG (Beyotime Biotechnology) was also utilized. ECL chemiluminescence kit was used for chemiluminescent detection followed by image analysis.

Zhang W., Li L., Zheng Y., Xue F., Yu M., Ma Y., Dong L., Shan Z., Feng D., Wang T, & Wang X. (2019). Schistosoma japonicum peptide SJMHE1 suppresses airway inflammation of allergic asthma in mice. Journal of Cellular and Molecular Medicine, 23(11), 7819-7829.

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Comprehensive Immunostaining Panel for Immune Cell Analysis

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Anti-Nono (Cat# 11058-1-AP and 66361-1-Ig) was from Proteintech. Anti-Elk3 (Cat# NBP1-83960) was from Novus Biologicals. Anti-Tmem241 (Cat# 203644-T32) was from Sinobiological. Anti-Lineage cocktail (Cat# 88-7772-72), Anti-CD127 (A7R34), anti-Sca-1 (D7), anti-Flt3 (A2F10), anti-α₄β₇ (DATK32), anti-Id2 (ILCID2), anti-PLZF (Mags.21F7), anti-Eomes (Dan11mag), anti-NKp46 (29A1.4), anti-NK1.1 (PK136), anti-CD45 (30-F11), anti-CD25 (PC61.5), anti-Gata3 (TWAJ), anti-RORγt (AFKJS-9), anti-Bcl11b (1F8H9), anti-CD3 (17A2), anti-CD19 (1D3), anti-KLRG1 (2F1), anti-CD90 (HIS51), anti-IL-22 (IL22JOP), anti-Ki67 (SolA15), anti-CD45.2 (104), anti-BrdU (BU20A), anti-PD-1 (J43) and anti-CD45.1 (A20) were purchased from eBiosciences (San Diego, USA). Anti-c-Kit (2B8), anti-CD150 (TC15-12F12.2), anti-CD48 (HM48-1), and anti-CD49a (HMα1) were purchased from Biolegend (California, USA). Anti-β-actin (Cat# RM2001) was purchased from Beijing Ray Antibody Biotech. Paraformaldehyde (PFA) and 4’,6-diamidino-2-phenylindole (DAPI) were from Sigma. IL-22 ELISA kit was purchased from Neobioscience.

Liu N., He J., Fan D., Gu Y., Wang J., Li H., Zhu X., Du Y., Tian Y., Liu B, & Fan Z. (2022). Circular RNA circTmem241 drives group III innate lymphoid cell differentiation via initiation of Elk3 transcription. Nature Communications, 13, 4711.

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Western Blot Analysis of Inflammatory Proteins

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Macrophages and total joint tissue proteins were extracted from CIA mice. For Western blotting, we used the following antibodies: anti-IL-17 (Abcam, Cambridge, MA, USA), anti-RORγt (eBioscience) and anti-β actin (Cell Signaling Technology, Danvers, MA, USA). Immune reaction bands were detected by using horseradish peroxidase–labeled, species-specific secondary antibodies (Cell Signaling Technology) and enhanced chemiluminescence analysis (EMD Millipore, Billerica, MA, USA). Immune reaction bands were scanned using Kodak Image Station 4000MM (Eastman Kodak, Rochester, NY, USA), and images were quantified using Quantity One 1-D Analysis software (Bio-Rad Laboratories).

Ye L., Wen Z., Li Y., Chen B., Yu T., Liu L., Zhang J., Ma Y., Xiao S., Ding L., Li L, & Huang Z. (2014). Interleukin-10 attenuation of collagen-induced arthritis is associated with suppression of interleukin-17 and retinoid-related orphan receptor γt production in macrophages and repression of classically activated macrophages. Arthritis Research & Therapy, 16(2), R96.

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Multiparametric Flow Cytometry of T-Cells

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Five replicates of splenocytes (10⁵) were plated in 200 µl of medium supplemented with 0.05 µg/ml PMA (Sigma-Aldrich), 0.001 µg/ml ionomycin calcium salt (Sigma-Aldrich) and 0.2 µl BD GolgiPlug (brefeldin A, BD Pharmingen). As control, the cells were stimulated with PMA and Ionomycin without GolgiPlug. After 4 h of incubation at 37°C and 5% CO₂, 100 µl of the supernatant was frozen at −20°C for ELISA. The cells were first stained with anti-CD8α and anti-CD4 before following the instruction for cytokines’ intracellular staining with a mix of anti-IL-2, anti-IL-4, anti-IL-10, anti-IL-17, anti-IL-22 and anti-IFN-γ, or intracellular staining for transcription factors with a mix of anti-Foxp3, anti-T-bet, anti-ROR-γt and anti-Gata-3 (eBioscience). The staining of intracellular cytokines and enzymes of CD8⁺ T-cells was done with a mix of anti-IL-2, anti-IFN-γ, anti-granzyme B and anti-perforin (reagents and isotypes from eBioscience). For FACS analysis, cells were resuspended in 50 µl PBS (1x).

Floderer M., Prchal-Murphy M, & Vizzardelli C. (2014). Dendritic Cell-Secreted Lipocalin2 Induces CD8+ T-Cell Apoptosis, Contributes to T-Cell Priming and Leads to a TH1 Phenotype. PLoS ONE, 9(7), e101881.

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Western Blot Analysis of Protein Signaling

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Proteins were extracted from the treated cells in RIPA lysis buffer [50 mM sodium fluoride, 0.5% Igepal CA-630 (NP-40), 10 mM sodium phosphate, 150 mM sodium chloride, 25 mM Tris pH 8.0, 1mM phenylmethylsulfonyl fluoride, 2 mM ethylenediaminetetraacetic acid (EDTA), 1.2 mM sodium vanadate] supplemented with protease inhibitor cocktail (Sigma-Aldrich). Equal amount of proteins was subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane. The membranes were blocked with 5% bovine serum albumin in TBST buffer (25 mM Tris-HCl, 125 mM NaCl, 0.1% Tween 20) for 2 hours and probed with the indicated primary antibodies overnight and then IRDye^®800CW- or IRDye^®680-conjugated secondary antibodies (LI-COR Biosciences) for 1 hour. The results were visualized by using an Odyssey Infrared Imager (LI-COR Biosciences). For loading control, the membranes were stripped and probed for GAPDH. The antibodies used are as follows: anti-RORγT (1:250 dilution, #14-6988, eBioscience), anti-REA (1:500 dilution, #07-234, Millipore), anti-STAT3 (1:1000 dilution, #9139, Cell Signaling Technology), and anti-GAPDH (1:10,000 dilution, #MAB374, Millipore).

Chen R.Y., Fan Y.M., Zhang Q., Liu S., Li Q., Ke G.L., Li C, & You Z. (2015). Estradiol Inhibits Th17 Cell Differentiation through Inhibition of RORγT Transcription by Recruiting ERα/REA Complex to the EREs of RORγT Promoter. Journal of immunology (Baltimore, Md. : 1950), 194(8), 4019-4028.

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Immunophenotyping of T-cell subsets

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FACS staining was conducted as described^{29 (link)} using the following antibodies: anti-mouse CD4 (L3T4, BD), anti-mouse TCR (553169, BD), anti-FoxP3 (17–5773–82, eBioscience), anti-T-bet (25–5825–82, eBioscience), anti-RORγt (17–6981–82, eBioscience), anti-CD69 (15–0691–82, eBioscience), pZAP-70 (557817, BD), anti-Ki-67 (612472, BD), and anti-ADAM12 7B8 (in-house).
Cells were acquired using FACSDiva after the exclusion of duplets. Dead cells were discriminated in all stains using the LIVE/DEAD Fixable Dead Cell Stain Kit at 405-nm excitation (L34955, Invitrogen). FlowJo 10.5.3 (Flowjo, LLC) was used for further analysis.

Liu Y., Bockermann R., Hadi M., Safari I., Carrion B., Kveiborg M, & Issazadeh-Navikas S. (2020). ADAM12 is a costimulatory molecule that determines Th1 cell fate and mediates tissue inflammation. Cellular and Molecular Immunology, 18(8), 1904-1919.

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Intracellular Cytokine Staining of T Cells

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For intracellular cytokine staining cultured cells were restimulated with PMA (50 ng/mL, Sigma Aldrich) and Ionomycin (1 μg/mL, Invitrogen, Life technologies) in the presence of Monensin (2.5 mM, Sigma Aldrich) for 5 h. To measure the cell viability (>80%) the cells were stained with Annexin-V FITC and 7-AAD according to the manufacturer's protocol (BD Bioscience). The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin-containing buffer before washing and staining in different combinations with anti-CD4 (Beckman Coulter, Krefeld, Germany), anti-IL-17A, anti-IL-22, anti-IL-9, anti-IL-8, anti-AHR, anti-RORγt (ebioscience), and anti-IFN-γ (BD Bioscience). All dotplots and histograms shown refer to gated CD4⁺ T cells. Data were acquired on FACSCantoII (BD Bioscience) and were analyzed with FlowJo Software (Tree Star Inc., Ashland, OR, USA).

Gasch M., Goroll T., Bauer M., Hinz D., Schütze N., Polte T., Kesper D., Simon J.C., Hackermüller J., Lehmann I, & Herberth G. (2014). Generation of IL-8 and IL-9 Producing CD4+ T Cells Is Affected by Th17 Polarizing Conditions and AHR Ligands. Mediators of Inflammation, 2014, 182549.

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Cytokine and Transcription Factor Analysis of T cells

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Splenocytes or CD4⁺ cells isolated from tumor tissue by anti-CD4 antibody-coated magnetic beads (BD IMag™), were cultured in RPMI supplemented with 10% FBS and 0.5 μg/ml concanavalin A (Con A; Sigma-Aldrich). Cell culture supernatants were used for estimation of cytokine production by stimulated cells (ELISA). For cytokine and transcription factor expression analysis, brefeldin A (10 μg/ml) and monensin (2 μM) (both from eBiosciences, San Diego, CA, USA) were added to the cell culture for the last 6 h. Cells were stained with anti-CD4 antibody (FITC, clone RM4-5), fixed and incubated with anti-mouse CD16/CD32 mAb, and stained for cytokine and/or transcription factor expression with the intracellular staining kit from eBiosciences. The following fluorochrome-conjugated mAbs were used: anti-T-bet (PE-Cy 7, clone 4B10), anti-GATA3 (PE, clone TWAJ), anti-RORγt (PE, clone AFKJS-9), anti-FoxP3 (APC or PE, clone FJK-16s), or their appropriate isotype control (all anti-human/mouse, all from eBiosciences). Flow cytometry was performed using FACSCalibur cytometer (BD Biosciences). All data were analyzed with Flowing software version 2.5.

WOJAS-TUREK J., SZCZYGIEŁ A., KICIELIŃSKA J., ROSSOWSKA J., PIASECKI E, & PAJTASZ-PIASECKA E. (2015). Treatment with cyclophosphamide supported by various dendritic cell-based vaccines induces diversification in CD4+ T cell response against MC38 colon carcinoma. International Journal of Oncology, 48(2), 493-505.

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Isolation and Analysis of Immune Cells from Murine Lymph Nodes

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Cervical lymph nodes were removed from mice and homogenized through a nylon mash (70 μM). The cells were then stained with fluorescence-conjugated anti-CD3, anti-CD4, anti-RORγt, and anti-Foxp3 (eBioscience). Transcription factor staining buffer set (eBioscience) was used for RORγt and Foxp3 intracellular staining. Stained cells were then acquired and analyzed on FACSCalibur using CellQuest (BD Biosciences) on FACSCelesta using Flowjo.

Hays A., Duan X., Zhu J., Zhou W., Upadhyayula S., Shivde J., Song L., Wang H., Su L., Zhou X, & Liang S. (2019). Down-regulated Treg Cells in Exacerbated Periodontal Disease during Pregnancy. International immunopharmacology, 69, 299-306.

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Intracellular Cytokine Profiling of T-cells

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Intracellular staining for IL-22, IL-17, IFN-γ, GM-CSF and IL-10 was performed on cells stimulated for 5 h with PMA and ionomycin in the presence of brefeldin A (all from Sigma-Aldrich) for the final 2.5 h of culture. Cells were fixed and permeabilized with Fix and Perm buffer (BD Biosciences) according to manufacturer’s instructions. Cells were stained with anti-IL-17 (eBioscience), anti-IL-22 (eBioscience), anti-IL-10 (Biolegend), anti-IFN-γ (Biolegend and BD Pharmingen), anti-GM-CSF (eBioscience). FACS data were analyzed with FlowJo (Tree Star). Intracellular staining for RORγt and T-bet were performed directly ex vivo or after in vitro cell expansion using anti RORγt (eBioscience) and anti-T-bet (Biolegend and eBioscience).

Duhen T, & Campbell D.J. (2014). IL-1β promotes the differentiation of polyfunctional human CCR6+CXCR3+ Th1/17 cells that are specific for pathogenic and commensal microbes. Journal of immunology (Baltimore, Md. : 1950), 193(1), 120-129.

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