Gata3 clone twaj

Manufactured by Thermo Fisher Scientific

GATA3 (clone TWAJ) is a laboratory reagent used for the detection of the GATA3 protein in biological samples. It is designed for use in immunohistochemistry applications.

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4 protocols using gata3 clone twaj

Comprehensive Immune Cell Phenotyping

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The following antibodies from BD, eBioscience and BioLegend were used from cell surface antigen staining: CD8α (clone 53-6.7; eBioscience and BD), CD5 (clone 53-7.3; eBioscience), CD25 (clone 7D4; BD), CD44 (clone IM7; eBioscience), CD69 (clone H1.2F3; BioLegend and eBioscience), CD62L (clone MEL-14; eBioscience and BD), CD98 (clone RL388; BioLegend), CD122 (clone 5H4; eBioscience), Vα2 (clone B20.1; eBioscience), CD107a (clone 1D4B; BD), and TCRβ (clone H57-597; BD).
For intracellular staining, the following antibodies from eBioscience were used: TNF (clone MP6-XT22), IFNγ (clone XMG1.2), and Themis (clone 1TYMS).
For nuclear staining, the following antibodies were used: Foxp3 (clone MF-14; BioLegend), GATA3 (clone TWAJ, eBioscience), and Eomes (clone Dan11mag; eBioscience).
For metabolic marker analysis, the following primary antibodies from Cell Signaling were used: c-Myc (clone D3N8F), pAkt T308 (clone D25E6), and p-S6 ribosomal protein S235/236 (clone D57.2.2E). Goat anti-rabbit F(ab’)2 fragment Alexa Fluor 488 (Invitrogen) was used as secondary antibody.

Gautam N., Wojciech L., Yap J., Chua Y.L., Ding E.M., Sim D.C., Tan A.S., Ahl P.J., Prasad M., Tung D.W., Connolly J.E., Adriani G., Brzostek J, & Gascoigne N.R. (2023). Themis controls T cell activation, effector functions, and metabolism of peripheral CD8+ T cells. Life Science Alliance, 6(12), e202302156.

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Multiparameter Flow Cytometric Analysis of Immune Cell Subsets

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Single-cell suspensions obtained from spleen, sdLNs, and skin were analyzed by flow cytometry using fluorochrome-conjugated mAbs listed in Table S1. Zombie Aqua or Zombie NIR (Biolegend) was used to distinguish live and dead cells. Intracellular staining was carried out on cells incubated with Brefeldin A (Biolegend) in all tissue digestion and FACS staining buffers, approximately for 4 hours. Cells were permeabilized and fixed with transcription factor staining buffer (Invitrogen) or Cytofix/Cytoperm (BD Biosciences) and subsequently incubated with fluorochrome-conjugated mAb to mouse IFN-γ (clone XMG1.2, eBioscience), IL4 (clone 11B11, Biolegend), Tbet (clone 4B10, Biolegend), or GATA3 (clone TWAJ, eBioscience). Flow cytometric analysis was carried out using a Cytek Aurora, and analysis was conducted with FlowJo software 9.7.6 (TreeStar).

Haddadi N.S., Mande P., Brodeur T.Y., Hao K., Ryan G.E., Moses S., Subramanian S., Picari X., Afshari K., Marshak-Rothstein A, & Richmond J.M. (2022). Th2 to Th1 Transition Is Required for Induction of Skin Lesions in an Inducible and Recurrent Murine Model of Cutaneous Lupus–Like Inflammation. Frontiers in Immunology, 13, 883375.

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Identification of Thymic ILC Populations

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Cell surface staining was performed at 4°C for 30 min in FACS buffer (10% FBS, 2.5mM EDTA in PBS). ILC were identified among CD8α⁻ (clone 5.3–6.7, Biolegend), CD3i⁻ (clone 17A2, Biolegend), IL‐7Rα⁺ (clone A7R34, Biolegend), B220⁻ (clone RA3‐6B2, eBioscience), CD11b⁻ (clone M1/70, eBioscience), CD11c⁻ (clone N418, eBioscience), CD3⁻ (clone 145‐2C11, eBioscience), and CD5⁻ (clone 53–7.3, eBioscience). Thymic iNKT cells were identified as mCD1d/PBS57⁺ and TCRβ⁺ (clone H57‐597, eBioscience). Intracellular cell staining was performed at room temperature for 1 h in permeabilization buffer (eBioscience). ILC2 and ILC3 subsets were identified using Abs against GATA3⁺ (clone TWAJ, eBioscience) and RORγt⁺ (clone AFKJS‐9, eBioscience). Staining for RANKL expression was performed in two steps using biotinylated RANKL (clone: IK22/5, eBioscience) and streptavidin‐PECy7 (Molecular Probes) at 4°C for 30 min in FACS buffer. Samples included Spherotech Accucount blank particles to enable calculation of cell frequency and were acquired using a Fortessa (BD). Samples were analyzed using FlowJo (FlowJo, LLC). Full gating strategy for all flow cytometry data identifying thymic ILC populations is shown in Supporting Information Fig. 1A.

Jones R., Cosway E.J., Willis C., White A.J., Jenkinson W.E., Fehling H.J., Anderson G, & Withers D.R. (2018). Dynamic changes in intrathymic ILC populations during murine neonatal development. European Journal of Immunology, 48(9), 1481-1491.

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Intracellular Cytokine Profiling by Flow Cytometry

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For analysis of intracellular cytokine production cells were re-stimulated for 4 hours at 37°C in RPMI media supplemented with 10% FCS, 2 mM L-glut, 100 U/ml P/S with 0.1 μg/ml Phorbol 12-myristate 13-acetate (PMA), 1 μg/ml Ionomycin and 10 μg/ml Brefeldin A. Cells were surface stained with mAbs diluted in PBS/0.1% BSA and Fc-block (Biolegend) for 30 min on ice. Fixable Viability Dye (eBioscience) was added to allow the exclusion of Additionally, biotinylated antibodies were used for the staining ST2 (IL-33R) (Mdb, clone DJ8) and TSLPR (Biolegend, 22H9). For intracellular staining, cells were fixed and permeabilized using the Foxp3/transcription factor staining kit (eBioscience), and incubated with the following fluorochrome-conjugated mAb: GATA3 (clone TWAJ), Foxp3 (clone FJK-16s), IL-5 (clone TRFK5), IFNg (XMG1.2), TGFB1-LAP (clone TW7-16B4), Ki67 (clone SolA15). Areg was detected with a biotinylated mAb (R&D Systems, BAF989). To identify ILC, a cocktail of biotinylated mAb against the following surface markers was used to allow gating on linage negative cells: NK1.1, CD19, CD5, F4/80, CD11c, Ter119, Cd3e, CD11b, LysG1, TCRgd. Binding of biotinylated antibodies was detected using fluorochromeconjugated streptavidin (Biolegend). Flow cytometry analyses were performed on a Fortessa flow cytometer (BD) and data were analyzed with FlowJo 9.9.4 software (BD).

Kabat A.M., Hackl A., Sanin D.E., Zeis P., Grzes K.M., Baixauli F., Kyle R., Caputa G., Edwards-Hicks J., Villa M., Rana N., Curtis J.D., Castoldi Â., Cupovic J., Dreesen L., Sibilia M., Pospisilik J.A., Urban J.F., Grün D., Pearce E.L., & Pearce E.J. (2022). Resident T cells orchestrate adipose tissue remodeling in a site peripheral to infection.

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