Gata3

Manufactured by BD

Sourced in United States

GATA3 is a laboratory equipment product designed for the detection and analysis of the GATA3 gene and protein. The GATA3 gene is a transcription factor that plays a crucial role in the development and differentiation of various cell types, including those found in the immune system and certain types of cancer. The GATA3 product provides researchers and scientists with the tools necessary to study the expression and function of this important biological marker.

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Lab products found in correlation

Facscanto 2, by BD (3 mentions) Interleukin 4 (il 4), by BD (3 mentions) Rorγt, by BD (3 mentions) Cytofix cytoperm, by BD (2 mentions) Phorbol myristate acetate (pma), by Merck Group (2 mentions) Ionomycin, by Merck Group (2 mentions) T bet, by Thermo Fisher Scientific (2 mentions) Golgistop, by BD (2 mentions) Il 10, by BioLegend (2 mentions) T bet, by BioLegend (2 mentions) Ifn γ, by BioLegend (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Ifn γ, by BD (2 mentions) Tgf β, by Thermo Fisher Scientific (2 mentions) Cd11b, by BD (2 mentions) Dylight 488 anti mouse igg, by Abcam (1 mentions) Diva 6, by BD (1 mentions) Foxp3 transcription factor staining kit, by Thermo Fisher Scientific (1 mentions) Anti socs3, by Abcam (1 mentions) Fcs express 4, by De Novo Software (1 mentions)

Spelling variants of this product

Anti-GATA3 (6 citations) GATA3 (L50-823) (6 citations) GATA3 PE (3 citations) GATA3 AF647 (2 citations) Gata3—PE-Cy7 (2 citations) GATA3 (clone L50-823), (2 citations) Anti-GATA3-PE-Cy7 (L50-823) (2 citations) Anti-Gata3-AF647 (2 citations) Anti-GATA3 PE (2 citations) GATA3-APC (2 citations)

14 protocols using gata3

Analysis of Th9 Cell Cytokine Profiles

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On day 5, cells were stimulated with 20 ng/mL phorbol 12-myristate 13acetate and 1 mmol/L ionomycin for 5 hours. Brefeldin A (10 mg/mL) was added after 2.5 hours. Cells were then fixed and permeabilized with Cytofix/ Cytoperm (BD Biosciences, Franklin Lakes, NJ), according to the manufacturer's protocol, and stained with Live/Dead Fixable Aqua dye; anti-human CD3, CD4, IL-9, IL-4, p-STAT1, p-STAT5, and GATA-3; anti-mouse CD3 and IL-9 (BD Biosciences); anti-human CD45RO ECD (Beckman Coulter, Fullerton, Calif); and anti-human IL-21 and T-bet and anti-mouse CD4 and CD44 IL-4 (eBioscience) antibodies. For SOCS3 staining, DyLight 488 Anti-Mouse IgG (Abcam, Cambridge, United Kingdom) was used after anti-SOCS3 (Abcam). PU.1 (BioLegend) and GATA-3 (BD Biosciences) antibodies were used for TF staining with the Foxp3/Transcription Factor Staining kit (eBioscience), according to the manufacturer's protocol.
Events were collected on an LSRFortessa with Diva 6.1.3 software (BD Biosciences) and analyzed with FlowJo 10 software (TreeStar, Ashland, Ore). All plots are gated on live singlet CD3 1 CD8 2 CD45RO 1 . All values used for analyzing proportionate responses are background subtracted.

Zhang Y., Siegel A.M., Sun G., Dimaggio T., Freeman A.F, & Milner J.D. (2019). Human T_H9 differentiation is dependent on signal transducer and activator of transcription (STAT) 3 to restrain STAT1-mediated inhibition. The Journal of allergy and clinical immunology, 143(3).

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Intracellular Cytokine Staining Protocol

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For intracellular detection of cytokines, cells were stimulated for 4 h at 37°C with 50 ng mL 1 PMA (Sigma-Aldrich, USA) and 1 μg mL 1 ionomycin (Sigma-Aldrich) in the presence of Brefeldin A (10 µg mL 1 , Enzo Life Science, USA). The Abs including anti-CD3, -CD8, -IL-4, -GATA-3, and -interferon-regulatory factor 4 (IRF4) mAbs were purchased from BDPharmingen (San Diego, USA) or eBioscience (San Diego). Cells were surface-stained with anti-CD3 and -CD8 mAbs in PBS+2% heat-inactivated fetal bovine serum (FBS, Gibco, USA) for 20 min at 4°C. Cells were resuspended in a fixation/permeabilization solution (Cytofix/Cytoperm; BD Pharmingen, USA) and incubated with anti-IL-4, -IL-9, -GATA-3 or -IRF4 mAbs for 30 min at 4°C. Cells were then washed with permeabilization buffer and resuspended in PBS+2% FBS for flow cytometric analysis (FACSCanto II; BD Bioscience, USA). Flow cytometry was performed on a FACS Canto II (BD Biosciences) and analyzed using BD FCSDiva Software and FCS Express 4 software (De Novo Software, USA).

Lu Y., Lin H., Zhai K., Wang X., Zhou Q, & Shi H. (2016). Interleukin-17 inhibits development of malignant pleural effusion via interleukin-9-dependent mechanism. Science China. Life sciences, 59(12).

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Multiparametric Immune Cell Profiling

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Specific cell-surface staining was performed using a standard procedure with anti-CD4, anti-PD1, anti-IgD, anti-GL7 (eBioscience), and anti-CXCR5 mAbs (BD Biosciences).
Intracellular c-Maf, GATA-3, FoxP3, BCL6, Ki67 (Ab from BD Biosciences), and T-bet (eBioscience), staining was performed according to the manufacturer’s protocol (FoxP3 staining set protocol, eBioscience). Cells were analyzed by flow cytometry with a FACS Canto II (BD Biosciences) and analyzed with the FlowJo Software. Live cells were analyzed within a FSC-A/FSC-H gate to exclude cell doublets and triplets.

Andris F., Denanglaire S., Anciaux M., Hercor M., Hussein H, & Leo O. (2017). The Transcription Factor c-Maf Promotes the Differentiation of Follicular Helper T Cells. Frontiers in Immunology, 8, 480.

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Murine Lung Immune Cell Profiling

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P1 and P21 male and female murine lungs were isolated as described above. Cells were blocked for 30 min with CD16/CD32 (Tonbo Biosciences). For intracellular analyses, cells were stimulated with PMA (50 ng/ml, Sigma-Aldrich), ionomycin (750 ng/mL, Sigma-Aldrich), and GolgiStop (BD Biosciences) for 5 hr, and then surface stained with fluorochrome-conjugated antibodies for 30 min: CD3 (clone: 145–2 C11, BD Biosciences), CD4 (RM4-5, BD Biosciences), and CD8a (clone: 53–6.7, Biolegend). Cells were then permeabilized with FOXP3 Fixation/Permeabilization Kit (BD Biosciences) as indicated by the manufacturer, and stained for TBET (clone: 4B10, Biolegend), GATA3 (clone: L50-823, BD Biosciences), FOXP3 (clone: FJK-16s, eBioscience), RORγt (clone: Q31-378, BD Biosciences), IFNγ (clone: XMG1.2, Biolegend), IL-4 (clone: 11B11, BD Biosciences), IL-10 (clone: JES5-16E3, Biolegend), and IL-17 (clone: TC11-18H10, Miltenyi Biotec) for 30 min. Cells were read using an LSRII flow cytometer using FACSDiva software. Flow data was analyzed using FlowJo (Tree Star Inc). Flow cytometry analysis for this project was done on instruments in the Stanford Shared FACS Facility.

Domingo-Gonzalez R., Zanini F., Che X., Liu M., Jones R.C., Swift M.A., Quake S.R., Cornfield D.N, & Alvira C.M. (2020). Diverse homeostatic and immunomodulatory roles of immune cells in the developing mouse lung at single cell resolution. eLife, 9, e56890.

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Western Blot Analysis of T Cell Lineage Transcription Factors

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Cells were lysed in radioimmunoprecipitation assay buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, and 1 mM EDTA) containing phosphatase inhibitor cocktail (Roche) and protease inhibitor cocktail (Roche), incubated on ice for 30 min, and collected by centrifugation at 13,000 rpm for 10 min. For immunoblotting, membranes were incubated with the T-bet (1:1,000; Thermo Fisher Scientific), GATA3 (1:1,000; BD Biosciences), and RORγt (1:1,000; BD Biosciences) primary antibody overnight at 4°C, followed by incubation with 1:4,000 dilution of HRP-linked secondary antibody for 1 h at room temperature. β-Actin was probed with the corresponding antibody to assure equal loading. Finally, immunoreactive proteins were detected using the enhanced chemiluminescence assay with HRP (Thermo Fisher Scientific).

Kim D., Kim M., Kim T.W., Choe Y.H., Noh H.S., Jeon H.M., Kim H., Lee Y., Hur G., Lee K.M., Shin K., Lee S.I, & Lee S.H. (2022). Lymph node fibroblastic reticular cells regulate differentiation and function of CD4 T cells via CD25. The Journal of Experimental Medicine, 219(5), e20200795.

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Western Blot Analysis of Protein Signaling

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Total protein was extracted using RIPA lysis buffer supplemented with EDTA-free Protease Inhibitor Cocktail (#4693132001; Roche, Germany), and were boiled in SDS loading buffer. 10 μg protein was separated by 10% SDS-PAGE, followed by transferring onto 0.45 μm nitrocellulose membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk in PBST and incubated with primary antibodies against ST3GAL6 (1/500, ab106527; Abcam), GATA3 (1/500, #558686; BD, USA), EGFR (1/1000, #4267; Cell Signaling Technology (CST), USA), phospho-EGFR (Tyr1068) (1/1000, #3777; CST), AKT (pan) (1/1000, #4691; CST), phospho-AKT (Ser473) (1/1000, #4060; CST), p44/42 MAPK (ERK1/2) (1/1000, #4695; CST), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1/1000, #4370; CST), STAT3 (1/1000, #9139; CST), phospho-STAT3 (Tyr705) (1/1000, #9145; CST), or GAPDH (1/1,000, sc-47724; Santa Cruz Biotechnology, USA) at 4℃ overnight. After rinse with PBST three times, the membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse antibodies (1/5,000; Jackson ImmunoResearch, USA; Table S1), followed by the detection with the Super Signal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, USA).

Dalangood S., Zhu Z., Ma Z., Li J., Zeng Q., Yan Y., Shen B., Yan J, & Huang R. (2020). Identification of glycogene-type and validation of ST3GAL6 as a biomarker predicts clinical outcome and cancer cell invasion in urinary bladder cancer. Theranostics, 10(22), 10078-10091.

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Isolation and Characterization of Immune Cells from Intestinal Biopsies

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For different experiments, LPMCs were isolated from intestinal biopsies of mice treated with DSS or from the colon of UC patients and cultured as described above. Prior to intracellular staining, cells were treated with a stimulation cocktail containing phorbol 12-myristate-13-acetate [PMA], Golgi-Stop and Ionomycin [eBioscience] for 4 h at 37°C. Cells were fixed and permeabilized using a transcription factor buffer set [BD Biosciences] according to the manufacturer’s instructions. Cells were stained for CD4 [BD Pharmingen], Tbet [Biolegend], IFNγ [BD Pharmingen], GATA3 [BD Pharmingen], IL4 [Biolegend], IL17A [Biolegend], RorγT [BD Pharmingen] and respective isotype controls. For other experiments cells were stained with CD4 [Biolegend], CD8 [Biolegend], CD14 [BD Pharmingen], CD15 [Biolegend], CD19 [BD Pharmingen], CD11c [BD Pharmingen], NK1.1 [Biolegend], CD68 [BD Pharmingen], CD80 [Biolegend], CD163 [Biolegend], CD206 [BD Pharmingen], CD25 [BD Pharmingen], FoxP3 [BD Pharmingen], CD49b [Biolegend], LAG3 [Biolegend] and IL10 [Biolegend]. Flow cytometry analysis was performed with FACS Calibur [BD Biosciences]. Cells were analysed using the FlowJo single cell analysis software [v.10.1r5, TreeStar].

Schmitt H., Ulmschneider J., Billmeier U., Vieth M., Scarozza P., Sonnewald S., Reid S., Atreya I., Rath T., Zundler S., Langheinrich M., Schüttler J., Hartmann A., Winkler T., Admyre C., Knittel T., Dieterich Johansson C., Zargari A., Neurath M.F, & Atreya R. (2019). The TLR9 Agonist Cobitolimod Induces IL10-Producing Wound Healing Macrophages and Regulatory T Cells in Ulcerative Colitis. Journal of Crohn's & Colitis, 14(4), 508-524.

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Quantification of Immune Markers in Murine Lungs

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Lung tissues collected from saline- or allergen-challenged mice were lysed with RIPA buffer (Cell signaling, Billerica, MA). The lysates (30 μg) were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% skimmed milk and incubated at 4 °C overnight with primary antibodies against GATA3 (0.5 μg/mL, BD Biosciences), Foxp3 (1.0 μg/mL, Abcam), and SOD2 (1:1000 dilutons, Cell signaling). After washing, the membranes were incubated with peroxidase-conjugated second antibody (Santa Cruz Biotechnology). Signals were developed with ECL select (GE Healthcare, Buckinghamshire, UK), detected using ChemiDoc (Bio-RAD, Hercules, CA), and quantified based on the intensities of the band, using Image J software (National Institute of Health, Bethesda, MD).

Seo Y.S., Kim H.S., Lee A.Y., Chun J.M., Kim S.B., Moon B.C, & Kwon B.I. (2019). Codonopsis lanceolata attenuates allergic lung inflammation by inhibiting Th2 cell activation and augmenting mitochondrial ROS dismutase (SOD2) expression. Scientific Reports, 9, 2312.

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Cytokine-Mediated T-Cell Activation Protocol

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The culture medium used was X-VIVO media (Lonza, USA) supplemented with 10% human AB serum, 1% GlutaMAX (Invitrogen, USA), 1% sodium pyruvate (Invitrogen, USA), and 1% penicillin/streptomycin (Invitrogen, USA). Fluorochrome-conjugated anti-CD4 was from BioLegend (USA). Fluorochrome-conjugated anti-Ki67, CTLA4, and GITR were from eBioscience (USA). Fluorochrome-conjugated anti-CD45RA, TGF-β, IL-10, IFN-γ, IL-4, IL-5, IL-13, FOXP3, and GATA3 were from BD (USA). PE Annexin V Apoptosis Detection Kit I, Cytofix/Cytoperm Kit, Pharmingen™ Leukocyte Activation Cocktail with BD GolgiPlug™, and Leukocyte Activation Cocktail with BD GolgiPlug were also from BD (USA). Anti-Human CD25 PerCP-Cyanine5.5 was from eBioscience (USA), and anti-Human CD25-PE was from BD (USA). Human Neuropilin-1 PerCP MAb was from R&D (USA). Anti-Human USP21 was purchased from Sigma-Aldrich Co. (USA), and anti-Human PIM2 was from Santa Cruz (USA); the secondary antibodies were from Sigma-Aldrich Co. (USA). Recombinant human cytokine IL-2 was purchased from R&D, and rIL-4 and TGF-β were from PeproTech (USA). Ficoll-Paque PLUS was purchased from GE Healthcare (UK). Human IL-10 ELISA Kit and Human TGF-β1 ELISA Kit were from RayBiotech (USA). Anti-CD3/CD8 Dynabeads were purchased from Invitrogen (USA).

Chen T., Hou X., Ni Y., Du W., Han H., Yu Y, & Shi G. (2018). The Imbalance of FOXP3/GATA3 in Regulatory T Cells from the Peripheral Blood of Asthmatic Patients. Journal of Immunology Research, 2018, 3096183.

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CD4+ T Cell Cytokine Profiling

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CD4⁺ T cells were positively or negatively selected from naïve and infected spleens (Miltenyi Biotec or Life Technologies) and cultured in X-Vivo 15 medium (Lonza, BioWhittaker) supplemented with l-glutamine and 2-mercaptoethanol, and then stimulated with PMA (10 ng/mL), ionomycin (1 μg/mL), and Golgistop (BD Biosciences; 1:1000) for 4–5 hours at 37°C, 5% CO₂. Cells were fixed, permeabilized and stained with antibodies against IFN-γ (Biolegend), IL-4 (BD Biosciences), and Gata-3 (BD Biosciences). For DNA methylation experiments, 6–10 spleens were pooled in order to obtain adequate numbers of cells for downstream analysis. Sorting was carried out using a BD FACS Aria. For statistical analysis, GraphPad Prism software was used to perform Student's t-test or ANOVA where appropriate.

Deaton A.M., Cook P.C., De Sousa D., Phythian-Adams A.T., Bird A, & MacDonald A.S. (2014). A unique DNA methylation signature defines a population of IFN-γ/IL-4 double-positive T cells during helminth infection. European Journal of Immunology, 44(6), 1835-1841.

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