Splenic Naive CD4 + T cells were obtained by the negative selection using Mojo Sort Mouse CD4 T Cell Isolation Kit (Biolegend) and positive selection using CD62L MicroBeads, mouse (Miltenyi). Naive CD4 + T cells were plated onto 24-well tissue culture plates (Costar) pre-coated with 10 mg/ml anti-TCRb antibody (clone H57-597) with 1 mg/ml anti-CD28 antibody (clone 37.51, BioLegend). T reg cell cultures contained IL-2 (15 ng/ml), recombinant human TGFb (10ng/ml) (Peprotech), anti-IFNg (Biolegend), and anti-IL-4 antibody (Biolegend). Th0 cell cultures contained IL-2 (15 ng/ml), anti-IL-4 (Biolegend) and anti-IFNg (Biolegend). Th2 cell cultures contained IL-2 (15 ng/ml), IL-4 (5 ng/ml) (Peprotech), and anti-IFNg (Biolegend). For ex vivo analysis, lung CD44 hi nT reg cells were cultured with IL-33 (10ng/ml) (Biolegend) with IL-2 (15 ng/ml) or TCRb with IL-2 (15 ng/ml) under control or fatty acid-free conditions.
Anti ifng
Manufactured by BioLegend
Anti-IFNg is a laboratory reagent used to detect and quantify Interferon gamma (IFN-γ) in biological samples. It functions as a specific antibody that binds to IFN-γ, allowing for its identification and measurement.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd8, by BioLegend (3 mentions)
Anti cd11b, by Thermo Fisher Scientific (2 mentions)
Facsaria 3, by BD (2 mentions)
Fortessa, by BD (2 mentions)
Dnase 1, by Roche (2 mentions)
Anti cd45, by BioLegend (2 mentions)
Anti foxp3, by BioLegend (2 mentions)
Anti f4 80, by BioLegend (2 mentions)
Fixation buffer, by BioLegend (2 mentions)
Collagenase d, by Roche (2 mentions)
Anti granzyme b, by BioLegend (2 mentions)
Intracellular staining permeabilization wash buffer, by BioLegend (2 mentions)
Anti cd11b, by BioLegend (2 mentions)
24 well tissue culture plate, by Corning (1 mentions)
Anti il 4 antibody, by BioLegend (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Anti cd28 antibody clone 37, by BioLegend (1 mentions)
Mojosort mouse cd4 t cell isolation kit, by BioLegend (1 mentions)
Il 33, by BioLegend (1 mentions)
Anti il 4, by BioLegend (1 mentions)
6 protocols using anti ifng
1
Isolation and Culture of Naive CD4+ T Cells
2
Flow Cytometry Analysis of Immune Cells
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Cells were suspended in PBS supplemented with 10% FBS and 0.02% sodium azide for cell surface staining. Cells were stained with: anti-CD103, anti-CD11c, anti-CD86 (BD Biosciences, Pharmingen, San Diego, CA, USA), anti-CD90.2, anti-CD4, anti-CD8α, anti-MHC-II, anti-XCR1 (BioLegend, San Diego, CA, USA), anti-NK1.1, anti-CD19 (Ablab, Vancouver, B.C., Canada), anti-CD11b (eBioscience, San Diego, CA, USA). For intracellular staining, cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (eBioscience). Antibodies used were: anti-IL-13 (eBioscience), anti-IFNg, and anti-IL-17A (BioLegend).
3
Tumor Infiltrating Immune Cell Analysis
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Briefly, 4 different group tumor tissues were digested at 37 °C for 30 minutes with 1 mg/mL Collagenase D and 0.1 mg/mL DNase I (Roche). Digestion was stopped by EDTA, and cells were filtrated through 70-mm cell strainers and washed twice with PBS containing 1 mM EDTA and 2% fetal bovine serum (FBS) (staining buffer). Cells were re-suspended in the staining buffer and stained with following antibodies on ice for 30 minutes: anti-CD45, anti-CD8, anti-IFNg, anti-Granzyme B, anti-CD11b, anti-CD11c, anti-FOXP3, anti-CD25, anti-F4/80, Ly6C were purchased from BioLegend. For intracellular staining, cells were fixed with fixation buffer (Biolegend) on ice for 15 minutes, and then washed twice with Intracellular Staining Permeabilization Wash Buffer (Biolegend). Antibodies against IFN-g (Clone XMG1.2) and Granzyme B (Clone: QA16A02) were added and incubated for 1 hour on ice. The cytokine producing cells were determined by flow cytometry. The flow cytometry data were collected on Fortessa (BD) and analyzed by FlowJo (Tree Star). For cell sorting, CD8+ T cells that were co-cultured with tumor cells for 6 hours were collected and washed with culture medium. Re-suspended cells were stained with anti-CD8a antibodies (Clone: 53-6.7) for 30 min on ice. After a washing step, cells were sorted on a BD FACS AriaIII (BD) and lysed in the buffer RLT plus (QIAGEN).
4
Ex vivo γδ T cell expansion
5
Multiparameter Immune Cell Analysis
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Single‐cell suspensions were washed with FACS buffer (PBS, 5% FBS, 2 mM EDTA) and resuspended in Fc block for 15 min. hMDMs were stained with the following antibodies during 45 min at 4°C: Fixable Viability Due eFluor 506 (eBioscience, 65‐0866‐18); anti‐CD14 (BD Pharmingen, 555397); anti‐CD11b (eBioscience, 48‐0112‐82); anti‐CD206(BD Biosciences, 740309); anti‐CD163 (BioLegend, 333622); anti‐HLA‐DR (Thermo Fisher Scientific, 17‐9956‐42); anti‐CD80 (BD Bioscience, 557227); anti‐CD115 (Sony Biotechnology, RT2336540).
CD8+ T cells were stained with the following surface markers: Fixable Viability efluor780 (Thermo Fisher Scientific, 65‐0866‐18) and anti‐CD8 (BioLegend, 980908). Subsequently, cells were incubated for 30 min with Fix/Perm buffer (eBioscience, 00−5523). Cells were washed with permeabilisation buffer (eBioscience, 00−5523) and stained ON (4°C) in permeabilisation buffer with anti‐IFNg (BioLegend, 502542); anti‐TNFa (BioLegend, 986802); anti‐IL‐2 (BioLegend, 500328) and anti‐GZMB (BioLegend, 515406). Cells were subsequently washed and resuspended in FACS buffer. FACS data were acquired using a FACS Fortessa or FACS Symphony (BD Biosciences) and data were analysed using the FlowJo (TreeStar) program.
CD8+ T cells were stained with the following surface markers: Fixable Viability efluor780 (Thermo Fisher Scientific, 65‐0866‐18) and anti‐CD8 (BioLegend, 980908). Subsequently, cells were incubated for 30 min with Fix/Perm buffer (eBioscience, 00−5523). Cells were washed with permeabilisation buffer (eBioscience, 00−5523) and stained ON (4°C) in permeabilisation buffer with anti‐IFNg (BioLegend, 502542); anti‐TNFa (BioLegend, 986802); anti‐IL‐2 (BioLegend, 500328) and anti‐GZMB (BioLegend, 515406). Cells were subsequently washed and resuspended in FACS buffer. FACS data were acquired using a FACS Fortessa or FACS Symphony (BD Biosciences) and data were analysed using the FlowJo (TreeStar) program.
6
Isolation and characterization of tumor-infiltrating CD8+ T cells
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Briefly, four different group tumor tissues were digested at 37 °C for 30 min with 1 mg/mL Collagenase D and 0.1 mg/mL DNase I (Roche). Digestion was stopped by EDTA and cells were filtrated through 70 mm cell strainers and washed twice with PBS containing 1 mM EDTA and 2% FBS (staining buffer). Cells were re-suspended in the staining buffer and stained with following antibodies on ice for 30 min: anti-CD45, anti-CD8, anti-IFNg, anti-Granzyme B, anti-CD11b, anti-CD33, anti-FOXP3, anti-CD25, anti-F4/80, Ly6C were purchased from BioLegend. For intracellular staining, cells were fixed with fixation buffer (Biolegend) on ice for 15 min, and then washed twice with Intracellular Staining Permeabilization Wash Buffer (Biolegend). Antibodies against IFN-g (Clone XMG1.2) and Granzyme B (Clone: QA16A02) were added and incubated for 1 h on ice. The cytokine producing cells were determined by flow cytometry. The flow cytometry data were collected on Fortessa (BD) and analyzed by FlowJo (Tree Star). For cell sorting, CD8+ T cells that were co-cultured with tumor cells for 6 h were collected and washed with culture medium. Re-suspended cells were stained with anti-CD8a antibodies (Clone: 53-6.7) for 30 min on ice. After a washing step, cells were sorted on a BD FACS AriaIII (BD) and lysed in the buffer RLT plus (QIAGEN).
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