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Anti tim 3 f38 2e2

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Sourced in United States

Anti-Tim-3 (F38-2E2) is a monoclonal antibody that binds to the T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3). Tim-3 is an inhibitory receptor expressed on various immune cells, including T cells, natural killer cells, and myeloid cells. The Anti-Tim-3 (F38-2E2) antibody can be used for the detection and study of Tim-3 expression and function.

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5 protocols using anti tim 3 f38 2e2

1

Comprehensive Immunological Antibody Panel

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The following antibodies were used in this study: anti-B7-H3 (FM276, Miltenyi Biotech), anti-GD2 (14.G2a, BD Biosciences), human Ig (polyclonal, Thermo Fisher Scientific), anti-mouse IgG (polyclonal, R&D), anti-CD3 (UCHT1, BioLegend), anti-HisTag (J095G45, BioLegend), anti-CD34 (QBEnd10, R&D), anti-ab-TCR (IP26, BioLegend), anti-CD107a (H4A3, BioLegend), anti-cD25 (BC96, BioLegend), anti-CD69 (FN50, BioLegend), anti-Tim3 (F38-2E2, BioLegend), anti-Lag3 (11C3C65, BioLegend), anti-PD-1 (EH12.1, BD Biosciences), anti-mouse CD45 (30-F11, BioLegend), anti-human CD45 (HI30, BioLegend), Ghost Red 780 (Tonbo Biosciences), Zombie Yellow Viability Dye (BioLegend), propidium iodide (Gibco), Cell Trace Violet (Thermo Fisher Scientific), and Precision Count Beads (BioLegend).
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2

Multiparameter Immune Profiling of Cells

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Cells were stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain kit (Invitrogen) and surface antibodies for 30 min at 4 °C. For intracellular cytokine staining, cells were treated with 50 nM phorbol-12-myristate-13-acetate (MilliporeSigma) and 250 nM ionomycin (MilliporeSigma) for 4 hours in the presence of Brefeldin A (BD Biosciences) before harvesting. Cells were washed and fixed with BD Cytofix™ Fixation Buffer (BD Biosciences) for 10 min at RT, then washed with PBS. Intracellular cytokines were stained in permeabilization buffer (eBioscience) for 30 min at 4 °C. The following antibodies were used: anti-LAG-3 (11C3C65, BioLegend), anti-PD-1 (EH12.1, BD Biosciences), anti-TIGIT (MBSA43, eBioscience), anti-Tim-3 (F38–2E2, Biolegend), anti-IFN-γ (4S.B3, eBioscience), and IL-10 (JES3–9D7, Biolegend). Cells were acquired on a BD Fortessa flow cytometer and data was analyzed with FlowJo software v10 (Threestar).
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3

Phenotypic Profiling of Immune Cells

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Cells were stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain kit (Invitrogen) and surface antibodies for 30 min at 4 °C. For intracellular cytokine staining, cells were treated with 50 nM phorbol-12-myristate-13-acetate (MilliporeSigma) and 250 nM ionomycin (MilliporeSigma) for 4 hours in the presence of Brefeldin A (BD Biosciences) before harvesting. Cells were washed and fixed with BD Cytofix™ Fixation Buffer (BD Biosciences) for 10 min at RT, then washed with PBS. Intracellular cytokines were stained in permeabilization buffer (eBioscience) for 30 min at 4 °C. The following antibodies were used: anti-LAG-3 (11C3C65, BioLegend), anti-PD-1 (EH12.1, BD Biosciences), anti-TIGIT (MBSA43, eBioscience), anti-Tim-3 (F38-2E2, Biolegend), anti-IFN-γ (4S.B3, eBioscience), and IL-10 (JES3-9D7, Biolegend). Cells were acquired on a BD Fortessa flow cytometer and data was analyzed with FlowJo software v10 (Threestar).
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4

Blocking Immune Checkpoint Receptors

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Cells were pre-coated with 10µg/ml blocking antibodies before co-culture. Antibodies used were anti-PD-L1 (6E11.1.9, Genentech), anti-PD-1 (EH12.2H7, BioLegend), anti-Tim-3 (F38-2E2, BioLegend), mouse IgG2a isotype (BioLegend), and mouse IgG1 isotype (BioLegend).
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5

NK Cell Purification and Functional Assay

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For separation of NK cells, anti-CD56 microbeads, columns, and filters were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). For purity analysis of NK cells, anti-CD56 monoclonal antibody (TULY 56)-FITC and anti-CD3 monoclonal antibody (UCHT1)-PE together with their relevant mouse IgG1-kappa isotype controls were purchased from eBioscience (California, United States). For stimulation of NK cells, human interleukin-2 (IL-2) recombinant protein and cell stimulation cocktail (×500) were purchased from eBioscience (California, United States). Blocking antibodies, anti-PD-1 (EH12-27) and anti-Tim-3 (F38-2E2) and their corresponding purified IgG1-kappa isotype controls were from BioLegend (California, United States). Anti-CD107a (LAMP-1) monoclonal antibody (eBioH4A3)-PE and Annexin V-FITC Apoptosis Detection Kit with PI were purchased from eBioscience (California, United States). For cytokines measurements, human IFN-γ and TNF-α ELISA kits were obtained from eBioscience (California, United States).
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