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3 protocols using igg1 rtk2071

1

Profiling T Cell Infiltration in Mammary Tumors

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For analysis of T cell infiltration and activation, MMTV-PyMT mammary tumor tissues were digested with collagenase D (1mg/mL) and DNase I (100μg/mL), and cell suspensions were filtered through a 70 μm cell strainer as previously described [75 (link)-76 (link)]. The viable mononuclear cells were isolated using the Histopaque (Sigma-Aldrich) gradient, and analyzed using a FACScaliber (BD Biosciences). To determinate the activation of CD8+ T cells, single cell suspension prepared from PyMT mammary tumors were stimulated with PMA plus ionomycin in the presence of Golgi-stop for 6 hours, followed by intracellular staining for IFN-γ or granzyme B-producing CD8+ cells. Fluorochrome-conjugated mouse mAbs, including FITC-CD8 (53-6.7), APC-CD4 (GK1.5) and PE-IFN-γ (XMG1.2), as well as CD16/CD32 (2.4G2), isotype control rat IgG2b (RTK4530), and IgG1 (RTK2071) were purchased from BioLegend (San Diego, CA).
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2

Flow Cytometry Analysis of Immune Cells

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Fluorochrome-conjugated mouse monoclonal antibodies (mAbs) for flow cytometry analysis of immune cells, including FITC-CD4 (GK1.5), Percp/Cy5.5–IL-17A (TC11-18H10.1), CD16/CD32 (2.4G2), isotype control rat IgG2b (RTK4530), and IgG1 (RTK2071) were purchased from BioLegend (San Diego, CA). Mouse TNF-α, IL-6, and IL-1β enzyme-linked immunosorbent assay (ELISA) kits were purchased from Biolegend. Monoclonal antibodies for ASC (clone 2EI-7, 04-147, EMD Millipore, Billerica, MA), and NLRP3 (clone D4D8T, Cell Signaling Biotechnology) were purchased from commercial resources as indicated. Anti-NLRP3 (Abcam, U.S.A.), anti-IL-1β (EMD Millipore, CA, U.S.A.), and anticaspase-1 (p20 and p10) (Santa Cruz Biotechnology, Santa Cruz, U.S.A.) for Western blotting analysis were purchased from commercial resources as indicated and used per manufacturer’s instruction.
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3

Cytokine-Mediated CD8+ T Cell Activation

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OVA257–264 (SIINFEKL) and gp10025–33 (KVPRNQDWL) peptides were purchased from AnaSpec Inc (Fremont, CA). Recombinant human IL-2, IL-7 and IL-15 cytokines were kindly provided by the National Cancer Institute (NCI, MD). For fluorescence-activated cell sorting (FACS) analysis, mouse monoclonal antibodies to CD4 (GK1.5), CD8a (53–6.7), CD3 (HL3), IFN-γ (XMG1.2), CD25 (PC61), CD90.1 (OX-7), isotype control rat IgG2b (RTK4530), and IgG1 (RTK2071) were purchased from BioLegend (San Diego, CA); FoxP3 antibody (FJK-16s) was purchased from eBioscience (San Diego, CA). For Western blotting analysis, anti-human MDA-7/IL-24 antibody was purchased from GenHunter (Nashville, TN, USA); anti-mouse MDA-7/IL-24 antibodies were purchased from R&D Systems (Minneapolis, MN); phosphor-STAT1 (Y701), phosphor-STAT3 (Y705), total STAT1 and total STAT3 antibodies were purchased from Cell Signaling Technology (Davers, MA). Cucurbitacin I was purchased from Sigma Aldrich (St. Louis, MO). BrdU Flow Kit was purchased from BD Biosciences (San Jose, CA). Polybrene was purchased from Sigma Aldrich (St. Louis, MO). CytoTox 96® Non-Radioactive Cytotoxicity Assay kits were purchased from Promega (Madison, WI). Cell Counting Kits 8 (CCK8) were purchased from Abcam (Cambridge, MA).
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