Fcγiii 2 receptor antibody

Cell suspensions were blocked with FcγIII/II receptor antibody (BD), and subsequently stained with antibodies from BD bioscience (San Jose, CA, USA), i.e., PerCP-Cy5.5-conjugated anti-CD45 (30-F11), FITC-conjugated anti-CD11c (HL3), PE-conjugated anti-SiglecF (E50-2440), PE-Cy7 conjugated anti-Gr-1 (cloneRB6-8C5), APC conjugated anti-CD3e (145-2C11). Cells were acquired on a LSRII flow cytometer (BD bioscience) with data analyzed using FlowJo software (v7.6.5, Tree Star Inc., Ashland, OR, USA). Cell surface marker expression patterns were used to identify the following cell types: T cells (CD45⁺ CD3⁺), neutrophils (CD45⁺ Gr-1^high), eosinophils (CD45⁺ SiglecF⁺ CD11c^low), and alveolar macrophages (CD45⁺SiglecF⁺CD11c^high) [28 (link),29 (link),30 (link)].

TILs were isolated from ID8 tumors by digestion of minced tumor for 30 minutes at 37°C in HBSS containing 50 KU of DNase I (Sigma-Aldrich) and 0.2 mg/mL collagenase II (Life Technologies) followed by discontinuous gradient centrifugation. The partially purified TILs were treated with Fcγ III/II receptor antibody (BD Biosciences) in PBS containing 0.5% BSA and 0.05% sodium azide and double-stained with FITC-conjugated anti-mouse CD3 and either PE-conjugated anti-mouse CD4 or PE-conjugated anti-mouse CD8 (BD Biosciences). The CD3+ T cell population was gated and analyzed for percentages of CD4+ and CD8+ T cells. Data collected on 30,000 total events were analyzed using FlowJo software (BD Biosciences).