Fc block antibody anti cd16 32

Murine mononuclear cells from oHSV-infected brains were isolated as previously described (17 ). To obtain splenocytes, spleens were collected and homogenized through a 70 mm strainer. Erythrocytes were lysed using RBC lysis buffer (Biolegend, San Diego, CA). Cells isolated from either brains or spleens were treated with Fc Block antibody (anti CD16/32, BD Biosciences). Cells were stained with mouse-specific immune cell surface markers for 30 min at 4°C. The following anti-mouse antibodies were used at a dilution of 1:200: CD3-APC, NK1.1-PE, CD69-FITC, CD27-V450, CD11b-PE, CD45-APC, CD3-PE-Cy7, CD107-APC, CD11b-PErCP-Cy5.5, and IFN-γ-FITC (Biolegend, San Diego, CA). For CD107a staining, mononuclear cells were cultured in 10% RPMI media with monensin (eBioscience, San Diego, CA) for 4 h before cell-surface staining. For staining of IFN-γ, we treated the cells with Cytofix/Cytoperm (BD) following initial cell-surface staining and then performed intracellular staining.

Murine mononuclear cells from livers were isolated as previously described (8 (link), 15 (link)). Erythrocytes were lysed using RBC lysis buffer (BioLegend, San Diego, CA). Cells isolated from livers were treated with Fc Block antibody (anti CD16/32, BD Biosciences). Cells were stained with mouse-specific immune cell surface markers for 30 min at 4°C. The following anti-mouse antibodies were used at a dilution of 1:200: CD3-APC, CD3-PerCP-Cy5.5, NK1.1-APC, NK1.1-PE, CD19-FITC, CD69-FITC, CD27-PE-Cy7, CD11b-PE, CD11b-PErCP-Cy5.5, Gr1 V450 and IFN-γ-FITC (Biolegend, San Diego, CA). For staining of IFN-γ, cells were treated with Cytofix/Cytoperm (BD) following initial cell-surface staining and then performed intracellular staining.