Anti fcr anti cd16 cd32 fc block

Cells were suspended with FACS buffer (1 mM EDTA, 0.01% sodium azide, 0.1% bovine serum albumin (BSA), 0.02 M phosphate, 0.15 M NaCl, pH 7.2) and then stained for 20 min with anti-FcR/anti-CD16+CD32/Fc Block (eBioscience) and fluorophore-conjugated antibodies: anti-CD45-PerCP Cy5.5 (BioLegend), anti-Ly6G-AF700 (Thermo Fisher Scientific), anti-CD19-APC (BioLegend), anti-CD11b-PE (BioLegend), anti-NK1.1-PE Cy7 (BioLegend), anti-TCR-beta-Pac Blue (BioLegend), anti-CD4-FITC (BioLegend), and anti-CD8-APC/Cy7 (BioLegend). After staining, cells were washed and then fixed with 1% PFA for 30 min. After fixation, cells were washed three times with 200-μl FACS buffer, and then 50 μl of 123count eBeads Counting beads (Thermo Fisher Scientific) were added to allow for quantification of total number of immune cell subtypes following manufacturer’s instructions. Data were acquired on a LSRII instrument (BD Biosciences). Analysis was performed using FlowJo software, version 10.0.8.

Splenocytes were suspended with FACS buffer (1 mM EDTA, 0.01% sodium azide, 0.1% Bovine Serum Albumin (BSA), 0.02 M phosphate, 0.15 M NaCl, pH 7.2) and then stained for 20 min with anti-FcR/anti-CD16 + CD32/Fc Block (eBioscience) and the following fluorophore-conjugated antibodies for NK cell receptor profiling: anti-CD19-APC (BioLegend), anti-CD3-Pac Blue (BioLegend), anti-NK1.1-PE Cy7 (BioLegend), anti-NKG2A-PE (BioLegend), anti-CD11b-PerCP (BioLegend), anti-CD27-AF700 (BioLegend), anti-CX3CR1-BV510 (BioLegend), anti-CD107a-APC Cy7 (BioLegend), and anti-NKG2D-FITC (BioLegend). After staining, cells were washed three times with 200 µl FACS buffer. Data were acquired on a LSRII instrument (BD Biosciences). Analysis was performed using FlowJo software, version 10.0.8.

Splenocytes were suspended with FACS buffer (1 mM EDTA, 0.01% sodium azide, 0.1% Bovine Serum Albumin (BSA), 0.02 M phosphate, 0.15 M NaCl, pH 7.2) and then stained for 20 min with anti-FcR/anti-CD16 + CD32/Fc Block (eBioscience) and the following fluorophore-conjugated antibodies for immune cell profiling: anti-CD45-PerCP Cy5.5 (BioLegend), anti-Ly6G-AF700 (Thermo Fisher Scientific), anti-CD19-APC (BioLegend), anti-CD11b-PE (BioLegend), anti-NK1.1-PE Cy7 (BioLegend), anti-TCR-β-Pac Blue (BioLegend), anti-CD4-FITC (BioLegend), and anti-CD8-APC/Cy7 (BioLegend). After staining, cells were washed three times with 200 µl FACS buffer, and then 50 µl of 123count eBeads Counting beads (Thermo Fisher Scientific) were added to allow for quantification of total number of immune cell subtypes following manufacturer’s instructions. Data were acquired on a LSRII instrument (BD Biosciences). Analysis was performed using FlowJo software, version 10.0.8.