Anti cd16 cd32 blocking antibody

BMMs (1 × 10⁶ cells/ml) were cultured in 48-well plates in OC medium supplemented with M-CSF (20 ng/ml) and RANKL (10 ng/ml) or M-CSF (20 ng/ml) alone. At day 1 of culture, BMMs were incubated with 10 or 75 μg/ml sCD83 to exclude possible toxic effects of the protein. After 24 h cells were detached from the plate by incubation with Accutase (Thermo Fisher Scientific) at 37°C for 10 min. Cells were then washed with PBS and incubated with anti-CD16/CD32 blocking antibody (BioLegend) for 10 min at RT, followed by staining with an antibody cocktail for 30 min at 4°C in the dark. The following antibodies were used to detect viability of preosteoclasts: APC-eFluor780-labeled anti-CD45 (eBioscience), APC-labeled anti-F4/80 (BioLegend), PE-labeled anti-OSCAR (Beckman coulter), DAPI (Roche), and added shortly before measurement. Data were acquired on the Gallios flow cytometer (Beckman Counter) and analyzed with the Kaluza software 1.5.

Aliquots of up to 3 × 10⁶ cells were blocked using an anti-CD16/CD32 blocking antibody (BioLegend, San Diego, CA) and labelled with fluorescently conjugated antibodies in the dark as described previously (Thomson et al. 2018). The following antibodies were purchased from BioLegend: CD4-AF488 (GK1.5), CD8α-PE/Cy7 (53-6.7), CD11b-PE (M1/70), CD44-BV421 (IM7), CD45-BV510 (30-F11), CD45-PE/Cy7 (30-F11), CD62L-PE (MEL-14), CD64-APC (X54-5/7.1), F4/80-FITC (BM8), Ly6G-AF700 (1A8), MHCII-BV510 (M5/114.15.2), NK1.1-BV605 (PK136) and TCRβ-PerCP/Cy5.5 (H57-597). The following were purchased from eBioscience (San Diego, CA): CD11b-AF700 (M1/70) and Ly6C-eFluor 450 (HK1.4). Dead cells were excluded from analysis using Fixable Viability Dye eFluor 780 (eBioscience, San Diego, CA). Cells were analysed using an LSRII (BD Biosciences, San Jose, CA). Data were analysed using FlowJo v10 (Tree Star, Ashland, OR).