Anti b220 fitc clone ra3 6b2

Bone marrow from the right femora was collected for flow cytometry as previously described (30) . Bone marrow cell suspensions were collected in 2% FCS in PBS and red and white blood cell differentials were analyzed using Mindray BC-5000 Auto Hematology Analyser (Vepalabs, QLD, AU). For flow cytometry analysis, cell suspensions were incubated with the appropriate antibody cocktail for 40 minutes in the dark, on ice with agitation. The osteoclast precursor antibody cocktail (all sourced from Biolegend) was made in Fc (fragment crystallizable antibody region) block and contained anti-B220-FITC (clone RA3-6B2), anti-CD3-FITC (clone 145-2C11), anti-Ter-119-biotin, anti-CD115-biotin (clone AFS98), anti-Ly6C-APCFire750 (clone HK1.4), and anti-CD11b-BV510 (clone M1/70). Cells were then washed and incubated with streptavidin-PE-BV785 for 30 minutes. Cells were washed and resuspended in 2% FCS in PBS for analysis. Specificity of staining was determined by comparison to unstained cells and appropriate isotype control cocktails. Five minutes prior to analysis 5 μg/ml 7-amino actinomycin D (Life Technologies, CA, USA) was added for exclusion of dead cells. Cells were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, CA) and FlowJo version 10 (Tree Star Data Analysis Software).