Multiparameter Analysis of BM Cell Subsets

BM cells prepared above were stained with lineage markers on FITC (CD4, CD5, CD8, CD11b, B220, Gr1, Ter119, and CD41, eBioscience, San Diego, CA, USA), CD117-APC-H7 (BD Bioscience, San Jose, CA, USA), Sca1-PE (eBioscience), CD16/32-PerCP5.5 (BD Bioscience), CD150-PE-Cy7 (BioLegend, San Diego, CA, USA), and CD105-BV421 (BD Bioscience) for 30 min on ice and gating as described by Pronk et al (34 (link)). and Singer et al. (12 (link)). Dead cells were excluded using 7-Amino-Actinomycin D (7-AAD; eBioscience) and AccuCheck counting beads (PCB100, Invitrogen) were used for quantification of absolute cell numbers. Multiparameter analysis was performed on a FACSSymphony A3 (BD Bioscience) using the FACSDiva software and data were analyzed using the FlowJo software (version 10, Tree Star Inc., Ashland, OR, USA).