Cd14 phycoerythrin

Human peripheral blood mononuclear cells (PBMCs) were isolated from patients receiving denosumab or zoledronic acid. PBMCs (5×105) were diluted in 100 μL fluorescence-activated cell sorting (FACS) buffer and stained with 1:100 dilutions of the following antibodies: CD14-phycoerythrin (product number 557154; BD Pharmingen^TM, San Diego, CA, USA), CD11b-allophycocyanin (product number 553312; BD Pharmingen^TM), vitronectin receptor (VNR)–fluorescein (product number 11-0519-42; Invitrogen; Thermo Fisher Scientific, lnc., Waltham, MA, USA), and 10 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) solution (product number 62248; Thermo Fisher Scientific, lnc.) for 20 min at room temperature. Finally, the cells were washed, resuspended in FACS Buffer (product number 07905; Thermo Fisher Scientific, lnc.), and analyzed using a BD LSRFortessa™ device (BD Biosciences, San Jose, CA, USA) with BD FACSDiva™ (BD Biosciences) software.
Live monocytes were first selected based on the DAPI-negative population and forward (FSC-A) and side scatter (SSC-A) analysis. Monocytes were further gated based on CD14 expression. Changes in OC population after denosumab or zoledronic acid treatment were compared based on CD14⁺/CD11b⁺ expression. CD14⁺/CD11b⁺/VNR⁻ and CD14⁺/CD11b⁺/VNR⁺ cells represent early and late OC precursors, respectively.