Multiparameter Analysis of Immune Cell Subsets

Blood was collected from tail veins in the presence of 5 mM EDTA, incubated with BD Fc Block for 10 min, and then stained with mAbs (BD Bioscience unless indicated otherwise) specific to CD45-APC-Cy7 (clone 30-F11), Ly6G-eFluor450 (1A8-Ly6g, eBioscience), CD11b-PerCP-Cy5.5 (M1/70), Ly6C-FITC (AL-21), NK1.1-APC (PK136), CD3-APC (145-2C11), CD19-APC (1D3), and TER119-APC (TER-119, eBioscience) for 20 min at room temperature. The samples were then fixed and lysed using BD FACS Lysing solution (BD Bioscience). After fixation, cells were washed and resuspended in 2% FBS/PBS. AccuCheck counting beads (PCB100, Invitrogen, Grand Island, NY, USA) 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) as described previously [10 (link)].