Differential Counting of Mouse Peripheral Blood Leukocytes

Peripheral blood was collected from the retro-orbital plexus of mice under isoflurane anesthesia. Peripheral blood was smeared on glass slides, stained with Wright-Giemsa reagent, and then 100 cells were counted differentially according to the type of WBC^{20 (link),21 (link)}. BM cell suspensions were prepared by repeatedly flushing the cells from femora using either Iscove-modified Dulbecco’s medium (IMDM: Invitrogen Corp., Carlsbad, CA, USA) or α-minimal essential medium (α-MEM: Thermo Fisher Scientific, Waltham, MA, USA); the cells then were dispersed by repeated passage through a 23-gauge hypodermic needle. BM cells were recovered from the femora of three mice per experimental group at each time point; the resulting individual cell suspensions then were counted. The number of cells was determined using a Sysmex PocH-100 iV Diff hematology analyzer (Sysmex Co., Kobe, Japan).