Flow Cytometry Analysis of Mouse WBCs

Flow cytometric analysis of white blood cells (WBC) isolated from C57BL/6NJ was performed, as described previously (Paul et al., 2017 (link)). Briefly, peripheral blood was collected and centrifuged at 2000×g for 15 min and plasma removed. Red blood cell lysis buffer at 1X dilution (eBioscience) was added and cells were incubated for 10 min at room temperature on an orbital shaker followed by addition of 1X Phosphate Buffered Saline Solution (PBS, Thermo Fisher Scientific). Cells were then centrifuged at 500×g for 5 min at 4°C to pellet out the WBC, fixed in 2% paraformaldehyde (Thermo Fisher Scientific) for 15 min on ice, washed, and incubated with Fc block (CD16/32 Block) for 20 min, followed by probing with anti-CD45-FITC, anti-CD4-PE, anti-CD8a-PerCP, anti-CD11b-PECy5, and anti-Ly6G-PE antibodies (all purchased from Thermo Fisher Scientific) for 1 h at room temperature in the dark. Samples were then washed twice in 1X PBS, and acquired using a Guava Flow Cytometer (Millipore). Unstained and single stained compensation controls were used during acquisition and FlowJo software (version 10.3.0) utilized for cytometric analysis.