Isolation and Analysis of Mouse Bone Marrow and Blood Cells

Mice were killed by cervical dislocation, and bone marrow cells were harvested by flushing the femur and tibia with phosphate buffered saline (PBS; Thermo Fisher) as described previously (Kipari et al. 2013 (link)). Erythrocytes were lysed (red blood cell lysis buffer; Sigma-Aldrich). 0.5 × 10⁶ singly-suspended cells were then blocked with 10% mouse serum for 20 min on ice, and then incubated for 30 min on ice with appropriate dilutions of antibodies of interest (Table 1) in PBS containing 10% mouse serum. Blood was collected in 10 mM sodium citrate buffer and antibodies were added directly to the blood. Intracellular staining of 11β-HSD1 in blood neutrophils was carried out as described previously (De Sousa Peixoto et al. 2008 (link)). Cells were fixed and permeabilized using a kit (Fix and Perm, Invitrogen; Thermo Fisher) to facilitate intracellular immunostaining of 11βHSD1. Cells were then washed in PBS before acquisition and analysis (BD FACS LSR Fortessa and FlowJo software; Oregon, USA). The gating strategy for analyzing blood neutrophils is shown in Supplementary Fig. 2.