Peripheral Blood and Bone Marrow Isolation

Peripheral blood (PB) and bone marrow (BM) samples were collected from all groups of mice [6 (link), 22 (link)]. Briefly, a 50 µl of PB was withdrawn from retro-orbital plexus or tail-vein injection in EDTA-coated microvette tubes (Sarstedt Inc., Newton, NC, USA) for hematologic analysis. The blood samples were run on HemaVet 950FS hematology analyzer (Drew Scientific Inc., Oxford, CT, USA) within 2 h of collection. PB was also collected from the posterior vena cava (with a 25-gauge needle and 1-ml syringe containing 250 U heparin). The red blood cells (RBCs) were lysed by hypotonic lysis buffer (BD Biosciences, CA, USA) and PB mononuclear cells (PBMNCs) were obtained by centrifugation. BM mononuclear cells (BMMNCs) were also flushed from tibias and femurs. RBCs present in BM were lysed, washed, and BMMNCs were resuspended in PBS or 2% FBS containing RPMI media as necessary.