Isolation and Analysis of Murine Bone Marrow

C57Bl/6 and FSTL-1 Hypomorphic (on C57Bl/6 background) mice were housed in accordance with the approved University of Pittsburgh School of Medicine Institutional Animal Care and Use Committee protocol^{12 (link)}. The bone marrow compartment of male and female wild-type and FSTL-1 Hypomorhic mice was isolated by femur flush protocol⁵⁰ . Isolated bone marrow cells were resuspended in sterile phosphate-buffered saline and counted using trypan blue stain and a hemocytometer. The cells were first blocked using anti-mouse CD16/CD32 (eBioscience) and subsequently surfaced stained in the presence of anti-mouse CD45 antibody (clone 30-F11, BD Bioscience). CD45-positive and CD45-negative cell populations were then sorted on a FACSAria via FACSDiva software (Becton-Dickinson) into phosphate-buffered saline. The sorted cell populations were pelleted and resuspended in RNA Lysis Buffer (Zymo Research). RNA was processed for gene expression analysis as previously described. These experiments were performed twice with n=3 or n=4 per group each time. Data shown include cells from all animals in the two experiments.