Effect of plasma from western diet fed mice on bone marrow derived macrophages (BMDM)

Due to their ligand profiles, it can be hypothesized that Stab1 and Stab2 may be involved in atherosclerosis. We treated bone marrow derived macrophages (BMDM) with plasma from Stabilin-deficient and Wildtype mice to account for gene changes. We used microarrays to detail the global programme of gene expression in BMDM during different plasma treatments. We identified distinct classes of up- and down-regulated genes in response to the treatment. Mouse femurs were dissected and flushed with PBS containing 10% FCS and filtered through a 70 μm Nylon cell strainer. The bone marrow was collected in a 50 ml tube. The bone marrow was pipetted up and down and centrifuged for 5 minutes with 400g. Supernatant was discarded and the cell pellet was resuspended in 10 ml red blood cell lysis buffer for 30 s. Immediately afterwards, 20 mL DMEM (Dulbecco's Modified Eagle Medium; Gibco) was added and cells were again centrifuged for 5 minutes with 400g. Supernatant was discarded and the cell pellet was resuspended in DMEM containing 10% FCS; 0,5% Penicillin; 0,5% Streptavidin and MCSF 30 ng/ml (Peprotech; cat. no. 315-02). At day four after BMDM isolation, the initial medium was replaced with DMEM containing 10% plasma of Wildtype (WT), ApoE-KO (ApoE), ApoE-Stab1-KO (AS1) and ApoE-Stab2-KO (AS2) mice after 8 weeks of Western Diet treatment. The RNA was isolated 48 hours after cultivation with the corresponding plasma.