Isolation of splenocytes and MDMs

Splenocytes were isolated by crushing spleens of healthy age-matched mice in 12-well plastic tissue culture plates using a 5 ml syringe. RBCs were lysed in RBC lysis (ACK) buffer at 37°C and for 1 min. Cells were washed 3–5 times with MACS buffer at 4 °C. Splenocytes were then either frozen and sent for ICP-MS studies or plated on 6, 12, 24, or 96 well Primaria plates (Fischer Scientific, Cat #08-772) in 10% FBS RPMI medium supplemented with 100 U/ml penicillin G and 100 mg/ml streptomycin sulfate, 6 mm HEPES, 1.6 mm l-glutamine, 50 mm β-mercaptoethanol. Within 3–4 days monocytes adhere to the surface and transform to MDMs⁶⁶. The suspension cells and antibiotics are washed away where indicated and the MDMs (kept in antibiotic free culture medium) are used for ex vivo infection models, Prussian blue staining for iron content, used for arginase/NOS activity assays, hemozoin assay or lysed for mRNA profiling, proteomics or western blots. Splenocytes for ICP-MS and ex vivo infection studies were obtained by harvesting spleens of sacrificed healthy mice.