Macrophage Infection Assay with Mycobacteria and Staphylococcus

RAW cells or BMDMs were transfected with siRNAs for 2d (RAW) or 3d (BMDM) prior to infection. 200 U/ml murine IFN-γ (Gibco) was added 24h before infection as indicated. For Mtb and M. smegmatis, macrophages were infected with a single cell suspension at an MOI of 3 with at least three replicates per experiment as previously described [26 (link)]. 4 hpi macrophages were extensively washed, lysed with 0.1% Triton X-100 at indicated time points, and serial dilutions were plated on 7H11. CFU were counted 15–21 days later for Mtb and 2–3 days later for M. smegmatis. For S. aureus infection, bacteria were opsonized with human serum for 1h prior to infection. Macrophages were infected at an MOI of 1, washed extensively 30 min post-infection, and lysed in 0.1% Triton-X-100 at indicated time points. S. aureus were plated on Tryptic Soy Agar, and CFU were quantified the following day.