Co-culture of Macrophages and Alveolar Cells

For co-culture experiments, primary macrophages isolated from mice fed standard diet, control diet, or DHA supplemented diet were plated in six well co-culture inserts (Falcon’s Transparent PET Membrane, 2.0 μm pore size, 1 × 10⁵ pores/cm²). The macrophages cultured in the transwell inserts were treated as described above, washed with fresh media, and placed over wells which contained confluent MLE12 cells (1 × 10⁵ cells/well) in HITES media, described elsewhere40 (link). The procedure was using an immortalized mouse alveolar macrophage cells line (MHS). MHS cells were cultured in RPMI-1640 (ATCC, Manassas, VA) under standard conditions. After 24 h of growth, the culture media was harvested from individual wells and the levels of HMGB1 and LTB₄ were measured (MHS cytokine responses, Supplemental Figure 1). The MLE12 cells were stained and evaluated for apoptosis, cl-caspase 3, and proliferation, Ki67, by flow cytometry.