Macrophage Polarization and miRNA Modulation

Murine macrophage cell line (RAW264.7) were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) and Penicillin-Streptomycin (Thermo fisher Scientific, Waltham, MA, USA) in 5% carbon dioxide (CO₂) at 37°C. Then the microRNA (miR) was transfected into 1.5x10⁶/ well of macrophages in 24-well plate following a previous protocol [38 (link)]. Briefly, the transfection mixture consisted of 5 pmol of miR (miR146a, miR223 or mimic control) (Ambion, Austin, TX, USA) and 1.5 μl Lipofectamin RNAimax reagent (Invitrogen, Carlsbad, CA, USA) in Opti-MEM (Gibco, Thermo Fisher Scientific) for one reaction was incubated with cells for 2 days before the verification of transfection-efficiency by quantitative polymerase chain reaction (qPCR) as demonstrated in Fig 1. In addition, M1 or M2 polarization was activated by LPS (10 ng/ml) or IL-4 (20 ng/ml), respectively, before cell collection and supernatant cytokines were measured by ELISA (ReproTech). For preparation of adoptive transfer, miR over-expressed RAW264.7 were treated with IL-4 (20 ng/ml) to activate M2 macrophage polarization, washed 2 times by PBS and intravenously administered into mice at 1 hour prior to LPS intra-peritoneal injection (4 mg/kg).