Microglia BV2 Cell Line Culture, Treatment, and Transfection, and Conditioned Medium Preparation

In this study, BV2 cells were cultured in DMEM with 10% FBS and penicillin-streptomycin at 37°C with 5% CO2. Then, the BV2 cells were pretreated with serum-free DMEM containing LPS (0.1 ug/ml). After incubation for 24 h, the conditioned medium was collected and denoted as MCM. Meanwhile, the BV2 cell complete culture medium was denoted as MCM-control. The BV2 cells (4 × 10⁵ cells per well) were seeded onto six-well plates overnight to reach 70% confluence for transfection. TREM2 (Myc-DDK-tagged) overexpression plasmid (MR202717) or pCMV6-Entry Mammalian Expression Vector (PS100001), which was purchased from OriGene Technologies, Inc. (Rockville, MD, United States), was transfected into cells for 4.5 h using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, United States) and then replaced with a complete medium to culture for another 48 h at 37°C, which was then collected for use in subsequent experiments. The C-Myc mouse monoclonal antibody (TA500002, OriGene Technologies, Inc., Rockville, MD, United States) was applied to confirm transduction efficiency. For combination treatments, LPS (0.1 ug/ml) was added to the BV2 cells after TREM2 (vector or overexpression) transfection for 24 h, and culturing was continued for another 24 h at 37°C. The above conditioned mediums containing LPS (0.1 ug/ml) and TREM2 (vector or overexpression) in BV2 cells were collected and were denoted as MCM + TREM2 (vector or overexpression).