Lyec rna

Primary cultured microglia (2 × 10⁵ cells/ml) in 96-well plates were incubated with miRNA complexed to LyoVec (InvivoGen #LYEC-RNA, San Diego, CA, USA) at a concentration of 5 μg/ml for indicated durations. Cell-conditioned medium was subsequently collected and analyzed via a customized mouse Procarta Plex Mix and Match 15-plex (eBioscience Inc., San Diego, CA, USA) panel following the manufacturer’s instructions. Briefly, 50 μl magnetic capture beads were plated on a 96-well plate. After washing, 50 μl of cell-conditioned medium were added to each well. The plate was shaken at room temperature for 30 min before being incubated overnight at 4 °C. On the following day, 30 μl of detection antibody mixture were added to each well, and the plate was placed on a shaker at 500 rpm for 30 min. Following washing, the plate was incubated with 50 μl streptavidin/well for 30 min on a shaker, and Reading Buffer was added. Read-out was performed on a Luminex 200 device Bio-Plex Software 4.0 (Bio-Rad Laboratories, Hercules, CA, USA).

Primary cultured mouse microglia or human-derived THP-1 cells were incubated with indicated concentrations of miRNAs complexed to LyoVec (InvivoGen #LYEC-RNA, San Diego, CA, USA) for indicated durations in 96-well plates (30,000 cells/well). Subsequently, supernatants were collected and stored at − 80 °C. TNF-α amounts were detected via Enzyme-Linked Immunoabsorbent Assay (ELISA; TNF alpha Mouse Uncoated ELISA Kit, Invitrogen, #88–7324-88, Carlsbad, CA, USA) or TNF alpha Human Uncoated ELISA Kit (Invitrogen, #88–7346-88, Carlsbad, CA, USA), according to the manufacturer’s instruction.

HEK-Blue TLR reporter and corresponding Null control cell lines were purchased from InvivoGen, San Diego, CA, USA. One day after seeding 50,000 cells/well in a 96-well plate, HEK-Blue SEAP reporter 293 cells expressing mTLR7, mTLR8, hTLR7 or hTLR8 and the inherent control cells were incubated with different miRNA mimics or control oligoribonucleotide (10 µg/mL) using LyoVec as tranfection agent (InvivoGen #LYEC-RNA, San Diego, CA, USA) according to the manufacturer’s protocol. Cells were stimulated with indicated agents dissolved in 90% HEK-Blue detection reagent and 10% culture media. Detection of the reporter protein secreted embryonic alkaline phosphatase (SEAP) was performed at 655 nm using Varioskan Flash (Thermo Fisher Scientific, Waltham, MA, USA). For each condition, four wells were individually measured, and the average was used for evaluation.

Primary cultures of microglia were generated from C57BL/6 mice as described above. Microglia were incubated with 5 µg/ml of let-7b or mutant oligoribonucleotide for indicated durations, or were exposed to increasing concentrations of let-7b or mutant oligoribonucleotide, as indicated, complexed to the transfection agent LyoVec (InvivoGen #LYEC-RNA, San Diego, CA, USA) for 12 h. Conditioned medium was collected and analyzed by enzyme-linked immunosorbent assay with hamster antibody against mouse TNF-α (BD Biosciences, Franklin Lakes, NJ, USA) as a capture antibody and biotin-labeled antibody against mouse as secondary antibody (BD Biosciences, Franklin Lakes, NJ, USA).