Lesion-remote astrocytes govern microglia-mediated white matter repair I

A 50 µl spot drop containing 50,000 microglia cells was seeded into the desired wells of a 24-well plate. Cells were cultured for 7 days, with a full media change given on day 1 followed by half media change on day 4. On day 7, cells were either left untreated or treated with CCN1 (Peprotech: 120-25) at 50 ng ml-1 or vehicle (BSA) for 24 h. Each condition was carried out in triplicate for each experiment. Cells were then removed using a cell scraper, replicates pooled, and then processed for RNA-seq. Overall design: Microglia were cultured for 7 days in defined microglia media under controlled conditions (37C/5%CO2)

将含有50,000个小胶质细胞（microglia）的50 μL滴状接种液接种至24孔板的指定孔内。细胞培养7天，于第1天进行全量换液，第4天进行半量换液。培养至第7天时，将细胞分为三组：不予处理，或用终浓度为50 ng·ml⁻¹的CCN1（Peprotech货号：120-25）处理，或用载体（BSA）处理，所有组别均处理24小时。每个实验条件均设置三次生物学重复。随后使用细胞刮铲收集细胞，合并所有重复样本后进行RNA测序（RNA-seq）。实验整体设计：将小胶质细胞置于成分明确的小胶质细胞专用培养液中，在可控培养条件（37℃、5%CO₂）下培养7天。