biostudies-literatureRuan ZNIA NIH HHS110791https://www.ebi.ac.uk/biostudies/studies/S-EPMC9133589biostudies-literatureUnknown39(6)Activated microglia release extracellular vesicles (EVs) as modulators of brain homeostasis and innate immunity. However, the molecules critical for regulating EV production from microglia are poorly understood. Here we establish a murine microglial cell model to monitor EV secretion by measuring the fluorescence signal of tdTomato, which is linked to tetraspanin CD63. Stimulation of tdTomato⁺ cells with ATP induces rapid secretion of EVs and a reduction in cellular tdTomato intensity, reflecting EV secretion. We generate a GFP⁺ tdTomato⁺ cell library expressing TurboGFP and barcoded short hairpin RNAs for genome-wide screening using next-generation sequencing. We identify Mcfd2, Sepp1, and Sdc1 as critical regulators of ATP-induced EV secretion from murineCell reportsFunctional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia.PMC9133589P30 AG013846R01 AG067763R01 AG054672R01 AG054199R01 AG072719RF1 AG054199R01 AG066429Ruan ZTakamatsu-Yukawa KIkezu SIkezu TLabadorf ATWang YEricsson MUshman MLfalseFunctional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia.Activated microglia release extracellular vesicles (EVs) as modulators of brain homeostasis and innate immunity. However, the molecules critical for regulating EV production from microglia are poorly understood. Here we establish a murine microglial cell model to monitor EV secretion by measuring the fluorescence signal of tdTomato, which is linked to tetraspanin CD63. Stimulation of tdTomato⁺ cells with ATP induces rapid secretion of EVs and a reduction in cellular tdTomato intensity, reflecting EV secretion. We generate a GFP⁺ tdTomato⁺ cell library expressing TurboGFP and barcoded short hairpin RNAs for genome-wide screening using next-generation sequencing. We identify Mcfd2, Sepp1, and Sdc1 as critical regulators of ATP-induced EV secretion from murine2022-01-01T00:00:00Z2022 May2026-05-09T18:55:30.269Z2025-05-18T12:06:19.777ZS-EPMC91335893554505210.1016/j.celrep.2022.110791