Generating Stable CD63-GFP-Expressing HeLa Cells

CD63 is a membrane marker tetraspanin protein of EVs, and we prepared HeLa cells stably expressing GFP-fused CD63 to secrete CD63-GFP-containing EVs (CD63-GFP-EVs) as previously reported9 (link)10 (link). HeLa cells (1 × 10⁵ cells) were plated on a 24-well microplate (Iwaki, Tokyo, Japan) and incubated for 1 day. They were transfected with CD63-GFP plasmid (pCT-CD63-GFP, pCMV, Cyto-Tracer, System Biosciences, Mountain View, CA) (800 ng) complexed with Lipofectamine LTX reagent (2 μl) and PLUS reagent (1 μl) (Invitrogen, Life Technologies Corporation) in α-MEM containing 10% FBS (200 μl). The cells were also treated with puromycin (3 μg/ml) (LKT Laboratories, St. Paul, MN) for the antibiotic selection of HeLa cells stably expressing CD63-GFP (CD63-GFP-HeLa).