Puromycin

CD63 is a membrane marker tetraspanin protein of exosomes, and we prepared HeLa cells stably expressing the GFP‐fused CD63 to secrete CD63‐GFP‐containing EVs (CD63‐GFP‐EVs) as previously reported [16]. 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, USA; 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⁻¹; LKT Laboratories, St. Paul, MN, USA) for the antibiotic selection of HeLa cells stably expressing CD63‐GFP (CD63‐GFP‐HeLa).

CD63-GFP-HeLa cells (2 × 10⁶ cells) were seeded on 100-mm dishes in α-MEM (10 ml) containing 10% FBS and puromycin (3 μg/ml) and incubated for 1 day at 37 °C under 5% CO₂. The cells were washed with serum-free α-MEM (five times, 5 ml) and incubated in α-MEM (10 ml) containing 10% exosome-free FBS (EXO-FBS, ATLAS biological, Fort Collins, CO, USA) and puromycin (3 μg/ml; LKT Laboratories) for 3 days. The cell culture medium was collected, and secreted exosomes were isolated using Total Exosome Isolation (from cell culture media) (Invitrogen, Austin, TX, USA). The concentrations of the isolated exosomes are described in terms of their protein concentrations, which were determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA).
Exosome isolation was conducted using ultracentrifugation63 . The collected cell culture medium was centrifuged (300 × g) for 10 min at 4 °C. The supernatant was centrifuged (2,000 × g) for 10 min at 4 °C and again (10,000 × g) for 30 min at 4 °C to remove cell debris. The supernatant was then centrifuged (100,000 × g) for 70 min at 4 °C using an ultracentrifuge (Himac CP65β, Hitachi, Tokyo, Japan) in duplicate, and the pellet was collected in Dulbecco’s phosphate buffered saline (PBS; Nacalai Tesque, Kyoto, Japan).

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).