Um uc 3

The UM-UC-3 cell line was obtained from the European Collection of Cell Cultures (ECACC). UM-UC-3 cells cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (FUJIFILM Wako Pure Chemical Corporation, Japan) supplemented with 10% FBS and 1% P/S at 37 °C in a humidified 5% CO₂ atmosphere for 3 days were used for the assay after confirming that they had reached 70 to 90% confluence. The cells were dissociated from the flask and suspended, and the cell suspension was centrifuged (1500 rpm × 5 min) to remove the old medium. The cells were seeded into 96-well plates (200 µL, 5000 cells/well). Cells were incubated for another 24 h after adding 2 µL of DMSO or DMSO extract per well. The experiment was performed according to the protocol of the assay kit. A total of 10 µL of CCK-8 reagents (DOJINDO, Japan) were added to each well after removing 100 µL of supernatant. The treated cells were incubated at 37 °C in a humidified 5% CO₂ atmosphere for 3.5 h. The absorbance at 450 nm was measured with a microplate reader. The cell viability was expressed as the ratio of absorbance between sample groups and the DMSO-treated control group.

Human bladder cancer cell lines UMUC3, J82, RT4 and immortalized urothelial cell line SV-HUC-1 were purchased from the American Type Culture Collection (Manassas, VA, USA), and bladder cancer cell line 5637 was kindly provided from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University. UMUC3 was cultured in Dulbecco's modified Eagle's medium (DMEM; Wako, Osaka, Japan), and 5637 and RT4 were cultured in RPMI 1640 medium (Wako). J82 was cultured in Eagle's minimum essential medium (EMEM; Wako), and SV-HUC-1 was cultured in Ham's F-12 (Wako), with 10% exosome-depleted fetal bovine serum (Thermo Fisher Scientific) and 5 μg/ml gentamaicin (MSD, NJ, USA) and 60 μg/ml tylosin (Sigma-Aldrich, St. Louis, USA), and incubated at 37°C in a humidified atmosphere containing 5% CO₂. Cells were grown to confluence for 72 h. Conditioned media were pooled and centrifuged to obtain EVs according to the same protocol as the urine samples.