Rpmi 1640

Primary epithelial cells were isolated as described previously [82 (link)] with slight modifications. Briefly, small intestines were harvested from 4 mice, opened longitudinally, and washed extensively with RPMI1640 medium (Nacalai, Kyoto, Japan) after mesentery, fats, Peyer’s patches, and luminal content were removed. The intestines were cut into pieces and shaken gently in RPMI-1640 containing EDTA (2 mM) and 10% fetal bovine serum (FBS) (Equitech-Bio, Kerrville, TX, USA). The tissue preparations were filtered with 70-μm mesh filters. Using 25%, 40%, and 75% Percoll (GE Healthcare Life Sciences, Chicago, IL, USA), the whole cells were spun in a centrifuge (AX-511) (Tomy, Tokyo, Japan) at 780× g for 20 min and IECs were obtained from the interface between the 25% and 40% layers. After verification of their expression of epithelial marker, the IECs were seeded in 12-well culture plates (Corning, Glendale, AZ, USA) at 4 × 10⁵ cells/mL in RPMI1640 containing penicillin/streptomycin and EV-depleted FBS and incubated at 37 °C with 5% CO₂ for 12 h. Then, the cells were treated with the indicated concentrations of EVs for 24 h after which RNA was extracted for further analysis.

The LNCaP, PC-3, DU145 and 22Rv1 human prostate cancer cell lines were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA). These cells were cultured at 37°C in a humidified incubator containing 5% CO₂ and 95% air. LNCaP, DU145 and 22Rv1 cells were cultured in RPMI-1640 (Sigma-Aldrich Corp. St. Louis, MO, USA) supplemented with 15% fetal bovine serum (Sigma-Aldrich Corp.), 50 μg/ml streptomycin and 50 IU/ml penicillin (Gibco, Grand Island, NY, USA). PC-3 cells were cultured in RPMI-1640 supplemented with 10% newborn calf serum (Equitech-Bio Inc., Kerrville, TX, USA), 50 μg/ml streptomycin and 50 IU/ml penicillin.
For androgen [dihydrotestosterone (DHT)] ablation, an androgen-independent prostate cancer cell line model LNCaP/AI was cultured in phenol red free RPMI-1640 (Sigma-Aldrich Corp.) supplemented with 15% charcoal/dextran-treated fetal bovine serum (HyClone, Logan, UT, USA), 50 μg/ml streptomycin and 50 IU/ml penicillin for 3 months.