Ev depleted fbs

LNCaP and PC-3 (ATCC, Manassas, VA, USA) were grown in RPMI 1640 and DMEM/F12 media (Sigma-Aldrich, St Louis, MO, USA), respectively. Both media were supplemented with 10% (v/v) EV-depleted FBS (Sigma-Aldrich). The FBS was EV-depleted by ultracentrifugation at 118,000 x g_avg for 18 h using a type 45 Ti rotor k-factor 178.6 (Beckmann Coulter, Brea, CA, USA), followed by filtration through a 0.22 μm filter (Merck Millipore, Billerica, Massachusetts, USA). Immortalized human benign prostate epithelial cells PNT2 (ECACC, Sigma-Aldrich) were grown in serum-free defined keratinocyte media, supplemented with bovine pituitary extract and human recombinant epidermal growth factor (Life Technologies, Carlsbad, CA, USA). All media were supplemented with 100 IU/mL of penicillin and 100 μg/mL streptomycin (HyClone, Logan, UT, USA). Cells were cultivated at 37 °C and 5% CO₂. Cell viability was measured by Trypan Blue solution (Sigma-Aldrich). Cells were routinely checked for mycoplasma contamination using the MycoAlert™ PLUS (Lonza Walkersville, MD, USA).

The human mast cell line, HMC1 cells and erythroleukemic cell line, TF1 cells (ATCC, Manassas, VA) were cultured in IMDM (HyClone, Logan, UT). Human embryonic kidney HEK293T (ATCC, Manassas, VA) and human melanoma cell, MML1 cells were cultured in RPMI1640 (Sigma Aldrich, St Louis, MO) media. Media was supplemented with 10% EV-depleted FBS (Sigma Aldrich), 2 mM L-glutamine (HyClone), and 1.2 U/ml 1-thioglycerol (Sigma Aldrich). The human MSCs from bone marrow were obtained as passage 1 from the MSC distribution of the Institute of Regenerative Medicine at Scott and White, USA, and cultured in alpha minimum essential medium (GIBCO® GlutaMAX™, Invitrogen, Carlsbad, CA) supplemented with 15% EV-depleted FBS (Sigma Aldrich). Three to four passages of MSCs were used for EV isolation. All media contained 100 U/ml penicillin and 100 µg/ml streptomycin (HyClone). For the EV depletion, FBS was ultracentrifuged at 118,000 × g_avg (Type 45 Ti rotor, Beckman Coulter, Miami, FL) for 18 h and filtered through a 0.22 µm filter as previously described [16].