Gentamicin tyrosine solution

Human kidney cancer cell lines 786‐O and Caki‐2 and the non‐malignant human kidney cell line HEK293T were purchased from ATCC (Manassas, VA, USA) and maintained in basal media (RPMI for 786‐O and Caki‐2, DMEM for HEK293T) supplemented with 10% FBS and 1% gentamicin–tyrosine solution (Invitrogen, Eugene, OR, USA) in a humidified incubator set to 37°C and 5% CO₂. For coculture experiments, the three cell lines were split into 35‐mm dishes (Becton Dickinson) and incubated for 2 days to reach 80–90% confluence. Transwell inserts (0.4‐μm pore size; Corning, Corning, NY, USA) were then placed into the wells and 5 × 10⁶ PBMCs from healthy volunteers were added to each Transwell insert. After 18 h, PBMCs were collected and lysed with Buffer RLT Plus with beta‐ME (RNeasy Plus Mini Kit; Qiagen, Venlo, Netherlands) and subjected to RNA extraction and qRT‐PCR.

We cultured PBMCs (1.0 × 10⁶) derived from healthy controls for 48 hours in Biotarget medium supplemented with 10% FBS and 4 mmol/L L‐glutamine (Biological Industries) in a humidified incubator set to 37°C and 5% CO₂. Human bladder cancer cell lines T24, J82, and 5637 were purchased from ATCC and maintained in basal media (Eagle's minimal essential medium [EMEM] for T24 and J82, Roswel Park Memorial Institute [RPMI] for 5637) supplemented with 10% FBS and 1% gentamicin‐tyrosine solution (Invitrogen) in a humidified incubator set to 37°C and 5% CO₂. PBMCs cultures were incubated with the three cytokines as follows: TNFα (1‐100 ng/mL), IL‐6 (10‐100 ng/mL), or IL‐1ra (10‐100 ng/mL) (FUJIFILM). We used R‐7050 (10 μmol/L) (Selleck Chemicals LLC), a TNFα receptor antagonist for the inhibition assay of TNFα. All mediums were centrifuged sequentially at 400 gravity for 5 minutes and 20 000 gravity for 10 minutes, and supernatants were stored at −80°C. cfDNA was isolated from 1.0 mL culture supernatants using the QIAamp^® Circulating Nucleic Acid Kit (QIAGEN) according to the manufacturer's protocol.