R 7050

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.