Cobas e601 system

Lentivirally transduced LNCaP cells were seeded at a density of 10⁵ cells/mL in 6‐well plates and were cultured in RPMI 1640 with 10% FCS for 48 hours. The culture medium was removed and replaced with RPMI 1640 medium with 10% CS‐FCS (Gibco Life Technologies) and cells were incubated with or without 50 nM T3 for another 3 days. The growth medium was collected at 24, 48 and 72 hours. For T3 analysis, PCa cells were transfected by lipofection with empty vector or CRYM expressing vector. After 48 hours, medium was replaced with RPMI medium with or without T3. The culture medium was collected at 24 and 48 hours. The level of secreted PSA and T3 in the growth medium was determined using a validated Elecsys electrochemiluminescence immune assay (Roche, Rotkreuz, Switzerland) on a Cobas e601system (Roche) that is routinely used for clinical applications.

The plasma concentration of CEA was measured by using the Roche High-sensitivity Assay kit performed on a Cobas e601 System (Roche). The cut-off point of CEA is 5 ng/ml. Samples were randomized for testing and blinded to the trained clinical laboratory technician who analyzed and interpreted the data.

Immediately after admission, urinary and blood samples were collected in nonheparinized tubes and then centrifuged at 1000× g at 4 °C for 15 min, before storage at −80 °C until assayed. We measured the urinary L-FABP levels by an enzyme-linked immunosorbent assay (ELISA) using the Human L-FABP ELISA Kit (CMIC, Tokyo, Japan). Plasma B-type natriuretic peptide (BNP) levels were measured using a chemiluminesence enzyme immunoassay for human BNP (Shionogi & Co., Ltd., Osaka, Japan). We measured serum high-sensitivity troponin T (hs-TnT) levels via an electrochemiluminescence immunoassay, using a Cobas® e601 system (Roche Diagnostics, Tokyo, Japan), and serum high-sensitivity C-reactive protein (hs-CRP) levels via a latex-enhanced hs-CRP immunoassay (N-Latex CRP II, Siemens Healthineers, Tokyo, Japan). Serum creatinine levels were determined by an enzyme method, using the Liquitech® Creatinine PAP II (Roche Diagnostics, Tokyo, Japan) upon admission, daily until day 3, and then on day 7.