Microplate luminometer

LNCaP and PC3 cell were plated on 12-well plates and transfected using Lipofectamine 2000 (3 mL per well; Invitrogen). The total amount of plasmid DNA was normalized to 1.5 μg per well. After 48 hr, cell were lysed in lysis buffer provided in the Luciferase Assay System (Beyotime Biotechnology). Luciferase activities were measured using the Dual-luciferase Reporter Assay System (Beyotime Biotechnology) with the aid of a microplate luminometer (Tecan, Switzerland), and Renilla luciferase activities of cell were used as internal control. All experiments were carried out in triplicate wells and repeated 3 times.

Blood was collected from the saphenous vein (50 μL) into heparinized collection vials (Sarstedt; Nümbrecht, Germany) at regular intervals (twice per week, up to 28 days) from SCID mice bearing NDF cells transduced with a GLuc reporter construct. Plasma was obtained following centrifugation at 10,000 x g for 7 minutes at 4°C, and stored at −80°C with no substantial loss of signal. For measurement of GLuc activity, plasma was diluted 1:10 in PBS containing 0.1% Triton X-100 in a solid white 96-well microplate. Next, two volumes of a 100 μM solution of coelenterazine in 1X PBS supplemented with 0.3M ascorbic acid and 0.1% Triton X100, pH 7.5, was added to the diluted plasma sample and luminescent signal was measured immediately on a microplate lumino-meter (Tecan) using a 500 ms integration time. Coelenterazine was obtained as a dry powder from NanoLight Technologies (Pinetop, AZ); stock solutions at 5 mg/ml in acidified ethanol were stored at −80°C.