Prl cmv renilla luciferase control vector

Cells were transfected using Neon™ Transfection System (Thermo Fisher Scientific, Waltham, MA, USA) with the following parameters (Table 4).
For two million cells in 100 μL of buffer R we added 0.5 μg of pRL-CMV Renilla luciferase control vector (Promega, Fitchburg, WI, USA), 1.2 pmol of the pGL3-based test vector, and salmon sperm DNA (Sigma 31149) to the total DNA amount of 26.5 μg. Luciferase activity was measured 24 h after electroporation using Dual-Luciferase Reporter Assay System (Promega, Fitchburg, WI, USA), Hidex Bioscan Plate Chameleon Luminometer and MicroWin 2000 software. Minimum of two independent experiments were performed, each including three to four technical replicates.

HepG2 cells (DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were cultured in RPMI 1640 GlutaMAX™-I supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Hep-3B cells (DSMZ, Braunschweig, Germany) and Huh7 cells (JCRB Cell Bank, Tokyo, Japan) were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin.
Cells were detached with TrypLE Express W/Phenol red (Life Technologies, Darmstadt, Germany). Media and additives were obtained from Gibco (Life Technologies). Cells were cultured under standard conditions at 37 °C in a humidified atmosphere supplemented with 5% CO₂.
For transfection experiments, 1.5 × 10⁵ Huh7 and Hep-3B cells, respectively, and 2 × 10⁵ HepG2 cells were plated per well of a 12-well plate (Nunc, Langenselbold, Germany) and grown for 24 h to reach approximately 80% confluence. Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany) was used to transfect the cells. Per well, 4 µL Lipofectamine, 1.6 µg plasmid DNA and 3 ng pRL-CMV Renilla luciferase control vector (Promega, Mannheim, Germany) were applied. The cells were harvested and luciferase activity was measured as described previously [24 (link)].