Lumat lb 9501 single tube luminometer

Chang Liver, HeLa, and normal human fibroblast (HFT) cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA). For reporter assays, cells were seeded at a density of 2 × 10⁵ cells/well in 6-well plates, incubated for 24 h, and then transfected with 1 µg DNA/well using Lipofectamine Plus^TM reagent (Invitrogen, Waltham, MA, USA), according to the supplier’s protocol. After transfection, the cells were incubated for an additional 48 h and harvested for luciferase assay. The cell pellet was disrupted in 100 µL reporter lysis buffer (Promega, Madosin, WI, USA), and luciferase activity was measured using the Promega Luciferase Assay System and Lumat LB 9501 Single Tube Luminometer (Berthold, Bad Wildbad, Germany). Renilla luciferase was used as an internal control to normalize transfection efficiency. Luciferase assays were performed in duplicate and each experiment was repeated at least twice.

Cells were washed with ice-cold PBS and lysed with 1X passive lysis buffer (#E4030, Promega) for 15 min with intermittent mixing. The cleared cell lysates were obtained by brief centrifugation and added with firefly luciferase assay substrate (#E1500, Promega) at the ratio of 1:4 (5 µl sample plus 20 µl substrate). The luciferase activity was measured using a Lumat LB 9501 Single Tube Luminometer (Berthold, Hartford, CT). The protein concentration of the cleared cell lysates was also determined using a Bio-Rad DC protein assay kit (Bio-Rad) and used to normalize the luciferase activity, which was expressed as relative luminescence/light unit (rlu).