Lb9507 luminometer

Luciferase reporter plasmids fused with Hspa5 3′UTR/Hspa5 3′UTR-Mutant (0.16 μg) and rno-miR-199a-5p mimic (5 pmol) were co-transfected into 293T cells. 48h later, cells were lysed. Luciferase activities were measured using a dual luciferase-reporter assay kit (Promega, USA) on a Lumat LB9507 luminometer (Berthold, Germany). Results were evaluated through normalization of the firefly luciferase activity with renilla luciferase activity as previously described [38 (link)].

SK-Mel28 cells stably expressing DsRed-LSF fusion protein and a control clone were grown to 70% confluence in 12-well plates and then transiently transfected with 900 ng of firefly luciferase reporter plasmid (human p21^CIP1 promoter cloned into pGL4.12) and 100 ng of Renilla luciferase control plasmid (EF-1α promoter cloned into the pRL-null vector) using Lipofectamine LTX (Invitrogen). For the siRNA experiment, 50 pmol of LSF siRNA (Invitrogen) was also co-transfected using Lipofectamine^®2000 (Invitrogen). The cells were lysed 48 hrs after transfection and luciferase activity was measured by using an LB9507 luminometer (Berthold) and the Dual-luciferase Assay Kit (Promega). All experiments were performed in triplicate.

A substrate for GLuc, coelenterazine h, was purchased from Wako Pure Chemicals. HEK293T cells (5 × 10⁵ cells per well) were cultured in 6-well dishes for 24 h, and transiently transfected with plasmids encoding CCR7-NGLuc and CCR7-CGLuc using PEI-MAX. After 24 h, the cells were cultured in fresh media for a further 24 h and subjected to the luminescence measurement. Five minutes after adding coelenterazine h (20 μM) to phenol red-free culture medium, luminescence signals were integrated over 2 sec using a LB9507 luminometer (Berthold Technologies, Bad Wildbad, Germany) or over 10 sec using an IVIS system (Perkin Elmer, MA, USA).