Td 20 20 luminometer

The Dual-Luciferase Reporter Assay System (Promega) was used to carry out transactivation experiments using BAX and MDM2 promoters cloned upstream of the Firefly luciferase reporter gene. SaOs2 cells were grown for 24 h and then transiently co-transfected using Lipofectamine 2000 (Life Technologies) with reporter vectors (500 ng of BAX promoter-LUC or MDM2 promoter-LUC and 100 ng of Renilla luciferase reporter) and expression vectors for p53, ZASP6 and Ankrd2. The cells were lysed in Passive Lysis Buffer (Promega) 16 h after transfection and luciferase activity was measured using the Dual-Luciferase Assay System (Promega) and TD-20/20 Luminometer (Promega). Firefly luciferase activity was normalized to that of Renilla luciferase. Plotted results are representative of three independent experiments performed in triplicate. Data were presented as means ± standard error of the mean. The Students t-test was done to determine the statistical significance (the accepted level was p<0.05).

An ATP assay was performed using an ATP Bioluminescence Assay Kit CLS II (Roche). The intensity of luminescence was measured with a TD-20/20 luminometer (Promega). The ATP content was normalized to the cell number. The amount of oligomycin A (Sigma-Aldrich)–insensitive intracellular ATP was used to calculate glycolysis-dependent ATP production. OXPHOS-dependent ATP production was calculated from the amounts of total intracellular ATP and glycolysis-produced ATP (Ojaimi et al, 2002 (link); Yang et al, 2014 (link)).

This assay was conducted according to the Promega Luciferase Assay Systems. Cells seeded into 96-well plates were used at around 90% confluence. PGL3-PIK3CD (the p110δ gene) reporter vectors p-GL3-R1, p-GL3-GN43 (or p-GL3 control) and pRL-RK were transfected overnight into ARPE-19 cells with lipofectamine 3000 (Life Technologies), followed by treatment with TGF-β2 for 24 h. After washing with PBS twice, cells were lysed with 1× passive lysis buffer, gently rocked for 15 min at room temperature, followed by addition of 50 μL LAR (luciferase reporter assay) to a 12 mm × 50 mm tube (Part #E2371, Promega. Disposable Cubettes), and the lysates were transferred from a well into the LAR substrate. These mixtures were first read for firefly luciferase activity in a TD-20/20 luminometer (Turner Designer, Promega). Finally, 20 μL 1× Stop and Glo substrate was added into the tubes, mixed and read again to obtain Renilla luciferase activity [28 (link)]. At least 3 independent experiments were performed.

NuPAGE 4–12% Bis-Tris protein gel (Thermo Fisher Scientific, Waltham, MA, USA) was used for SDS-PAGE and Western blotting analyses. The chemiluminescence signals were detected using the LAS-3000 device (FUJIFILM, Tokyo, Japan). For detection of FLAG-tagged Met-IR and untagged full-length replication proteins of TMV, anti-DYKDDDDK antibody (clone 1E6; FUJIFILM Wako Pure Chemical, Osaka, Japan) and antisera against tomato mosaic virus replication proteins [8 (link)] were used, respectively. Luciferase activity was measured using the Renilla Luciferase Assay System (Promega) and the TD-20/20 luminometer (Promega). Radiolabeled RNA products were separated by 8 M urea–2.4% PAGE and detected using the Typhoon FLA 7000 scanner (GE Healthcare, Chicago, IL, USA). Band intensity of viral genomic RNA was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

The luciferase expression was checked by measuring the luciferase activity in Luc-DC by using the luciferase assay system kit (Promega) and the TD-20/20 Luminometer (Promega). The 2 × 10⁶ Luc-DC or parental MuTuDC lines were used for the experiment. The light emission was measured as relative light units (RLU). The luciferase activity in Luc-DC was also measured by using the Xenogen imaging system (Xenogen/Caliper life science, Platform of the Cellular Imaging Facility (CIF), University of Lausanne). From 1575 to 100,000 Luc-DC or parental MuTuDC lines were plated into a 96-well plate and the bioluminescence was monitored after addition of 0.15 mg/ml of d-luciferin and quantified as photons/s/cm²/sr.