Firefly luciferase substrate

FLP-IN T REX 293-derived GILT-expressing cell lines were transfected with plasmids encoding ACE2, APN or DPP4 to express viral receptor, and seeded into 96-well plates with black wall and clear bottom. THP-1-derived cell lines, 0.8 × 10⁵ cells per well were seeded into black wall 96-well plates and treated with PMA (10 ng/ml) for 24 h to induce differentiation. The differentiated cells were infected with desired pseudotyped lentiviral particles for 4 h, and then replenished with fresh media. Two days post infection, the media were removed, and cells were lysed with 30 µl/well of cell lysis buffer (Promega) for 15 min, followed by adding 50 µl/well of firefly luciferase substrate (Promega). The firefly luciferase activities were determined by luminometry in a TopCounter (Perkin Elmer).

The method for detecting viral infectivity has been described previously [45 (link)]. Briefly, virus samples were mixed with 1 × 10⁴ TZM-bl cells in 200 µL DMEM containing 10 µg/mL of DEAE-dextran in 96-well plates. After 48 h of incubation, 150 µL of supernatant was removed and 50 µL of firefly luciferase substrate (Promega, Madison, WI, USA) was added into each well to test the relative luciferase units (RLUs), which represent the infection of the virus.

To draw conclusions about G_q/11 signaling, phospholipase C (PLC) activation was assessed. Luciferase-based reporter gene assays were performed that use responsive elements in the promotor region of the gene encoding a firefly luciferase [37 (link)]. Equal amounts of MC4R-WT and MC4R stop mutations and nuclear factor of activated T-cells (NFAT) reporter plasmid were cotransfected. In the case of G418 treatment, 125 µg/mL G418 was added to cells at the time of transfection. After 48 h, cells were treated with alpha-MSH or SM, then incubated in supplement-free MEM at 37 °C with 5% CO₂. After 6 h, the reaction was terminated by discarding the medium. Cells were lysed at room temperature using 50 µL passive lysis buffer (PLB; Promega, Fitchburg, WI, USA), then frozen at −80 °C for 10 min. Afterwards, 10 µL lysate was transferred onto a white opaque 96-well plate. Automatic injection of 40 µL firefly luciferase substrate (Promega, Mannheim, Germany) and determination of luminescence were performed with the plate reader Mithras LB940.

Vero cells were seeded in 96-well microtiter plates (20,000 cells/well) and cultured for 24 h. All compounds of the TocriScreen Kinase Inhibitor Toolbox were diluted 1:1000 in GMEM medium containing 5% FBS to give a final concentration of 10 µM and incubated with the cells for 2 h at 37 °C and 5% CO₂. The cells were washed and inoculated for 90 min at 37 °C with VSV*∆G(FLuc) using an m.o.i. of 0.5 ffu/cell. The cells were washed again and incubated for 6 h with GMEM medium containing the respective inhibitor. Finally, 30 µL of cell lysis buffer (Promega, Madison, WI, USA) was added to each well and 6 µL of lysate transferred to a white 96-well microtiter plate. Firefly luciferase substrate (Promega) was automatically injected (30 µL/well) into each well and luminescence recorded for 1 s with the Centro LB 960 luminometer (Berthold Technologies, Bad Wildbad, Germany). All kinase inhibitors were subject to at least two rounds of analysis. Those compounds that led to suppression of luciferase activity by at least 70% were selected for a third round of analysis. In some experiments, viruses expressing the sNLuc reporter protein were used. Analysis of sNLuc activity in the cell culture supernatant was performed as described previously [29 (link)].

Hs578 cells were transfected in batches in 48-well plates using 2.5 ng ERα36 or ERα66 and 50 ng tk-ERE-luc vector per well, as well as ALDH1A1 wild-type/mutant promoter-Luc for 48 h. The cells were then incubated with indicated ER ligands for 24 h. The cells were then lysed, and firefly luciferase emission was detected upon addition of firefly luciferase substrate (Promega) on a PerkinElmer Victor 3-V plate reader. β-gal was analyzed using the Tropix β-gal actosidase detection kit (Tropix), and emission was detected on a PerkinElmer Victor 3-V plate reader. Luciferase counts were normalized to β-gal counts obtained in each well.

RAW264.7 cell lines stably transfected with a G418-selected construct that express firefly luciferase driven by promoters containing either NFkB- or an NFAT-response element-containing promoters (NFkB-RAW and NFAT-RAW cells, respectively) were employed in reporter assays as previously described [27 (link)]. Briefly, reporter cells were seeded (4×10⁴ cells/6mm diameter culture well) in MEM/FBS, incubated overnight, treatments added in triplicate wells over 24 h then lysed with Passive Lysis Buffer (Promega, Madison, WI) for 24 h at 4°C. Signal was measured using firefly luciferase substrate (Promega) as per manufacturer's instruction using a Clariostar multifunctional microplate reader (BMG Labtech).