One glo luciferase assay substrate

The in vitro anti-inflammatory activity of the aqueous extract was evaluated using a cell model that has been previously described [55 (link)]. Briefly, R3/1-Nf-κB cells were seeded (5000 cells/well) in a white 96-well plate (BRANDplates^®, cell grade). Cells were pretreated with different concentrations of the extracts (1, 10, 50, and 100 µg/mL) for 18 h in complete medium (DMEM 10% FBS, 1% L-glutamine, 1% Penicillin/Streptomycin). This process was followed by a 6 h stimulation with 10 ng/mL TNFα. To avoid components’ interference with the reading of the luciferase assay, cells were washed once with 100 µL of warm PBS before 50 µL of DMEM was added. Subsequently, 50 µL ONE-Glo™ Luciferase Assay Substrate (purchased from Promega Corporation, Madison, WI, USA) was directly added to the wells, followed by a luciferase measurement performed using a luminometer (Wallac Victor2 1420, Perkin-Elmer™ Life Science, Monza, Italy). Experiments were performed with biological and technical replicates and the results are shown as mean ± SD compared to untreated control cells. Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple comparisons test (p < 0.05 was considered significant). The cell viability for all the concentrations tested in the anti-inflammatory assay was verified by MTT assay on R3/1-Nf-κB cells.

After treatment, R3/1-NF-κBcells were washed twice with cold PBS followed by a freeze-thaw cycle with reporter lysis buffer (purchased from Promega Corporation, Madison, WI, USA) for complete cell lysis. After the freeze-thaw cycle, 100 µL ONE-Glo™ Luciferase Assay Substrate (purchased from Promega Corporation, Madison, WI, USA) was directly added to the wells, followed by a luciferase measurement performed using a luminometer (Wallac Victor2 1420, Perkin-Elmer™ Life Science, Monza, Italy).

Luciferase emission spectrum was determined as follows: the medium in wells containing transfected cells was replaced with phosphate-buffered saline, and d-luciferin (Synchem, Germany) was added at a final concentration of 0.5 mM. Luciferase activity was measured using an IVIS Spectrum (Caliper, Alameda, CA, USA). The instrument stage was kept at 37 °C, and the imaging setup was field of view (FOV) C. Light output was measured using an open filter and a series of band pass filter (20 nm) ranging from 500 to 700 nm each for 5 s, 5 min after substrate addition to live cells. For in vitro fluorescence measurements, the instrument was set with an excitation filter of 570 nm and an emission filter of 640 nm. All the data are expressed in photon flux and analyzed with Living Image Software 4.0 (Caliper, Alameda, CA, USA). For the analysis of bioluminescence from cells transfected with pMNTurboLuc or MINI-pMNTurboLuc, cells were analyzed at different days after transfection using One-Glo luciferase assay substrate (Promega) and a plate luminometer (SpectraMax L luminometer, Molecular Devices, Sunnyvale, CA, USA).