Luciferase substrate

HeLa/STAT3 cells stably express firefly luciferase reporter gene under the control of the STAT3 response element (Signosis). Cells were grown to confluence on square petri dishes in DMEM (high glucose+sodium pyruvate+L-glutamine) (Gibco) supplemented with 1% v/v PS, 10% v/v/ h.i.FBS, 100 μg/ml Hygromycin B in a humidified incubator at 37°C with 5% CO₂ for 5–6 days. The day before the assay the cells were trypsinised and plated in 96 well plates at 5 x 10⁴ cells per well and incubated as above. Promastigote secretory gel, recombinant human Oncostatin M (Sigma) and anti-IGF1R antibodies (Peprotech) were added to the wells as required and incubated for a further 24 hours. Following this, wells were aspirated and gently washed in PBS once before adding 50 μl of the lysis buffer. Cells were allowed to lyse for 5 mins at room temperature with gentle rocking. Lysing cells were passed up and down a pipette tip before being freeze-thawed once at -80°C and room temperature. Twenty microliters of the lysate was transferred to a new 96 well plate containing 100 μl of a Luciferase substrate (Promega) and mixed gently 2–3 times by pipette. Relative light intensity was measured in a Spectramax M3 plate reader.

Forty-eight hours after luciferase construct transfection, the cells were lysed. Luciferase substrate (Promega, Fitchburg, WI, USA) was added to the lysates, and the luciferase activity of the lysates was measured using a luminometer (Promega).

To detect the tissue distribution of mRNA-LNP formulation upon intravenous administration in mice, an FLuc reporter mRNA-LNPs was used for BLI as described previously.^{16 (link)} Briefly, 6–8-week-old female BALB/c mice (n = 3) were inoculated with 10 μg of FLuc mRNA-LNP via the i.v. route. Six hours after injection, animals were given an intraperitoneal injection of luciferase substrate (Promega), and fluorescent signals were collected for 60 s with an IVIS Spectrum instrument (PerkinElmer). The heart, liver, spleen, lung, and kidney tissues were collected, and the fluorescence signal of each tissue was detected for 60 s. The fluorescence signal of the region of interest (ROI) was quantified using Living Image 3.0.

Pseudovirus inhibition assays were performed using TZM-bl cells and PBMC as previously described [162 (link), 163 (link)]. Briefly, serial dilutions of inhibitors were pre-incubated with target cells or viruses depending on target epitope, for 1hr at 37°C, then virus, cells, and inhibitor were added together and incubated for 48–72 hours. Infection supernatants were then aspirated, cells lysed, luciferase substrate (Promega, Madison Wisconsin, USA) added and the presence of luciferase quantified by measuring relative light units (RLU) using a Dynex MLX 96-well plate reader.

The MH7A cells were transfected with pGL3-hBAFF-Luc and pcDNA-lacZ for monitoring transfection efficiency by β-galactosidase assay. Luciferase activity was determined by incubating cell extracts with luciferase substrate (Promega). Luminescence was measured using luminometer (Berthold Technologies, Oak Ridge, TN, USA). Luciferase units of experimental vector were normalized to the control vector in each sample.^{31 (link), 32 (link), 48 (link)}