Bright glo lysis buffer

Reporter mice were i.v. injected with 150 mg/kg of D-luciferin diluted in PBS (Intrace Medical SA), anesthetized with 2.5% Isofluran (Abbot), and monitored using an IVIS Spectrum CT (PerkinElmer). Photon flux was quantified using the Living Image 4.5.4 software (Caliper). For ex vivo luciferase activity measurements tissues were homogenized in BrightGlo lysis buffer (Promega) using a FastPrep24 homogenizer (MP Biomedicals). Lysates were mixed with a BrightGlo luciferase assay substrate (Promega), and luminescence was quantified using a Synergy 2 microplate reader (BioTek).

Vero cells, in 6-well plates at 90% confluency, were co-transfected with 2 μg of different pCI-F constructs, 1 μg of the various pCI-H plasmids with 9 μl of TransIT-LT1 (Mirus). Vero cells in 24 wells were transfected with 1μg of the various pCI-H plasmids with 3 μl of TransIT-LT1 (Mirus). All transfections were performed according to the manufacturer’s protocol. In some experiments, phase contrast pictures were taken 24 h post-transfection with a confocal microscope (Olympus, Fluoroview, FV1000).
The quantitative fusion assay was performed as described previously [73 (link), 74 (link)] with subtle modification. Briefly, Vero cells were co-transfected with the F and H expression plasmids and 0.1 μg of pTM-Luc (kindly provided by Laurent Roux, University of Geneva). In parallel, separate 6-well plates of Vero-cSLAM cells at 30% confluency were infected with MVA-T7 at a multiplicity of infection (MOI) of 1. After overnight incubation, both cell populations were mixed and incubated at 37°C. 2.5 hours later, the cells were lysed using Bright Glo Lysis Buffer (Promega), and the luciferase activity was determined using a luminescence counter (PerkinElmer Life Sciences) and the Britelite reporter gene assay system (PerkinElmer Life Sciences).

HeLa cells were seeded onto 10 cm² dishes at 50,000 cells per well in CSM and left to adhere overnight. Cells were transiently transfected with luciferase tagged-mouse mammary tumour virus (MMTV-Luc; 2 µg) or luciferase tagged-nuclear factor-κB response element (NRE-Luc; 2 µg) using Fugene 6 reagent (3:1 volume-to-weight ratio with DNA) for 24 h. Cells were re-seeded onto 24-well plates at 50,000 cells per well in CSM and left to adhere overnight at 37°C/5% CO₂. Cells were treated as specified in the results, and 18 h later each well was washed twice with 1× PBS. 100 µl of Bright Glo lysis buffer (Promega, E2620) was added to each well and left to lyse on ice for 30 min. Cell lysates were transferred to a white, flat-bottomed 96-well plate and luciferase absorbance was read using a luminometer (Glomax, Promega). Ten 1-s reads were taken from each well and the average relative light units (RLUs) determined. Background wells were included that only contained lysis buffer. IC₅₀ and EC₅₀ values were extrapolated from the resulting dose–response curves using non-linear regression analysis in GraphPad Prism software, with the following equation: Y=Bottom+{Top-Bottom)/[1+10^(LogIC₅₀-X)×HillSlope]}. Where X, log of dose or concentration; Y, response; Top and Bottom, plateaus; LogIC₅₀ interchangeable with LogEC₅₀; HillSlope, slope factor or Hill slope, unitless.

Lentiviruses pseudotyped with the SARS-CoV-2 spike were generated in HEK293T cells as described previously [28 (link),29 (link)]. Briefly, HEK293T cells were co-transfected with a SIV-based self-inactivating vector encoding luciferase (pGAE-LucW), the SIV-based packaging plasmid (pAdSIV3), and a WT spike-encoding plasmid. Pseudoviruses were harvested 72 h after transfection and used for the infection of 293T-DSP-mix cells transfected with an ACE2 expression construct or empty vector (mock control). At 48 hpi, cells were lysed through the addition of 100 µL Bright Glo lysis buffer (Promega) for 15 min at 37 °C. Three minutes later, after the addition of 25 µL Bright Glo substrate (Promega), the luciferase signals were measured using a microplate luminometer (VICTOR X5, PerkinElmer) and the PerkingElmer 2030 Manager software.