Spectramax m3 plate reader

Full-length SARS-CoV-2 WA1/2020, B.1.351 and B.1.1.7, viruses were designed to express nanoluciferase (nLuc) and were recovered via reverse genetics¹⁹ . One day before the assay, Vero E6 USAMRID cells were plated at 20,000 cells per well in clear-bottom black-walled plates. Cells were inspected to ensure confluency on the day of assay. Serum samples were tested at a starting dilution of 1:20 and were serially diluted threefold up to nine dilution spots. Serially diluted serum samples were mixed in equal volume with diluted virus. Antibody–virus and virus-only mixtures were then incubated at 37 °C with 5% CO₂ for 1 h. After incubation, serially diluted sera and virus-only controls were added in duplicate to the cells at 75 plaque-forming units at 37 °C with 5% CO₂. Twenty-four hours later, the cells were lysed, and luciferase activity was measured via Nano-Glo Luciferase Assay System (Promega) according to the manufacturer specifications. Luminescence was measured by a Spectramax M3 plate reader (Molecular Devices). Virus neutralization titres were defined as the sample dilution at which a 50% reduction in RLU was observed relative to the average of the virus control wells.

Logarithmically grown cells were inoculated into SC medium containing different concentrations of P_i as described above. At different time points (0, 2, 4, 6, and 8 h), 1 mL of cell culture was transferred into a microcentrifuge tube and sedimented by centrifugation at 13,000 × g for 1 min. The supernatant was transferred into a new tube and diluted with P_i-free SC medium to be within a linear range of detection (200-fold dilution for 10 mM samples, 10-fold dilution for 0.5 mM samples, and no dilution for 0 mM samples). Fifty microliters of diluted samples was mixed with 32 μL of 0.1 mM malachite green solution containing 0.35% (mass/vol) of polyvinyl alcohol (molecular mass 85,000 to 124,000 Da) and 43 μL of 4.48 mM ammonium molybdate solution containing 12.5% (vol/vol) H₂SO₄. After incubation for 15 min at room temperature, the absorbance was measured at 620 nm on a SpectraMax M3 plate reader (Molecular Devices, USA) in a 96-well clear plate with a flat bottom.

Glucose, lactate, glutamine, and glutamate concentration were measured using Glucose-Glo™ Assay, Lactate-Glo™ Assay and Glutamine/Glutamate-Glo™ Assay (Promega), respectively. Culture medium dilution of 1/500, 1/100 and 1/50 in PBS were used to measure glucose, lactate, and glutamine/glutamate concentration, respectively. Cell ATP concentration was measured using CellTiter-Glo® Luminescent Cell Viability Assay (Promega), following the manufacturer’s instructions. Cell DNA content was measured using CyQUANT™ Cell Proliferation Assay (Invitrogen) by resuspending cell pellets in 250 μL CyQUANTTM GR dye/cell-lysis buffer. Finally, cell protein content was measured by resuspending cell pellets in PathScan® Sandwich ELISA Lysis Buffer (Cell Signalling Technology) then by using Pierce™ 660 nm Protein Assay Reagent (Thermo Scientific™) following the manufacturer’s instructions. For all these assay, absorbance, fluorescence, and luminescence were read with SpectraMax® M3 plate reader (Molecular Devices).

HeLa (human cervix adenocarcinoma cells) (Cellonex) were cultured in Dulbecco’s Modified Eagle Medium (DMEM)-(Lonza) supplemented with 10% w/w fetal calf serum and antibiotics (penicillin/streptomycin/amphotericin B) at 37 °C in a 5% CO₂ incubator. HeLa cells were transferred to 96-well plates at a cell density of 1 × 10⁴ cells per well in 150 µL culture medium and grown overnight. A single concentration of 50 µM of the test compound was incubated with the cells for an additional 48 h, and cell viability in the wells assessed by adding 20 µL 0.54 mM resazurin in PBS for an additional 2–4 h. The numbers of cells surviving drug treatment were determined by reading resorufin fluorescence (excitation 560 nm, emission 590 nm) with a SpectraMax^® M3 plate reader (Molecular Devices, San Jose, CA, USA). Fluorescence readings for the individual wells were converted to percent (%) cell viability relative to the average readings from untreated control wells. Plots of % cell viability vs. log(compound) were used to determine IC₅₀ values by non-linear regression using version 5.02 GraphPad Prism (GraphPad Holdings LLC, La Jolla, CA, USA).
The compounds investigated were the optimized NCC, the individual API and a physical mixture of the API in stoichiometric ratios identical to those used to produce the NCC.