Dulbecco s modified eagle s medium low glucose 1 g l dmem

After treatment, the samples were mixed into 320 µl of Gibco Difco™ Beef Extract (1.5% w/v beef extract in ddH₂O; Life Technologies) and rolled for 15 min to chelate any free ions in the solution. The samples then were serially diluted (10⁰–10^–7) in infectivity media (Dulbecco’s modified Eagle’s medium low glucose 1 g/L (DMEM, Life Technologies) supplemented with 2.5% fetal bovine serum (Merck), 3% tryptose phosphate broth (Merck), 1 × non-essential amino acids (Gibco), 1 × antibiotic–antimycotic (Thermo Fisher Scientific) and 1 × L-Glutamine (Gibco]) and added in triplicate to 17Cl-1 cell plates. For each sample or control there were 4 replicate treatments (in light or dark); each treatment replicate was plated in triplicate. Infected 17Cl-1 plates were incubated at 37 °C in a 5% CO₂ atmosphere for 24 h. Plates were scored for cytopathic effect (CPE) by microscopy and viral titres were determined by the Reed and Muench 50% tissue culture infectious dose (TCID₅₀) end point method^{55 (link)}. Outliers were removed from the four replicates using an outlier test. The full protocol was repeated in triplicate on different days to account for any variability in the assay.

17Cl-1 murine fibroblasts cells and murine hepatitis virus A59 strain (MHV-A59) were kindly gifted by Professor Ian Goodfellow’s lab (Department of Pathology, University of Cambridge, UK). 17Cl-1 cells were resurrected from cold-storage cell banks and propagated using routine cell culture protocols in complete growth medium (Dulbecco’s modified Eagle’s medium low glucose 1 g/L (DMEM, Life Technologies) supplemented with 5% foetal bovine serum (Merck), 6% tryptose phosphate broth (Merck), 1 × non-essential amino acids (Gibco), 1 × antibiotic–antimycotic (Thermo Fisher Scientific) and 1 × L-Glutamine (Gibco). 17Cl-1 cells were seeded (100 μl per well) into 96-well plates (untreated, flat bottom) at 1 × 10⁴ cells/well at 37 °C for 24 h in 5% CO₂ prior to the day of testing.