Ht901 8foz

Live virus neutralization assays were performed by PRNT at Bioqual (Rockville, MD), on sera samples collected at pre-challenge (Weeks 0, 6, 15) and post-challenge. Vero 76 cells (ATCC No. CRL-1586) were plated at 175,000 cells/well in DMEM + 10% FBS + gentamicin, overnight at 37°C, 5% CO₂. Serially diluted serum samples were mixed with SARS-CoV-2 virus (isolate USA-WA1/2020) for a final 30 pfu/well virus per well and final starting 1:20 serum dilution, then incubated at 37°C, 5% CO₂ for 1 h. Media was removed from cell cultures and virus-serum dilution mixtures were added to cells in duplicate and then incubated at 37°C, 5% CO₂ for 1 h for virus infection. After incubation, 0.5% methylcellulose media (Sigma #M0512-100G) was added to each well and plates were incubated at 37°C, 5% CO₂ for 3 days. Each well was then washed and fixed with methanol, then stained with 0.2% crystal violet (Sigma #HT901-8FOZ) in 20% MeOH for 30 min at room temperature. Plates were washed, dried and plaques were recorded to calculate the number of infectious units.

A549 holoclone and paraclone cells, as also parental A549 cells transfected with either control siRNA (siCTRL) or siCDA, were seeded into 6-well plates at a density of 10′000 cells/well. Different concentrations of 5′-DFCR or 100 μM tetrahydrouridine (THU) were added into plates after cells attached for 6-day treatment. Subsequently, the media were removed, and cell colonies were gently washed with PBS and stained by 1% crystal violet solution (in 50% Ethanol), which was prepared from 2.3% crystal violet solution (HT901-8FOZ, Sigma). After 30 min-staining, the dye solution was pipetted back for recycling and excess dye was gently rinsed off with tap water. After the plates were air-dried, pictures were captured with a standard photo camera. Crystal violet-stained cells were dissolved in 10% acetic acid solution (2 mL/ well) for 30 min on a shaker and the concentration of the extracted dye (100 μL/ well) was quantified with a spectrophotometer at 590 nm [39 (link)].