Cellbind costar

Viral kinetics in HRECs were performed by seeding 1 × 10⁵ cells per well in collagen-coated 24-well plates (Greiner Bio-one) using growth medium #2; for staining 96-well clear flat bottom black polystyrene surface-treated microplate (CellBIND Costar; Corning) was used, as described previously [15 (link)]. After 24 h of incubation, cells were washed twice with 500 μL of LHC base medium (Gibco, Thermo Fisher Scientific), and inoculated at MOI-2 and MOI-1 in triplicate with HCoV-NL63 or SARS-CoV-2 or mock inoculated with infection medium #2 (airway epithelial cell basal medium [PCS-300-030; ATCC] supplemented with 2% Ultroser G [Sartorius, Göttingen Germany], 1% MEM nonessential amino acids solution [Gibco, Thermo Fisher Scientific], 1% HEPES [Gibco, Thermo Fisher Scientific], 1% GlutaMax [Gibco, Thermo Fisher Scientific], 100 IU/mL penicillin, and 100 μg/mL streptomycin) and incubated at 37 °C with 5% CO₂ for 2 h. Cells were rinsed with LHC basal medium, fresh infection medium #2 was added to each well, and plates were incubated at 37 °C with 5% CO₂ for 96 h.

For virus titration assays, ~20,000 cells (Vero-E6/HRECs/Deer-RECs) per well were seeded in a 96-well plate (CellBIND Costar; Corning) and, 24 h prior to infection, the cells were washed once with LHC medium and pre-incubated with an infection medium containing ATCC airway epithelial cell basal medium, 2% Ultroser-G (Sartorius Stedim Biotech GmbH, Goettingen, Germany), 1 × 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Thermo Fisher Scientific), 1× MEM non-essential amino acids (Thermo Fisher Scientific), 1× Glutamax (Thermo Fisher Scientific), Pen-Strep, and AmpB (Thermo Fisher Scientific). For transcriptomic analysis, six-well plates with a seeding density of 300,000 cells per well on the day of infection were washed once with LHC medium and inoculated with infection medium containing different doses of SARS-CoV-2 (10⁵, 10⁴, 10³, 10², 10, 1 PFU/mL) or mock inoculated with infection medium only. After 2 h incubation at 37°C and 5% CO₂, the inoculum was removed, cells were washed once with LHC medium, replaced with fresh infection media, and incubated for 6, 24, and 48 h. Following infection, virus-induced CPE, including rounding of cells, cell detachment, clumping, and dead cells, were recorded. For imaging, the cells on plates were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA).