Asys expert 96 uv microplate reader

To determine SARS-CoV-2 infection, 30,000 Caco-2 cells were seeded in 96-well plates. The next day, the compound of interest was added and the cells were infected with a multiplicity of infection of 0.001 in a total volume of 180 μL. Two days later immunodetection assay was performed as previously described³² (link); cells were fixed in 4% paraformaldehyde (PFA), permeabilized using 0.1% Triton-X, and stained with 1:5000 diluted anti-SARS-CoV-2 S protein antibody 1A9 (Biozol GTX-GTX632604) in antibody buffer (10% FCS and 0.3% Tween 20 in phosphate-buffered saline [PBS]) for 1 hour at 37°C. After 3 washes, the secondary horseradish peroxidase–conjugated antibody (#A16066; Thermo Fisher Scientific, Waltham, MA) (1:15,000) was incubated for 1 hour at 37°C. Following 4 times of washing, the TMB peroxidase substrate (#52-00-04; Medac, North Augusta, SC) was added for 5 minutes and the reaction stopped using 0.5 M H₂SO₄. The optical density was recorded at 450–620 nm using the Asys Expert 96 UV microplate reader (Biochrom, Cambridge, United Kingdom). Values were corrected for the background signal derived from uninfected cells and untreated controls were set to 100% infection.