Quantifying SARS-CoV-2 Infection by Immunofluorescence

Fixed cells were washed three times with Dulbecco-modified PBS containing 0.2% BSA (DPBS/BSA), permeabilized with 0.1% Triton X-100 in DPBS/BSA and processed for immunodetection of viral N protein, automated fluorescence imaging, and image analysis. Briefly, viral NP was detected with an in-house-developed rabbit polyclonal antibody^{22 (link)} counterstained with Alexa Fluor 647-conjugated goat anti-rabbit secondary antibody (ThermoFisher Scientific, catalogue number A32733); nuclear staining was done using Hoechst DNA dye (ThermoFisher Scientific, catalogue number H3570). Automated fluorescence imaging was done using a Molecular Devices Image-Xpress Nano high-content epifluorescence microscope equipped with a 10× objective and a 4.7-megapixel CMOS camera (pixel size, 0.332 μm). Image analysis was performed with CellProfiler-4 software (www.cellprofiler.org). Automated detection of nuclei was performed using the Otsu algorithm inbuilt in the software. To automatically identify infected cells, an area surrounding each nucleus (5-pixel expansion of the nuclear area) was used to estimate the fluorescence intensity of the viral NP immunolabeled protein, using an intensity threshold such that <0.01% of ‘positive cells’ were detected in noninfected wells.