Biotek cytation 7 cell imaging multi mode reader

Each cell line was seeded into wells in 96-well plates one day before the assay. Cells were fixed with 4% paraformaldehyde for 1 h at room temperature and blocked by 2% (w/v) bovine serum albumin in phosphate-buffered saline (PBS) with 0.5% Tween (PBST) for 1 h at room temperature. The cells were then incubated with the primary antibody, binding either to the ACE2 [rabbit anti-ACE2 polyclonal antibody (1:500) (Abcam, USA)] or TMPRSS2 [mouse anti-TMPRSS2 monoclonal antibody (1:50) (Santa Cruz Biotechnology, USA)] proteins, at 37 °C for 1 h. After that, the cells were washed with PBST three times, and incubated with the secondary antibody; goat anti-rabbit Alexa 488 (1:500; Invitrogen, USA) or goat anti-mouse Alexa 568 (1:500; Invitrogen, USA). After washing, Hoechst dye (Thermo Fisher Scientific, USA) was used to stain cells’ nuclei. The fluorescent signals were detected by BioTek Cytation 7 Cell Imaging Multi-Mode Reader (Agilent Technologies, USA).

Neutralization assays were performed with live ancestral SARS-CoV-2 viruses in a biosafety level 3 laboratory. VeroE6 cells were seeded in 96-well black plates (SPL Life Sciences, Gyeonggi-do, Republic of Korea). Samples were serially diluted in plain medium and 1000 PFU/well viruses were added for incubation at 37 °C for 1 h. Sample–virus mixtures were then added to the seeded VeroE6 cells and incubated for 1 h at 37 °C. Cells were washed with PBS and replenished with culture medium containing 1% FBS. Inoculated cells were further incubated for 6 h, and then were fixed with 4% formalin. The plates were washed, permeabilized with 0.1% NP40, blocked with 2% BSA, and stained with in-house rabbit anti-SARS-CoV-2 nucleoprotein polyclonal antibody and detected by anti-rabbit Alexaflour 488 (Abcam plc, Cambridge, UK). Fluorescent positive cells were detected by a Biotek Cytation 7 Cell Imaging Multi-Mode Reader (Agilent Technologies, Inc., Santa Clara, CA, USA) with data captured by Gen5 Image Prime version 3.11.19. Neutralization titers were calculated and determined in GraphPad Prism 9 by performing 4-parameter logistical fitting on the detected foci. The 50% focus reduction neutralization titer (FRNT50) was determined as the interpolated reciprocal of the dilution having 50% reduction in infected fluorescent loci compared to control wells.