Cellinsight cx7 hcs

Caco-2 cells were seeded at a concentration of 10,000 cells/well in 96-well plates the day before transduction. Cells were transduced with two BacMam baculovirus vectors (Molecular Montana), one vector expressing the recombinant SARS-CoV-2 3CLpro (enzyme BacMam) and the other expressing an intracellular biosensor–red fluorescent protein (RFP) and mNeonGreen (mNG) fluorescent protein with an inhibitor tag. Three h after transduction, the cells were treated with a serial dilution of compounds and then incubated for 28 h, followed by fixation of the cells with 3.7% formalin for 30 min. The fixative was removed, the cells were washed with PBS, and the cells were nuclear stained with Hoechst 33,342 at 1.5 µg/mL for 30 min at room temperature in the dark. After washing with PBS, the plates were kept in the dark at 4°C until imaging was performed on a high-content screening (HCS) platform (CellInsight CX7 HCS, Thermo Fisher Scientific) with a 10X objective.

All infections with HCoV-229E (ATCC® VR-740) were carried out in a Biosafety Level 2 (BSL2) facility in accordance with Public Health Agency of Canada regulations (UBC BSL2 Permit # B17-0024). Viral stocks used in the experiments were propagated in Caco-2 cells, and all HCoV-229E infections were performed at 33°C. Caco-2 cells were seeded at a concentration of 8,000 cells/well in 96-well plates the day before infection. Cells were pretreated with compounds for 3 h, then incubated with the virus (diluted to an MOI of 1 in EMEM w/o FBS) for 48 h, followed by fixation of the cells with 3.7% formalin for 30 min to inactivate the virus. The fixative was removed, and the cells were washed with PBS, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 1% BSA for 1 h, followed by immunostaining with the mouse primary antibody J2 (dsRNA) at working dilutions of 1:1000 for 1 h at room temperature. A secondary antibody goat anti-mouse IgG Alexa Fluor 488 was used at a 1:2000 dilution with the nuclear stain Hoechst 33,342 at 1.5 µg/mL for 1 h at room temperature in the dark. After washing with PBS, the plates were kept in the dark at 4°C until imaging was performed on an HCS platform (CellInsight CX7 HCS, Thermo Fisher Scientific) with a 10X objective (Supplementary Materials and Methods).