key: cord-0784799-5gh9tklm
authors: Li, Pengfei; Wang, Yining; Lavrijsen, Marla; Lamers, Mart M.; de Vries, Annemarie C.; Rottier, Robbert J.; Bruno, Marco J.; Peppelenbosch, Maikel P.; Haagmans, Bart L.; Pan, Qiuwei
title: SARS-CoV-2 Omicron variant is highly sensitive to molnupiravir, nirmatrelvir, and the combination
date: 2022-01-20
journal: Cell Res
DOI: 10.1038/s41422-022-00618-w
sha: 8964337990e63113f5f26498a47b554e5859b1ae
doc_id: 784799
cord_uid: 5gh9tklm

nan

Human lung epithelial cell line Calu-3 was cultured in advanced DMEM/F12 supplemented with 1% (vol/vol) GlutaMAXTM Supplement (Gibco, Grand island, USA), 10 mM HEPES, 100 IU/mL Penicillin and 100 mg/mL Streptomycin (Gibco, Grand Island, USA). Wild type (WT) SARS-CoV-2 (isolate BetaCoV/Munich/BavPat1/2020) was originally obtained from European Virus Archive Global. SARS-CoV-2 Delta and Omicron variants were isolated from infected patients at Department of Viroscience, Erasmus MC, The Netherlands. Cell lines were analyzed by genotyping and confirmed to be mycoplasma negative.

Adult lung tissues were obtained from residual, tumor-free, material obtained at lung resection surgery for lung cancer for culturing organoids. and 10 µM Y27632 (Sigma-Aldrich). To differentiate airway organoids into proximal phenotype, culture medium was changed into complete base medium (CBM; Stemcell

Pneumacult-ALI) supplemented with 10 uM DAPT (Tocris). 

Calu-3 cells were seeded into 96-well tissue culture plates (1×10 4 cells/well), and then treated with the indicated compounds for 48 hours. Cells were incubated with 10 µL 5 mg/mL 3-(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT) for 3 hours, then replaced with 100 µL DMSO medium and incubated at 37°C for 30 minutes. The absorbance at 490 nm was recorded using a microplate absorbance reader (Bio-Rad, CA, USA).

Cell culture supernatants from virus infected Calu-3 cells were collected at indicated time points. Virus titer was quantified by using a 50% tissue culture infectious dose (TCID50) assay. Briefly, ten-fold dilutions of viruses were inoculated onto Calu-3 cells, grown in a 96well tissue culture plate at 2,000 cells/well. The plate was incubated at 37 °C for 3-4 days, and each well was examined under a light microscope for cytopathic effect (CPE). The TCID50 value was calculated by using the Reed-Muench method.

First, Calu-3 cells that were cultured in 8-well slides (Ibidi GmbH, Germany) or hAOs were fixed in 4% paraformaldehyde for 30 minutes. Subsequently, hAOs were added into the CytoSpin II Cytocentrifuge (Shandon Scientifi Ltd, Runcorn, England) and spun down into slides at 800 rpm for 5 min. The 8-well slides (Calu-3 cells) and slides containing organoids were then rinsed 3 times with PBS, followed by permeabilizing with PBS containing 0.2% (vol/vol) tritonX100 for 10 min. Then the slides were twice rinsed with PBS for 5 min, followed by incubation with blocking solution (5% donkey serum, 1% bovine serum albumin, 0.2% tritonX100 in PBS) at room temperature for 1 hour. Next, slides were 

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