PMC:7321036 / 76081-79167 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/7321036","sourcedb":"PMC","sourceid":"7321036","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7321036","text":"Experimental Model and Subject Details\n\nCells\nAfrican green monkey kidney epithelial Vero E6 (Vero 76, clone E6, Vero E6, ATCC® CRL-1586) authenticated by ATCC and tested negative for mycoplasma contamination prior to commencement were maintained in a humidified atmosphere at 37°C with 5% CO2, in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS, Invitrogen). ACE2-expressing A549 cells, a human lung epithelial cell line, were a kind gift from Brad Rosenberg. A549-ACE2 cells were cultured in DMEM supplemented with 10% FBS and maintained at 37°C with 5% CO2. Caco-2 (ATCC® HTB-37) human colon epithelial cells were maintained in a humidified atmosphere at 37°C with 5% CO2 in DMEM-containing 20% (v/v) FBS.\n\nViruses (Institut Pasteur, Paris)\nThe SARS-CoV-2 isolate BetaCoV/France/IDF0372/2020 was supplied by the National Reference Centre for Respiratory Viruses hosted by Institut Pasteur (Paris, France) and headed by Pr. Sylvie van der Werf. The isolate originated from a human sample provided by Dr. X. Lescure and Pr. Y. Yazdanpanah from the Bichat Hospital, Paris, France. The isolate was supplied through the European Virus Archive goes Global (EVAg) platform. Viral stocks were prepared by propagation in Vero E6 cells in DMEM supplemented with 2% FBS. Viral titers were determined by plaque assay in Minimum Essential Media (MEM) supplemented with 2% (v/v) FBS (Invitrogen) and 0.05% agarose. All experiments involving live SARS-CoV-2 were performed in compliance with Institut Pasteur Paris’s guidelines for Biosafety Level 3 (BSL-3) containment procedures in approved laboratories. All experiments were performed in at least three biologically independent samples.\n\nViruses (Mount Sinai, New York)\nFor infection experiments in ACE2-A549 cells, SARS-CoV-2, isolate USA-WA1/2020 (NR-52281), which shares 99.983% sequence identity with the BetaCoV/France/IDF0372/2020 isolate, was deposited by the Center for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH. SARS-CoV-2 was propagated in Vero E6 cells in DMEM supplemented with 2% FBS, 4.5 g/L D-glucose, 4 mM L-glutamine, 10 mM Non-Essential Amino Acids, 1 mM Sodium Pyruvate and 10 mM HEPES as described previously (Blanco-Melo et al., 2020). All work involving live SARS-CoV-2 was performed in the CDC/USDA-approved BSL-3 facility of the Global Health and Emerging Pathogens Institute at the Icahn School of Medicine at Mount Sinai in accordance with institutional biosafety requirements.\n\nViruses (University of Freiburg, Germany)\nFor infection experiments in Caco-2 cells, SARS-CoV-2 isolate Muc-IMB-1/2020, kindly provided by the Bundeswehr Institute of Microbiology, Munich, Germany, was used. SARS-CoV-2 was propagated in Vero E6 cells in DMEM supplemented with 2% FBS. All work involving live SARS-CoV-2 was performed in the BSL-3 facility of the Institute of Virology, University Hospital Freiburg, and was approved according to the German Act of Genetic Engineering by the local authority (Regierungspraesidium Tuebingen, permit 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