Cell Lines and Virus Propagation for COVID-19 Research

Cercopithecus aethiops kidney cells (Vero-76, ATCC CRL 1587) and human lung adenocarcinoma Calu-3 (ATCC HTB-55) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Vero-76 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Microtech, Naples, Italy) supplemented with 10% Fetal Bovine Serum (FBS, Microgem, Naples, Italy) and antibiotic solution (10,000 U penicillin + 10 mg streptomycin, Himedia, Mumbai, India). Calu-3 cells were grown in Eagle’s Minimum Essential Medium (EMEM, Microtech) supplemented with 10% FBS antibiotic solution. All the coronaviruses used in this study, which are (i) HCoV-229E (ATCC VR-740); (ii) HCoV-OC43 (ATCC VR-1558); and (iii) SARS-CoV-2 (clinical isolate, strain VR PV10734, kindly donated by the Lazzaro Spallanzani Hospital, Rome, Italy), were propagated in the Vero-76 cell line and their concentration was determined via plaque assay [20 (link)]. For HCoV-OC43, the viral concentration was expressed as the median tissue culture infectious dose (TCID₅₀). All experimental work involving SARS-CoV-2 was performed in a biosafety level (BSL) 3 laboratory.