Penicillin streptomycin antibiotics

African green monkey kidney (VERO) epithelial cells (ATCC CCL-81 sex of cell Female) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% inactivated fetal calf serum (FCS) (EuroClone, Milan, Italy), 1 mM glutamine (EuroClone, Milan, Italy), 1% streptomycin - penicillin antibiotics (EuroClone, Milan, Italy) and incubated in a humidified atmosphere (5% CO₂ at 37°C) as reported elsewhere (Ammerman et al., 2008 ). Cells were washed with sterile warm phosphate buffered saline (PBS), trypsinized and counted. Cells were replated in 48-well plates (Wuxi NEST Biotechnology Co., Ltd, China) at 7 x 10⁴ cells/mL. Cells were infected with SARS-CoV-2 virus when > 90% confluent monolayer was observed comprising approximately 1 x 10⁶ cells/well after 72 hours. A549 cells (ATCC sex of cell male) were maintained in DMEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, EuroClone), 2% penicillin-streptomycin (Sigma-Aldrich) and 2% L-glutamine (Sigma-Aldrich). Cells were cultivated in T75 flasks and kept at 37°C in 5% CO₂ humidity. Biocompatibility test has been performed also on VERO cells using cell culture conditions specified above.

The human ovarian cancer SKOV3 cell line was purchased from ATCC (American Type Culture Collection, Manassas, Virginia United States). SKOV3 culture cells were maintained in RPMI (Thermo Fischer Scientific, Waltham, Massachusetts, USA) supplemented with 10% fetal bovine serum (Euroclone, Milan, Italy) and 1% penicillin/streptomycin antibiotics (Euroclone, Milan, Italy) at 37°C in a humidified incubator with 5% CO₂. In order to achieve stable expression of the luciferase gene, SKOV3 cells were transfected with pcDNA3.1-luciferase vector using Lipofectamine 2000 Transfection Reagent, following the manufacturer’s instructions (Thermo Fischer Scientific, Waltham, Massachusetts, USA). We selected the SKOV3 positive clones (SKOV3-luc) in medium containing G418 neomycin (Euroclone, Milan, Italy). To evaluate the luciferase expression, we placed the serial dilutions of SKOV3-luc (500, 1000, 2500, 5000 cell/well) in 96 well plate (Sarstedt, Nümbrecht, Germany) and determined the luciferase activity with ONE-GloTMLuciferase Assay System kit (Promega Madison, WI) using Glomax MultiDetection System instrument (Promega Madison, WI). All images of cell cultures were acquired using CCD camera (DFC420, Leica, Wetzlar, Germany) with light microscopy (DMIL, Leica, Germany) at 20x magnification.