Dm6000 inverted light microscope

Vero E6 cells (ATCC CRL-1586) cultures were maintained in DMEM (Dulbecco´s Modified Eagle Medium) supplemented with 10% foetal bovine serum (FBS) and 100U/mL of penicillin–streptomycin and cultured at 37ºC and 5% CO₂ (Gibco, Gran Island, NY, USA) in 6 and 24 well plates (Nunc, ThermoFisher, Madrid, Spain) until an almost confluent monolayer was formed (10⁶ cells/ml). For infection, monolayers were washed twice with phosphate buffered saline (PBS) and inoculated with a SARS-CoV-2 infected PPE sample kept in non-supplemented DMEM. Non-infected control cultures (mock/negative control) were prepared using non-supplemented DMEM as inoculum. A positive control of infection was carried out using the Human coronavirus 229E ATCC ® VR-740 ™ strain (ATCC, LG Promochem, Barcelona, Spain). Cell monolayers were checked daily under a Leica DM6000 inverted light microscope for the presence of cytopathic effects (CPE) for up to 48 h post infection. All procedures were performed in a biosafety level 3 laboratory at our Institution as mentioned before. Cell lysate was collected from wells by gentle scrapping and pipetting, for further RT-qPCR assays. All the experiments were done in duplicate.

Briefly, VERO E6 cells (ATCC CRL-1586) were cultured and maintained in DMEM (Dulbecco´s Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS) and 100U/mL of penicillin-streptomycin. Cells were cultured at 37°C and 5% CO₂ (Gibco, Grand Island, NY, USA) in 6 and 24 well plates (Nunc, ThermoFisher, Madrid, Spain) until an almost confluent monolayer was formed (10⁶ cells/ml). To infect cultured cells with the virus, monolayers were washed twice with phosphate-buffered saline (PBS) and inoculated with SARS-CoV-2 from a naturally infected face mask sample kept in non-supplemented DMEM. Non-infected control cultures (mock/negative control) were prepared using non-supplemented DMEM as inoculum. Positive control of infection was carried out using the human coronavirus 229E ATCC ® VR-740 ™ strain (ATCC, LG Promochem, Barcelona, Spain). All the experiments were done in duplicate. Cell monolayers were checked daily under a Leica DM6000 inverted light microscope for the presence of cytopathic effects (CPE) for up to 48 h post-infection. The cell lysate was collected from wells by gentle scraping and pipetting, for further RT-qPCR assays.