Reagents and infection with SARS-CoV-2

PBMCs Isolation

The PBMCs were obtained by using a density-gradient centrifugation technique with a 1.077 g 3 mL-1 Ficoll gradient. In this method, the Buffy coats were mixed gently and then diluted (1:1) with PBS before being carefully transferred to a 50-mL tube containing 10 mL of Ficoll. This mixture was centrifuged at 2700 rpm for 20 minutes at room temperature. The resulting PBMCs were cultured as adherent monolayers (at a concentration of 1,53106 cell3mL-1) in RPMI 1640 medium. If necessary, CD14+ cell sorting could be performed to enhance the purity of the cells. After allowing the cells to adhere for 2-3 hours, they were washed with PBS. Then they incubated in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin at 37 C with 5% CO2 atmosphere until they were ready for infection.

Viruses and Cell Lines

The SARS-CoV-2 virus stocks were propagated using the Vero cell line, and the supernatant was collected at 2-3 dpi. Plaque assays determined viral titers on Vero cells. Vero CCL-81 cells were grown in MEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% Penicillin-Streptomycin and were incubated at 37 C with a 5% CO2 atmosphere. 

Reagents and Infection 

The cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 and incubated under continuous agitation at 15 rpm for 1 hour to allow for virus absorption. Following the infection, the cells were washed twice with pre-warmed PBS and then incubated for 24 hours in RPMI medium containing 10% FBS and 1% Penicillin-Streptomycin at 37 C with a 5% CO2 atmosphere.