Direct cellular cytotoxicity (DCC) of PBMCs was determined using Vero E6 cells infected with pNL4–3Δenv_SARS-CoV-2-SΔ19(G614)_Ren virus as target, as previously described (33 (link)). Briefly, Vero E6 cells were infected with pNL4–3Δenv_SARS-CoV-2-SΔ19(G614)_Ren (100ng p24-Gag) for 48 hours and then co-cultured for 1 hour with PBMCs from the participants (ratio 1:2). Vero E6 cell monolayer was dissociated with trypsin-EDTA (Sigma Aldrich-Merck, Darmstadt, Germany) and caspase-3 activity in these cells was measured by chemiluminescence using Caspase-Glo 3/7 Assay system (Promega, Madison, WI) and a luminometer Centro XS3 LB 960 with MikroWin 2010 software (Berthold Technologies) as a measure of PBMCs cytotoxic activity against target cells (34 (link)). PBMCs were collected from the supernatants and analyzed by flow cytometry to characterize the cytotoxic cells. The following controls were used: target cells alone as negative control, target cells infected with pseudotyped SARS-CoV-2 as basal control for viral replication, and infected target cells co-cultured with NK cell line NKL (CVCL_0466) during the same time that PBMCs from the participants as positive control for viral replication and caspase-3 activity.
Free full text: Click here
