Mikrowin 2010

Proliferation was assessed using the Click-iT EdU Proliferation Assay for Microplates (C10499, Invitrogen). Briefly, cells were plated at 48% confluency in 96 well black microplate (CLS3603, Corning, MERCK), labeled with 10 µM EdU for 24 h, then fixed and clicked according to the manufacturer’s instructions. Fluorescence was analyzed with a Mithras LB 940 reader (Berthold technologies, Bad Wildbad, Germany) and MikroWin 2010 software (Berthold technologies) using excitation filter 560 nm and emission filter 590 nm.

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.

The total protein concentration was determined by Lowry’s method using the DC Protein Assay Kit, according to the manufacturer’s instructions (Bio-Rad) with serial dilution series of Bovine Serum Albumin (BSA, Sigma) used as calibration standard. The optical density was measured at 750 nm using a plate reader (Berthold) with MikroWin 2010 software. Proteins contained in the whole-cell lysate (WCL) or the eluates were separated on a 4–15% gradient sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pre-casted gel (Bio-Rad). For Mn²⁺-Phos-tag SDS-PAGE gel, the acrylamide-pendant Phos-tag ligand (50 μM, Phos-tag^TM Acrylamide, FUJIFILM Wako Chemicals USA Corporation) and MnCl₂ (100 mM) were added to the 7% separating gel before polymerization. The Phos-tag gel was soaked in a transfer buffer containing 5 mM EDTA (10 min × 3) followed by washing in a transfer buffer without EDTA (20 min). Proteins were transferred on a nitrocellulose membrane and then detected with appropriate antibodies. Immune-complexes were revealed with HRP-conjugated secondary antibodies and enhanced chemoluminescence (Pierce™ ECL Western Blotting Substrate). Uncropped and unprocessed scans of representative blots are provided as a Source Data file.