Fbs rpmi 1640 medium

Blood samples, collected in a BD Vacutainer Plus plastic serum tube, were processed to separate plasma and peripheral blood mononuclear cells (PBMCs), as previously described (26 (link)). Plasma was obtained by centrifugation of whole blood at 400g for 10 min and storage at −20°C until use. Plasma was then employed for neutralization assay, while PBMCs were resuspended at the concentration of 1,5×10⁶ cells/150μl in 10% FBS RPMI 1640 medium (Euroclone, Milan, Italy) and subsequently stimulated with anti-CD28 (1 μg/mL) and 180 µg/mL of SARS-CoV-2 spike (S) recombinant peptides, a mixture of 181 17-/13-mers peptides spanning the full length of the protein (NR-52402, BEI Resources, NIAID, NIH). Unstimulated PBMCs were cultured as negative controls. Cells were harvested 18 hours post-stimulation for flow cytometric analyses.

PBMCs were resuspended at the concentration of 1×10⁶ cells/mL in 10% FBS RPMI 1640 medium (Euroclone, Milan, Italy) and subsequently stimulated with 500 μg/mL of S (spike) and N (nucleocapsid) recombinant peptides of SARS-CoV-2 (NovateinBio, Woburn, MA, USA). Unstimulated PBMCs were cultured as control as well. Cells were harvested 10- or 24-hours post-infection (hpi) for RNA or flow cytometric analyses, respectively.