Costar flat bottom luminometer plates

SARS or MERS pseudovirus bearing SARS-CoV or MERS-CoV S protein, respectively, and a defective HIV-1 genome that expresses luciferase as reporter was prepared by co-transfecting 293T cells with the plasmid pNL4-3.luc.RE (encoding Env-defective, luciferase-expressing HIV-1) and pcDNA3.1-MERS-CoV-S plasmid^{61 (link),62 (link)}. To detect the inhibitory activity of a peptide on infection by SARS or MERS pseudovirus, ACE2-transfected 293T (293T/ACE2) cells and Huh-7 cells (10⁴ per well in 96-well plates) were respectively infected with SARS or MERS-CoV pseudovirus, in the presence or absence of a peptide at indicated concentration. The culture was re-fed with fresh medium 12 h post-infection and incubated for an additional 72 h. Cells were washed with PBS and lysed using lysis reagent included in a luciferase kit (Promega). Aliquots of cell lysates were transferred to 96-well Costar flat-bottom luminometer plates (Corning Costar), followed by the addition of luciferase substrate (Promega). Relative light units were determined immediately in the Ultra 384 luminometer (Tecan US)^{63 (link)}.