Irdye800cw goat anti human igg secondary antibodies

HCVpp was generated by cotransfection of 293T cells with pNL4-3.lucR−E− and the corresponding expression plasmid encoding wildtype or mutant E1E2 genes as described previously [35 (link)]. Cell lysates and culture supernatants were harvested 72 hours post-transfection for immunoblotting, infectivity assay and purification of virions. HCVpp was pelleted by centrifugation of culture supernatant at 16,000 rpm for 1 hour, resuspended, and purified over a 20% (wt/vol) sucrose cushion [35 (link), 76 (link)]. Envelope glycoproteins E1 and E2 were detected by immunoblotting using biotinylated mouse mAb A4 [28 (link)] and mAb HCV1 [24 (link)] and the IRDye680RD Streptavidin (1:2,000) and IRDye800CW goat anti-human IgG secondary antibodies (1:10,000) (LI-COR Biosciences), respectively. HIV-1-p24 was detected using biotinylated mouse monoclonal antibody (diluted to 1:1,000; Aalto Bio Reagents). The immunoblots were analyzed with the Odyssey Infrared Imaging System and Image Studio software (LI-COR Biosciences).

Sub-confluent Vero cells in 24-well plates were infected in duplicate with ten-fold serially diluted viruses. After 2 h incubation at 37 °C, cells were washed with Opti-MEM (Thermo Fisher Scientific) and overlaid with 1 ml per well of 0.8% methylcellulose dissolved in Opti-MEM containing 1% L-glutamine, 2.5% penicillin-streptomycin, 0.1% gentamicin, 0.5% amphotericin B and 2% TrypLE Select (Thermo Fisher Scientific). On day 4 post-infection, cells were fixed using ice-cold 80% methanol. For dual-staining assays to detect expression of APMV3 and SARS-CoV-2 antigens, wells were incubated for 2 h at room temperature (RT) with a primary chicken anti-APMV3 serum (1:2,000) and a human anti-SARS-CoV-2 S RBD antibody (CR3022, 1:2,000) (or, as a negative control, was replaced with a combination of normal chicken IgY control (R&D Systems, cat# AB-101-C, 1:1,000) and human IgG1 isotype control (BioLegend, cat# 403501, 1:1,000)). After extensive washing with PBS, wells were incubated for 1 h at RT with 1:2,000 diluted infrared dye (IRDye)-conjugated 680LT donkey anti-chicken IgY (LI-COR, cat# 926-68028) and IRDye 800CW goat anti-human IgG secondary antibodies (LI-COR, cat# 926-32232). After washing with PBS, plates were scanned with an Odyssey CLx imager (LI-COR).