Glomax navigator system g2000

Pseudotyped VSVs bearing the S protein, the 19 aa-truncated S protein of SARS-CoV-2, or VSV-G were generated as described below. Briefly, 293 T cells were grown to 80% confluence on collagen-coated tissue culture plates and then transfected with each expression vector: pCAG-SARS-CoV-2 S-full, pCAG-SARS-CoV-2 S-t19, and pCAG-VSV-G. After 24 h of incubation, the cells transfected with each plasmid were infected with G-complemented (*G) VSV∆G/Luc (*G-VSV∆G/Luc) [16 (link)] at a multiplicity of infection (MOI) of 0.5 per cell. Then, the virus was adsorbed and extensively washed four times with 10% FBS DMEM. After 24 h of incubation, to remove cell debris, the culture supernatants containing pseudotyped VSVs were centrifuged, and then, they stored at − 80 °C until ready for use. The pseudotyped VSV bearing SARS-CoV-2 S protein or SARS-CoV-2 truncated S protein are referred to as Sfullpv or St19pv, respectively. The infectivity of Sfullpv, St19pv, or VSVpv to 293 T cells was assessed by measuring the luciferase activity. The value of the relative light unit (RLU) of luciferase was determined using a PicaGene Luminescence Kit (TOYO B-Net Co., LTD, Tokyo, Japan) and GloMax Navigator System G2000 (Promega Corporation, Madison, WI), according to the manufacturer’s protocol.

To examine the effect of trypsin after viral infection, VeroE6 cells seeded in 96-well culture plates were treated with DMEM containing 10% FBS on ice for 10 min. Approximately 100 μL of SARS-CoV-2pv (Wuhan, alpha, delta, and omicron) or VSVpv were added to the cells. After viral adsorption on ice for 60 min, the virus was removed, and the infected cells were treated for 5 min with various concentrations of trypsin (Sigma, T1426) in DMEM that was prewarmed at room temperature. After trypsin solution was removed, cells were cultured in DMEM containing 10% FBS at 37 °C for 24 h.
To examine the effect of trypsin on viral particles, SARS-CoV-2pv or VSVpv was exposed to various concentrations of trypsin in DMEM for 5 min at 37 °C. To inactivate trypsin, FBS was added at a final concentration of 20%. Then 20 μL of sample was added to each well, and the cells were incubated at 37 °C for 24 h. The infectivity of both SARS-CoV-2pv and VSVpv was assessed separately by measuring luciferase activity. The relative light unit value of luciferase was determined using the PicaGene Luminescence Kit (TOYO B-Net Co., Ltd., Tokyo, Japan) and GloMax Navigator System G2000 (Promega Corporation, Madison, WI), according to the manufacturer’s protocol.

BmNPV-luciferase virus, which carries the luciferase gene under the control of the polyhedrin promoter, was obtained from Nihon Nosan Co. Ltd. The virus was amplified in BmN cells, and the titer was determined using the limiting dilution method. A group of day 0 WT or cocoon-free silkworm pupae (n = 5) was injected with 50 μL of BmNPV-luciferase at a concentration of 2 × 10¹³ pfu/mL. Five days after the injection, each of the infected silkworm pupa was homogenized in 5 mL of 1× PBS and centrifuged at 4°C for 30 min at 4,400 ×g to remove the large debris. The resulting supernatant was assayed for luciferase activity using the PicaGene BrillianStar-LT luminescence kit (Toyo Ink). An aliquot of 50 μL from the 5000× diluted supernatant was mixed with a 50 μL of luciferin substrate reagent and incubated at 25°C for 5 min. The luminescence was measured using the GloMax Navigator System G2000 (Promega), and the relative luminescence unit value for the female cocoon-free silkworm pupae was expressed as a percentage of the female WT silkworm pupae.