In this assay, ACE2h cells (1 × 106 cells/mL) were seeded into 96-well plates, 50 μL per well, and cultured with DMEM in an incubator set at 37 °C and containing 5% CO2 for 2 h. A volume of 25 μL medium was removed carefully and the same volume of tested compound dissolved in medium was added and incubated for 2 h. Five microliters of SARS-CoV-2 spike pseudovirus (Sino Biological, PSV001) was added followed by 4 h’s incubation, and then, 100 μL of complemented DMEM was added. After 6–8 h’s further infection, all the culture medium containing virus was removed and cultured in 200 μL of fresh medium for 48 h. Luciferase Assay System (Promega™ E1500) was used to assess the entrance of virus, briefly, cell culture lysis reagent was added to each well after the medium was removed, then with the luminescence solution added, chemiluminescence was detected by a microplate reader at 560 nm.
Psv001
Manufactured by Sino Biological
The PSV001 is a laboratory instrument designed for the purification and separation of biological samples. It utilizes a centrifugal force to separate components based on their density and size. The core function of the PSV001 is to provide a reliable and efficient method for isolating and concentrating specific biomolecules or cellular components from complex mixtures.
Automatically generated - may contain errors
2 protocols using psv001
1
SARS-CoV-2 Spike Pseudovirus Inhibition Assay
2
SARS-CoV-2 Ultrastructure and Spike Protein Localization
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Vero E6 and Calu-3 cells infected with SARS-CoV-2 for 30 min and 24 h were harvested by centrifugation at 500×g for 5 min. Cells were fixed with 2.5% glutaraldehyde without re-suspending the pellet at 4 °C overnight. Sections were made and stained with 2% uranium acetate and lead citrate after cells were embedded in epoxy resin and ultrathin. Slides were imaged under an electron microscope (JEM 1400Plus).
Immuno-electron microscopy was used to observe the co-localization of TfR and the spike protein in HEK293/hACE2 (OEC001, Sino Biological) and BHK-21/hTfR cells infected with SARS-CoV-2-s pseudovirus (PSV001, Sino Biological), with the sections prepared as described above. Sections were incubated with corresponding primary antibodies (mouse anti-human TfR antibody (11020-MM04, Sino Biological); rabbit anti-SARS-CoV-2-spike antibody (40150T62-COV2, Sino Biological) for 2 h at room temperature overnight after blocking with 1% BSA for 30 min. Subsequently, the sections were washed with PBS and incubated with 1% BSA for another 30 min. Secondary antibodies (5-nm gold-colloid labeled goat anti-mouse IgG (G7527, Sigma-Aldrich) and 15-nm gold-colloid labeled goat anti-rabbit IgG (ab27236, Abcam) were used to determine the localization of target proteins. Slides were imaged under an electron microscope as described above.
Immuno-electron microscopy was used to observe the co-localization of TfR and the spike protein in HEK293/hACE2 (OEC001, Sino Biological) and BHK-21/hTfR cells infected with SARS-CoV-2-s pseudovirus (PSV001, Sino Biological), with the sections prepared as described above. Sections were incubated with corresponding primary antibodies (mouse anti-human TfR antibody (11020-MM04, Sino Biological); rabbit anti-SARS-CoV-2-spike antibody (40150T62-COV2, Sino Biological) for 2 h at room temperature overnight after blocking with 1% BSA for 30 min. Subsequently, the sections were washed with PBS and incubated with 1% BSA for another 30 min. Secondary antibodies (5-nm gold-colloid labeled goat anti-mouse IgG (G7527, Sigma-Aldrich) and 15-nm gold-colloid labeled goat anti-rabbit IgG (ab27236, Abcam) were used to determine the localization of target proteins. Slides were imaged under an electron microscope as described above.
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