In 96-well tissue culture plates, 2.4 × 104 Vero E6 cells were distributed in each well and incubated overnight at a humidified 37 °C incubator under 5%CO2 condition. The cell monolayers were then washed once with 1× PBS and subjected to virus adsorption (hCoV-19/Egypt/NRC-03/2020 (Accession Number on GSAID: EPI_ISL_430820)) for 1 h at room temperature (RT). The cell monolayers were further overlaid with 100 μL of DMEM containing varying concentrations of the test compounds. Following incubation at 37 °C in 5% CO2 incubator for 72 h, the cells were fixed with 100 μL of 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet in distilled water for 15 min at RT. The crystal violet dye was then dissolved using 100 μL absolute methanol per well and the optical density of the color is measured at 570 nm using Anthos Zenyth 200rt plate reader (Anthos Labtec Instruments, Heerhugowaard, Netherlands). The IC50 of the compound is that required to reduce the virus-induced cytopathic effect (CPE) by 50%, relative to the virus control.
Anthos zenyth 200rt plate reader
Manufactured by Anthos Labtec
Sourced in Netherlands
The Anthos Zenyth 200rt is a multimode plate reader designed for absorbance, fluorescence, and luminescence measurements. It features a monochromator-based optical system and supports a wide range of microplate formats. The Zenyth 200rt is capable of performing various endpoint and kinetic assays.
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6 protocols using anthos zenyth 200rt plate reader
1
Evaluating Antiviral Compound Efficacy
2
Evaluating Anti-Inflammatory Effects of Marine Nutraceuticals
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The levels of TNFα produced by LPS stimulated RAW 264.7 macrophages in the cell culture supernatant were measured using a mouse TNFα ELISA kit (R&D Systems, Minneapolis, MN, USA) and performed as per the manufacturer’s instructions. All extracts were tested in five different concentrations 50 µg/mL, 25 µg/mL, 12.5 µg/mL, 6.25 µg/mL, and 3.125 µg/mL. Dexamethasone was used as a reference anti-inflammatory drug, and untreated, stimulated cells (LPS + ethanol) were used as a positive control. Absorbance was read at 450 nm using an Anthos Zenyth 200rt plate reader (Anthos Labtec Instruments, Heerhugowaard, Netherlands). The commercially available marine nutraceuticals Lyprinol® (BLACKMORES®, Alexandria, Australia) and Deep Sea Krill oil (Swisse, Collingwood Melbourne, Australia) were used as reference anti-inflammatory nutraceuticals. The assays were repeated in triplicate.
3
Antiviral Cytopathic Effect Assay
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In a humidified 37 °C incubator under a 5% CO2 condition, 2.4 × 104 Vero-E6 cells were placed in 96-well plate and then incubated overnight. The cell monolayers were then washed once with PBS and subjected to virus adsorption for 1 h at room temperature. Afterwards, the cells were further overlaid with 50 μL of DMEM mixed with the tested compunds and extracts. The cells were incubated for 72 h, fixed with 4% paraformaldehyde for 20 min, and then stained for 15 min with 0.1% crystal violet. Subsequently, 100 μL of methanol were used to dissolve the crystal violet, and the optical density was measured using Anthos Zenyth 200rt plate reader (Anthos Labtec Instruments, Heerhugowaard, The Netherlands) at 570 nm. The IC50 of the compound is the concentration that reduces the virus-induced cytopathic effect (CPE) by 50% relative to a virus control.
4
Antiviral Screening in Vero-E6 Cells
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In 96-well tissue culture plates, 2.4 × 104 Vero-E6 cells were distributed in each well and incubated overnight in a humidified 37 °C incubator under 5% CO2 condition. The cell monolayers were then washed once with 1× PBS and subjected to virus adsorption for 1 h at room temperature (RT). The cell monolayers were further overlaid with 50 μL of DMEM containing varying concentrations of the selected test compounds, Azithromycin, Niclosamide, and Nitazoxanide. Following incubation at 37 °C in 5% CO2 incubator for 72 h, the cells were fixed with 100 μL of 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet in distilled water for 15 min at RT. The crystal violet dye was then dissolved using 100 μL absolute methanol per well and the optical density of the color measured at 570 nm using Anthos Zenyth 200 rt plate reader (Anthos Labtec Instruments, Heerhugowaard, Netherlands). The IC50 of the compound is that required to reduce the virus-induced cytopathic effect (CPE) by 50%, relative to the virus control.
5
SARS-CoV-2 Antiviral Compound Screening
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The IC50 values for the tested compounds were determined as previously described [2 (link),3 (link)], with minor modifications. Briefly, in 96-well tissue culture plates, 2.4 × 104 Vero-E6 cells were distributed in each well and incubated overnight at a humidified 37°C incubator under 5% CO2 condition. The cell monolayers were then washed once with 1× PBS. An aliquot of the SARS-CoV-2 “NRC-03-nhCoV” virus [40 (link)] containing 100 TCID50 was incubated with serial diluted compounds and kept at 37 °C for 1 h. The Vero-E6 cells were treated with virus/compound mix and co-incubated at 37 °C in a total volume of 200 µL per well. Untreated cells infected with virus represented the virus control; however, cells that were not treated and not infected were the cell control. Following incubation at 37 °C in 5% CO2 incubator for 72 h, the cells were fixed with 100 μL of 10% paraformaldehyde for 20 min and stained with 0.5% crystal violet in distilled water for 15 min at RT. The crystal violet dye was then dissolved using 100 μL of absolute methanol per well and the optical density of the color was measured at 570 nm using the Anthos Zenyth 200 rt plate reader (Anthos Labtec Instruments, Heerhugowaard, the Netherlands). The IC50 of the compound was that required to reduce the virus-induced cytopathic effect (CPE) by 50%, relative to the virus control.
6
Antiviral Assay Protocol for Vero-E6 Cells
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2.4 x 104 Vero-E6 cells were seeded into each well of a 96-well plate, and the plates were incubated at 37 °C with 5 % CO2 for 24 h. After one wash with PBS, we let viruses adhere to cell monolayers at room temperature for an hour. Then, 50 μL of DMEM containing the test compounds at varying doses was applied to the cell monolayers. After being fixed in 100 mL of 4 % paraformaldehyde for 20 min, cells were stained for 15 min with 0.1 % crystal violet. By utilizing an Anthos Zenyth 200 rt plate reader (Anthos Labtec Instruments, Heerhugowaard, The Netherlands), we were able to determine the produced color at 570 nm. Each well had 100 μL of crystal violet dissolved in methanol put to it. The "IC50" of the drug is the concentration at which the virus-induced cytopathic effect (CPE) is reduced by 50 %. Remdesivir was used as a positive control.
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