For transduction experiments, target cells were seeded in 96-well plates 24 h prior to transduction. For transduction, the culture medium was aspirated, and equal volumes of VSV pseudotypes were added to the cells. At 16–18 h posttransduction, transduction efficiency was quantified by measuring the virus encoded firefly luciferase (fLuc) activity in cell lysates using a commercial kit (Beetle-Juice; PJK) and a Hidex Sense plate luminometer (Hidex).
Sense plate luminometer
Manufactured by Hidex
Sourced in Germany
The Sense plate luminometer is a compact and efficient instrument designed for the measurement of luminescence-based assays. It provides rapid and accurate detection of light output from multi-well microplates, enabling users to conduct a variety of luminescence-based experiments and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Celltiter glo luminescent cell viability assay kit, by Promega (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
Triton x 100, by Carl Roth (1 mentions)
Prism 7, by GraphPad (1 mentions)
Cell culture lysis reagent, by Promega (1 mentions)
Gal screen, by Thermo Fisher Scientific (1 mentions)
8 protocols using sense plate luminometer
1
VSV Pseudotype Transduction Efficiency
2
Camostat Mesylate Cytotoxicity Assay
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Cell viability following treatment of Calu-3 cells with camostat mesylate was analyzed using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega). In brief, Calu-3 cells grown to 50% confluency in 96-well plates were incubated for 24 h in the absence or presence of different concentrations (1-500 μM) of camostat mesylate. Next, the culture medium was aspirated and 100 μl of fresh culture medium was added before an identical volume of the assay substrate was added. Wells containing only culture medium served as a control to determine the assay background. After 2 min of incubation on a rocking platform and additional 10 min without movement, samples were transferred into white opaque-walled 96-well plates and luminescent signal were recorded using a Hidex Sense plate luminometer (Hidex).
3
Cell Viability Assay for Calu-3 Cells
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For the analysis of cell vitality of Calu-3 cells treated with camostat mesylate or FOY-251 the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega) was used. For this, Calu-3 cells were grown in 96-well plates to reach ~50% confluency, before they were incubated in the presence of different concentrations of camostat mesylate or FOY-251 for 24 h. Cells treated with DMSO (solvent control) served as controls. Following incubation, 100 μl of CellTiter-Glo substrate were added per well and the samples were incubated for 30 min on a rocking platform. In addition, fresh culture medium (without cells) was also incubated with CellTiter-Glo substrate in order to define the assay background. Following incubation, the samples were transferred into white opaque-walled 96-well plates and luminescence was recorded (200 msec/sample) using a Hidex Sense plate luminometer (Hidex).
4
Pseudotype-Based SARS-CoV-2 Entry Assay
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For analysis of S protein-driven cell entry, target cells were seeded in 96-well plates. In case of experiments addressing S protein usage of different ACE2 orthologues as receptor, cells were transfected with the respective ACE2 expression plasmids or empty vector. At 24 h post seeding (or transfection), the culture medium was removed and cells were inoculated with equal volumes of pseudotype preparations. At 16-18 h post inoculation, pseudotype entry was quantified by measuring the activity of virus-encoded luciferase in cell lysates. For this, cells were lysed using PBS supplemented with 0.5% triton X-100 (Carl Roth) for 30 min at RT. Subsequently, cell lysates were transferred into white 96-well plates, mixed with luciferase substrate (Beetle- Juice, PJK) and luminescence measured with a Hidex Sense Plate luminometer (Hidex).
5
SARS-CoV-2 Spike Pseudovirus Neutralization Assay
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Neutralizing activities of antibodies were assessed in a pseudovirus neutralization assay as described [42 (link)]. Briefly, 1 × 104 Vero cells were seeded per well of a 96‐flat bottom plate 1 day before the infection. Antibodies were pre‐diluted in cell culture medium and pre‐incubated for 30 min with vesicular stomatitis virus‐based pseudoviruses bearing the SARS‐CoV‐2 spike protein in a final volume of 100 μl per replicate (four replicates per experiment) before being inoculated on Vero cells. Pseudoviruses incubated in culture medium without antibody served as control (100% cell entry). At 16–18h postinoculation, the culture medium was aspirated and pseudovirus cell entry was analyzed by measuring the activity of virus‐encoded firefly luciferase in cell lysates using a commercial substrate (Beetle‐Juice, PJK GmbH) and a Hidex Sense plate luminometer (Hidex). IC50 values were calculated by plotting the virus entry in percent against the antibody concentrations and using the normalized response vs. inhibitor equation (variable slope) of GraphPad Prism 7.02.
6
Cell Viability Assay for Calu-3 Cells
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For the analysis of cell vitality of Calu-3 cells treated with Camostat mesylate or FOY-251 the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega) was used. For this, Calu-3 cells were grown in 96-well plates to reach ~50% confluency, before they were incubated in the presence of different concentrations of Camostat mesylate or FOY-251 for 24 h. Cells treated with DMSO (solvent control) served as controls. Following incubation, 100 µl of CellTiter-Glo substrate were added per well and the samples were incubated for 30 min on a rocking platform. In addition, fresh culture medium (without cells) was also incubated with CellTiter-Glo substrate in order to define the assay background. Following incubation, the samples were transferred into white, opaque-walled 96-well plates and luminescence was recorded (200 m/sample) using a Hidex Sense plate luminometer (Hidex).
7
SARS-CoV-2 Spike Pseudotype Entry Assay
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Vesicular stomatitis virus (VSV) pseudotypes harbouring the SARS-CoV-2 spike protein (S) were generated as described previously [22 (link)]. BHK-21 cells were transfected for the expression of human [53 (link)] or cat ACE2 that has been cloned from cDNA generated from trachea samples as described above, using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA). At 24 h post transfection (p.t.), cells were inoculated with SARS-CoV-2 S-pseudotyped VSV for 16–18 h. ACE2 expressing Vero E6 cells and BHK-21 cells transfected with empty plasmid were used as positive and negative controls, respectively. To quantify the efficiency of S-mediated pseudotype entry, cells were lysed with 1× Cell Culture Lysis Reagent (Promega, Madison, WI, USA) for 30 min at room temperature. Lysates were transferred into white 96-well plates and firefly luciferase activity was measured using Beetle-Juice substrate (PJK, Kleinblittersdorf, Germany) and a Hidex Sense plate luminometer (Hidex, Turku, Finland).
8
SARS-CoV-2 S Protein Effector Function Assay
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293T effector cells grown to 75% confluency in 12-well plates were cotransfected with expression plasmids for the respective S protein or empty vector (1.5 µg/well) and the beta-galactosidase alpha fragment (0.5 µg/well) using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Subsequently, effector cells were washed, resuspended in 500 µl and added to 293T target cells (96-well format, 100 µl/well, four technical replicates) that were transfected with plasmids encoding ACE2 (0.1 µg/well) and the beta-galactosidase omega fragment (0.1 µg/well), or A549-ACE2 target cells (96-well format, 100 µl/well, four technical replicates) that were transfected with plasmid encoding the beta-galactosidase omega fragment (0.1 µg/well). Beta-galactosidase substrate (Gal-Screen, Thermo Fisher Scientific) was added (100 µl/well) after an additional 24 h of incubation, and samples were incubated for 90 min in the dark at room temperature before they were transferred into white 96-well plates and luminescence was measured using a Hidex Sense plate luminometer (Hidex).
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