Cells were seeded into 96 well white opaque plates (Greiner) at 2000 cells per well and incubated at 37°C and 5% CO2 overnight. Cells were treated with selected drugs at different final concentrations and incubated for another 72 hr except for the initial studies of Aurora Kinase inhibitors in FLX1, where incubation was 120 hr. After incubation, plates and CellTiter-Glo (CTG, Promega) reagent were allowed to equilibrate at room temperature on the bench for 30 min. The CTG assay was performed following the manufacturer’s instructions and measured with a SpectraMax i3 Multi-Mode Platform (Molecular Devices). All experiments were done in at least biological duplicate with three technical replicates per condition. When multiple individual siRNA were used, the results are shown averaged in a standard dose response curve. In addition, AUC is calculated for each siRNA using GraphPad Prizm and shown as a separate point.
96 well white opaque plate
Manufactured by Greiner
Sourced in France
The 96-well white opaque plate is a laboratory equipment designed for various scientific applications. It features a 96-well format with a white opaque material. The core function of this product is to provide a platform for various types of assays and experiments.
Automatically generated - may contain errors
Lab products found in correlation
S30 t7 high yield protein expression system, by Promega (2 mentions)
Renilla luciferase assay kit, by Biotium (2 mentions)
Victor2, by PerkinElmer (2 mentions)
Celltiter glo ctg, by Promega (1 mentions)
Spectramax i3 multi mode platform, by Molecular Devices (1 mentions)
Caspase glo 8 reagent, by Promega (1 mentions)
Cytation 3 cell imaging multi mode reader luminometer, by Agilent Technologies (1 mentions)
Genesys 30, by Thermo Fisher Scientific (1 mentions)
Veritas microplate luminometer, by Promega (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Synergy h1, by Greiner (1 mentions)
Quanti luc, by InvivoGen (1 mentions)
7 protocols using 96 well white opaque plate
1
Cell Viability Assay Using CellTiter-Glo
2
Caspase-8 Assay for TRAIL and Nimesulide
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The caspase-8 assay was used to assess the effects of TRAIL only, nimesulide only, and nimesulide+TRAIL on caspase-8 activity, and nimesulide effective concentration. Cancer cells were seeded in 96-well white opaque plates (Greiner Bio-One) at 7,500 cells/well and incubated for 24 hours at 37°C. Cells were incubated for 24 hours at 37°C with TRAIL alone (0.000001–10 μg/mL) or nimesulide alone (0.001–200 μM) or with nimesulide (50 μM) + TRAIL (0.000001–10 μg/mL) or DMSO only. An equal volume of Caspase-Glo 8 reagent (Promega) was added to each well, and the luminescence was measured after 45 minutes using a Cytation 3 Cell Imaging Multi-Mode Reader luminometer (BioTek). To determine the effective concentration (EC50) of nimesulide, Jurkat cells were seeded in 96-well white opaque plates at 7,500 cells/well and incubated for 24 h at 37°C. Cells were treated for 2 hours with nimesulide (0.001–200 μM) before being treated for 24 hours with TRAIL (0.01–0.1 μg/mL) at 37°C. An equal volume of Caspase-Glo 8 reagent (Promega) for each sample was added to each well, and the luminescence was measured after 45 min.
3
In Vitro High-Yield Protein Expression Screening
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In this study, we used a S30 T7 high-yield protein expression system (Promega Corporation). We performed screening assays of our compounds in small-scale in vitro transcription–translation reactions using the included control DNA template containing the Renilla reniformis luciferase gene as previously described [21 (link)]. From each reaction, 2.5 μL samples were taken and diluted by adding 97.5 μL of the lysis buffer from the Renilla Luciferase Assay kit (Biotium) used. The mix was thoroughly mixed, then 50 μL were placed into a 96-well white opaque plate (Greiner). Right before the measurement, 50 μL of the assay reagent were added to all the samples, mixed and immediately placed in a luminometer (Perkin Elmer Victor2) for measurement.
4
Luminescent Bacterial Growth Assay
5
Renilla Luciferase Protein Expression Assay
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In this study, the S30 T7 high-yield protein expression system (Promega Corporation) was used. Screening assays of our compounds were performed in small-scale in vitro transcription/translation reactions, using the included control DNA template containing the Renilla reniformis luciferase gene as previously described [24 (link)]. From each reaction, 2.5 μL samples were taken and diluted by adding 97.5 μL of the lysis buffer from the Renilla Luciferase Assay kit (Biotium) used. The mix was thoroughly mixed and 50 μL was then placed into a 96-well white opaque plate (Greiner). Right before the measurement, 50 μL of the assay reagent was added to all the samples, which were then mixed and immediately placed in a luminometer (Perkin Elmer Victor2) for measurement. Data were presented as percent of control (without antibiotic).
6
Transient Transfection of HuH7 Cells
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HuH7 human hepatocarcinoma cells were kindly provided by J. Rosenbaum (INSERM U889, Bordeaux University, Bordeaux, France) and were maintained in Dulbecco's modified Eagle's medium-high glucose (Cat. No. D6429; Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum, 100 units mL -1 penicillin and streptomycin.
Twenty hours before transfection, cells were seeded at a density of 500,000 cells/well in 6well dishes. Transient transfections were performed using polyethylenimine (PEI, linear, Mr 25,000; Cat. No. 23966 Polysciences, Warrington, PA) with a PEI/DNA ratio of 4:1, as explained in Ruigrok et al. (2018) (Ruigrok et al. 2018) . Typically, transient transfections were performed using 8 µg PEI, 1 µg of the BRET probe of interest and 1 µg empty vector.
After overnight incubation, cells were then detached, and plated at a density of 10 5 cells into white-opaque 96-well plate (Greiner Bio One, les Ulis, France).
Twenty hours before transfection, cells were seeded at a density of 500,000 cells/well in 6well dishes. Transient transfections were performed using polyethylenimine (PEI, linear, Mr 25,000; Cat. No. 23966 Polysciences, Warrington, PA) with a PEI/DNA ratio of 4:1, as explained in Ruigrok et al. (2018) (Ruigrok et al. 2018) . Typically, transient transfections were performed using 8 µg PEI, 1 µg of the BRET probe of interest and 1 µg empty vector.
After overnight incubation, cells were then detached, and plated at a density of 10 5 cells into white-opaque 96-well plate (Greiner Bio One, les Ulis, France).
7
Quantitative Luciferase Assay for IFN Response
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RAW-Dual,
THP1-Dual, A549-Dual, and RAW-Dual ISG-KO-RIG-I cells
were seeded at 50,000 cells/well in 100 μL media in clear cell
culture-treated 96-well plates (Greiner Bio-One). When adherent cells
became ∼80% confluent or suspension cells reached a density
of 1.5 × 106 cells/mL, SLR-LNPs or controls were added
to wells at 2x concentration in 100 μL media. Supernatant was
collected 24 h after treatment, and the Quanti-Luc (Invivogen) assay
was used to determine the amount of secreted luciferase per the manufacturer’s
instructions. Briefly, 50 μL of Quanti-Luc solution was injected
into each well of a white opaque 96-well plate (Greiner Bio-One) containing
20 μL of collected supernatant per well on a Synergy H1 multimode
microplate reader, and each well was immediately read upon injection.
The average luminescence value of a negative control group (PBS-treated)
was subtracted from all other read luminescence values to take into
account the background. Finally, each dose–response curve was
fit using the GraphPad Prism software (log(agonist) vs response–four
parameter fit) to estimate EC50 values.
THP1-Dual, A549-Dual, and RAW-Dual ISG-KO-RIG-I cells
were seeded at 50,000 cells/well in 100 μL media in clear cell
culture-treated 96-well plates (Greiner Bio-One). When adherent cells
became ∼80% confluent or suspension cells reached a density
of 1.5 × 106 cells/mL, SLR-LNPs or controls were added
to wells at 2x concentration in 100 μL media. Supernatant was
collected 24 h after treatment, and the Quanti-Luc (Invivogen) assay
was used to determine the amount of secreted luciferase per the manufacturer’s
instructions. Briefly, 50 μL of Quanti-Luc solution was injected
into each well of a white opaque 96-well plate (Greiner Bio-One) containing
20 μL of collected supernatant per well on a Synergy H1 multimode
microplate reader, and each well was immediately read upon injection.
The average luminescence value of a negative control group (PBS-treated)
was subtracted from all other read luminescence values to take into
account the background. Finally, each dose–response curve was
fit using the GraphPad Prism software (log(agonist) vs response–four
parameter fit) to estimate EC50 values.
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