HeLa and U2OS cells (500/well) were seeded in 96-well plate in 50 µl of complete media and grown for 24 hours. Then, serial dilutions of doxorubicin and etoposide (Selleck Chemicals) were prepared in DMSO and each combined to enoxacin in complete media containing 2X RealTime-Glo reagents (Promega), such that each compound was at 2X the final concentration. 50 µl of drug combinations were dispensed on cells and luminescence was read after 24 hours on a Tecan Infinite F200 plate reader (at 37 °C). Half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism® software for the two chemotherapeutics in the absence or presence of increasing amounts of enoxacin. All experiments were performed in triplicate wells for each condition and repeated twice.
Realtime glo reagent
Manufactured by Promega
Sourced in United States
The RealTime-Glo™ reagent is a bioluminescent cell-based assay system that measures cell viability in real-time. It utilizes luciferase-based technology to generate a luminescent signal proportional to the number of viable cells present in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Realtime glo mt cell viability assay, by Promega (2 mentions)
Etoposide, by Selleck Chemicals (1 mentions)
Doxorubicin, by Selleck Chemicals (1 mentions)
Prism 7 and 8, by GraphPad (1 mentions)
Ensight multimode plate reader, by PerkinElmer (1 mentions)
Victor 1430, by PerkinElmer (1 mentions)
Synergy h4, by Agilent Technologies (1 mentions)
Ultra low attachment 96 well plate, by Corning (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
8 protocols using realtime glo reagent
1
Synergistic Chemotherapy Potentiation by Enoxacin
2
Quantifying Breast Cancer Cell Viability
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Cell viability in
human breast cancer cells (MDA-MB-231) was quantified using Real-Time-Glo
MT Cell Viability Assay (Promega, G9712) as previously described.34 (link) Cells (2000 cells) were seeded into 96-well
white clear-bottom plates (Corning, 3903) in full serum media overnight
before washing and changing to suboptimal media (RPMI + 5% FBS + 1%
P/S) containing Real-Time-Glo reagents according to the Promega protocol.
A baseline luminescence read (prior to treatment) was taken using
an EnSight Multimode Plate Reader (PerkinElmer) after an hour of incubation
at 37 °C. Cells were then treated with compounds or vehicle control
(PBS + 0.05% DMSO) daily. Results were normalized to vehicle-treated
controls as 100% viable (Graphpad Prism 7 and 8).
human breast cancer cells (MDA-MB-231) was quantified using Real-Time-Glo
MT Cell Viability Assay (Promega, G9712) as previously described.34 (link) Cells (2000 cells) were seeded into 96-well
white clear-bottom plates (Corning, 3903) in full serum media overnight
before washing and changing to suboptimal media (RPMI + 5% FBS + 1%
P/S) containing Real-Time-Glo reagents according to the Promega protocol.
A baseline luminescence read (prior to treatment) was taken using
an EnSight Multimode Plate Reader (PerkinElmer) after an hour of incubation
at 37 °C. Cells were then treated with compounds or vehicle control
(PBS + 0.05% DMSO) daily. Results were normalized to vehicle-treated
controls as 100% viable (Graphpad Prism 7 and 8).
3
RealTime-Glo™ MT Cell Viability Assay
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Prior to the RealTime-Glo™ MT cell viability assay (Promega Corporation, Madison, US), HT-29 cells (1,500/well) were plated in a 96-well plate supplemented with 200 μL DMEM high glucose medium containing 10% fetal bovine serum (FBS) and antibiotic–antimycotic. The plate was incubated for 12 h at 37°C in a humidified 5% CO2 atmosphere to promote cell adherence. Test compounds (bortezomib, 5-KF, and D -fructose) were diluted in a medium at a concentration 2-fold higher than the target concentration desired in the subsequent assay. The RealTime-Glo reagent (Promega Corp.) was prepared as a 2x stock in the test compound diluent. Following incubation, the medium was removed and the cells were treated with 100 μL of medium containing the respective test compound and 100 μL test compound diluent containing the 2x RealTime-Glo™ reagent. Afterward, cells were incubated at 37°C in a humidified 5% CO2 atmosphere. In a discontinuous mode, the luminescence of each well was measured at 37°C using a Tecan Infinite 200 M Plex plate reader (Tecan Group AG). Per test compound, six biological replicates were performed.
4
HL-60 Cell Viability Assay
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The HL-60 cells (2000) were seeded into UV-C sterilized opaque 96-well plates (Tomtec, Budapest, Hungary) in 40 μL media. RealTime-Glo™ reagent (Promega) (2X) was added according to the instructions of the manufacturer. The effect of DU325 was examined in concentrations of 5 μM, 1 μM, 0.2 μM, and 0.04 μM in final volume 100 μL/well. We measured luminescence on a multimode microplate reader (Victor 1430, Wallac, Perkin Elmer) corresponding to live cell number after 1 min to register the baseline and subsequently samples were measured daily.
5
Tumor Organoid Viability Assay
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Tumor slices were prepared as described previously (Sivakumar et al. 2019 (link); Nishida-Aoki et al. 2020 (link)). Briefly, dissected tumor tissues were cut into 400-µm organotypic tumor slices using the Leica VT1200S vibratome microtome (Nusslock) with HBSS as the cutting medium. The slices were then cut into 400-µm cuboids using a McIlwain tissue chopper (Ted Pella) as described previously (Horowitz et al. 2021 (link)). Cuboids were immediately placed into 96-well ultralow-attachment plates (Corning) and incubated with Williams’ medium containing 12 mM nicotinamide, 150 nM ascorbic acid, 2.25 mg/mL sodium bicarbonate, 20 mM HEPES, 50 mg/mL additional glucose, 1 mM sodium pyruvate, 2 mM L-glutamine, 1% (v/v) ITS, 20 ng/mL EGF, 40 IU/mL penicillin, and 40 ug/mL streptomycin containing RealTime Glo reagent (Promega) according to the manufacturer's instructions. After 48 h, baseline cell viability of cuboids was measured by RealTime Glo bioluminescence using the Synergy H4 instrument (Biotek). Cuboids were exposed to either DMSO (control), staurosporine (200 nM), verteporfin, or Vivace Therapeutics compounds VT104 and VT107, and overall tumor tissue viability was measured daily, up to 7 d after treatment.
For more details, see theSupplemental Material .
For more details, see the
6
Cell Growth Kinetics Assay
7
Cell Viability Assay with Drugs
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Cells were seeded in IMDM plus 15% FBS in 96-well plates. Stocks of drugs were diluted, titrated into wells, and incubated for 24-72 h. Viability was measured by Promega Real-Time Glo reagent with fluorescence (SpectraMax i3x). Data was analyzed using GraphPad Prism.
8
Cell Viability Assay with RealTime-Glo
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Around 20 μL of the treated cells were added to the 96 well plate containing 55 μL of media. About 25 μL of RealTime-Glo™ reagent (Promega, USA) was added to assess the viability at 24 h. As per the manufacturer's protocol, the final concentration of MT reagent was maintained at 1X. Luminescence was measured for 1000 ms integration time using a SpectraMaxM5 multiplate reader (Molecular Devices, USA). To determine the number of viable cells, obtained experimental luminescence was normalized with respect to control luminescence using (4). [ 43 ]
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