The increase in ROS levels results in a decrease in the levels of antioxidants. To confirm the presence of intracellular ROS, the levels of the antioxidant, glutathione (GSH), was measured using the GSH-Glo™ Glutathione assay kit (V6911, Promega, Wisconsin, USA), according to the manufacturer's instructions. Briefly, cells (2500-4500 cells per well) were seeded in opaque 96 well plates. Cells were treated for 24 h with either vehicle or MMV652103. Thereafter, the cell culture medium was removed and 100 μl of 1X GSH-Glo™ Reagent (prepared using Luciferin-NT substrate and Glutathione S-Transferase each diluted 1:100 in GSH-Glo™ Reaction Buffer) was added to each well. The plate was then mixed briefly on a shaker and incubated for 10 min at RT. Thereafter, 100 μl reconstituted Luciferin Detection Reagent was added to each well. The plate was then incubated for 15 min RT in order to stabilize the luminescence signal. The plate was read using the GloMax® Explorer Multimode plate reader (Promega, Wisconsin, USA).
Glomax explorer multimode plate reader
Manufactured by Promega
Sourced in United States
The GloMax® Explorer Multimode plate reader is a versatile instrument designed for detection and quantification of various luminescent, fluorescent, and absorbance-based assays in a microplate format. It provides accurate and reliable measurements across a range of applications.
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Lab products found in correlation
4 protocols using glomax explorer multimode plate reader
1
Measuring Intracellular Oxidative Stress
2
Measurement of Oxidative Stress in Breast Cancer Cells
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To determine whether MMV652103 induced oxidative stress in breast cancer cells, the levels of H2O2 were measured using the ROS-Glo™ H2O2 assay kit (G8820, Promega, Wisconsin, USA), according to the manufacturer's instructions. Briefly, cells (2500-4500 cells per well) were seeded in opaque 96 well plates. Cells were treated for 24 h with either vehicle or MMV652103 in a volume of 80 μl per well. After 18 h of incubation, 20 μl H2O2 Substrate Solution was added to each well. After 24 h of incubation 100 μl ROS-Glo™ Detection Solution (prepared by mixing 10 ml Luciferin Detection Reagent, 100 μl D-Cysteine and 100 μl Signal Enhancer Solution) was added to each well and incubated at RT for 20 min. After incubation, the luminescence was read using the GloMax® Explorer Multimode plate reader (Promega, Wisconsin, USA).
3
Dual-Luciferase Assay for Protein Synthesis
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HeLa and HEK293T cells were seeded in 96-well plates with 100 µL of medium. After 24 h, cells (∼50% confluency) were transfected with 100 ng of plasmid DNA (50 ng of nLuc plasmid, 50 ng of pGL4.13 [FFLuc]) using Viafect (Promega) at a 2:1 (reagent:DNA) ratio following the manufacturer's protocol. IMR90 cells were seeded in 96-well plates with 100 µL of medium, grown until ∼50% confluent (often 4–5 d), and then transfected using Viafect at a 4:1 ratio. Twenty-four hours after transfection, cells were either lysed in well with 100 µL of Glo lysis buffer (Promega) for 10 min and frozen at −80°C (for the control) or treated with 100 µg/mL CHX (or other inhibitors as described in the figure legends) for the indicated amounts of time before lysis/sample freezing. Luciferase assays were then performed on thawed samples by mixing equal volumes of cell lysate with either ONE-Glo (Promega) for FFLuc or Nano-Glo (Promega) for nLuc in black 96-well plates for 5 min. Luminescence was measured on a GloMax Explorer multimode plate reader (Promega). The cotransfected FFLuc was used as an internal control to confirm protein synthesis inhibition but was not used for normalization except where indicated. In assays using NSC119893, fresh medium with inhibitors was replaced 1.5 h after the initial treatment, as described previously (Robert et al. 2006 (link)).
4
Cytotoxicity and Proliferation Assay of H9c2 Cells
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The cytotoxicity and proliferative effect of H9c2 cells were determined colorimetrically using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. H9c2 cells were seeded at 2 × 104 cells/well in 96-well plates. For the cytotoxicity assay, H9c2 cells were treated with the serial dilution of carpaine ranging from 10−4–102 µM for 24 h to determine the 50% inhibitory concentration (IC50) of carpaine. For the proliferation assay, cells were treated with carpaine for 8, 24, and 48 h, and images were taken at each time point using an Olympus CKX41 inverted microscope. Following the incubation period, a 0.5 mg/mL final concentration of MTT solution was added to each well and incubated for 1–4 h at 37 °C. The supernatants from each well were removed, and precipitated formazan crystals were dissolved in 100 µL of DMSO. The absorbance was read at 560 nm using a GloMax Explorer Multimode plate reader (Promega).
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