ATP-based measurements of cellular viability were performed by plating cells in 200 μL of media in 96-well plates. The number of cells and biological replicates seeded varied depending on the cell line and the duration of the experiment. At the indicated times, 40 μL of CellTiter-Glo reagent (Promega) was added to each well, mixed for 5 min, after which the luminescence was measured on the SpectraMax M5 Luminometer (Molecular Devices). For the drug treatment experiments, FRAX-597, Ruxolitinib, and Selumetinib were obtained from Selleckchem and Quizartinib from LC Laboratories.
Spectramax m5 luminometer
Manufactured by Molecular Devices
The SpectraMax M5 luminometer is a multi-mode microplate reader that can measure luminescence, fluorescence, and absorbance in microplates. It is capable of quantifying various biochemical and cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Celltiter glo reagent, by Promega (2 mentions)
Selumetinib, by Selleck Chemicals (1 mentions)
Frax597, by Selleck Chemicals (1 mentions)
Ruxolitinib, by Selleck Chemicals (1 mentions)
Quizartinib, by LC Laboratories (1 mentions)
Bioglo luciferase assay, by Promega (1 mentions)
Atp bioluminescence assay kit hs 2, by Roche (1 mentions)
Ready to glow secreted luciferase reporter system, by Takara Bio (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
6 protocols using spectramax m5 luminometer
1
Measuring Cellular Viability with ATP Assay
2
Cellular Proliferation Assay via ATP
3
Jurkat-PD-1 Cell Line for Inhibitory Checkpoint Assay
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The Jurkat-PD-1 cell line was firstly developed by stable expression of human PD-1 by puromycin-resistant lentivirus system and high-level PD-1 expressed cells were sorted by FACS. Then Jurkat-PD-1-NFAT cell line was generated by stable co-expression of pGL3 luciferase vector under control of NFAT response elements from the IL-2 promoter (#17870, Addgene). Cancer cells (5 × 105 cells) were seeded into a white 96-well plate and cultured at 37 °C and 5% CO2 for 12 h. After removing the medium, 1 × 106 Jurkat-PD-1-NFAT luciferase cells in 50 μL medium was added. The cells were stimulated with 5 µL anti‑CD3/anti‑CD28 activator (25 IU, #10,971, STEMCELL Technologies) and treated with each antibody (30 μg/mL). The plate was incubated at 37 °C 5% CO2 incubator for 6 h and 100 μL luminescence substrate (Bio-Glo™ Luciferase Assay, Promega) was added, and relative luciferase units were scored using a SpectraMax®M5 luminometer (Molecular Devices).
4
Gaussia Luciferase Assay for Gas Vesicles
5
Measuring Intracellular ATP Levels
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ATP Bioluminescence Assay Kit HS II (Roche Applied Science) was employed to measure intracellular ATP level. 50 μL of Cell lysate was mixed with 50 μL of luciferase reagent followed by thoroughly mix. Then signal was measured and integrated for 10 s by using a SpectraMax M5 luminometer (Molecular Devices).
6
NF-κB Promoter-Metridia Luciferase Assay
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The pNFκB-Metridia luciferase reporter plasmid contains an NF-κB promoter element upstream of the secreted Metridia luciferase (MetLuc) gene (Clontech Laboratories, Mountain View, CA). The pMetridia luciferase reporter plasmid contains several cloning sites that allow the insertion of promoter and/or enhancer elements upstream of the secreted MetLuc gene (Clontech Laboratories), which serves as an internal standard of transfection. Transient transfection was achieved using Lipofectamine® LTX (Life Technologies) according to the manufacturer’s instructions. Briefly, sub-confluent cells in 24-well plates were transiently transfected with 0.5 μg of the reporter construct and then stimulated 24 h later with whole P. gingivalis cells for 48 h in E-MEM containing 5% FBS. Secreted Metridia luciferase activity in culture supernatants was analyzed using Ready-To-Glow™ Secreted Luciferase Reporter Systems (Clontech Laboratories) and a SpectraMax M5 luminometer (Molecular Devices, Menlo Park, CA).
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