A total of 1 × 104 HAK‐1B cells/well were seeded in a white 96‐well plate (Thermo Fisher Scientific, Waltham, MA, USA) 2 days before the analysis. ROS levels were measured using the ROS‐Glo H2O2 Assay kit (Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions. Briefly, the cells were incubated with ROS‐Glo Detection Solution for 20 minutes before treatment with different concentrations of SASP at (0, 50, and 100 μM) and CDDP (0 and 20 μM) with or without 1 mM NAC for 6 hours. Cell‐derived ROS in the plates were analyzed using the GLOMAX‐Multi+ Luminescence System (Promega). ROS levels were determined as values relative to untreated control. Each assay was carried out in triplicate.
Glomax multi luminescence system
Manufactured by Promega
The GLOMAX‐Multi+ Luminescence System is a multi-mode microplate reader designed for the detection and quantification of various luminescent assays. It offers versatile measurement capabilities, including luminescence, fluorescence, and absorbance, within a single compact instrument.
Automatically generated - may contain errors
Lab products found in correlation
White 96 well plate, by Thermo Fisher Scientific (1 mentions)
Ros glo h2o2 assay kit, by Promega (1 mentions)
Gsh glo glutathione assay kit, by Promega (1 mentions)
Xfect mesc transfection reagent, by Takara Bio (1 mentions)
Prl sv40, by Promega (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
3 protocols using glomax multi luminescence system
1
Quantifying Cellular ROS Levels
2
Intracellular GSH Quantification in CDDP-Treated Cells
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Intracellular levels of GSH were measured using the GSH‐Glo Glutathione Assay kit (Promega) according to the manufacturer's instructions. First, 1 × 104 HAK‐1B cells/well were cultured in a white 96‐well plate overnight and treated with 20 μM CDDP, with DMSO vehicle as a control, with or without SASP at concentrations of 0, 50, and 100 μM for 3 hours. The cells were washed with water and GSH‐Glo reagent was added to the plate. After incubation, luciferin detection reagent was added and luminescence was measured using the GLOMAX‐Multi+ Luminescence System (Promega). All measured GSH concentrations were normalized by the cell number counted at the time of measurement. Total GSH concentration was determined from a standard curve prepared using known GSH concentrations. All experiments were conducted in triplicate.
3
Tagln Promoter Luciferase Assay
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The Tagln promoter [14] was amplified from the mouse genome by polymerase chain reaction (PCR) using the following primers: forward 5'-GCTCGCTAGCCTCGA AGTCAAGACTAGTTCCCACC-3' and reverse 5'-AGGCCAGATCTTGATGGGGCGCCGGCTGGGTGAGG-3' (the In-Fusion sites are underlined). The PCR product was inserted into the multi-cloning site of a pGL4.15 firefly luciferase reporter vector (Promega, Madison, WI, USA) using the In-Fusion HD Cloning Kit (Takara Bio Inc.). A pGL4.15 plasmid was co-transfected with pRL-SV40 (Promega) into MOVAS or UV2 cells using Xfect mESC Transfection Reagent (Clontech, Palo Alto, CA, USA) according to the instructions provided by the manufacturer. Following transfection, the cells were incubated in serumfree medium for two days and prepared using the Dual-Luciferase Reporter Assay System (Promega).
Luciferase activity was measured using the GloMax-Multi Luminescence System (Promega).
Luciferase activity was measured using the GloMax-Multi Luminescence System (Promega).
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