Ros glotm h2o2 assay

The cells were seeded in 96-well microplates (1.5 × 10³ cells/well) and, after 24 hours, treated with metformin. Hydrogen peroxide (H₂O₂) levels were measured using the ROS-Glo^TM H₂O₂ assay (Promega, Fitchburg, WI, USA) in accordance with the manufacturer’s instructions. The bioluminescent readings were obtained from three independent experiments using a microplate reader (TECAN Group Ltd., Männedorf, Switzerland).

The human lung adenocarcinoma cell line A549 and all the components used for cell cultures were purchased from Sigma-Aldrich (Milan, Italy). The CellTiter 96® AQueous One Solution Cell Proliferation Assay and ROS-Glo^TM H₂O₂ Assay were purchased by Promega (Madison, MI, United States). All components used for RNA extraction and RT-PCR, including TaqMan primers, were purchased from Invitrogen, Life Technologies (California, United States). The Bradford Assay reagent and iScript cDNA synthesis kit were purchased from Bio-Rad (Hercules, CA, United States). Nanoparticles based on polystyrene (800 nm) (PS-NPs) were obtained from Sigma-Aldrich (Milan, Italy).

The NADP/NADPH-Glo^TM assay (Promega, Madison, WI) was used to detect total oxidized and reduced nicotinamide adenine dinucleotide phosphates. The ROS-Glo^TM H₂O₂ assay (Promega, Madison, WI) was used to measure the level of hydrogen peroxide (H₂O₂) in culture. All assays were performed as directed in the protocol. Briefly, for NADP/NADPH measurements, tumor cells were seeded at the same concentration (10⁴ per well) in triplicate in 24 well plates for 6 hours prior to the addition of an equal volume of the NADP/NADPH-Glo ^TM detection reagent to each well. The luminescence is read after incubation with the detection reagent for 60 min at room temperature. For H₂O₂ measurements, tumor cells were seeded at the same concentration (10⁴ per well) in triplicate in 24 well plates for 24 hours (aerobic incubator). 25μM of the H₂O₂ substrate was added for the last 6 hours of incubation and 50μl of media samples per well were removed and mixed with detection solution (50μl). Luminescence was measured after incubation with the detection solution for 20 minutes at room temperature.

The ROS‐GloTM H₂O₂ assay (Promega) was used to assess cellular H₂O₂ levels according to the manufacturer's instructions. The plate was read using a Glo‐Max luminometer with the Cell‐titre Glo in built protocol (PMT activated). Menadione (20 μM) was used as a positive experimental control.

The ROS-Glo^TM H₂O₂ assay (Promega, Milan, Italy) was used to detect hydrogen peroxide (H₂O₂) levels in cell culture according to the manufacturer’s instructions. The ROS assay was performed by plating 1 × 10⁴ cells into each well of a 96-well plate. Cells were treated with compounds, incubated with H₂O₂ substrate solution for 6 h, after which ROS-Glo detection solution was added. Luminescence units were measured using GloMax^® 96 microplate luminometer (Promega, Milan, Italy). Values were normalized to protein concentration.