4.7. Hydrogen Peroxide Measurements in Cell Culture

Cells were plated in a 96-well plate at 5000 cells in 80 µL of solution per well. For this assay, cells were plated in Gibco MEM media without phenol red and glutamine (51200038, Thermo Fisher Scientific, Waltham, MA, USA) with glutamine (Millipore Sigma Aldrich, G7513, Burlington, MA, USA) added to a final concentration of 1 mM. In addition, media contained 10% fetal bovine serum, 300 mg/mL streptomycin, 100 U/mL penicillin, and 1× non-essential amino acids. Cells were grown for 24 h. Instructions for the Promega ROS-Glo™ H₂O₂ assay (G8820, Promega, Madison, WI, USA) were followed. For the assay, 20 µL of substrate mix was added to each well and cells remained in CO₂ incubator for 4 h prior to adding 100 µL of ROS-Glo detection solution, incubating for 20 min at room temperature, and measuring the bioluminescence using a plate reader (FLUOStar Omega, BMG Labtech, Cary, NC, USA).