Ros glo h2o2 luminescence assay

Sodium nitroprusside dihydrate (Na₂[Fe(CN)₅NO]·2H₂O, SNP, ≥99%) (SNP), silver nitrate (AgNO₃), and anhydrous methanol (99.8%) were purchased from Sigma-Aldrich (St. Louis, USA). A Millipore Milli-Q Biocell A10 water purifying system was used to prepare ultrapure water. A2780, A2780cis, U-87 MG (Sigma-Aldrich, St. Louis, MO, USA), MDA-MB-231 (Cell Biolabs, CA, USA) SK-OV-3, MCF-7, MRC-5, W1-38 (ATCC, Manassas, VA, USA) cells were cultured according to the manufacturers’ instruction. LysoTracker Green DND-26, Hoechst 33342, and rhodamine B were purchased from (ThermoFisher Scientific, Waltham, MA, USA). CellTiter-Glo® from (Promega, Madison, WI, USA), PE-Annexin V Apoptosis Detection Kit from (Becton-Dickinson Franklin Lakes, NJ, USA). ROS-Glo™ H₂O₂ luminescence assay from (Promega, Madison, WI, USA), DAX-J2 PON Green Kit was purchased from (AAT Bioquest, Sunnyvale, CA, USA), FluorSave™ reagent (Millipore, Burlington, MA, USA). qPCR Primers from (Integrated DNA Technologies, Bologna, Italy).

For quantitative determination of H₂O₂ level, A2780, SK-OV-3 and MRC-5 were plated in 96 multi well at a density of 1x10⁴ per well and were cultured at 37.0 °C for 24h. Then cells were treated with 10 µg/ml of AgNP, AgNO₃, SNP and cisplatin (CisPt) (3 µg/ml: 10 μM) for different time points (6, 12 and 24h). Oxidative stress was evaluated using ROS-Glo™ H₂O₂ luminescence assay (Promega, Madison, WI, USA) corresponding to manufacturer's guidelines and then read with a Tecan M1000 instrument.