Amplex red hydrogen peroxidase assay kit

Cellular peroxidase activity was measured using the Amplex Red Hydrogen Peroxidase assay kit (Invitrogen, CA, USA) according to the manufacturer’s protocol. Briefly, Raji cells (2 × 10⁵ cells/ml) were incubated with or without a 400-cycle ZnO chip for the indicated time, and the cellular peroxidase activity then was measured at _Ex530 nm and _Em590 nm in a Wallac EnVision microplate reader (PerkinElmer, MA, USA).

An Amplex® Red Hydrogen Peroxidase assay kit (Invitrogen) was used to determine whether AgGom or HiGom caused intracellular toxicity in MM96L cells by generation of ROS, a known marker of intracellular oxidative stress in response to environmental noxious conditions. Carboxy-H2DCFDA (Invitrogen), a fluorescence probe that detects intracellular hydrogen peroxide, was added 30 min prior to harvesting cells to measure ROS production in MM96L cells. Fluorescent cells were first washed twice with PBS before being analysis with a BD FACSCalibur™ flow cytometer (BD Biosciences) using excitation and emission wavelengths of 492 nm and 517 nm, respectively. Approximately 10,000 events were recorded per sample and the read-out was analysed using FlowJo v10.06. In a separate series of experiments, MM96L cells were pre-treated with 10 µM mitoTEMPO, a mitochondrially-targeted superoxide scavenger, for 2 h prior to gomesin addition.