Quantifying Extracellular Hydrogen Peroxide

Hemocytes were plated in 96-well plates containing treatments in their respective culture mediums in a total volume of 70–100 µL with 5 replicates of each treatment. The plates were sealed with sealing tape and incubated for 3 h at 20 °C. Extracellular hydrogen peroxide was quantified using Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen™, Waltham, MA, USA, catalog no. A22188). A 100 μM Amplex^® Red reagent and 0.2 U/mL HRP working solution in 1× reaction buffer were prepared according to the manufacturer’s protocol. Hydrogen peroxide standards were prepared in 1× reaction buffer with concentrations between 0 and 10 µM. Plates were centrifuged using ThermoScienfic™ Megafuge 16R centrifuge at 1000× g rcf for 3 min, and 50 µL of the supernatant was transferred to a new 96-well plate. The working solution was transferred in 50 µL volume to each well containing the supernatant and the hydrogen peroxide standards, then incubated for 30 min in the dark. Absorbance was measured using a spectrophotometer (BioTek™, Winooski, VT, USA, PowerWave XS2) at λ = 560 nm and the results were normalized to the absorbance of the culture medium, expressed as relative to the control group with no pesticide exposure.