Quantifying Hydrogen Peroxide in Drosophila

Hydrogen peroxide was quantified using the Amplex Red hydrogen peroxide/peroxidase assay kit (Invitrogen catalog no. A22188) as previously demonstrated but with minor modifications (30 (link)). Three female adult D. melanogaster flies were collected and homogenized in 300 μl of PBS with three 2-mm glass beads beating for 10 s at 4,200 rpm. For Canton-S flies, heads were removed because of the intense red eye pigment. Samples were centrifuged at 12,000 × g for 3 min (room temperature), and 50 μl of supernatant was used for the assay following the manufacturer’s protocol with spectrophotometry quantification at 560 nm or excitation/emission of 535/595 nm using a BioTek Eon microplate reader or Eppendorf PlateReader AF2200, respectively. Hydrogen peroxide concentrations were normalized to total protein and plotted as relative H₂O₂ to the vehicle. Total protein was quantified using a bicinchoninic acid (BCA) protein assay kit (Invitrogen catalog no. 23227) following the manufacturer’s microplate protocol. Protein was measured from samples that were obtained from the H₂O₂ determination protocol and used to normalize H₂O₂ quantification. Samples were centrifuged at 12,000 × g for 3 min (room temperature), and 25 μl was used for quantification as per the manufacturer’s microplate protocol using a BioTek Eon microplate reader at 562 nm.