Quantification of Total Free Radicals in Drosophila Using a Fluorescence-Based Biochemical Assay.

Abstract

Free radicals, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), induce oxidative stress. This stress plays crucial roles in cellular signaling, stress response, and disease progression, making the quantification of free radicals essential for understanding oxidative stress mechanisms. Here, we present a high-throughput fluorescence-based protocol for measuring the presence of total free radicals, including ROS and RNS, in the whole adult Drosophila melanogaster (fruit fly). The protocol involves homogenizing whole adult flies in PBS and treating only the supernatant of the lysate with dichlorodihydrofluorescein-DiOxyQ (DCFH-DiOxyQ), which then converts into a fluorescent molecule, dichlorofluorescein (DCF), upon reacting with free radicals. The level of fluorescence is directly proportional to the amount of free radicals present in the sample. This protocol offers simplicity, scalability, and adaptability, making it ideal for studying oxidative stress in the model organism Drosophila and its different tissues under different dietary regimes, environmental stresses, genetic mutations, or pharmacological treatments. It is to be noted that the protocol uses a kit from Abcam, which has been used to measure free radicals in mice, rats, human blood, and cell lines. It can also be applied to biofluids, culture supernatants, and cell lysates, making it suitable for a wide range of sample types beyond whole organisms or tissues. However, due to our research focus and expertise, here we describe a detailed protocol to measure free radicals responsible for inducing oxidative stress only in fruit flies. Key features • Quantifies total free radicals including ROS and RNS levels in adult Drosophila melanogaster using a fluorescence-based approach for oxidative stress studies. • Suitable for high-throughput analysis with a 96-well black plate format, simultaneously enabling efficient handling of multiple samples and standards. • Adaptable to different experimental conditions, including diverse ROS-inducing treatments and mutations in Drosophila. • Offers detailed instructions for reagent preparation, sample homogenization, fluorescence measurement, normalization, and statistical analysis of data to ensure reproducibility and accuracy across research settings. Graphical overview Schematic workflow of the assay. Whole adult fruit flies are homogenized in PBS buffer and centrifuged. The clear supernatant is carefully transferred into new tubes for further treatment with different reagents, loaded into a clear-bottom black 96-well plate, and treated with another set of reagents. The plate is then incubated, and fluorescence is measured using the Agilent BioTek Synergy H1 plate reader.