Eosinophil Reactive Oxygen Species Measurement

For each examined individual experiment, a fresh Dihydrorhodamine (DHR)-123 (Sigma-Aldrich, Chemie GmbH, Taufkirchen, Germany) dye working solution was prepared. DHR-123 is a fluorescence probe commonly used for measuring ROS [56 (link)]. It can passively diffuse through cell membranes and then, after being exposed to intracellular nitric oxide and peroxynitrite, DHR-123 is oxidized to rhodamine-123, which exhibits green fluorescence and stains mitochondria inside a living cell [57 (link)], thus allowing detection by flow cytometer. After isolating eosinophil subtypes, flow cytometer test tubes (Corning Falcon, Newport, Tennessee, USA) were prepared (5 × 10⁴ of an eosinophil subtype in each tube), and 1x PBS was added to a final volume of 200 μL. A separate test tube with 200 μL DHR-123 was prepared to evaluate reagent contamination. Each experimental tube was supplemented with DHR-123 working solution (final concentration 750 ng/mL), mixed by gentle vortexing, and incubated for 45 min, a sufficient amount of time for fluorescence evaluation [58 (link)], at standard conditions (5% CO₂ in air at 37 °C). For flow cytometry calibration, a test tube with 5 × 10⁴ eosinophils in PBS, but without DHR-123, was prepared. After incubation, the relative amount of ROS formed was evaluated in a flow cytometer by measuring the mean fluorescence intensity (MFI) of the investigated eosinophil subtype population.