ROS Generation Measurement in Mosquito Tissues

To determine the level of ROS generation, MRO (MAG, Testis and seminal vesicles) from HPX12 silenced or control mosquitoes were collected in PBS and incubated with a 2 mM solution of the oxidant-sensitive fluorophores, CM-H2DCFDA [5-(and-6)-chloromethyl-29,79-dichloro-dichlorofluorescein diacetate, acetyl ester] (Sigma) for 20 min on a microscopic glass slide. After a series of experimental trial, the final concentration of DCFDA was optimized to 0.2 mM^{41 (link)}. Following, incubation for 20-min at room temperature in the dark, the MRO was washed three times with 1xPBS. Next, the MRO was transferred to a new glass slide in a drop of fresh PBS and checked the fluorescence at 400 × magnification under a fluorescence microscope, which emits a mercury excitation beam to assess green fluorescence at 480 nm and 530 nm, respectively. Fluorescent images were acquired using an inverted fluorescent microscope (BX61 Olympus), with a CC12 fluorescence camera (Olympus Co., Tokyo, Japan). Pixel intensity within microphotographs was determined and analyzed using Image J 1.240 software, and the redox state measurements were expressed as arbitrary units.