5 and 6 chloromethyl 29 79 dichloro dichlorofluorescein diacetate acetyl ester

To unravel the origin of the oxidative stress response in the blood-fed brain, we performed a reactive oxygen species (ROS) determination assay by incubating the brain tissue dissected from naïve and blood-fed mosquitoes with a 2 mM solution of the oxidant-sensitive fluorophores, CM-H2DCFDA [5-(and-6)-chloromethyl-29,79-dichloro-dichlorofluorescein diacetate, acetyl ester] (Sigma, St. Louis, MO, USA). After a 20-min incubation at room temperature in the dark, the brain tissues were washed thrice with PBS, and then transferred to a glass slide in a drop of PBS and checked the fluorescence intensity at wavelength 490 nm under a fluorescent microscope.

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.