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INSIGHTS ON LOCALIZED ROS FORMATION AND PH INISOLATED BIVALVE GILLS: A LIVE-IMAGING APPROACH

Georgina A. Rivera‐Ingraham,Iara Rocchetta,Ulf Bickmeyer,Doris Abele-2013-09-01
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TL;DRAbstract

Live-imaging techniques (LIT), using specific dyes and quantifying the resulting fluorescence signals under a confocal microscope, have traditionally been used with isolated cells and tissues, to study various physiological parameters in-vitro. In the present study we report the successful application of LIT to complete gill filaments for the analysis and characterization of reactive oxygen species (ROS) and nitric oxide (NO) formation. Our results indicate that H2O2 (hydrogen peroxide), HOO• (peroxyl radical) and ONOO- (peroxynitrate anion) radicals (assessed through C-H2DFFDA staining) are mainly formed in the blood sinus of the filaments. Contrary, in the periphery of the filaments, with the lateral cilia and highest mitochondrial densities (MTK Deep Red 633 staining), staining with O2•- (DHE) and NO (DAF-2) sensitive dyes was highest. These peripheral areas also show the most acidic pH values (as observed with the dye ageladine-a) and mitochondria with low membrane p

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Live-imaging techniques (LIT), using specific dyes and quantifying the resulting fluorescence signals under a confocal microscope, have traditionally been used with isolated cells and tissues, to study various physiological parameters in-vitro. In the present study we report the successful application of LIT to complete gill filaments for the analysis and characterization of reactive oxygen species (ROS) and nitric oxide (NO) formation. Our results indicate that H2O2 (hydrogen peroxide), HOO• (peroxyl radical) and ONOO- (peroxynitrate anion) radicals (assessed through C-H2DFFDA staining) are mainly formed in the blood sinus of the filaments. Contrary, in the periphery of the filaments, with the lateral cilia and highest mitochondrial densities (MTK Deep Red 633 staining), staining with O2•- (DHE) and NO (DAF-2) sensitive dyes was highest. These peripheral areas also show the most acidic pH values (as observed with the dye ageladine-a) and mitochondria with low membrane p

Keywords

StainingHydrogen peroxideChemistryReactive oxygen speciesBiophysicsVesicleFluorescence microscopeMitochondrion

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