In the presence of cellular oxidants, nonfluorescent DCF is converted into the fluorescent 5-(and-6)-chloromethyl-2,7-dichlorofluorescein (CM-DCF) and can be observed by confocal microscopy.
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In addition, fresh frozen left ventricle myocardium (8-μm slices) was incubated with 4 μmol/L 2′,7′-dichlorofluorescein (DCF) diacetate for 1 hour at 37°C.
Images were taken by confocal microscopy (Nikon PCM-2000).
The analyses were performed using tetra-methyl-rhodamine ester (TMRE) dye reagent (Invitrogen, Carlsbad, Calif).
Isolated mitochondria were incubated with 100 nmol/L TMRE and observed under a fluorescence microscope at an excitation wavelength of 549 nm and emission wavelength of 574 nm.
These were fit to a monoexponential [ ROS activity was measured using 2 different methods.
Peroxidase and XO activity were determined in whole-cell homogenates using Amplex Red reagent (Invitrogen) according to the protocol of the manufacturer.
Oxypurinol also largely restored normal mitochondrial morphology.
Surprisingly, however, cardiac contractile function and cardiac compliance were unimproved, indicating that the contractile deficit might be independent of mitochondrial dysfunction and the initiation of apoptosis.
Despite these improvements, subsequent developing deficits in cardiac function were unaffected, suggesting that additional factors intrinsic to the contractile apparatus and/or its milieu were responsible for the contractile dysfunction observed in the Cry AB-overexpressing transgenic mice (TGox) and compared them to controls (nontransgenic [NTG]), and an untreated transgenic (TG) group.