After overnight incubation in M199 with 0.1% BSA, confluent cultures were incubated in hypoxia (1% O2) or normoxia (20% O2) in α-mangostin or vehicle (DMSO) for 8 hours. Some cultures were also treated with SOD (superoxide dismutase, 400 units, Sigma-Aldrich) or catalase (400 units, Sigma-Aldrich). DHE (10 μM, Invitrogen, Carlsbad, CA) or DCF (25 μM, Sigma-Aldrich) in phenol red free M199 with 25 mM HEPES was added for 15 minutes. For DHE staining, images were taken at 200X magnification by using an inverted fluorescent microscope (Axiovert 135, Carl Zeiss, excitation, 480 nm; emission, 567 nm). The fluorescence intensity was quantified by MetaMorph Image System (Molecular Devices). For DCF staining, the fluorescence intensity was detected by microplate reader (Bio-Tek) with excitation 504 nm and emission 529 nm.
Metamorph image system
Manufactured by Molecular Devices
Sourced in United States
The Metamorph Image System is a comprehensive software solution designed for advanced image acquisition, processing, and analysis. It provides a versatile platform for a wide range of microscopy techniques, including fluorescence, brightfield, and live-cell imaging. The system offers robust features for image capture, customizable analysis workflows, and integrated data management, enabling researchers to efficiently explore and interpret complex biological phenomena.
Automatically generated - may contain errors
3 protocols using metamorph image system
1
Hypoxia-Induced Oxidative Stress Evaluation
2
Quantifying Retinal Superoxide Production
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Superoxide production was evaluated in retinal frozen sections by the dihydroethidium (DHE) method, as described previously. [43] (link) Briefly, frozen sections were preincubated in NADPH (100 µM) with or without PEG-SOD (400 U/mL) or NOS inhibitor L-NG-nitroarginine methyl ester (L-NAME, 100 µmol, 1 mM) for 20 minutes, followed by reaction with DHE (2 µM) for 20 minutes at 37°C. DHE images from serial sections treated with or without inhibitors were obtained using a fluorescence microscope (AxioVision; Carl Zeiss, Thornwood, NY). DHE was excited at 470 nm with an emission spectrum of 610 nm. The images were analyzed for reaction intensity using computer assisted morphometry (Metamorph Image System; Molecular Devices, Downingtown, PA).
3
Superoxide Quantification in Frozen Tissue Sections
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Dihydroethedium (DHE) method was used to evaluate superoxide formation as described previously.15 (link) Briefly, fresh frozen sections were preincubated with or without SOD-polyethylene glycol (400 U/ml, Sigma-Aldrich, St. Louis, MO, USA) for 30 min, followed by reaction with DHE (10 μM) for 15 min at 37 °C. Superoxide oxidizes DHE to ethidium bromide, which binds to DNA and fluoresces red.60 (link) The fluorescence microscope (AxioVision; Carl Zeiss) was used to obtain the DHE images immediately after incubation. DHE was excited at 488 nm with an emission spectrum of 610 nm. Six images per slide were taken and computer-assisted morphometry (Metamorph Image System; Molecular Devices, Sunnyvale, CA, USA) was used to analyze images for fluorescence intensity.
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