Cyto-Grx1-roGFP2 [34 (link)] adenovirus was added to the media on the second day of culture. Experiments were done on the 4th–5th day of culture. Images were taken using a spinning disk confocal microscope (Andor Revolution XD, Olympus) at 10× magnification and analyzed using ImageJ and MATLAB. For each data point, two images were taken using laser excitation at 405 nm and 488 nm and recording the emission at 525 ± 25 nm. The excitation ratio (405 nm / 488 nm) indicates the redox status of the GSH/GSSG couple. Cyto-Grx1-roGFP2 ratios were normalized to between 0–1, with 1 and 0 representing the maxim and minimum levels of oxidation, respectively, during the ischemic or reperfusion phases.
Andor revolution xd
Manufactured by Olympus
The Andor Revolution XD is a high-performance scientific imaging system designed for advanced microscopy applications. It features a state-of-the-art scientific camera with high sensitivity, resolution, and speed, making it suitable for a wide range of imaging techniques.
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4 protocols using andor revolution xd
1
Measuring Cellular Redox Status with Cyto-Grx1-roGFP2
2
Spinning-Disk Confocal Microscopy for Live-Cell Imaging
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The fluorescent images were acquired by a spinning-disk confocal microscope Andor Revolution XD with 100 × N.A. 1.4 oil immersion or 60 × N.A. water immersion objective on the Olympus IX-71 inverted optical microscope. Generally, the z-stacks were acquired before and after the acquisition of an AFM image of the same cell. The spacing between the optical sections was chosen based on the Nyquist criterion (0.21 μm for the used setup). The imaging parameters (laser intensity and exposition time) were adjusted in preliminary experiments to decrease the acquisition time and still preserve high signal-to-noise ratio and low phototoxicity. The fluorescent images were analyzed by Fiji (NIH, Bethesda, MD).
3
Fluorescence Imaging of Bacterial Cultures
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The densities of the fresh overnight bacterial cultures were adjusted to the 0.5 McFarland standard, and 100 µL was placed in a sterile 96-well plate. The plates were incubated at room temperature for 2 h before the amount of fluorescently labeled F-PEP or F-P-PEP construct corresponding to its MIC was added to the appropriate wells. Images were captured immediately using a spinning disc confocal microscope (Andor xD revolution) on the Olympus IX81 platform and analyzed with iQ3 software, the excitation wavelength was 488 nm and the emission wavelength was 525 nm.
4
Calcium Imaging of BMMCs
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BMMCs in RPMI were loaded with 1 μM Fluo-4 AM (Life Technologies) with 0.02% Pluronic F-127 for 10 minutes at 37°C, washed twice in Tyrode’s buffer, and recovered in Tyrode’s buffer (135 mM NaCl, 5.5 mM glucose, 5 mM KCl, 1 mM MgCl2, 1.3 mM CaCl2, 0.5% BSA, pH 7.4) for 15 minutes at 37°C before imaging. Images were acquired every 5 seconds on an Andor XD revolution spinning disk confocal microscope (Olympus), and fluorescence was quantitated using MetaMorph software (Molecular Devices). Data were expressed as fold over baseline fluorescence. Cells that moved out of focus during the experiment or did not respond to a final administration of ionomycin were excluded from analysis.
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