Cells ectopically expressing a mitochondrial GFP tag (3xHA-EGFP-OMP25; Addgene #83356) were seeded in a glass bottom, 4 compartment dish and incubated in DMEM containing TMRE (25 nM; Invitrogen) and verapamil (10 μM; Caymen Chemicals), to facilitate the loading of TMRE into cells, 40 min prior to imaging. Cells were additionally treated with carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; 10 μM; Sigma) for 30 min before imaging. Hypoxic cell samples were sealed in a portable microscope stage sealed within the hypoxia chamber. Further transfer of the stage to the microscope was achieved by flooding the stage with a pre-made gas tank of 0.1% O2 and 5% CO2 maintained at positive pressure. Images were collected with a Nikon Ti microscope with a Plan Apo 20x N.A. 0.75 DIC lens and an Andor Zyla 5.5 sCMOS camera. Using Fiji software (http://fiji.sc/ ), TMRE fluorescence was calculated by subtracting basal fluorescence (FCCP treated) and normalized to mitochondrial content by dividing GFP fluorescence intensity. The number of visual fields analyzed per experimental condition are outlined in the figure legends.
Plan apo 20x n a 0.75 dic lens
Manufactured by Oxford Instruments
The Plan Apo 20x N.A. 0.75 DIC lens is a high-performance objective lens designed for use in microscopy applications. It features a 20x magnification and a numerical aperture of 0.75, providing excellent resolution and image quality. The lens is optimized for Differential Interference Contrast (DIC) imaging, which enhances the contrast of transparent samples. The Plan Apo design ensures a flat image field and high image quality across the entire field of view.
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2 protocols using plan apo 20x n a 0.75 dic lens
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Mitochondrial Function Under Hypoxia
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Mitochondrial Function Under Hypoxia
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Cells ectopically expressing a mitochondrial GFP tag (3xHA-EGFP-OMP25; Addgene #83356) were seeded in a glass bottom, 4 compartment dish and incubated in DMEM containing TMRE (25 nM; Invitrogen) and verapamil (10 μM; Caymen Chemicals), to facilitate the loading of TMRE into cells, 40 min prior to imaging. Cells were additionally treated with carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; 10 μM; Sigma) for 30 min before imaging. Hypoxic cell samples were sealed in a portable microscope stage sealed within the hypoxia chamber. Further transfer of the stage to the microscope was achieved by flooding the stage with a pre-made gas tank of 0.1% O2 and 5% CO2 maintained at positive pressure. Images were collected with a Nikon Ti microscope with a Plan Apo 20x N.A. 0.75 DIC lens and an Andor Zyla 5.5 sCMOS camera. Using Fiji software (http://fiji.sc/ ), TMRE fluorescence was calculated by subtracting basal fluorescence (FCCP treated) and normalized to mitochondrial content by dividing GFP fluorescence intensity. The number of visual fields analyzed per experimental condition are outlined in the figure legends.
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