Computational protocol: Alterations in c-Myc phenotypes resulting from dynamin-related protein 1 (Drp1)-mediated mitochondrial fission

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Protocol publication

[…] Mitochondria were visualized in cells expressing pEGFP-Mito or pDsRed2-Mito vectors. Cells were grown to ∼50% confluency in six-well, glass-bottom tissue culture plates. Fluorescent images of magnification × 20 were obtained with a Zeiss Axiovert fluorescent microscope (Wake Forest, NC, USA). Images of magnification × 60 were obtained on a Zeiss LSM710 confocal microscope (Wake Forest, NC, USA). Confocal stacks of the mitochondria were analyzed using Imaris software (Bitplane Scientific, South Windsor, CT, USA) and cellular 3D reconstructions were obtained as previously described. Mean fluorescence intensities were obtained on at least 50 individual cells and quantified using ImageJ software (http://rsbweb.nih.gov/ij/). Live-cell confocal microscopy was performed on cells stained with MitoTracker Green FM at a concentration of 1 μℳ for 30 min before the start of experiment. Cells were counterstained with Hoechst 33342 at a concentration of 2 μg/ml. During the imaging, cells were maintained in phenol red-free DMEM in which the MitoTracker and Hoechst dyes were added at 1/10 the original concentration. Images were captured on the LSM710 microscope approximately every 45 s. […]

Pipeline specifications

Software tools Imaris, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Rattus norvegicus
Diseases Neoplasms, Muscular Disorders, Atrophic, Cytochrome-c Oxidase Deficiency
Chemicals Adenosine Monophosphate, Adenosine Triphosphate