Computational protocol: Slower Dynamics and Aged Mitochondria in Sporadic Alzheimer's Disease

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Protocol publication

[…] Cells were infected at an approximate multiplicity of infection (MOI) of 5–10 with a lentivector encoding DsRed2-Mito construct obtained from Clontech (632421) that was kindly provided by Dr. Ismael Santa-María (Columbia University, NY). Cells were treated with 20 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP, Sigma, St. Louis Missouri) for 6 h and, in the reversible condition, CCCP was removed after 1 h and the medium was replaced for DMEM 10% FCS. After the treatments cells were fixed with 4% paraformaldehyde (PFA) and the mitochondrial pattern was observed with an Axioskop2 plus microscope coupled to a CCD color camera (Zeiss). For analysis, we used the Mitochondrial Network Analysis (MiNa) toolset, a combination of different ImageJ macros that allows the semiautomated analysis of mitochondrial networks in cultured mammalian cells []. Briefly, the image was converted to binary by thresholding following the conversion to a skeleton that represents the features in the original image using a wireframe of lines of one pixel wide. All pixels within a skeleton were then grouped into three categories: end point pixels, slab pixels, and junction pixels. The plugin analyzes how the pixels are spatially related and defined to measure the length of each branch and the number of branches in each skeletonized feature as well as the mitochondrial network morphology. The parameters used in the study were (1) individuals, punctate, rods, and large/round mitochondrial structures; (2) networks, mitochondrial structures with at least a single node and three branches; (3) the mean number of branches per network; and (4) the average of length of rods/branches. Ten randomly chosen fields containing between 10 and 15 cells were used to quantify the pattern of mitochondria. We classify the mitochondrial morphology into three different subtypes according to the length of the branches: filamentous (long and spaghetti-like shape; branch > 2.3 μm), fragmented (completely dotted; branch < 1.8 μm), and intermediate pattern (when both filamentous and fragmented mitochondria were found; 1.8 μm ≥ branch ≥ 2.3 μm). [...] Colocalization analysis was performed with ImageJ software (Bethesda, MD). The background of different channels was edited with the subtract background tool with a rolling ball radius of 30 pixels, and binary images were obtained by a threshold intensity. The logical operation AND of the Image Calculator tool was used to generate an image harboring only overlapping structures of both channels. Colocalization measurement was obtained by quantifying the area occupied by the overlapping elements per cell. At least 200 cells were measured for each cell line. […]

Pipeline specifications

Software tools MiNA, ImageJ, Coloc
Application Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Alzheimer Disease, Mitochondrial Diseases