Mitochondria are cellular organelles that play a central role in cellular (patho)physiology and fulfil a wide range of functions in cellular metabolism. Within living cells, individual mitochondria are motile and display fission/fusion events. The balance between these processes, in combination with mitochondrial swelling/shrinking and removal of mitochondria by mitophagy determines net mitochondrial morphology. Mitochondrial structure and metabolism are tightly interfaced with the rest of the cell. Therefore, physiological analyses ideally are carried out using the natural mitochondrial “habitat”, being the cytosol of an intact living cell. In this respect, fluorescence microscopy is the most direct method to assess live-cell mitochondrial morphofunction. Mitochondria play a central role in cellular (patho)physiology and display a highly variable morphology that is probably coupled to their functional state. Here we present a protocol allowing unbiased and automated quantification of mitochondrial “morphofunction” (i.e. morphology and membrane potential), cellular parameters (size, confluence) and nuclear parameters (number, morphology) in intact living Primary Human Skin Fibroblasts (PHSFs). Cells are cultured in 96-well plates and stained with tetramethyl rhodamine methyl ester (TMRM), Calcein-AM and Hoechst 33258. Next, multispectral fluorescence images are acquired using automated microscopy and processed to extract 44 descriptors. Subsequently, the descriptor data is subjected to a quality control (QC) algorithm based upon Principal Component Analysis (PCA) and interpreted using univariate, bivariate and multivariate analysis. The protocol requires a time investment of about 4 hours distributed over 2 days. Although specifically developed for PHSFs that are widely used in preclinical research, the protocol is portable to other cell types and can be up-scaled for implementation in High-Content Screening (HCS).
The above study was carried out within the RIMLS and Radboud Center for Mitochondrial Medicine (RCMM) of the Radboudumc in a collaborative effort between the Department of Biochemistry and the SME Khondrion.
The paper was published as:
Iannetti, E.F., Smeitink, J.A.M., Beyrath, J., Willems, P.H.G.M., Koopman, W.J.H. (2016) Multiplexed high-content analysis of mitochondrial morphofunction using live-cell microscopy. Nature Protoc. 11:1693-1710.
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