title: Multiplexed high-content analysis of mitochondrial morphofunction using live-cell microscopy.
abstract: Mitochondria have a central role in cellular (patho)physiology, and they display a highly variable morphology that is probably coupled to their functional state. Here we present a protocol that allows 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 (acetoxy-methyl ester) and Hoechst 33258. Next, multispectral fluorescence images are acquired using automated microscopy and processed to extract 44 descriptors. Subsequently, the descriptor data are 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 ∼4 h distributed over 2 d. Although it is specifically developed for PHSFs, which are widely used in preclinical research, the protocol is portable to other cell types and can be scaled up for implementation in high-content screening.
authors: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Nijmegen, the Netherlands.|Khondrion BV, Nijmegen, the Netherlands.
authors: Eligio F
authors: EF
authors: Iannetti
authors: Khondrion BV, Nijmegen, the Netherlands.|Department of Pediatrics, Radboud Center for Mitochondrial Medicine, Radboudumc, Nijmegen, the Netherlands.|Centre for Systems Biology and Bioenergetics, Radboudumc, Nijmegen, the Netherlands.
authors: Jan A M
authors: JA
authors: Smeitink
authors: Khondrion BV, Nijmegen, the Netherlands.
authors: Julien
authors: J
authors: Beyrath
authors: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Nijmegen, the Netherlands.|Khondrion BV, Nijmegen, the Netherlands.|Centre for Systems Biology and Bioenergetics, Radboudumc, Nijmegen, the Netherlands.
authors: Peter H G M
authors: PH
authors: Willems
authors: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Nijmegen, the Netherlands.|Khondrion BV, Nijmegen, the Netherlands.|Centre for Systems Biology and Bioenergetics, Radboudumc, Nijmegen, the Netherlands.
authors: Werner J H
authors: WJ
authors: Koopman
mesh_terms: D002460:Cell Line; D002470:Cell Survival; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008853:Microscopy / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism*; D012867:Skin / Q000166:cytology