Rotenone is a highly lipophilic compound and an inhibitor of the electron transport system's complex 1 (NADH dehydrogenase) (Hoglinger et al., 2005 Jayaraj, Tamilselvam, Manivasagam, & Elangovan, 2013). In vitro rotenone-based PD models represent a reliable and reproducible tool for investigating disease characteristics through analysis of subcellular organelle morphology (Betarbet et al., 2000 Sherer et al., 2002). Dysfunction in mitochondrial dynamics in neurons is recognized as a measurable phenotype of in vitro and in vivo models of Parkinson's disease (PD) and other neurodegenerative diseases such as Alzheimer's disease and Huntington's disease (Franco, Li, Rodriguez-Rocha, Burns, & Panayiotidis, 2010 Haque et al., 2008 Pozo Devoto & Falzone, 2017). In eukaryotic cells, mitochondria continuously undergo flux between fission and fusion, resulting in an observable difference in morphology (Scott & Youle, 2010). Here, we describe a protocol for the analysis of mitochondria. Many protocols (or pipelines) developed can be readily adapted by biologists to analyze myriad cell biology contexts. As a result, the routine utilization of such techniques by biology researchers has accelerated (Kamentsky et al., 2011).
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The existence of software allows the successful automation of data-extraction processes to liberate investigators from hours of image-by-image manual data capture. In the last few decades, the development of microscopy technology and the blending of routine biology and computer science skills have resulted in the development of intuitive open-source image analysis software to support laboratory experiments. The extraction of substantial quantitative data from fluorescence and light-microscopic images of in vitro and ex vivo biological samples is beyond the ability of most day-to-day laboratory researchers. © 2020 The Authors.īasic Protocol 1: SN4741 neuron culture and treatment in a rotenone-based model of Parkinson's diseaseīasic Protocol 2: Identification of cell nuclei, measurement of mitochondrial membrane potential, and measurement of mitochondrial fragmentation in mouse-derived midbrain dopaminergic neurons In this protocol, we describe the utilization of cell culture techniques, high-content imaging (HCI), and the subsequent open-source image analysis pipeline for the quantification of mitochondrial fragmentation in the context of a rotenone-based in vitro Parkinson's disease model. These developments allow routine biology researchers to rapidly turn hypotheses into results. Advances in microscopy and the availability of intuitive open-access software have accelerated the rate of image acquisition and analysis, respectively. Delineating the dysfunction in mitochondrial dynamics found in diseased states can aid our understanding of underlying mechanisms of disease progression and possibly identify novel therapeutic approaches. For example, government-provided land, water, electricity at concessional rate in New Mumbai area to attract people and to reduce the pressure of the population in Mumbai city.Neuronal mitochondrial fragmentation is a phenotype exhibited in models of neurodegeneration such as Parkinson's disease. Sometimes government policies also affect population distribution. For example, agriculture or mining activities support a large population since they provide a source of living to many people. The distribution of population also depends upon human factors such as agriculture, mining, transportation, urbanization, etc. The Himalayan region in India is thinly populated but the Gangetic plains are thickly populated and the Deccan plateau region is moderately populated. Plateau regions are moderately populated and plains are densely populated. For example, if you consider the relief factors, mountains and hilly areas are thinly populated. Distribution of the population in the world is very uneven because the distribution of population depends upon many physical factors such as relief, climate, availability of water supply, soil, etc.
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