Recent proteomic, metabolomic, and transcriptomic studies have highlighted a connection between changes in mitochondria physiology and cellular pathophysiological mechanisms. Secondary assays to assess the function of these organelles appear fundamental to validate these -omics findings. Although mitochondrial membrane potential is widely recognized as an indicator of mitochondrial activity, high-content imaging-based approaches coupled to multiparametric to measure it have not been established yet. In this paper, we describe a methodology for the unbiased high-throughput quantification of mitochondrial membrane potential in vitro, which is suitable for 2D to 3D models. We successfully used our method to analyze mitochondrial membrane potential in monolayers of human fibroblasts, neural stem cells, spheroids, and isolated muscle fibers. Moreover, by combining automated image analysis and machine learning, we were able to discriminate melanoma cells from macrophages in co-culture and to analyze the subpopulations separately. Our data demonstrated that our method is a widely applicable strategy for large-scale profiling of mitochondrial activity.

Neural progenitor cells (NPCs) are often used to study the subcellular mechanisms underlying differentiation into neurons in vitro. Works published to date have focused on the pathways that distinguish undifferentiated NPCs from mature neurons, neglecting the earlier and intermediate stages of this process. Current evidence suggests that mitochondria interaction with the ER is fundamental to a wide range of intracellular processes. However, it is not clear whether and how the mitochondria–ER interactions differ between NPCs and their differentiated counterparts. Here we take advantage of the widely used NPC line LUHMES to provide hints on the mitochondrial dynamic trait changes that occur during the first stage of their maturation into dopaminergic-like neurons. We observed that the morphology of mitochondria, their interaction with the ER, and the expression of several mitochondria–ER contact site resident proteins change, which suggests the potential contribution of mitochondria dynamics to NPC differentiation. Further studies will be needed to explore in depth these changes, and their functional outcomes, which may be relevant to the scientific community focusing on embryonic neurogenesis and developmental neurotoxicity.