Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput. Video LinkThe video component of this article can be found at

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Of all of the organs in the human body, the heart is one of the most vital. Should the heart or the blood vessels around the heart fail to develop properly, congenital heart defect occurs. Apoptosis is a highly controlled and regulated process known to occur throughout embryogenesis and is especially critical in heart development. However, very little is known about the specific apoptotic changes that occur during development. Therefore, our research investigates the molecular mechanisms of heart development. We hypothesize that heart cells isolated from a newly developing heart have greater apoptotic potential than heart cells of a fully developed heart. Utilizing whole hearts dissected from chick embryos, cardiomyocytes and cardiac fibroblasts are isolated from hearts harvested at different developmental stages. Next, these cells are treated with various concentrations of hydrogen peroxide for 2 hours and then stained with Annexin‐V‐FITC. The amount of apoptosis induced, as quantified by flow cytometry, is then used as a measure of susceptibility to apoptosis. Our data indicate that the amount of apoptosis induced by hydrogen peroxide treatment is no different for either cardiomyocytes or cardiac fibroblasts isolated from the same developmental stage. However, cell death for both types of cells at E5, as heart development is initially occurring, is significantly greater as compared to cells at E10, once heart development is complete. These results indicate that once heart development is done, both cardiomyocytes and fibroblasts develop resilience to apoptosis. This is critical, as it is important for heart cells to be susceptible to apoptotic signals throughout development, but also necessary for resistance to apoptosis once formative events have occurred.