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

Sepsis, a condition that affects 750,000 people per year, is characterized as a severe inflammatory response to infection. Patients exhibit highly increased concentrations of circulating cytokines as a result of their immune response. Consequently, end‐organ failure observed in many septic patients has been linked to these increased cytokine levels, with tissue damage resulting, at least in part, via apoptosis. Specifically, acute kidney injury (AKI) induced by sepsis is a serious problem, and is poorly characterized. This study aims to determine the cellular mechanism of apoptosis during sepsis using an in vitro model of sepsis‐induced AKI. Cultured kidney cells were treated with either cytokines secreted from THP1 cells exposed to LPS, or recombinant cytokines. Then, MTT assays, at various times post‐exposure, were used to measure changes in cell viability. Furthermore, flow cytometry of whole cells stained with Annexin‐V‐FITC determined specifically the amount of apoptosis incurred. Our results show a small, yet significant, decrease in cell viability for cultured cells treated with recombinant cytokines versus untreated cells. Correlated flow cytometry results indicate that the majority of the cell death occurring is in fact due to apoptosis. Additionally, Western blotting, immunoprecipitation, and BH3 profiling assays were conducted to determine which BCL‐2 family proteins may be important in cytokine‐mediated apoptosis. Data from these analyzes suggest anti‐apoptotic BCL‐2 and pro‐apoptotic BIM are key contributors. Further experiments are currently underway using primary kidney cells, and eventually the in vivo cecal ligation and puncture (CLP) model in mice will be employed to give additional insight.