Dr Matthew McKenzie
We were invited by the Journal of Visualized Experiments to submit our protocol for measuring mitochondrial calcium and mitochondrial membrane potential simultaneously in live cells using confocal microscopy. This is a video journal, and provides the protocol in an easy to follow video as a resource for the research community.
Centre for Genetic Diseases
Molecular Basis of Mitochondrial Disease
Journal and article title
Our protocol has multiple advantages over other methods that are used to measure mitochondrial calcium and membrane potential. Instead of requiring complicated genetically encoded probes, it uses readily available fluorescent dyes, allowing for fast and simple cell staining. In addition, the protocol allows for the use of dyes with very different spectral properties, meaning that the mitochondrial calcium and membrane potential signals are easily separated.
Mitochondria act as local calcium buffers to regulate intracellular calcium concentrations and the energy requirements of the cell. When this
process is disrupted, excessive mitochondrial calcium uptake can cause the collapse of the mitochondrial membrane potential, releasing pro-apoptotic molecules that trigger cell death induction.
Our protocol presents a fast and straight-forward method for examining this process, and can be used to determine how mitochondrial calcium and membrane potential are affected by genetic defects, environmental toxins and therapeutic drugs. This can be used to investigate how these mitochondrial parameters are involved in wide range of human diseases, including diabetes and age-related conditions such as Alzheimer's and Parkinson's Disease.
Other points of interest
Confocal imaging to measure mitochondrial calcium and membrane potential can be readily performed here at the MHTP. Dr Kirstin Elgass and Dr Sarah Creed at Monash Micro Imaging helped us to develop this technique and established the downstream image analysis. Please speak with them and/or myself if you are interested in this protocol, or check out the video at doi:10.3791/55166 !