Using flow cytometry for mitochondrial assays

The understanding of how different cell types adapt their metabolism in the face of challenges has been attracting the attention of researchers for many years. Recently, immunologists also started to focus on how the metabolism of immune cells can impact the way that immunity drives its responses. The presence of a pathogen or damage in a tissue changes severely the way that the immune cells need to respond. When activated, immune cells usually shift their metabolism from a high energy demanding status using mitochondria respiration to a glycolytic based rapid ATP production. The diminished amount of respiration leads to changes in the mitochondrial membrane potential and, consequently, generation of reactive oxygen species. Here, we show how flow cytometry can be used to track changes in mitochondrial mass, membrane potential and superoxide (ROS) production in live immune cells. ● This protocol suggests a quick way of evaluating mitochondrial fitness using flow cytometry. We propose using the probes MitoTraker Green and MitoTracker Red/ MitoSOX at the same time. This way, it is possible to evaluate different parameters of mitochondrial biology in living cells. ● Flow cytometry is a highly used tool by immunologists. With the advances of studies focusing on the metabolism of immune cells, a simplified application of flow cytometry for mitochondrial studies and screenings is a helpful clarifying method for immunology.