Quantification of mitochondrial reactive oxygen species in macrophages during sepsis

Sepsis is the leading causes of death globally, arising from an imbalanced host response to severe infection. It leads to multi-organ failure and poor outcomes in septic patients due to compromised glucose and lipid oxidation, reduced oxygen consumption, elevated levels of circulating substrates, and impaired mitochondrial function. Mitochondria, essential cellular organelles, play a vital role in regulating various cellular activities and the host immune response to infection. Pathogens, particularly bacteria, often disrupt mitochondrial functions to dysregulate host immunity. Additionally, the mitochondrial function is closely associated with most host immune responses, making mitochondria crucial in maintaining host homeostasis during infection. The intrinsic inflammatory response triggered by pathogens in sepsis impairs mitochondrial function, resulting in excessive production of mitochondrial reactive oxygen species (ROS) and subsequently damage to multiple organs. Here, we present a simple protocol for assessing mitochondrial ROS levels in bone marrow-derived macrophages (BMDMs) isolated from mice. We observed a higher level of ROS generation in lipopolysaccharide (LPS)-treated BMDMs, indicating the effectiveness and efficiency of our designed protocol for assessing mitochondrial ROS generation in vitro.