Experimental Setup for Investigation of Acute Mitochondrial Oxygen Sensing in Primary Cells

The ability to sense and respond to acute changes in oxygen is essential for the viability of cells and organisms. To study molecular mechanisms of acute oxygen sensing, we established a setup for the adjustment of acute hypoxic conditions in cultured cells, exemplified here for the use of primary pulmonary arterial smooth muscle cells (PASMCs). The mitochondrial electron transport chain (ETC) is the main consumer of oxygen but recently also emerged as essential oxygen sensor suggesting that the ETC itself adapts its electron flux to oxygen availability. To test this assumption and to experimentally manipulate electron flux through the ETC, we used alternative oxidase (AOX), which bypasses the cytochrome pathway of the ETC when blocked. The described combination of our experimental setup and AOX allowed us in previous publications unprecedented insights into the role of the ETC in cellular oxygen sensing and cellular response mechanisms in living cells. Against this background, we here describe and discuss this method in detail, which will allow transfer to other cell types and research questions.