Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay

Mitochondria are organelles, approximately the size of bacteria, found in eukaryotic cells. They are unique organelles because they contain DNA and have two membranes, an outer and inner one. Mitochondria's outer and inner membranes are separated by an intermembrane space, and the inner membrane folds into structures called cristae around the innermost compartment, called the matrix. These cristae increase the inner membrane's surface area so that multiple processes that use the cristae can occur simultaneously. While mitochondria are involved in many cellular functions such as controlling apoptosis and housing multiple metabolic pathways, their vital role in the production of ATP is essential for cell survival. In fact, 90% of a cell's energy is derived from mitochondria¹. ATP production involves the generation of an electrochemical difference across the outer and inner membranes, termed the mitochondrial membrane potential (Δψ), which arises as H⁺ ions are pumped from the matrix into the intermembrane space. ATP production is ultimately harnessed during the oxidation of reducing equivalents via electron movement through the mitochondrial respiratory chain (ETC). The final electron acceptor is molecular oxygen (O₂). As oxygen is consumed, the H⁺ concentration differential builds up to its maximum, at which point H⁺ ions move down their concentration gradient from the intermembrane space to the matrix by passing through the ATP synthase complex. The movement of H⁺ ions causes a conformational change in ATP synthase, and ADP is brought into proximity with inorganic phosphate to react and generate ATP. Finally, ATP is translocated out of the mitochondrial matrix into the cytosol and can either be stored or used to facilitate reactions due to the large amount of free energy released during the hydrolysis of its phosphates. This whole process is termed oxidative phosphorylation, and since oxygen is consumed, mitochondria are said to respire².

The buildup and strength of Δψ, the amount of O₂ reduced (termed oxygen consumption), as well as the generation of ATP can all be used as indications of cell health. Mitochondrial functional studies, such as measurement of Δψ, total ATP content and production, and oxygen consumption can be quantified either by traditional biochemical methods or fluorescence and luminescence in plate-based assays. For example, mitochondrial membrane potential can be compared among different samples using fluorescent dyes such as tetramethylrhodamine ethyl ester, which binds specifically to mitochondria. ATP generation can be monitored by adding a luminescent protein to a reaction whose changes correlate to ATP concentration. Quantification of oxygen consumption rates, or absolute rates of respiration, during OXPHOS, can help elucidate the causes of disparities in mitochondrial function and energy metabolism. Oxygen consumption assessment can be used to calculate respiratory control ratios (RCRs). RCR values describe the ability of mitochondria to make ATP in response to the influx of ADP, which is the main function of mitochondria. RCR values signify the overall condition of isolated mitochondria and allow for the comparison of responses to different experimental treatments. Differences in RCR values may represent mitochondrial dysfunction or indicate a biological difference between different mitochondria isolated from two or more sources. Another important measure of function in isolated mitochondria is mitochondrial efficiency defined as moles of ATP synthesized per moles of O₂, or the P/O ratio³.

Given the amount of information that can be gathered from measuring mitochondrial parameters and various instances in which this information can be utilized, the ability to efficiently gather functional data can be useful in many different research areas. Mitochondrial oxygen consumption measurements have been performed for decades with very specific instrumentation—using a Clark electrode, which can be limited by the sample size necessary to carry out measurements, and more recently, sophisticated instruments that can measure mitochondrial respiration and multiple other parameters but can be cost-prohibitive. This protocol is an adapted alternative approach using an oxygen-sensitive phosphorescent probe (MitoXpress)^4,5. The probe signal is detected with a plate reader in time-resolved fluorescence mode for continuous measurements over time. Phosphorescence has a larger energy difference between the absorbed and emitted photon compared to fluorescence and therefore, is better suited for continuously monitoring changes in signal. This enables almost any lab to perform these measurements, not just those that focus on mitochondrial metabolism or who can afford highly specialized equipment. The model system we utilize is isolated mitochondria from three tree lizards, two parental species and one introgressed (containing nuclear DNA from one parental species and mitochondria from the other—hybrids). These lizards were chosen because we hypothesized there are metabolic and energy consequences for hybrids having different nuclear and mitochondrial DNA sources. We utilized a commercially available assay kit with a multi-mode plate reader that can increase access to this type of assay to more researchers and research fields.