Assessing bioenergetic functions from isolated mitochondria in Drosophila melanogaster

Abstract

Mitochondria are involved in generating more than 90 percent of cellular energy and are responsible for many cellular processes such as metabolism, cell signalling, apoptosis and ageing. Currently, there are a number of different experimental approaches employed to measure mitochondrial health and function. Here, we demonstrate a novel approach that quantifies substrate induced mitochondrial respiration from Drosophila. This protocol is optimized for mitochondria isolated from third instar larvae, and can also be used for mitochondria isolated from adult thoraces. This procedure outlines how to perform high throughput and high resolution mitochondria specific measurements for state II, state III, state IV_O respiration and residual oxygen consumption.

Keywords: Drosophila; Seahorse XF; bioenergetics; complex I; larvae; mitochondrial isolation.

Figure 1.

Optimization of mitochondrial concentration for oxygen consumption rate (OCR). A. Isolated mitochondria are visualized under the microscope before and after experiment. The mitochondria appear as a monolayer at the base of the well. B. Oxygen consumption rate in pmoles.min⁻¹. The graph shown above is a typical plot obtained using the methods outlined in this protocol. The four injections are reported in blue. C. State II and III respiration rates are linear from 1.25 to 20 µg of mitochondrial protein. The number above each data point represents the RCR value calculated by state III/II and state III/IVo (value inside the bracket) for each mitochondrial concentration.

Figure 2.

Optimization of injections. A. ADP is saturated at 1 mM concentration. B. BAM 15 is saturated at 16 µM and C. FCCP is saturated at 8 µM.