Analysis of regional brain mitochondrial bioenergetics and susceptibility to mitochondrial inhibition utilizing a microplate based system

Abstract

The analysis of mitochondrial bioenergetic function typically has required 50–100 μg of protein per sample and at least 15 min per run when utilizing a Clark-type oxygen electrode. In the present work we describe a method utilizing the Seahorse Biosciences XF24 Flux Analyzer for measuring mitochondrial oxygen consumption simultaneously from multiple samples and utilizing only 5 μg of protein per sample. Utilizing this method we have investigated whether regionally based differences exist in mitochondria isolated from the cortex, striatum, hippocampus, and cerebellum. Analysis of basal mitochondrial bioenergetics revealed that minimal differences exist between the cortex, striatum, and hippocampus. However, the cerebellum exhibited significantly slower basal rates of Complex I and Complex II dependent oxygen consumption (p < 0.05). Mitochondrial inhibitors affected enzyme activity proportionally across all samples tested and only small differences existed in the effect of inhibitors on oxygen consumption. Investigation of the effect of rotenone administration on Complex I dependent oxygen consumption revealed that exposure to 10 pM rotenone led to a clear time dependent decrease in oxygen consumption beginning 12 min after administration (p < 0.05). These studies show that the utilization of this microplate based method for analysis of mitochondrial bioenergetics is effective at quantifying oxygen consumption simultaneously from multiple samples. Additionally, these studies indicate that minimal regional differences exist in mitochondria isolated from the cortex, striatum, or hippocampus. Furthermore, utilization of the mitochondrial inhibitors suggests that previous work indicating regionally specific deficits following systemic mitochondrial toxin exposure may not be the result of differences in the individual mitochondria from the affected regions.

Resolution of Mitochondrial Oxidative Stress Rescues Coronary Collateral Growth in Zucker Obese Fatty Rats

“…A XF24 extracellular flux analyzer (Seahorse Bioscience, North Billerica, MA) was used to measure intact isolated mitochondria function/bioenergetics according to protocol established by Rogers et al (2011) 14 and Sauerbeck et al (2011) 15 with modifications. First, a 24-well dual-analyte sensor cartridge was primed with calibration solution at 37 °C incubator without CO 2 overnight.…”

“…For most measurements, either the highest or lowest point-to-point rates were taken. For oligomycin rate, an average of the last three point-to-point rates were taken since the effect of oligomycin requires at least 1 min to reach maximal effect and an average of the last 30 s of the measure provides the most consistent result 15 .…”

Standardized bioenergetic profiling of adult mouse cardiomyocytes

“…Classically, mitochondrial function has been determined by using isolated organelles and measuring maximal respiration rates by polarographic methods (4,18,22) or by measuring activity of the individual complexes (19,22). Although such techniques represent powerful reductionist approaches for assessing and making assumptions about mitochondrial "health," they cannot account for extramitochondrial regulation of metabolism (i.e., substrate uptake).…”

Phenelzine Mitochondrial Functional Preservation and Neuroprotection after Traumatic Brain Injury Related to Scavenging of the Lipid Peroxidation-Derived Aldehyde 4-Hydroxy-2-Nonenal

“…[14][15][16][17][18] The XF24 involves a transient, 7 mL chamber in specialized microplates that allows for the determination of oxygen and proton concentrations in real time. 18,19 Based upon our previous studies, 5.0 mg mitochondrial protein was used to study the 4-HNE-induced mitochondrial dysfunction.…”

“…The OCR measurements were performed as previously described. 14 Briefly, the area under the curve was generated by an algorithm executed by the Seahorse device and these values were compared between experimental and control groups. Using the Excel software package (Microsoft), pointby-point rates are generated using the AKOS algorithm written by AKOS Gerencser in collaboration with Seahorse Bioscience.…”