Bezuidenhout 2016b Abstract MitoFit Science Camp 2016
|Analysis and comparison of mitochondrial function using cooling plate or ice during mechanical separation of mouse skeletal muscle fibers.
Permeabilized muscle fibers is the most widely used sample preparation technique when assessing mitochondrial function in skeletal and cardiac muscle tissue. A cooling plate was developed in an attempt to standardize mechanical separation of fibers, through close regulation of temperature and optimization of visualization. Here, we wanted to investigate if using the cooling plate would influence mitochondrial function.
Mitochondrial oxygen consumption was measured using high resolution respirometry (HRR, Oroboros Oxygraph-2K) in skeletal muscle permeabilized fibers from adult male mice (1.5-2.5 mg tissue/2 ml chamber). MiR05 + catalase + creatine (MiR06Cr) respiration medium was used, the oxygen (O2) range was maintained between 400-250 µM, and mitochondrial respiratory capacities were determined using a SUIT (substrate-uncoupler-inhibitor-titrations) protocol. Data were normalized to wet weight (Ww) (mg tissue), complex IV (COX) and citrate synthase (CS) activities.
No significant differences were found in any of the respiratory states determined with fibers prepared on ice versus the cooling plate when data were normalized using Ww and COX activity, whereas data normalized using CS activity were significantly different across all respiratory states between the groups when compared to data normalized to Ww and COX activity. The use of the cooling plate for preparing permeabilized fibers maintains mitochondrial function and it improves various practical aspects of mechanical separation. These improvements contributed to faster, more accurate sample preparation and increased picture quality. Further studies have been carried out using a combination of ice and the cooling plate, and suggestions have been made for improvements of the cooling plate to allow its sole use when preparing fibers.
1-Univ Cape Town, Dept Human Biol, South Africa; 2-Oroboros Instruments, Innsbruck, Austria; 3-D.Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria. - [email protected]
The project was supported by K-Regio project MitoFit, funded in part by the Tyrolian Government and the European Regional Development Fund (ERDF).
Labels: MiParea: Respiration, Instruments;methods
Organism: Mouse Tissue;cell: Skeletal muscle Preparation: Permeabilized tissue
Coupling state: LEAK, OXPHOS, ET Pathway: N, S, ROX HRR: Oxygraph-2k
MitoFit Science Camp 2016