Preparation of purified perikaryal and synaptosomal mitochondrial fractions from relatively small hypothalamic brain samples
| Autor: | Dávid Sándor Kiss, Laszlo V. Frenyo, Tibor Bartha, Gergely Jocsak, István Tóth, Attila Zsarnovszky, Ágnes Sterczer |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Isolation of mitochondrial fraction for oxygen-consumption measurements Gradient fractionation Clinical Biochemistry Small brain Fractionation CELL DEBRIS Mitochondrion Biology law.invention 03 medical and health sciences Medical Laboratory Technology Myelin 030104 developmental biology medicine.anatomical_structure Percoll Biochemistry law Respiration measurement Respiratory measurements medicine Clark-type electrode Electron microscope Neuroscience |
| Zdroj: | MethodsX |
| ISSN: | 2215-0161 |
| DOI: | 10.1016/j.mex.2016.05.004 |
| Popis: | In order to measure the activity of neuronal mitochondria, a representative proof of neuronal processes, physiologically relevant mitochondrial samples need to be gained as simply as possible. Existing methods are, however, either for tissue samples of large size and/or homogenous microstructures only, or are not tested for mitochondrial function measurements. In the present article we describe a gradient fractionation method to isolate viable and well-coupled mitochondria from relatively heterogeneous histological microstructures such as the hypothalamus. With this new method, we are able to isolate a sufficient amount of functional mitochondria for determination of respiratory activity, in a short period of time, using affordable equipment. • Verified by electron microscopy, our method separates highly enriched and well-preserved perikaryal and synaptosomal mitochondria. Both fractions contain minimal cell debris and no myelin. Respiratory measurements (carried out by Clark-type electrode) confirmed undisturbed mitochondrial function providing well-evaluable records. The demonstrated protocol yields highly viable mitochondrial subfractions within 3 h from small brain areas for high-precision examinations. Using this procedure, brain regions with relatively heterogeneous histological microstructure (hypothalamus) can also be efficiently sampled. • Up to our present knowledge, our method is the shortest available procedure with the lowest sample size to gain debris-free, fully-viable mitochondria. |
| Databáze: | OpenAIRE |
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