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首页> 外文期刊>Brain research. Brain research protocols >An improved cell isolation technique for studying intracellular Ca(2+) homeostasis in neurones of the cochlear nucleus.
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An improved cell isolation technique for studying intracellular Ca(2+) homeostasis in neurones of the cochlear nucleus.

机译：一种改进的细胞分离技术，用于研究耳蜗核神经元中的细胞内Ca（2+）稳态。

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Neurones isolated from various parts of the brain are used extensively for electrophysiological and immuncytochemical studies, as well as to investigate their Ca(2+) homeostasis. In this work we report on an isolation technique that yielded neurones suitable for functional studies targeting the investigation of their Ca(2+) handling mechanisms. The cell isolation involved enzymatic dissociation with combined collagenase/pronase treatment and gentle mechanical trituration. At the end of the isolation the cells were incubated in a cell culture incubator (CO2 concentration = 5.1%) at 37 degrees C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated horse serum. The vitality of the isolated cells was indicated by their low intracellular Ca(2+) concentrations (17.2 +/- 0.5 nM; n = 38) and by their ability to produce large Ca(2+) transients on depolarization. These Ca(2+) transients were rapidly terminated and the resting intracellular Ca(2+) concentration was quickly restored proving that isolation did not compromise the Ca(2+) homeostatic mechanisms of the nerve cells. The technique allowed reliable, long (45-60 min) and reproducible measurements of Ca(2+) currents on these neurones as well as the recording of their intracellular Ca(2+) concentration. Our results indicate that incubation in DMEM with horse serum markedly increases the number of surviving neurones after the enzyme treatment, and their Ca(2+) homeostasis can be studied for significantly longer periods of time.
机译：从大脑的各个部位分离出的神经元被广泛用于电生理和免疫细胞化学研究，以及调查其Ca（2+）稳态。在这项工作中，我们报告了一种隔离技术，该技术可产生适用于针对其Ca（2+）处理机制进行研究的功能研究的神经元。细胞分离涉及酶解与胶原酶/链霉蛋白酶组合处理和轻度机械研磨。分离结束时，将细胞在细胞培养箱（CO2浓度= 5.1％）中于37摄氏度，补充有10％热灭活的马血清的Dulbecco改良的Eagle培养基（DMEM）中进行孵育。低细胞内Ca（2+）浓度（17.2 +/- 0.5 nM; n = 38）以及去极化时产生大Ca（2+）瞬态的能力表明了分离细胞的活力。这些Ca（2+）瞬变被迅速终止，并且静止的细胞内Ca（2+）浓度被快速恢复，证明了隔离不会损害神经细胞的Ca（2+）稳态机制。该技术允许对这些神经元上的Ca（2+）电流进行可靠，长时间（45-60分钟）和可重复的测量，并记录其细胞内Ca（2+）的浓度。我们的结果表明，在DMEM中与马血清一起孵育显着增加了酶处理后存活神经元的数量，并且可以研究其Ca（2+）稳态的时间更长。

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