探讨单细胞记录和神经细胞培养时神经元的分离方法

To explore the suitable methods to isolate cortical neurons for single neuronal recording or neuronal culture

目的探讨针对不同实验目的皮层神经元的最佳分离方法。方法针对单细胞记录分别采用蛋白酶E分离法和胰蛋白酶分离法分离神经元,针对神经细胞培养分别采用胰蛋白酶分离法和机械分离法,之后通过形态学、膜片钳和染色法等方法分别比较上述分离方法对神经元活性的影响。结果经蛋白酶E或胰蛋白酶分离法得到的细胞在进行单细胞记录时虽然细胞的死亡率没有区别,但形态学、膜片钳等实验均表明蛋白酶E分离法分离的神经元活性更好;在神经细胞培养中经形态学、膜片钳和台盼蓝染色法和凝胶电泳等方法都表明胰蛋白酶分离法分离的神经元活性好,更适于神经细胞培养。结论单细胞记录时蛋白酶E分离法更为合适,神经细胞培养时胰蛋白酶分离法更为合适。

Objective To explore the suitable methods for cortical neurons in different experiments including single neuronal recording and neuronal culture. Methods Cortical neurons were isolated by pronase E or trypsin for single cell record, or by trypsin or mechanical methods for neuronal culture, and the activeness of neurons were examined by morphological observation, pateh-elamp techniques, trypan blue dyeing and gel electrophoresis techniques. Results Neurons isolated by pronase E were suitable for single record as examined by morphological observation and pateh-elamp method, even though the death rates of neurons were no differenee; neurons isolated by trypsin were suitable for culture as examined by morphological observation, pateh-elamp techniques, trypan blue method and gel electrophoresis techniques. Conclusion Neurons isolated by pronase are suitable for a single cell recording, and for neuronal culture neurons isolated trypsin are mueh more suitable.