用Fura-2双波长荧光法测定小鼠海马细胞内游离钙

Determination of intracellular free Ca^(2+) in hippocampal neurons of mice with Fura-2 double-wavelength fluorecent technique

目的:探讨小鼠海马细胞分离及细胞内钙离子浓度([Ca2+]i)的测定。方法:低浓度胰蛋白酶消化、分离海马细胞,采用Ca2+荧光指示剂Fura2双波长荧光法测定[Ca2+]i。结果:海马细胞存活率超过95%。细胞外Ca2+1.0mmol/L时,静息状态下海马细胞[Ca2+]i为(210±10.7)nmol/L。30mmol/LKCl可显著增加[Ca2+]i,并且KCl的这种效应呈一定的剂量依赖关系。结论:低浓度胰蛋白酶消化分离小鼠海马细胞简单、可靠;Fura2建立的双波长荧光法灵敏、可靠。

Objective: To explore dissociation method for the hippocampal neurons of mice and measurement of free intracellular calcium. Methods: Digesting with low concentration of trypsin and gently triturating mode were used to dissociate hippocampal neurons.Concentration of Ca^(2+) was measured with Fura-2 double wavelength fluoremetry. Results: The cellular viability rate was over 95%, Concentration of Ca^(2+)in the resting hippocapal neuron was (210±10.7)nmol/L on condition when concentration of Ca^(2+) outside the cells was 1.0 mmol/L. Potassium chloride of 30 mmol/L markedly increased the concentraion of Ca^(2+) in the hippocampal neurons, and the effect was concentraion-dependent. Conclusion: The method to dissociate hippocampal neurons by digesting with low concentration of trypsin was useful and easy, and the adoption of Fura-2 double wavelength fluoremetry in dissociated mice hippocampal neurons was useful in monitoring change of intracellular Ca^(2+).