应用Fura—2/AM测定兔视网膜细胞内游离钙的方法

目的：建立适宜条件下，用荧光指示剂Fura—2测定乳兔视网嘻细胞内游离钙(简称Ca2+)的方法． 方法：用酶解制备视网膜细胞悬液．Fura-2/AM负载进行荧光测定． 结果：经0.05%胰蛋白酶消化10分钟，可使细胞存活率达90%以上．在37°C条件下与Fura-2/AM温育40分钟，于30分钟内测定为最佳条件。 结论：酶息状态下Ca2+]i水平为(223±27)nmol/L，其值在文献报道范围内．高钾去极化，在K+为25mmol/L和50mmol/L时．分别使Ca2+]i增加了59%和148%．由此证明视网膜细胞悬液制备和测定方法是可行的． （中华眼底病杂志，1996，12：108-110）

THE DETERMINATION OF INTRACELLULAR FREE Ca2+ CONCENTRATIONOF DISSOCIATED RABBIT RETINA CELLS BY Fura-2/AM

PURPOSE:To approach the establishment of t be optimal method for determining the intracellular free Ca2+ concentrationCa2+]i of dissociated newborn rabbit retina cells by using fluorescent indicator-Fura-2/AM. METHODS:Trypsin was employed to prepare the retlna cell suspensions which were then loaded with Fura-2/AM followed by fluorescence determination. RESULTS:The cellular viability rate of retina cell suspensiotls prepared by 0.05% trypsin 10 minutes at 37°C was over 90%. Loading the retina cell suspensions with Fura-2/AM 40 minutes at 37°C and then measurlng the fluorescent intensity of the suspensions within 30 minutes were proved to be the optimum. CONCLUSIONS:The resting Ca2+]i of retina cell suspension was (223±27)nmol/L whlch was within the expected range of Ca2+]i level. 25mmoI/L and S0mmol/L K+ increased the Ca2+Ji 59% and 148% respectively. These results indicate that the preparation of retina cell suspensions and the method of Ca2+Ji determination are reliable and feasible. （Chin J Ocul Fundus Dis,1996,12: 108-110 ）