Halide fluxes in epithelial cells measured with an automated cell plate reader.
Halide fluxes in epithelial cells measured with an automated cell plate reader.
复制标题
使用自动细胞板读取器测量上皮细胞中的卤化物通量。
DOI：
10.1016/s0003-2697(03)00142-8
复制
发表时间：
2004
影响因子：
2.9
通讯作者：
Dix,JamesA
中科院分区：
文献类型：
--
作者：
Mahlangu,DickAFD;Dix,JamesA
介绍了一种用6-甲氧基-N-（3-磺丙基）喹啉（SPQ）和6-甲氧基-N-乙基喹啉碘化喹啉（MEQ）作为氯离子荧光探针测定培养的上皮细胞氯离子渗透性的方法。该方法包括在多孔板中培养细胞，用SPQ或MEQ孵育细胞，然后用硝酸盐交换细胞内或细胞外的卤离子。SPQ或MEQ荧光的所得时间过程之后是用多孔荧光板读数器的重复读数。交换时间通过用时间的单个指数函数拟合时间过程来提取。通过测量A6和MDCK细胞中氯通道激活剂和阻断剂的作用来验证该方法。基线碘/硝酸盐交换时间为200- 300秒。异丙（cAMP激活氯离子通道的调节剂）使交换速率增加1.4±0.1倍; A23187（钙激活氯离子通道的调节剂）增加速率3.4±0.4;缓激肽（也是钙激活氯离子通道的调节剂）使速率增加2.0±0.4; forskolin（腺苷酸环化酶的直接刺激剂）使速率增加2.7±0.3。二苯胺-2-羧酸盐（一种氯离子通道阻滞剂）使速率降低0.12±0.03。这些结果表明，我们的方法是培养的上皮细胞中的卤化物-硝酸盐交换的有效指标。
A method is introduced to measure chloride permeability in cultured epithelial cells using 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) and 6-methoxy-N-ethylquinolinium iodide quinolinium (MEQ) as fluorescent chloride-sensitive probes. The method involves growing cells in multiwell plates, incubating cells with SPQ or MEQ, and then exchanging intracellular or extracellular halide ions with nitrate. The resulting time course of SPQ or MEQ fluorescence is followed by repetitive readings with a multiwell fluorescence plate reader. Exchange times are extracted by fitting the time course with a single exponential function of time. The method was validated by measuring the effect of chloride channel activators and blockers in A6 and MDCK cells. The baseline iodide/nitrate exchange time was 200–300s. Isoproterenol (a modulator of cAMP-activated chloride channels) increased the exchange rate by a factor of 1.4±0.1; A23187 (a modulator of calcium-activated chloride channels) increased the rate by 3.4±0.4; bradykinin (also a modulator of calcium-activated chloride channels) increased the rate by 2.0±0.4; forskolin (a direct stimulator of adenylate cyclase) increased the rate by 2.7±0.3. Diphenylamine-2-carboxylate (a chloride channel blocker) decreased the rate by 0.12±0.03. These results indicate that our method is a valid indicator of halide–nitrate exchange in cultured epithelial cells.