Measurement of intracellular pH in cultured cells by flow cytometry with BCECF-AM
Measurement of intracellular pH in cultured cells by flow cytometry with BCECF-AM
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DOI：
10.1016/0168-1656(95)00189-1
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发表时间：
1996-05-15
影响因子：
4.1
通讯作者：
Nabet, P
中科院分区：
文献类型：
--
作者：
Franck, P;Petitipain, N;Nabet, P
本研究评价了荧光染料BCECF流式细胞术测定培养细胞内pH（pH（i））的适用性，并监测了小鼠杂交瘤分批培养和鸡胚成纤维细胞单层培养（第5代）中pH（i）的变化。该技术产生了高度可重复、可重复的结果。校准曲线的理论灵敏度为0.0004 pH单位。但对绿色/红色荧光比率的直方图的标准偏差的分析表明平均灵敏度为0.08（0.07-0.09）pH单位。通过用细胞建立校准曲线，使由于细胞大小、荧光染料掺入和酯酶引起的干扰最小化，所述细胞的pH（i）将在488 nm激发后使用525/610 nm荧光比率测量。在培养开始时，指数生长的分批培养的杂交瘤的pH（i）为7.50。pH（i）在指数生长期期间增加，并朝向细胞死亡下降。单层培养的鸡成纤维细胞的pH（i）为7.30。
This study evaluates the suitability of flow cytometry with the fluorochrome BCECF for measuring the intracellular pH (pH(i)) of cultured cells, and monitors the changes in pH(i) in murine hybridoma in batch culture and chick embryo fibroblast in monolayer culture (5th passage). The technique produced highly reproducible, repeatable results. The theoretical sensitivity from the calibration curve was 0.0004 pH units. But analysis of the standard deviation of the histogram of the green/red fluorescence ratios indicated a mean sensitivity of 0.08 (0.07-0.09) pH units. Interference due to cell size, fluorochrome incorporation and esterases were minimized by establishing a calibration curve with the cells whose pH(i) was to be measured using the 525/610 nm fluorescence ratio after excitation at 488 nm. The pH(i) of exponentially growing, batch cultured hybridomas was 7.50 at the start of culture. pH(i) increased during the exponential growth phase and dropped towards cell death. The pH(i) of the chick fibroblasts in monolayer culture was 7.30.