Yu J, Zheng J J, Ong B Y, Bose R
Department of Pharmacology, University of Manitoba, Winnipeg, Canada.
Blood Vessels. 1991;28(6):464-74. doi: 10.1159/000158893.
The intracellular pH (pHi) of basilar artery rings was determined with 2',7'-bis-(carboxyethyl)-5,(6)-carboxyfluorescein (BCECF) by measuring the ratio of emitted (540 nm) fluorescence intensities (FI) at excitation wavelengths of 500 and 440 nm. There was a dye loss from the rings in 90 min (39.3 +/- 3.6%, p less than 0.001). We found that the ratio of fluorescence intensities does not adequately correct for dye loss; hence, we derived a method to correct for dye loss during pHi determinations. Calibration curves of the ratio versus pHi were constructed for the artery rings. The slope and intercept of the calibration curves depended on FI440. Linear regression lines for the slope and intercept versus FI440 were: [formula; see text] In solutions with different pH and different concentrations of free BCECF, the slope of the ratio versus pH of the solution was steeper at high concentrations of BCECF. Thus, pHi was calculated from a calibration curve in which the slope and intercept were determined from FI440 with the above formula. The corrected pHi was 7.37 +/- 0.05 (n = 25) at pHo 7.4 and 37 degrees C.
通过测量在500和440nm激发波长下发射（540nm）荧光强度（FI）的比率，使用2'，7'-双（羧乙基）-5，（6）-羧基荧光素（BCECF）测定基底动脉环的细胞内pH（pHi）。在90分钟内环中有染料损失（39.3 +/- 3.6%，p<0.001）。我们发现荧光强度比率不能充分校正染料损失；因此，我们推导了一种在pHi测定过程中校正染料损失的方法。为动脉环构建了比率与pHi的校准曲线。校准曲线的斜率和截距取决于FI440。斜率和截距与FI440的线性回归线为：[公式；见原文]在具有不同pH和不同游离BCECF浓度的溶液中，在高浓度BCECF下，溶液的比率与pH的斜率更陡。因此，pHi是根据校准曲线计算的，其中斜率和截距用上述公式由FI440确定。在pHo 7.4和37℃时，校正后的pHi为7.37 +/- 0.05（n = 25）。
