APPLICATION OF A NEW PH-SENSITIVE FLUOROPROBE (CARBOXY-SNARF-1) FOR INTRACELLULAR PH MEASUREMENT IN SMALL, ISOLATED CELLS
APPLICATION OF A NEW PH-SENSITIVE FLUOROPROBE (CARBOXY-SNARF-1) FOR INTRACELLULAR PH MEASUREMENT IN SMALL, ISOLATED CELLS
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DOI：
10.1007/bf00370705
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发表时间：
1990-10-01
影响因子：
4.5
通讯作者：
VAUGHANJONES, RD
中科院分区：
文献类型：
--
作者：
BUCKLER, KJ;VAUGHANJONES, RD
我们报告使用一种新的pH敏感的双发射荧光探针，羧基-sNARF-1（羧基-SNARF-1）的比例记录细胞内的pH值（pHi）在小的分离细胞。该方法用单个1型细胞（细胞直径约为0.5 μ m）中的pHi测量来说明。10 μ m）。羧基-SNARF-1使用乙酰氧基甲基酯的浴涂进行负载。当在540 nm处激发时，荧光探针在590 nm和640 nm处产生强的、负相关的发射信号。稳定的比率记录的pHi可以实现从一个单一的细胞（pHi 8.5-6.5）长达50分钟。光漂白的探针是最小化照明在相对较低的光强度（50 W氙灯0.2%的传输中性密度过滤器）。可以使用尼日利亚菌素技术原位校准探针，并且这与Reynner等人（1989）的独立零点技术（细胞外弱酸/弱碱应用）具有良好的定量一致性。该荧光探针提供了优于其他常用探针的某些优点，用于pHi 2“，7”-双-（2-羧乙基）-5（和-6）-羧基荧光素（BCECF）：（i）由于其两个强的pH敏感峰发射，SNARF在低光强度下显示出用于比率记录的良好信噪比;（ii）与BCECF不同，SNARE的双重发射不需要激发滤光器的顺序机械切换，因此简化了落射荧光设置;（iii）因为羧基-SNARF-1发射信号在可见光谱的黄/红端，所以荧光药物如阿米洛利、乙基-异丙基-amibride（EIPA）、4，4“-二异硫氰基芪2，2“-二磺酸（DIDS）和肉桂酸类似物不会干扰pHi记录，即使在高浓度下使用。
We report the use of a new pH-sensitive dual-emission fluoroprobe, carboxy-seminaphthorhodafluor-1 (carboxy-SNARF-1) for ratiometric recording of intracellular pH (pHi) in small isolated cells. The method is illustrated with pHi measurement in single type-1 cells (cell diameter .apprx. 10 .mu.m) isolated from the carotid body of the neonatal rat. Carboxy-SNARF-1 is loaded using bath application of the acetoxymethyl ester. When excited at 540 nm, the fluoroprobe gives strong, inversely related emission signals at 590 nm and 640 nm. Stable ratiometric recordings of pHi can be achieved from a single cell (pHi 8.5-6.5) for up to 50 min. Photobleaching of the probe is minimised by illuminating at relatively low light intensity (50 W xenon lamp with 0.2% transmission neutral density filter). The probe can be calibrated in situ using the nigericin technique and this is in good quantitative agreement with the independent null-point technique (extracellular weak acid/weak base application) of Eisner et al. (1989). This fluoroprobe offers certain advantages over the other commonly used probe for pHi 2'',7''-bis-(2-carboxyethyl)-5(and -6)-carboxyfluorescein (BCECF): (i) because of its two strong pH-sensitive peak emissions, SNARF displays a good signal-to-noise ratio for ratiometric recording at low light intensities; (ii) unlike BCECF, the dual emission of SNARE requires no sequential mechanical switching of excitation filters, thus simplifying the epifluorescence set-up; (iii) because carboxy-SNARF-1 emission signals are at the yellow/red end of the visible spectrum, fluorescent drugs like amiloride, ethyl-isopropyl-amibride (EIPA), 4,4''-diisothiocyanostilbene 2,2''-disulphonic acid (DIDS) and cinnamate analogues do not interfere with the pHi recording, even when used at high concentrations.