Buckler K J, Vaughan-Jones R D
University Laboratory of Physiology, Oxford, UK.
Pflugers Arch. 1990 Oct;417(2):234-9. doi: 10.1007/BF00370705.
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 approximately 10 microns) 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. Photo-bleaching 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).(ABSTRACT TRUNCATED AT 250 WORDS)
我们报告了一种新型pH敏感双发射荧光探针——羧基半萘并罗丹明氟-1（carboxy-SNARF-1）的应用，用于对小的分离细胞内的细胞内pH（pHi）进行比率记录。该方法通过对新生大鼠颈动脉体分离出的单一1型细胞（细胞直径约10微米）进行pHi测量得以说明。通过在浴液中应用乙酰氧基甲酯来加载羧基-SNARF-1。当在540nm激发时，该荧光探针在590nm和640nm处给出强烈的、呈反比关系的发射信号。对于单个细胞（pHi 8.5 - 6.5），可以实现长达50分钟的稳定pHi比率记录。通过在相对低光强度下照射（50W氙灯搭配0.2%透过率的中性密度滤光片），可将探针的光漂白降至最低。该探针可使用尼日利亚菌素技术进行原位校准，并且这与Eisner等人（1989年）的独立零点技术（细胞外弱酸/弱碱应用）在定量上具有良好的一致性。（摘要截短于250字）
