Use of a pH-sensitive fluorescent probe for measuring intracellular pH of Caco-2 cells

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• 作者：Earvin Liang ; Puchun Liu ; Steven Dinh
• 关键词：Intracellular pH ; Caco-2 cells ; pH-sensitive fluoroprobes ; BCECF ; Delivery agents
• 刊名：International Journal of Pharmaceutics
• 出版年：2007
• 出版时间：29 June 2007
• 年：2007
• 卷：338
• 期：1-2
• 页码：104-109
• 全文大小：828 K

文摘

This paper describes the application of a pH-sensitive fluorescent probe [2′,7′-bis(2-carboxylethyl)-5(6)-carboxyfluorescein or BCECF] to measure intracellular pH (pH_i) changes in Caco-2 cells. As a function of BCECF's ionization, the fluorescence was monitored at λ_ex = 440 and 503 nm, and λ_em = 535 nm. Time course studies were conducted with the addition of two weak acid delivery agents, one weak base delivery agent, oleic acid, or tetradecylamine. When applicable, 10 μM bovine serum albumin or 10 mM ammonium chloride was added into the cell suspension to hinder the pH gradient effect. Adding a weak acid at 2, 10, or 50 mM to the cell suspension, the pH_i dropped substantially from 7.4 to 7.1, 6.9, or 6.7, respectively. The pH_i then increased gradually over a 10-min period but did not return to its initial value. Conversely, the pH_i increased instantaneously after the addition of a weak base. When Caco-2 cells were placed in solutions with different bulk pH (7.0, 7.5, and 8.0), the lower the pH in which the cells were exposed, the larger the pH_i drop occurred with the addition of an acid. The results suggest that these weak acids or bases are transported transcellularly across Caco-2 cells.