Monitoring intracellular pH changes in response to osmotic stress and membrane transport activity using 5-chloromethylfluorescein

Abstract

Intracellular free H⁺ concentration (pHi) responds to numerous extracellular stimuli. The use of fluorescent indicator dyes to measure pHi is strongly influenced by the ability of target cells to retain activated dye within the cytoplasmic compartment. Here, 3 pH-sensitive indicator dyes—acetoxymethyl (AM) esters of SNARF-1 and BCECF, and the thiol-reactive 5-chloromethyfluorescein (CMFDA)—were examined for monitoring pHi. The stability of pH measurements was strongly affected by temperature, cell type, indicator dye, and use of transport inhibitors to prevent dye export. Cellular retention of CMFDA, which forms covalent complexes, was sufficient to permit monitoring of transient pHi changes over extended time periods in a multi-well plate assay format. In human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells, increasing osmotic pressure caused a significant rise in pHi. In contrast, activation of native or transfected β-adrenergic, cholinergic, and d and m opioid receptors did not measurably affect pHi in HEK293 cells. Decreases in pHi were observed in CHO cells expressing the human H⁺/peptide transporter PEPT1 upon addition of dipeptide substrates. The use of CMFDA in multi-well formats should facilitate study of osmotic and transport activity and screening for drugs that affect pHi.