Fluorescent pH probes, Fluorescent Proteins, and Intrinsic Cellular Fluorochromes are Tools to Study Cytosolic pH (pH~(cyt)) in Mammalian Cells

摘要

Our understanding of intracellular pH homeostasis in eukaryotic systems has been enhanced since the introduction of carboxyfluorescein diacetate as a useful pH probe more than 20 years ago. BCECF, a derivative of this earlier fluoroprobe has dominated the field. In the past 10 years, SNARF-1 has emerged as an alternative pH probe. Recently, a novel derivative of BCECF, BCPCF has been developed. Green Fluorescent Proteins (GFP's) have also been used recently to monitor pH in a "non invasive manner" in several cell types. Here, we report that human mammary epithelial cells can be transfected with the gene encoding for cyan (CFP), green (GFP), and yellow (YFP), to study cytosolic pH. The novel red fluorescent protein (DsRed) is not sensitive to pH. Multidrug resistance (MDR) has been associated with altered cytosolic pH homeostasis. We show that experimental maneuvers that decrease pH~(in) enhance the efficacy of chemotherapeutic drugs. We also show that short pulses of UV-B light elicited acidosis in cells, as evaluated by ratio ion cell imaging, and confocal/spectral imaging microscopy. During the course of these experiments we noticed that cells exhibit intrinsic fluorochromes that can be used to monitor pH in living cells.