Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live <em>Drosophila</em> Ovarian Tissue

Abstract

SHORT ABSTRACT We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values. LONG ABSTRACT Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating of pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin.1 Here, we describe a protocol we routinely use for culturing and imaging Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage from mCherry::pHluorin transgenic wild type and mutant lines; however the methods we describe can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, culturing ovarian tissue, and acquiring and analyzing images to obtain pHi values.