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

Summary

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.

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 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. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here 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, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Introduction

Recent studies revealed a role for changes in pHi during cellular differentiation and dysplasia in vivo^1,2. These studies found that pHi is remarkably consistent in cells of the same type at the same stage of differentiation, but that it changes as cells transition from one stage to another. In some cases, blocking the changes in pHi partially disrupts differentiation, suggesting that the change in pHi is not just a consequence of changes in cell fate but instead helps to promote the change in cell fate, perhaps through effects on pH-sensitive regulatory proteins or chemical reactions required for dif....

Protocol

NOTE: To measure pHi in the FSC lineage, we calculate the ratio of fluorescence intensities of pHluorin to mCherry in FSCs, pFCs, and follicle cells in physiological conditions, and convert the ratios into pHi values with standard calibration curves for each cell type⁷. First, live imaging experiments are performed to measure fluorescence intensities of pHluorin and mCherry in germaria dissected into a buffer containing NaHCO₃, which mimics physiological conditions^1,7. Next, standard curves are generated by measuring pHluorin and mCherry fluorescence intensities in a Na^+....

Results

Here we have described the process of measuring pHi in the follicle epithelium, which involves several steps. First, the ovaries are dissected from flies of the appropriate genotype using tools for dissecting and mounting (Figure 1). The ovarioles are then imaged using quantitative fluorescence microscopy and the images are analyzed to obtain measurements of pHi. For each image, the cell types of interest are identified as described in Section 3.1 (

Discussion

Here, we describe a method for measuring the pHi of cells in the FSC lineage within wildtype tissue. This protocol has been developed and refined over the past five years, since we first began to study pHi in Drosophila ovarian tissue. During that time, the protocol has been used successfully by multiple investigators in our lab and on at least four different spinning disc and laser scanning microscopes. The reproducibility of our original observation, that pHi increases as cells in the FSC lineage differentiate.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fly Stocks
UAS-mCherry::pHluorin[1]
y1 w*;P{GawB}10930/CyO	Bloomington Stock Center	7023
Act-Gal4 flipout stock	Bloomington Stock Center	4409
Name	Company	Catalog Number	Comments
Chemicals for Buffer preparation
NaCl	Sigma Aldrich	S5886
KCl	Sigma Aldrich	P-3911
glucose	Mallinckrodt	4912
HEPES	Thermo Fisher Scientific	BP310
MgSO4	Thermo Fisher Scientific	M63
CaCl2	Sigma Aldrich	C-5080
HCO3	Sigma Aldrich	S-5761
MgCl2	Sigma Aldrich	M-9272
NMDG+	Sigma Aldrich	M-2004
K2HPO4	Mallinckrodt	7088	Use to Make KHPO4 pH 7.4
KH2PO4	Thermo Fisher Scientific	BP362	Use to Make KHPO4 pH 7.4
Concanavalin A, Alexa Fluor 647 Conjugate	Thermo Fisher Scientific	C21421	0.25 mg/ml dilution
Nigericin	Thermo Fisher Scientific	N1495
Name	Company	Catalog Number	Comments
Dissection and mounting tools
2 Dumont Inox forceps (Size 5)	Thermo Fisher Scientific	NC9473431
2 23-gauge syringe needles	Sigma Aldrich	Z192457
9-well glass dissecting dish	Thermo Fisher Scientific	13-748B
Vacuum Grease	Dow Corning	1018817
22 X 40 mM glass coverslips	Thermo Fisher Scientific	12545C
Round Glass Coverslips, 12mm diameter, 0.13-0.16mm thickness	Ted Pella, Inc.	26023
3-D mounting chamber	custom manufactured		.stl and .ipt files for 3-D printer included as supplemental files
Name	Company	Catalog Number	Comments
Other equipment
pH meter	Thermo Fisher Scientific	13-620-183A	Model: Accumet AB15
Dissection microscope	Olympus Corporation	0H11436	Model: SZ2-ST
Confocal Microscope	Leica Biosystems		SP5 or SP8 laser-scanning confocal microscope with a 40× objective with a numerical aperture of 1.3