%0 Journal Article
%T A measure of endosomal pH by flow cytometry in Dictyostelium
%A Anna Marchetti
%A Emmanuelle Lelong
%A Pierre Cosson
%J BMC Research Notes
%D 2009
%I BioMed Central
%R 10.1186/1756-0500-2-7
%X We have revisited these classical protocols to measure more accurately endosomal pH, making use of a fluorescent probe (Oregon green) more adequate for very acidic pH values. This pH-sensitive probe was combined with a pH-insensitive marker, in order to visualize simultaneously endosome dynamics and pH changes. Finally, a flow cytometer was used to measure endosomal pH in individual cells.Using these simple protocols the endosomal pH of endocytic compartments can be assessed accurately, revealing the extreme acidity of Dictyostelium lysosomes (pH <3.5).Dictyostelium amoebae are widely used to study the organization and function of the endocytic pathway. Due to their small, haploid, and fully sequenced genome [1], these cells are easily amenable to genetic studies. Analysis of specific knockout mutants has been instrumental in revealing the role of various gene products in diverse aspects of endocytosis, and this analysis relies on the use of well-documented protocols describing the various facets of endosome biology. In Dictyostelium, endocytosed material is found within minutes in lysosomes, which are acidified by the action of a V-type H+-ATPase [2]. Endocytosed material is then transferred more slowly (>30 minutes) to post-lysosomes devoid of H+-ATPase, which are less acidic [3,4]. The dynamics and organization of the endocytic pathway can be analyzed in a given mutant cell by determining the number and size of lysosomes and post-lysosomes, and the speed of transfer between these two compartments [5,6], or by measuring the pH of endosomal compartments.In Dictyostelium, the endosomal pH has usually been measured by following the fluorescence of endocytosed fluorescein isothiocyanate (FITC)-coupled dextran in a spectrofluorimeter (reviewed in [7]). FITC has a pKa of approximately 6.5 and its ionization equilibrium leads to pH-dependent fluorescence over a range of 5 to 9. The values measured in early endocytic compartments (lysosomes) were as low as pH 4.7 [3,4], w
%U http://www.biomedcentral.com/1756-0500/2/7