CE analysis of the acidic organelles of a single cell

Abstract

CE analysis of the acidic organelles of a single cellThe properties of organelles within a cell have been shown to be highly heterogeneous. Until now, it has been unclear just how much of this heterogeneity is endemic to the organelle subpopulations themselves and how much is actually due to stochastic cellular noise. An attractive approach for investigating the origins of heterogeneity among the organelles of a single cell is CE with LIF detection (CE-LIF). As a proof of principle, in this report we optimize and use a single cell CE-LIF method to investigate the properties of endocytic (acidic) organelles. Our results show that the properties of individual acidic organelles containing Alexa Fluor ® 488 Dextran suggest that there are two groups of CCRF-CEM cells: a group with a high dextran content per cell, and a group with a low dextran content per cell. Furthermore, the individual organelle measurements of the single cells allow us to compare in each group the distributions of doxorubicin content per acidic organelle and electrophoretic mobilities of these organelles. Keywords IntroductionHeterogeneity among cells of the same type is known to result partly from intracellular processes, such as pinocytosis [1], the expression of proteins participating in DNA replication [2], and cellular responses to drug treatments [3], and partly from the microenvironment in which the cells reside [4]. Within the cell, heterogeneity at the subcellular level, which refers to variations in the properties of organelles of a given type, has remained practically uncharacterized up to this point. There is a great need to develop and apply single cell approaches capable of measuring the different facets of cellular heterogeneity at the subcellular level. Among the existing techniques for investigating subcellular heterogeneity, chemical imaging has been used to probe the heterogeneity of subcellular properties such as organellar pH, ion concentration, and protein interactions [5,6]. Flow cytometry [7] and CE with LIF detection (CE-LIF) [7,8] have both been used to analyze individual organelles in samples prepared from thousands to millions of cells. With all of these techniques, it has been impossible to track down which organelles originate from the same cell, information that is needed if we are to determine cell-to-cell variations.In order to determine cell-to-cell variations, single cells have been analyzed by CE [9], chromatography [10], MS [11], enzymatic radiolabeling [12], sensitive voltammetric detection [13], and flow cytometry [14]. CE has been used to describe the contents and functions of single cells (e.g., protein expression [15], release of neurotransmitters, and cell secretion [16]). In regard to the subcellular analysis of single cells, CE has been used to sample small subcellular regions, and to measure properties of the whole nucleus of a single cell [17,18]. The ability to detect individual mitochondrial particles by CE-LIF after their release from single cells has been recently described [19]. This report sho...

A PDMS sheath flow cuvette for high‐sensitivity LIF measurements in CE

“…The sheath flow cuvette has also begun to reprise its role in cytometry but in a new field termed ''molecular cytometery'', where individual cells are lysed and the cellular components are separated and Cameron D. Skinner detected [15][16][17][18]. The cuvette's ability to focus the effluent from the separation capillary, including particles, allows cellular species and organelles to be characterized optically and according to their electrophoretic mobility [19,20].…”

Recent advances in the analysis of biological particles by capillary electrophoresis

“…These organelles were separated using -300 V/ cm and detected with a two-channel post-column LIF detector. Chen et al [53] used the same approach to investigate a cell-to-cell variation in DOX distribution among the acidic organelles in CCRF-CEM cells. The cells were incubated with Alexa 488-labeled dextran and DOX.…”

Electrochemical Probes for Detection and Analysis of Exocytosis and Vesicles

“…This group has had success in applying the CE-LIF methodology to investigate a number of subcellular components including cell nuclei, [52] mitochondria, [53] and various acidic organelles. [54] A long-standing assumption in the study of exocytosis is that release is quantal in nature and that each quantum is comprised of the contents of a single vesicle. [13][14]55] With this premise, amperometric detection of release has, for instance, been used to quantify the chemical content of vesicles from different cell types.…”