Flow Cytometric Quantification of Glucose-Stimulated -Cell Metabolic Flux Can Reveal Impaired Islet Functional Potency

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Abstract:

The objective of this study was to develop a multiparametric flow cytometry assay to simultaneously quantify isolated pancreatic islet cell viability, apoptosis, and glucose-induced metabolic flux. INS-1 and rat islet -cells were stained with fluorescent probes for cell viability (ToPro3), apoptosis (Annexin V and VADFMK), and intracellular calcium (Ca2+i) (Fura Red), stimulated with glucose, and analyzed on a FACS VantageTM flow cytometer. Glucose-induced metabolic activity was indicated by changes in Fura Red fluorescence and the autofluorescence of the pyridine [NAD(P)H] and flavin (FAD/FMN) nucleotides. Rat islets cultured under conditions of proinflammatory cytokine-induced oxidative stress were evaluated by flow cytometry and transplantation into diabetic mice. INS-1 and rat islet -cell health and metabolic activity were quantified in response to elevated glucose dose and inhibitors of glycolysis and mitochondrial function. Changes in metabolite fluorescence were converted to an area under the curve (AUC) value. Rat islets cultured under oxidative stress conditions showed decreased viability, increased apoptosis, and decreased glucose-induced metabolic activity indicated by reduced AUC for pyridine and flavin nucleotides and Ca2+i. Reduced metabolite AUC measured by flow cytometry correlated with the inability to reverse diabetes in mice. Single cell flow cytometry can simultaneously quantify both overall islet cell health and -cell glucose responsiveness as indicators of functional potency.

Keywords: Diabetes; Flow cytometry; Glucose; Islet; Mitochondria; Pancreas; Transplantation; -Cell

Document Type: Research Article

DOI: http://dx.doi.org/10.3727/096368908787648038

Publication date: December 1, 2008

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  • Cell Transplantation publishes original, peer-reviewed research and review articles on the subject of cell transplantation and its application to human diseases. To ensure high-quality contributions from all areas of transplantation, separate section editors and editorial boards have been established. Articles deal with a wide range of topics including physiological, medical, preclinical, tissue engineering, and device-oriented aspects of transplantation of nervous system, endocrine, growth factor-secreting, bone marrow, epithelial, endothelial, and genetically engineered cells, among others. Basic clinical studies and immunological research papers are also featured. To provide complete coverage of this revolutionary field, Cell Transplantation will report on relevant technological advances, and ethical and regulatory considerations of cell transplants. Cell Transplantation is now an Open Access journal starting with volume 18 in 2009, and therefore there will be an inexpensive publication charge, which is dependent on the number of pages, in addition to the charge for color figures. This will allow work to be disseminated to a wider audience and also entitle the corresponding author to a free PDF, as well as prepublication of an unedited version of the manuscript.
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