A novel fluorescence imaging approach for comparative measurements of pancreatic islet function in vitro.
Pancreatic islet dysfunction is a key element in the development of type 2 diabetes. Determining possible early warning signs of dysfunction is thus important to determining the underlying causes of diabetes. We describe an improved fluorescent imaging approach to detect potential islet dysfunction. Using Cell Tracker Red (CTR, a mildly thiol-reactive fluorescent probe) to positively label particular islets, we measured intracellular free calcium with fura-2 AM in both CTR-labeled and unlabeled sets of pancreatic islets simultaneously in vitro. This approach enhances sensitivity by controlling for differences in background fluorescence, temperature, and perifusion dynamics. We confirmed that 200 nM CTR produced no spectral overlap with fura-2 and no significant physiological effects in selective tests of islet function. To demonstrate the utility of dual-labeling, we compared untreated islets with islets pretreated with low-dose pro-inflammatory cytokines (IL-6 + IL-1B) to induce mild dysfunction. We alternated CTR-labeling between control and test islets and identified consistent reductions in the amplitude and trajectory of glucose-stimulated calcium responses (GSCa) among cytokine-treated islets that were independent of labeling. Observations were verified using a MATLAB program specifically designed to identify key features in the GSCa. Our findings thus demonstrate the utility of CTR-labeling in identifying islet dysfunction and propose that this technique can be adapted for other cells and tissues.
Department of Medicine, Division of Endocrinology and Metabolism, University of Virginia, Charlottesville, VA, USA.
Kathryn L
KL
Corbin
Thomas E
TE
Hall
Ruth
R
Haile
Craig S
CS
Nunemaker
D000818:Animals; D002118:Calcium / Q000378:metabolism; D058948:Cell Tracking / Q000379:methods; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000493:pharmacokinetics / Q000494:pharmacology; D016257:Fura-2 / Q000493:pharmacokinetics / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D007515:Islets of Langerhans / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D008163:Luminescent Measurements / Q000379:methods*; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence
D016207:Cytokines; D005456:Fluorescent Dyes; D005947:Glucose; D002118:Calcium; D016257:Fura-2
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