Isolated Pancreatic Islet Treatment and Apoptosis Measurement.

Abstract

This study investigates the effect of pro-inflammatory cytokines on pancreatic islets, particularly insulin-producing β-cells, using a combination of fluorescence staining techniques and confocal microscopy to assess cell viability, apoptosis, and β-cell-specific death. Isolated mouse islets were treated with varying concentrations of a cytokine cocktail, including TNF-α, IL-1β, and IFN-γ, to mimic immune-mediated apoptosis during the development of type 1 diabetes. The viability of islet cells was evaluated with FDA/PI dual staining, where FDA conversion to fluorescein indicated viable cells, and PI marked membrane-compromised cells. YOPRO-1 and nuclear staining provided additional data on apoptosis, with Annexin-V confirming early apoptotic cells. Quantitative analysis revealed significant increases in apoptosis and cell death rates in cytokine-treated islets. To specifically assess effects on β-cells, Zn²⁺ selective indicator staining was used to label insulin-producing cells through the zinc association in insulin granules, revealing substantial β-cell loss following treatment of islets with pro-inflammatory cytokines for 24 h. These multi-staining protocols effectively capture and quantify the extent of cytokine-induced damage in islets and can be used to evaluate therapeutics designed to prevent β-cell apoptosis in early type 1 diabetes.