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Alanazi and Alghali: Investigating calcium signalling in pancreatic beta cell lines

Introduction: The overall aim of the research was to assess the effect of different cystic fibrosis transmembrane conductance regulator mutations on calcium signalling in the pancreatic beta cell. This is important as an increase of cytosolic Ca2+ concentration is critical in the signal transduction mechanisms that cause pancreatic beta cells to elicit exocytosis of insulin-containing vesicles.

Objectives: To introduce fluo-5N dye into BRIN-BD11 cell lines to assess Ca2+ concentration within intracellular calcium stores in this cell line.

Materials and methods: BRIN-BD11 cells were maintained in RPMI medium, washed with phosphate-buffered saline (PBS) and trypsinized. BRIN-BD11 cells were then preincubated with 1–5 µM fluo-5N/AM for 1–3 hours at 37°C. Cells were recovered by centrifugation and the supernatant was removed by aspiration. Cells were resuspended in PBS containing 3 mM glucose and 1 mM CaCl2. Spectrophotometric measurements were recorded from 2 ml aliquots of magnetically stirred cell suspensions (between 5 × 104 and 1 × 106 cells/ml) at 37°C using a Cairn Research Fluorescence Spectrophotometer (Cairn Research, Kent, UK). Fluo-5N fluorescence was measured using an excitation wavelength of 480 nm and emission set at 515 nm.

Results: See Figure 1.

FIGURE 1

Passive depletion of intracellular Ca2+ stores of BRIN-BD11 cells. Fluo-5N-loaded BRIN-BD11 cells (1 × 106 cells/ml) were resuspended in a Ca2+-containing PBS. Extracellular Ca2+ was chelated by addition of 2 mM ethylene glycol tetraacetic acid, followed 1 minute later by addition of thapsigargin (1 µM) and Ionomycin (1 µM) to deplete the endoplasmic reticulum Ca2+ stores. Three minutes later, nigericin (5 µM) was added to elicit the depletion of the acidic Ca2+ stores.

Suppl-1-45-fig1.jpg

Conclusions: These data indicate that we can load fluo-5N into intracellular calcium stores and that we may be able to use this approach to measure changes in Ca2+ stored within this compartment. However, further validation is needed.

Acknowledgements: Dr Alan Harper and Dr Catriona Kelly for supervising this project.




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