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Investigating calcium signalling in pancreatic beta cell lines

K Alanazi, A Alghali
Published in : HAMDAN MEDICAL JOURNAL ; Vol 8, No 4 (2015): Supplement Issue
DOI : 10.7707/hmj.529

Abstract


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– 5μM fluo-5N/AM for 1– 3hours at 37°C. Cells were recovered by centrifugation and the supernatant was removed by aspiration. Cells were resuspended in PBS containing 3mM glucose and 1mM CaCl2 . Spectrophotometric measurements were recorded from 2ml aliquots of magnetically stirred cell suspensions (between 5Å~104 and 1Å~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 480nm and emission set at 515nm.

Results: See Figure 1.

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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