A practical and robust method to evaluate metabolic fluxes in primary pancreatic islets

• DOI: 10.1016/j.molmet.2024.101922
• Publication Date: 2024-03-21T08:45:19Z
• Authors: Debora S. Rocha; Antonio C. Manucci; Alexandre Bruni-C...; Alicia J. Kowalto...; Eloisa A. Vilas-Boas
• Keywords: Pancreatic Islets; Primary (astronomy)

Evaluation of mitochondrial oxygen consumption and ATP production is important to investigate pancreatic islet pathophysiology. Most studies use cell lines due difficulties in measuring primary respiration, which requires specific equipment consumables, expensive poorly reproducible. Our aim was establish a practical method assess metabolic fluxes using standard commercial consumables. Pancreatic islets were isolated from mice/rats, dispersed with trypsin, adhered pre-coated Seahorse or Resipher microplates. Oxygen evaluated Extracellular Flux Analyzer Real-time Cell Analyzer. We provide detailed protocol all steps optimize isolation high yield functionality. few per replicate; both rat mouse present robust basal respiration proper response modulators glucose. The technique validated by other functional assays, show these cells conserved calcium influx insulin secretion also that our maintain levels, addition maintaining up 89% viability after five days cultures. Furthermore, OCRs can be measured analyzers plate respirometry systems, materials. Overall, we established oxidative phosphorylation, valuable tool uncover basic β-cell mechanisms as well for translational investigations, such pharmacological candidate discovery transplantation protocols.