Measurement of pancreatic islet beta-cell proliferation by flow cytometry v1

This protocol describes the steps to assess pancreatic beta-cell proliferation by flow cytometry. It is suitable for islets isolated from both rodents and humans. We routinely apply this protocol to quantify total and proliferating beta-cells but, with the appropriate antibody-fluorophore conjugates, can be easily adapted to assess other islet cells types (eg. alpha and delta) and markers (eg. apoptosis). Briefly, islets labelled with the proliferation marker EdU are dispersed, dead cells labeled and fixed. EdU is detected using the Click-iT Plus EdU Flow Cytometry Assay Kit and beta-cells immunostained with fluorophore-coupled anti-insulin antibodies. Beta-cell proliferation is then determined by flow cytometry by calculating the percentage of double-positive cells for EdU and insulin over the corresponding total insulin-positive cell population.