Purification of replicating pancreatic ?-cells for gene expression studies.

ABSTRACT: ?-cell proliferation is a rare event in adult pancreatic islets. To study the replication-related ?-cell biology we designed a replicating ?-cells sorting system for gene expression experiments. Replicating ?-cells were identified by EdU incorporation and purified by flow cytometry. For ?-cell separation islet cells were sorted by size, granularity and Newport Green fluorescence emission that was combined with emitted fluorescence for EdU-labelled replicating cells sorting. The purity of the resulting sorted populations was evaluated by insulin staining and EdU for ?-cell identification and for replicating cells, respectively. Total RNA was isolated from purified cell-sorted populations for gene expression analysis. Cell sorting of dispersed islet cells resulted in 96.2% purity for insulin positivity in the collected ?-cell fraction and 100% efficiency of the EdU-based cell separation. RNA integrity was similar between FACS-sorted replicating and quiescent ?-cells. Global transcriptome analysis of replicating vs quiescent ?-cells showed the expected enrichment of categories related to cell division and DNA replication. Indeed, key genes in the spindle check-point were the most upregulated genes in replicating ?-cells. This work provides a method that allows for the isolation of replicating ?-cells, a very scarce population in adult pancreatic islets.

SUBMITTER: Carballar R 

PROVIDER: S-EPMC5727529 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature