Publication:20180105164153

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Publication
URL https://www.ncbi.nlm.nih.gov/pubmed/29235543
Title Purification of replicating pancreatic β-cells for gene expression studies

Authors Reyes Carballar, Maria de Lluc Canyelles, Claudia Fernández, Yasmina Martí, Sarah Bonnin, Esther Castaño, Eduard Montanya, Noèlia Téllez
Date 12 13, 2017

Publisher Scientific Reports
DOI 10.1038/s41598-017-17776-2
Tag Alternative Splicing, Animals, Cell Proliferation, Cell Separation, Cells, Cultured, Fluorescent Antibody Technique, Gene Expression, Gene Expression Profiling, Insulin-Secreting Cells, Male, Rats, Wistar, Transcriptome



Abstract:
β-cell proliferation is a rare event in adult pancreatic islets. To study the replication-related β-cell biology we designed a replicating β-cells sorting system for gene expression experiments. Replicating β-cells were identified by EdU incorporation and purified by flow cytometry. For β-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 β-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 β-cell fraction and 100% efficiency of the EdU-based cell separation. RNA integrity was similar between FACS-sorted replicating and quiescent β-cells. Global transcriptome analysis of replicating vs quiescent β-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 β-cells. This work provides a method that allows for the isolation of replicating β-cells, a very scarce population in adult pancreatic islets.


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