Abstract
The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.
Highlights
The overall goal of this method is to isolate and culture mixed murine intestinal cells to enable the study of gut peptide secretion and live cell imaging of enteroendocrine cells
Enteroendocrine cells secrete an array of gut peptides including glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), cholecystokinin (CCK) and peptide YY (PYY)[2]
Using cultures generated from transgenic mice expressing the calcium fluorescent sensor, GCaMP3, under the control of the proglucagon promoter[11], L cells are readily identifiable dependent and interspersed within the culture (Figure 2B). pathways stimulated the secretion of GLP-1
Summary
The overall goal of this method is to isolate and culture mixed murine intestinal cells to enable the study of gut peptide secretion and live cell imaging of enteroendocrine cells. Gut peptide secretion can be studied using a variety of in vitro and ex vivo experimental models, each with advantages and disadvantages (for a comprehensive recent review covering the use of in vivo models see Svendsen et al.[4]). Ex vivo techniques such as the isolated perfused intestine[5] and Ussing chambers[6] are currently being used to explore gut peptide secretion in response to various substances. All animal procedures were approved by the University of Cambridge Animal Welfare and Ethical Review Body and conformed to the Animals (Scientific Procedures) Act 1986 Amendment Regulations (SI 2012/3039)
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