title: Effective Isolation of Functional Islets from Neonatal Mouse Pancreas.
abstract: Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks' Balanced Salt Solution (HBSS) at 37 °C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.
authors.affiliation: Department of Cell and Development Biology, Vanderbilt University Medical School.
authors.forename: Chen
authors.initials: C
authors.lastname: Huang
authors.affiliation: Department of Cell and Development Biology, Vanderbilt University Medical School; Guoqiang.gu@vanderbilt.edu.
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Gu
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000737:chemistry; D002469:Cell Separation / Q000379:methods*; D017364:Collagenases / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008274:Magnesium / Q000737:chemistry; D051379:Mice; D008808:Mice, Inbred CBA; D010179:Pancreas / Q000166:cytology*
chemical_list: D003470:Culture Media; D007328:Insulin; D017364:Collagenases; D008274:Magnesium; D005947:Glucose; D002118:Calcium