Isolation and Culture of Mouse Pancreatic Islets for Ex Vivo Imaging Studies with Trappable or Recombinant Fluorescent Probes
Isolation and Culture of Mouse Pancreatic Islets for Ex Vivo Imaging Studies with Trappable or Recombinant Fluorescent Probes
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DOI：
10.1007/978-1-59745-019-5_12
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发表时间：
2010-01-01
期刊：
影响因子：
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通讯作者：
Rutter, Guy A.
中科院分区：
文献类型：
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作者：
Ravier, Magalie A.;Rutter, Guy A.
内分泌胰腺包含1,000-2,000个神经内分泌细胞的小簇，称为朗格汉斯胰岛。胰岛根据情况分泌胰岛素、胰升糖素或其他激素，在控制哺乳动物体内的葡萄糖稳态方面起着核心作用。胰岛分布于整个外分泌组织，仅占整个器官体积的1-2%；人胰腺约有10(6)个胰岛，而啮齿类动物约有2×10(3)个胰岛。胰岛与外分泌组织的分离通常始于用胶原酶消化胰腺。含有胶原酶的培养液要么注射到胰管内，器官留在原位消化，要么在分离胰腺后加入，并在体外将其解剖成小块。然后，胰岛可以通过梯度密度或手工采摘从外分泌组织中分离出来。所获得的胰岛可以原封不动地使用，例如，用于测量胰岛素或胰升糖素的分泌，也可以用无钙培养液或胰蛋白酶/胰淀粉酶分散成单个细胞。后者有助于引入重组或可捕获的探针和显微成像研究，例如，细胞内游离钙浓度的变化或单个细胞器或蛋白质的动态变化。
The endocrine pancreas contains small clusters of 1,000-2,000 neuroendocrine cells termed islets of Langerhans. By secreting insulin, glucagon, or other hormones as circumstances dictate, islets play a central role in the control of glucose homeostasis in mammals. Islets are dispersed throughout the exocrine tissue and comprise only 1-2% of the volume of the whole organ; human pancreas contains about 10(6) islets whereas rodents have approximately 2 x 10(3) islets. The isolation of islets from the exocrine tissue usually begins with digestion of the pancreas with collagenase. Collagenase-containing medium is either injected into the pancreatic duct, and the organ left to digest in situ, or added after isolation of the pancreas and its dissection into small pieces ex vivo. Islets can then be separated from the exocrine tissue by gradient density or by handpicking. The islets obtained can either be used intact, for example, to measure insulin or glucagon secretion or be dispersed into single cells with a Ca2+-free medium or with trypsin/dispase. The latter facilitates the introduction of recombinant or trappable probes and microimaging studies of for example, changes in cytosolic-free Ca2+ concentration or the dynamics of individual organelles or proteins.