A protocol for islet isolation from mouse pancreas

Li, Dongsheng; Yuan, Yahong; Tu, Han-Jun; Liang, Qingle; Dai, Long‐Jun

doi:10.1038/nprot.2009.150

Cited by 299 publications

(228 citation statements)

References 11 publications

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“…Isolation and culture of islets of Langerhans Murine islets were isolated from 6-to 9-week-old male C57/BL6J mice using collagenase digestion, followed by Ficoll gradient purification, and manually harvested as described by Li et al [16]. The purified islets were collected using a glass pipette under a stereo microscope (Olympus SZ51, Bilbao, Spain) and maintained in Petri dishes.…”

Section: Methodsmentioning

confidence: 99%

Cardiotrophin 1 protects beta cells from apoptosis and prevents streptozotocin-induced diabetes in a mouse model

et al. 2013

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Aims/hypothesis Cardiotrophin 1 (CT-1) is a recently described cytokine originally isolated from the heart where it has been shown to play an important role in apoptotic protection of cardiomyocytes and heart hypertrophy. Its beneficial properties have also been described in other organs such as liver and neuromuscular tissue. In the present study, we investigated whether CT-1 can confer protection against pro-apoptotic stimuli in pancreatic beta cells, and its role in insulin secretion and diabetes development.Methods The effects of CT-1 on apoptosis and function were studied using MIN6B1 cells and freshly isolated murine pancreatic islets. The impact on the development of diabetes was evaluated in Ct1-null (Ct1 −/− ) mice (the gene Ct1 is also known as Ctf1) using two streptozotocin (STZ)-induced models of diabetes. Results CT-1 has a protective effect in MIN6B1 cells and murine islets under the pro-apoptotic stimulus of serum deprivation, which correlates with the expression of B cell lymphoma-extra large, or following exposure to a mixture of cytokines. In addition, CT-1 enhances glucose-stimulated insulin secretion in MIN6B1 cells and this was repressed by inhibitors of phospholipase C. Furthermore, Ct1 −/− mice were more prone to develop diabetes, and their glucose tolerance test showed impaired plasma glucose clearance which correlated with decreased pancreatic insulin secretion. Conclusions/interpretation The results obtained from both in vitro and in vivo experiments show that CT-1 improves beta cell function and survival, and protects mice against STZ-induced diabetes.

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Section: Methodsmentioning

confidence: 99%

Cardiotrophin 1 protects beta cells from apoptosis and prevents streptozotocin-induced diabetes in a mouse model

et al. 2013

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“…Islets were isolated from wild-type and TRPM2 − / − mice as described previously [25]. Briefly, pancreata were dissected from mice, minced into pieces (∼ 1 mm 3 ) and digested with collagenase [type IA; 1 mg · ml − 1 in Hanks balanced salt solution (HBSS)] for 10 min with gentle agitation at 37 • C. After washing with RPMI 1640, islets were hand-picked under a dissecting microscope and incubated overnight in the RPMI 1640 medium prior to drug treatments.…”

Section: Mouse Islets and Streptozotocin-induced Cell Deathmentioning

confidence: 99%

TRPM2-mediated intracellular Zn2+ release triggers pancreatic β-cell death

Manna

Munsey

Abuarab

et al. 2015

Biochemical Journal

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Reactive oxygen species (ROS) can cause pancreatic β-cell death by activating transient receptor potential (melastatin) 2 (TRPM2) channels. Cell death has been attributed to the ability of these channels to raise cytosolic Ca 2 + . Recent studies however revealed that TRPM2 channels can also conduct Zn 2 + , but the physiological relevance of this property is enigmatic. Given that Zn 2 + is cytotoxic, we asked whether TRPM2 channels can permeate sufficient Zn 2 + to affect cell viability. To address this, we used the insulin secreting (INS1) β-cell line, human embryonic kidney (HEK)-293 cells transfected with TRPM2 and pancreatic islets. H 2 O 2 activation of TRPM2 channels increases the cytosolic levels of both Ca 2 + and Zn 2 + and causes apoptotic cell death. Interestingly, chelation of Zn 2 + alone was sufficient to prevent β-cell death. The source of the cytotoxic Zn 2 + is intracellular, found largely sequestered in lysosomes. Lysosomes express TRPM2 channels, providing a potential route for Zn 2 + release. Zn 2 + release is potentiated by extracellular Ca 2 + entry, indicating that Ca 2 + -induced Zn 2 + release leads to apoptosis. Knockout of TRPM2 channels protects mice from β-cell death and hyperglycaemia induced by multiple low-dose streptozotocin (STZ; MLDS) administration. These results argue that TRPM2-mediated, Ca 2 + -potentiated Zn 2 + release underlies ROS-induced β-cell death and Zn 2 + , rather than Ca 2 + , plays a primary role in apoptosis.

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“…In this sense, in our hands the filtration protocol described by Li et al (2009) achieved only 71.96 ± 3.60 % of purity (unpublished results) in contrast to their claim of almost 100 % purity in most cases, making this protocol unfeasible for the purification of islets by filtration alone with a 70 lm cell strainer. Therefore, we decided to filter preparations through a 100 lm cell strainer, as reported by Salvalaggio et al (2002).…”

Section: Discussionmentioning

confidence: 74%

“…Islets were purified according to the protocol by Li et al 2009 with some modifications (Collagenase P instead of Collagenase XI, filtering through a 100 lm cell strainer instead of a 70 lm one).…”

Section: Filtrationmentioning

confidence: 99%

“…In the current study, we first assessed the yield and quality of mouse islets isolated by two protocols of Histopaque and filtration purification, the latter with some modifications (Li et al 2009), taking the handpicking as the gold standard method for islet purification. Besides, we made a balance considering also the cost and the time consumed by each procedure, which are also determinant in the election of a method for a laboratory routine.…”

Section: Introductionmentioning

confidence: 99%

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Filtration is a time-efficient option to Histopaque, providing good-quality islets in mouse islet isolation

Ramírez-Domínguez

Castaño

2014

Cytotechnology

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Pancreatic islet transplantation is a promising therapy for Type I Diabetes. For many years the method used worldwide for islet purification in both rodent and human islet isolation has been Ficoll-based density gradients, such as Histopaque. However, it is difficult to purify islets in laboratories with staff limitations when large scale isolations are required. We hypothesized that filtration could be a more simple and fast alternative to obtain good quality islets. Four separate islet isolations were performed per method, comparing filtration and Histopaque purification with handpicking as the gold standard method for islet purity. Different parameters of quality were assessed: yield in number of islets per pancreas, purity by dithizone staining, viability by Fluorescein Diacetate/ Propidium Iodide vital staining and in vitro functionality assessed by Glucose Stimulated Insulin Secretion. Time efficiency and cost were also analyzed. The overall quality of the islets obtained both by Histopaque and filtration was good. Filtration saved almost 90 % of the time consumed by Histopaque purification, and was also cheaper. However, one-third of the islets were lost. Since human and rodent islets share similar size but different density, filtration appears as a purification method with potential interest in translation to clinic.

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A protocol for islet isolation from mouse pancreas

Cited by 299 publications

References 11 publications

Cardiotrophin 1 protects beta cells from apoptosis and prevents streptozotocin-induced diabetes in a mouse model

Cardiotrophin 1 protects beta cells from apoptosis and prevents streptozotocin-induced diabetes in a mouse model

TRPM2-mediated intracellular Zn2+ release triggers pancreatic β-cell death

Filtration is a time-efficient option to Histopaque, providing good-quality islets in mouse islet isolation

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