A Practical Guide to Rodent Islet Isolation and Assessment

Carter, Jeffrey D.; Dula, Stacey B.; Corbin, Kathryn L.; Wu, Runpei; Nunemaker, Craig S.

doi:10.1007/s12575-009-9021-0

Cited by 236 publications

(197 citation statements)

References 53 publications

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“…However, islets in this work were healthy, with a SI between 2 and 20 (Carter et al 2009) not showing any harmful effect of Histopaque on the islets, as supported also by more recent studies performed with Ficoll-based gradients (McCall et al 2011;Mita et al 2010;Lamb et al 2011), nor were any statistical differences between methods observed. However, although GSIS is an important predictor of islet function it does not have a correlation with clinical transplantation outcomes (Ricordi et al 2001;Street et al 2004).…”

Section: Discussionsupporting

confidence: 86%

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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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Section: Discussionsupporting

confidence: 86%

“…Islet function is defined by its ability to regulate the release of insulin and other hormones in response to changes in extracellular glucose concentration (Carter et al 2009). Therefore, GSIS provides an accepted measure of islet function and, consequently, of the quality of the preparation.…”

Section: Discussionmentioning

confidence: 99%

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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“…The cell suspension was centrifuged at 1,500 rpm for 4 min, the precipitated cells were re -suspended in HBSS solution, passed through a 100-μm cell strainer (BD Biosciences, San Jose, CA, USA), and collected into a 10 -cm dish. Pancreatic islets were handpicked and cultured at 37" !under a humidified atmosphere of 5% CO2 and 95% air in islet medium (RPMI-1640 medium containing 2.0 g/l NaHCO3, 10 mM HEPES solution, 50 μM 2 -mercaptoethanol, 1 mM sodium pyruvate, 10% FBS, 100 IU/ml penicillin, and 50 μg/ml streptomycin) (22).…”

Section: Pancreatic Islet Isolationmentioning

confidence: 99%

Protein kinase C-δ signaling regulates glucagon secretion from pancreatic islets

et al. 2017

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: Accumulating evidence supports the "glucagonocentric hypothesis", in which antecedent α-cell failure and inhibition of glucagon secretion are responsible for diabetes progression. Protein kinase C (PKC) is involved in glucagon secretion from α-cells, although which PKC isozyme is involved and the mechanism underlying this PKC-regulated glucagon secretion remains unknown. Here, the involvement of PKCδ in the onset and progression of diabetes was elucidated. Immunofluorescence studies revealed that PKCδ was expressed and activated in α-cells of STZ-induced diabetic model mice. Phorbol 12-myristate 13-acetate (PMA) stimulation significantly augmented glucagon secretion from isolated islets. Pre-treatment with quercetin and rottlerin, PKCδ signaling inhibitors, significantly suppressed the PMA-induced elevation of glucagon secretion. While Go6976, a Ca 2+ -dependent PKC selective inhibitor did not suppress glucagon secretion. Quercetin suppressed PMA-induced phosphorylation of Tyr 311 of PKCδ in isolated islets. However, quercetin itself had no effect on either glucagon secretion or glucagon mRNA expression. Our data suggest that PKCδ signaling inhibitors suppressed glucagon secretion. Elucidation of detailed signaling pathways causing PKCδ activation in the onset and progression of diabetes followed by the augmentation of glucagon secretion could lead to the identification of novel therapeutic target molecules and the development of novel therapeutic drugs for diabetes.

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“…Islets were isolated from pancreas of rats by collagenase V digestion and Ficoll 400 step density gradient separation as described (Cherif et al 1998, Carter et al 2009, Li et al 2009). …”

Section: Cell Culture and Treatmentsmentioning

confidence: 99%

Low-level phenolic estrogen pollutants impair islet morphology and β-cell function in isolated rat islets

Song¹,

Xia²,

Zhao³

et al. 2012

Journal of Endocrinology

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Phenolic estrogen pollutants, a class of typical endocrinedisrupting chemicals, have attracted public attention due to their estrogenic activities of imitating steroid hormone 17b-estradiol (E 2 ) effects. Exposure to these pollutants may disrupt insulin secretion and be a risk factor for type 2 diabetes. In this study, we investigated the direct effects of phenolic estrogen diethylstilbestrol (DES), octylphenol (OP), nonylphenol (NP), and bisphenol A (BPA) on rat pancreatic islets in vitro, whose estrogenic activities were DESONPOOPOBPA. Isolated b-cells were exposed to E 2 , DES, OP, NP, or BPA (0, 0 . 1, 0 . 5, 2 . 5, 25, and 250 mg/l) for 24 h. Parameters of insulin secretion, content, and morphology of b-cells were measured. In the glucose-stimulated insulin secretion test, E 2 and DES increased insulin secretion in a dose-dependent manner in a 16 . 7 mM glucose condition. However, for BPA, NP, or OP with lower estrogenic activity, the relationship between the doses and insulin secretion was an inverted U-shape. Moreover, OP, NP, or BPA (25 mg/l) impaired mitochondrial function in b-cells and induced remarkable swelling of mitochondria with loss of distinct cristae structure within the membrane, which was accompanied by disruption of mRNA expression of genes playing a key role in b-cell function (Glut2 (Slc2a2), Gck, Pdx1, Hnf1a, Rab27a, and Snap25), and mitochondrial function (Ucp2 and Ogdh). Therefore, these phenolic estrogens can disrupt islet morphology and b-cell function, and mitochondrial dysfunction is suggested to play an important role in the impairment of b-cell function.

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A Practical Guide to Rodent Islet Isolation and Assessment

Cited by 236 publications

References 53 publications

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

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

Protein kinase C-δ signaling regulates glucagon secretion from pancreatic islets

Low-level phenolic estrogen pollutants impair islet morphology and β-cell function in isolated rat islets

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