Bovine Serum Albumin Density Gradient Isolation of Rat Pancreatic Islets1

Lake, S. P.; Anderson, Jill; Chamberlain, J.; Gardner, Sue J.; Bell, P. R. F.; James, R. F. L.

doi:10.1097/00007890-198706000-00006

Cited by 70 publications

(34 citation statements)

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“…On the other hand, a number of studies have reported toxic effects of Ficoll to islets during the isolation process (Lake et al 1987;Salvalaggio et al 2002;Scharp et al 1973). 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.…”

Section: Discussionsupporting

confidence: 76%

“…Since the 1960s, the most common method for islet purification, both in rodents and humans, is density gradient centrifugation, based on the different densities of islets and exocrine tissue (Lacy and Kostianovsky 1967;McCall et al 2011;Mita et al 2010;van der Vliet et al 1988;Lake et al 1987). However, alternative methods based on other physical properties have been suggested in the last years, such us filtration or osmotic shock (Atwater et al 2010;Salvalaggio et al 2002).…”

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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Section: Discussionsupporting

confidence: 76%

Section: Introductionmentioning

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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“…Islets of Langerhans were isolated from mice, as previously described (34). In brief, the common bile duct was cannulated, and the pancreas was distended with 3 ml of Dulbecco's modified Eagle's medium containing 1.3 U/ml collagenase P (Boehringer Mannheim, Indianapolis, IN).…”

Section: Methodsmentioning

confidence: 99%

Virus-induced autoimmune diabetes: most beta-cells die through inflammatory cytokines and not perforin from autoreactive (anti-viral) cytotoxic T-lymphocytes.

et al. 2000

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Autoimmune diabetes is caused by selective loss of insulin-producing pancreatic ␤-cells. The main factors directly implicated in ␤-cell death are autoreactive, cytotoxic (islet-antigen specific) T-lymphocytes (CTL), and inflammatory cytokines. In this study, we have used an antigen-specific model of virally induced autoimmune diabetes to demonstrate that even high numbers of autoreactive CTL are unable to lyse ␤-cells by perforin unless major histocompatibility complex class I is upregulated on islets. This requires the presence of inflammatory cytokines induced by viral infection of the exocrine pancreas but not of the ␤-cells. Unexpectedly, we found that the resulting perforin-mediated killing of ␤-cells by autoreactive CTL is not sufficient to lead to clinically overt diabetes in vivo, and it is not an absolute prerequisite for the development of insulitis, as shown by studies in perforin-deficient transgenic mice. In turn, destruction of ␤-cells also requires a direct effect of ␥-interferon (IFN-␥), which is likely to be in synergy with other cytokines, as shown in double transgenic mice that express a mutated IFN-␥ receptor on their ␤-cells in addition to the viral (target) antigen and do not develop diabetes. Thus, destruction of most ␤-cells occurs as cytokine-mediated death and requires IFN-␥ in addition to perforin. Understanding these kinetics could be of high conceptual importance for the design of suitable interventions in prediabetic individuals at risk to develop type 1 diabetes. Diabetes

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“…Islets of Langerhans were isolated from mice as previously described (21,22). Briefly, the pancreas was perfused via the common bile duct with collagenase P (Roche Molecular Biochemicals, Indianapolis, IN) followed by purification of islets on a Histopaque-1077 gradient (Sigma-Aldrich, St. Louis, MO).…”

Section: Islet Isolationmentioning

confidence: 99%

Fas Is Detectable on β Cells in Accelerated, But Not Spontaneous, Diabetes in Nonobese Diabetic Mice

Darwiche

Chong

Santamaría

et al. 2003

The Journal of Immunology

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Fas (CD95) is a potential mechanism of pancreatic β cell death in type 1 diabetes. β cells do not constitutively express Fas but it is induced by cytokines. The hypothesis of this study is that Fas expression should be measurable on β cells for them to be killed by this mechanism. We have previously reported that up to 5% of β cells isolated from nonobese diabetic (NOD) mice are positive for Fas expression by flow cytometry using autofluorescence to identify β cells. We have now found that these are not β cells but contaminating dendritic cells, macrophages, and B lymphocytes. In contrast β cells isolated from NODscid mice that are recipients of T lymphocytes from diabetic NOD mice express Fas 18–25 days after adoptive transfer but before development of diabetes. Fas expression on β cells was also observed in BDC2.5, 8.3, and 4.1 TCR-transgenic models of diabetes in which diabetes occurs more rapidly than in unmodified NOD mice. In conclusion, Fas is observed on β cells in models of diabetes in which rapid β cell destruction occurs. Its expression is likely to reflect differences in the intraislet cytokine environment compared with the spontaneous model and may indicate a role for this pathway in β cell destruction in rapidly progressive models.

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Bovine Serum Albumin Density Gradient Isolation of Rat Pancreatic Islets1

Cited by 70 publications

References 0 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

Virus-induced autoimmune diabetes: most beta-cells die through inflammatory cytokines and not perforin from autoreactive (anti-viral) cytotoxic T-lymphocytes.

Fas Is Detectable on β Cells in Accelerated, But Not Spontaneous, Diabetes in Nonobese Diabetic Mice

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