Quantification of cells in islets of langerhans using DNA determination

Beckmann, J.; Lenzen, Sigurd; Holze, Susanne; Panten, U.

doi:10.1007/bf02056106

Cited by 14 publications

(4 citation statements)

References 11 publications

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“…The validation of the nuclei counts obtained through flow cytometry has been compared with DNA concentration measurements and found to be linear. The advantage with both DNA content measurements and nuclei counting is that they are less subjective or prone to error 73 , 74 . However, neither differentiate between islet and non-islet cells.…”

Section: Non-ieq Methodsmentioning

confidence: 99%

The Flaws and Future of Islet Volume Measurements

Huang

Harrington²,

Stehno‐Bittel

2018

Cell Transplant

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When working with isolated islet preparations, measuring the volume of tissue is not a trivial matter. Islets come in a large range of sizes and are often contaminated with exocrine tissue. Many factors complicate the procedure, and yet knowledge of the islet volume is essential for predicting the success of an islet transplant or comparing experimental groups in the laboratory. In 1990, Ricordi presented the islet equivalency (IEQ), defined as one IEQ equaling a single spherical islet of 150 μm in diameter. The method for estimating IEQ was developed by visualizing islets in a microscope, estimating their diameter in 50 μm categories and calculating a total volume for the preparation. Shortly after its introduction, the IEQ was adopted as the standard method for islet volume measurements. It has helped to advance research in the field by providing a useful tool improving the reproducibility of islet research and eventually the success of clinical islet transplants. However, the accuracy of the IEQ method has been questioned for years and many alternatives have been proposed, but none have been able to replace the widespread use of the IEQ. This article reviews the history of the IEQ, and discusses the benefits and failings of the measurement. A thorough evaluation of alternatives for estimating islet volume is provided along with the steps needed to uniformly move to an improved method of islet volume estimation. The lessons learned from islet researchers may serve as a guide for other fields of regenerative medicine as cell clusters become a more attractive therapeutic option.

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Section: Non-ieq Methodsmentioning

confidence: 99%

The Flaws and Future of Islet Volume Measurements

Huang

Harrington²,

Stehno‐Bittel

2018

Cell Transplant

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“…Amylase was measured by a modification of the method of Bernfeld (1955), with soluble starch as substrate and maltose monohydrate as standard. DNA was measured with diaminobenzoic acid (Beckmann et al, 1981).…”

Section: Assaysmentioning

confidence: 99%

Insulin resistance is accompanied by impairment of amylase-gene expression in the exocrine pancreas of the obese Zucker rat

Trimble¹,

Bruzzone²,

Belin³

1986

Biochemical Journal

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Insulin plays a major role in the control of pancreatic amylase biosynthesis. In this study we determined glucose metabolism by pancreatic acini as well as the pancreatic content of both amylase protein and amylase mRNA during development of insulin resistance in the obese Zucker rat. At age 4 weeks there were no abnormalities detected in the above parameters, although the obese animals were already hyperinsulinaemic. At 6 weeks glucose metabolism was decreased by 50% in acini from obese rats, whereas pancreatic amylase-gene expression was only slightly impaired. At 22 weeks glucose metabolism was decreased by 50%, amylase content by 55% and amylase mRNA by 60% in acinar tissue of obese rats. As expected, hyperinsulinaemia increased markedly with age. Thus development of severe insulin resistance was associated with impairment of amylase-gene expression. To decrease insulin resistance, one group of adult obese rats was treated with Ciglitazone for 4 weeks. A lowered plasma insulin concentration without alteration of food intake was taken as evidence of decreased insulin resistance. This was associated with normalization of glucose metabolism and a marked increase of both amylase content of pancreatic tissue and amylase mRNA. In conclusion, both the increase of insulin resistance with age and its partial reversal by Ciglitazone treatment appear to modulate pancreatic amylase-gene expression in the obese Zucker rat.

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“…total volume or number of cells) and (2) the fraction that is islet tissue, from which the amount of islet tissue is calculated. Methods to measure total amount of tissue include the change in electrical resistance as a tissue particle passes through an aperture and assay of DNA content, total protein, and dry weight (10, 29, 30). Measurements of the packed cell volume (PCV) are routinely made with human islet preparations (31) but have not been used to estimate the volume of tissue.…”

mentioning

confidence: 99%

Enumeration of islets by nuclei counting and light microscopic analysis

Pisania

Papas

Powers

et al. 2010

Laboratory Investigation

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Islet enumeration in impure preparations by conventional dithizone staining and visual counting is inaccurate and operator dependent. We examined nuclei counting for measuring the total number of cells in islet preparations, and we combined it with morphological analysis by light microscopy (LM) for estimating the volume fraction of islets in impure preparations. Cells and islets were disrupted with lysis solution and shear, and accuracy of counting successively diluted nuclei suspensions was verified with: (1) visual counting in a hemacytometer after staining with crystal violet, and automatic counting by (2) aperture electrical resistance measurement and (3) flow cytometer measurement after staining with 7-aminoactinomycin-D. DNA content averaged 6.5 and 6.9 pg DNA/cell for rat and human islets, respectively, in agreement with literature estimates. With pure rat islet preparations, precision improved with increasing counts, and samples with about 160 or more islets provided a coefficient of variation of about 6%. Aliquots of human islet preparations were processed for LM analysis by stereological point counting. Total nuclei counts and islet volume fraction from LM analysis were combined to obtain the number of islet equivalents (IE). Total number of IE by the standard method of dithizone staining/manual counting was overestimated by about 90% compared to LM/nuclei counting for 12 freshly isolated human islet research preparations. Nuclei counting combined with islet volume fraction measurements from light microscopy is a novel method for achieving accurate islet enumeration.

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Quantification of cells in islets of langerhans using DNA determination

Cited by 14 publications

References 11 publications

The Flaws and Future of Islet Volume Measurements

The Flaws and Future of Islet Volume Measurements

Insulin resistance is accompanied by impairment of amylase-gene expression in the exocrine pancreas of the obese Zucker rat

Enumeration of islets by nuclei counting and light microscopic analysis

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