Rapid assessment of islet viability with acridine orange and propidium iodide

Bank, Harvey L.

doi:10.1007/bf02628826

Cited by 156 publications

(105 citation statements)

References 35 publications

Supporting

Mentioning

102

Contrasting

Unclassified

Order By: Relevance

“…This screening assay allows a judgment to be made on the condition of a population of islets prior to experiments. The conformation that the fluorescent colours are related to the physiological condition of intact islets will be reported separately [21]. Briefly, the insulin content of green type A islets are identical to untreated islets and these islets have normal ultrastructural morphology.…”

Section: Discussionmentioning

confidence: 99%

Assessment of islet cell viability using fluorescent dyes

Bank

1987

Diabetologia

View full text Add to dashboard Cite

Summary.A rapid fluorometfic method has been developed to evaluate the viability of isolated islet cells. The assay differentiates between viable and nonviable cells by the simultaneous use of the inclusion and exclusion dyes acridine orange and propidium iodide. When viewed by fluorescent microscopy, viable cells fluoresce green, while nonviable cells fluoresce bright red. Although the acridine orange and propidium iodide assay measures membrane integrity, the results of this assay correlate with other measures of cell viability. Compared to trypan blue exclusion, this assay is easier to read, more stable, and has fewer staining artifacts. The assay enables the rapid estimation of the viability of a population of islet cells prior to time-consuming experiments rather than retrospectively. This assay can also be used with intact islets. Stained islets can be divided into three distinct groups: green fluorescing islets contain insulin, red fluorescing islets contain little or no insulin and a third class of islets containing some non-viable cells fluoresce red, green, and yellow. The yellow colour is due to the superimposition of red and green fluorescing cells. Key words:Islet viability, fluorochromes, B cells, acridine orange, propidium iodide, trypan blue exclusion, membrane integrity.A reliable method for rapidly determining the viability of isolated islet cells would be useful in studies on transplantation, cryopreservation, insulin release, and biosynthesis. Functional assays to determine insulin content or release and morphological assays including histochemistry or electron microscopy are time-consuming and expensive for routine viability assays.An alternative approach uses inclusion and/or exclusion dyes to test the integrity of the plasma membrane. Several fluorometric dyes were tested as indicators of membrane integrity. Fluorescein diacetate (FDA) has been used as a fluorometric assay of cell viability. FDA is a nonpolar ester which passes through plasma membranes and is hydrolysed by intracellular esterases to produce free fluorescein. The polar fluorescein is confined within cells with an intact plasma membrane and can be observed under appropriate excitation conditions [1][2][3][4]. However, staining of islet cells with FDA results in high background fluorescence. This fluorescence is presumably due to extracellular hydrolysis of the FDA or to fluorescein that leaked through damaged membranes.Acridine orange (AO) is a membrane-permeable, monovalent, cationic dye [5,6] which binds to nucleic acids. A low concentration of AO causes a green fluorescence, while a high concentration causes a red fluorescence. Propidium iodide (PI) is impermeable to intact plasma membranes, but it easily penetrates the plasma membrane of dead or dying cells [7] and intercalates with DNA or RNA forming a bright red fluorescent complex [8][9][10][11]. Both intracellular and extracellular background fluorescence is minimal after staining with optimal concentrations of these dyes.A combination of low concentrations of AO and...

show abstract

Section: Discussionmentioning

confidence: 99%

Assessment of islet cell viability using fluorescent dyes

Bank

1987

Diabetologia

View full text Add to dashboard Cite

show abstract

“…This method allows the morphological observation of apoptotic cells and distinguishes between apoptotic and necrotic cells. [12] Figure 2 shows viable cells in green and non-viable cells in red under fluorescence microscopy. Results of apoptotic characteristics were shown on cells treated with low concentration; (30 ug/mL) and IC 50 (60 ug/mL), which were stained in green at cytoplasm and nucleus.…”

Section: Resultsmentioning

confidence: 99%

Crude ethyl acetate extract of marine microalga,Chaetoceros calcitrans, induces Apoptosis in MDA-MB-231 breast cancer cells

et al. 2014

View full text Add to dashboard Cite

Background:Marine brown diatom Chaetoceros calcitrans and green microalga Nannochloropsis oculata are beneficial materials for various applications in the food, nutraceutical, pharmaceutical and cosmeceutical industries.Objective:This study investigated cytotoxicity of different crude solvent extracts from C. calcitrans and N. oculata against various cancer cell lines.Materials and Methods:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was carried out to screen the cytotoxic effects of hexane (Hex), dichloromethane (DCM), ethyl acetate, and methanol extract from C. calcitrans and N. oculata toward various cancer cell lines. Flow cytometry cell cycle was used to determine the cell cycle arrest while the mode of cell death was investigated through acridine orange/propidium iodide (AOPI) staining, Annexin V-Fluorescein Isothiocyanate (FITC) and Terminal deoxynucleotidyl transferase-mediated d-UTP Nick End Labeling (TUNEL) assays. Expression profile of apoptotic and proliferative-related genes was then determined using the multiplex gene expression profiler (GeXP).Results:Crude ethyl acetate (CEA) extract of C. calcitrans inhibited growth of MDA-MB-231 cells, with IC50 of 60 μg/mL after 72 h of treatment. Further studies were conducted to determine the mode of cell death at various concentrations of this extract: 30, 60 and 120 μg/mL. The mode of cell death was mainly apoptosis as shown through apoptosis determination test. The expression data from GeXP showed that caspase-4 was upregulated while B-cell leukemia/lymphoma 2(Bcl-2) was down regulated. Thus, caspase-4 induction endoplasmic reticulum death pathway is believed to be one of the mechanisms underlying the induction of apoptosis while Bcl-2 induced S and G2/M cell cycle phase arrest in MDA-MB-231 cells.Conclusion:CEA extract of C. calcitrans showed the highest cytotoxicity on MDA-MB-231 via apoptosis.

show abstract

“…Embedded islets, defined as islets with Ͼ 50% of their surface covered with intact nonendocrine tissue, were counted. Islet viability was evaluated before and after culture based on acridine orange (10 mmol/L) and propidium iodide (15 mmol/L) (AO/PI) fluorescent stains (27)(28)(29)(30)(31). The ratio of living to dead cells was determined for 50 randomly selected islets, and was then used to calculate average islet viability.…”

Section: Macaca Nemestrina Islet Evaluationmentioning

confidence: 99%

Improved Islet Yields From Macaca Nemestrina and Marginal Human Pancreata After Two‐Layer Method Preservation and Endogenous Trypsin Inhibition

Matsumoto

Rigley

Reems

et al. 2003

American Journal of Transplantation

View full text Add to dashboard Cite

We tested whether two-layer method (TLM) pancreas preservation and trypsin inhibition (Pefabloc) during processing allows longer preservation while retaining or improving viable islet recovery. Non-marginal primate (Macaca nemestrina) and marginal human (ischemic or preservation-injured) pancreata were processed with a research-oriented pan technique (Seattle method). Organs were processed upon arrival (∫ Pefabloc), or after TLM or University of Wisconsin solution (UW) preservation (π Pefabloc). Islet yield, viability, and function were assessed. Pefabloc increased M. nemestrina islet yields from 9696 ∫ 1749 IE/g to 15822∫ 1332 IE/g (p ∞ 0.01). Twolayer method preservation (∞ 6 h) further increased yields, to 23 769 ∫ 2773 IE/g (vs. π Pefabloc; p ∞0.01). Similarly, Pefabloc increased marginal human islet yields from 2473 ∫ 472 IE/g to 4723∫ 1006 IE/g (p ∞ 0.04). This increase was maintained after lengthy TLM preservation (Ͼ 30 h; 4801 ∫ 1066 IE/g). We also tested the applicability of TLM preservation (23.5 ∫ 3.2 h) to the processing of marginal human pancreata by the Edmonton/Immune Tolerance Network clinical protocol. Islet yield and function approached published results of pancreata processed 4.8 ∫ 0.8 h after organ recovery (p Ω 0.06). Pefabloc, and TLM vs. UW preservation, prolonged the tolerable interval between organ recovery and islet isolation. Islet yield, viability, and functionality improved from both marginal and nonmarginal pancreata.

show abstract

Rapid assessment of islet viability with acridine orange and propidium iodide

Cited by 156 publications

References 35 publications

Assessment of islet cell viability using fluorescent dyes

Assessment of islet cell viability using fluorescent dyes

Crude ethyl acetate extract of marine microalga,Chaetoceros calcitrans, induces Apoptosis in MDA-MB-231 breast cancer cells

Improved Islet Yields From Macaca Nemestrina and Marginal Human Pancreata After Two‐Layer Method Preservation and Endogenous Trypsin Inhibition

Contact Info

Product

Resources

About