Islet Cell Suspensions Research Articles

Trypan Blue as a marker of plasma membrane permeability in alloxan-treated mouse islet cells

Suspensions of pancreatic islet cells from noninbred ob/ob-mice were incubated with Trypan Blue. Microscope photometry showed that apparently viable cells excluded the dye completely, whereas the nuclei of nonviable cells accumulated Trypan Blue by a saturable process. The nucleus-to-medium dye gradient was more then 30:1 in media containing 0.1% or less Trypan Blue. The apparent affinity constant for nuclear binding of the dye was 3.1 X 10(4)l/mol. Albumin partially inhibited the nuclear staining. More than 0.5% Trypan Blue in the medium was toxic per se. In the absence of albumin, 0.5 or 20 mmol/l alloxan, 1 mmol/l N-ethylmaleimide, or 0.1 mmol/l chloromercuribenzene-p-sulphonic acid, but not 20 mmol/l streptozotocin, increased the frequency of islet cells stained with 0.1% Trypan Blue. The absorbance of nuceli was also increased in cells treated with alloxan or N-ethylmaleimide, but not in those treated with chloromercuribenze-p-sulphonic acid. It is concluded that alloxan rapidly increases the permeability of the plasma membrane in mouse beta-cells. This action of alloxan appears to be more acute than any such effect of streptozotocin.

Chlorotetracycline as a fluorescent Ca2+ probe in pancreatic islet cells.

Pancreatic islets, or suspensions of islet cells, from noninbred ob/ob-mice were incubated with chlorotetracycline and analyzed for Ca2+-dependent fluorescence in a microscope. Unless logarithmically transformed, signals from islets were asymmetrically distributed with unstable variance. Signals from cells pelleted in glass capillaries were more homogeneous and depended linearly on the thickness of the sample. The effect of sample thickness and a significant enhancement of fluorescence by alloxan suggest that beta-cells were involved in producing the signal from whole islets. The signal from dispersed cells was probably diagnostic of Ca2+ in beta-cell plasma membranes because it was suppressed by La3+ and had a spectrum indicative of an apolar micromilieu; fluorescent staining of cell surfaces was directly seen at high magnification. Fluorescence from cells was enhanced by 0.5-10 mM Ca2+ in a dose-dependent manner, whereas less than 0.5 mM Ca2+ saturated the probe alone in methanol. The signal from islets or dispersed cells was suppressed by 5 mM theophylline; that from cells was also suppressed by 0.5 mM 3-isobutyl-1-methylxanthine, 1.2 or 15 mM Mg2+, 3-20 mM D-glucose, and, to a lesser extent, 20 mM 3-O-methyl-D-glucose. D-glucose was more inhibitory in the absence than in the presence of Mg2+, as if Mg2+ and D-glucose influenced the same Ca2+ pool. L-glucose, D-mannopheptulose, or diazoxide had no noticeable effect and 20 mM bicarbonate was stimulatory. The results suggest that microscopy of chlorotetracycline-stained cells can aid in characterizing calcium pools of importance for secretion. Initiation of insulin release may be associated with an increas

Possible toxic effects of normal and diabetic patient serum on pancreatic B-cells

Serum from normal blood-donors and juvenile diabetic patients inhibited Rb+ accumulation and stimulated release of 51Cr and insulin in suspensions of dispersed pancreatic islet cells prepared from ob/ob mouse islets, which are rich in B-cells. The effects indicate the presence of a B-cytotoxic factor in human serum. Serum from mouse and fetal calf also inhibited the islet cell accumulation of Rb+. Toxicity was not suppressed by treating serum with protein A-Sepharose and did not correlate with islet cell binding of fluorescent antibodies to human immunoglobulin. Whereas all sera inhibited Rb+ accumulation, 3 of 6 diabetic patient sera, but no blood-donor serum, made the cells fluoresce on exposure to the fluorescent antibodies. Supporting a dependence on complement, toxicity remained after dialysis, but was destroyed by treating serum with zymosan-A or heating at 56 degrees for 30 min.

Alloxan cytotoxicity in vitro. Microscope photometric analyses of Trypan Blue uptake by pancreatic islet cells in suspension

Suspensions of islet cells were prepared by shaking pancreatic islets from non-inbred ob/ob mice in a Ca2+-free buffer. The cells were incubated with or without 20 mM-alloxan, and subsequently with Trypan Blue. The uptake of Trypan Blue by cell nuclei was analysed by microscope photometry and by counting the frequency of cells appearing stained on visual inspection. Cells classified as stained or unstained by inspection showed no overlap in nuclear absorbance. Suspensions not exposed to alloxan contained 70-80% of unstained cells. Alloxan markedly decreased the frequency of unstained cells, an effect counteracted by 5 or 20 mM-D-glucose. The spectrum of Trypan Blue in islet-cell nuclei was red-shifted by about 20 nm. A similar red-shift was observed on adding the dye to solutions of albumin or histones, but not on mixing the dye with DNA. Binding to basic proteins may explain the concentrative uptake of Trypan Blue in dead cells and contribute to the oncogenic transformation of phagocytotically active cells. Beta-Cells in vitro are killed by alloxan and hence represent a valid model for studying the diabetogenic action of the drug.

The use of dispersed pancreatic islet cells in measurements of transmembrane transport

Suspensions of dispersed islet cells were prepared by shaking collagenaseisolated pancreatic islets of ob ob - mice in Ca 2+-free buffer. The dispersed cells exhibited a glucose uptake with stereospecificity for the d isomer and concentrated Rb + about 30-fold from a medium containing 70 μ m RbCl. These results compare well with previous observations on unbroken islets and indicate that the dispersion procedure does not cause serious damage to the plasma membranes of the β-cells. By double isotope labeling and centrifuging the incubated cells through oil, incubation times as short as only a few seconds can be used. The elimination of the extracellular tissue space and the short incubation times should facilitate the study of transport kinetics in the pancreatic islet cells.

The preparation of, and studies on, free cell suspensions from mouse pancreatic islets.

Pancreatic islets, isolated from the pancreas of obese-hyperglycemic mice, were used to prepare free islet cells in suspension. Batches of 100 islets were disrupted by mechanical shaking for 10 sec in a Ca2+-free HEPES-buffered Krebs-Ring'er medium containing 1 mM EGTA. From 200–500 islets, about 2.2×106 cells could be obtained in suspension, corresponding to a yield of roughly 55% as calculated from the content of DNA in cells and islets. The isolated islet cells appeared well-preserved in the light and the electron microscope and seemed to exhibit a high degree of viability as judged by viable cell counts, a radioactive assay for lysis and insulin release. Insulin release from islet cell suspensions, as calculated per cell, per content of DNA or insulin or per packed cell volume, was stimulated by glucose alone or in combination with theophylline. The glucose response was low compared with that of intact isolated islets.