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

Abstract

Summary. 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 meeha.nieal shaking for 10 see in a Ca~+-free HEPES-buffered Krebs-Ringer 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 wellpreserved 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 calcuIated per cell, per content of DNA or insulin or per packed cell volume, was stimulated by glucose alone or iu combination with theophylline. The glucose response was low compared with that of intact isolated islets.

Long-Chain CoA esters activate human pancreatic beta-cell K ATP channels: potential role in Type 2 diabetes

“…Islet volume and purity were determined by microscopical sizing on a grid after staining with diphenylthiocarbazone [16] and viability was assessed by membrane integrity testing (trypan blue exclusion). Islets were shipped to the department of Molecular Medicine at Karolinska Institutet, Stockholm, Sweden, where a cell suspension was prepared [17]. The dispersed beta cells were seeded into Petri dishes (Nunc, Roskilde, Denmark) and incubated at 37°C under 5% CO 2 using RPMI 1640 culture medium (Flow Laboratories, Irvine, UK), supplemented with 10% foetal bovine calf serum and antibiotics (100 IU/ml pencillin, 100 µg/ml streptomycin and 60 µg/ml gentamycin).…”

Protein kinase C activity affects glucose‐induced oscillations in cytoplasmic free Ca2+ in the pancreatic B‐cell

“…The islets of these micecontain more then 90% Bcel!r [I I]. A cell suspension was prepared and washed essentially as previously described [12]. The cells were resuspended in RPM1 1640 culture medium (Flow Laboratories, Scotland, UK).…”

Ca2+-induced Ca2+ release by activation of inositol 1,4,5-trisphosphate receptors in primary pancreatic β-cells

“…Human islets were obtained from the Nordic Network for Clinical Islet Transplantation in Uppsala after isolation from cadaver donors (under a protocol approved by the local ethics committee) as previously described [27]. Free cells were prepared by shaking the islets in a Ca 2+ -deficient medium [28]. The cells were suspended in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 μg/ml streptomycin and 30 μg/ml gentamicin and allowed to attach to circular 25 mm cover slips (poly-L-lysine coated in the case of rat cells) during 1-3 days culture at 37°C in a humidified atmosphere of 5% CO 2 .…”