Preparation of Rat Islet B-Cell-Enriched Fractions by Light-Scatter Flow Cytometry

Abstract

Flow cytometry has been examined as a method to separate islet cells into homogeneous subpopulations. Collagenase-isolated rat islets were dissociated into single cells and these were analyzed and sorted according to their low forward angle light scattering properties by using automated flow cytometry. Light scatter histograms showed two peaks of viable cells. Radioimmunoassay of hormone content in cell fractions collected across the the two peaks showed that glucagon-containing cells were concentrated towards the left side of the left peak and somatostatin-containing cells were concentrated towards the right side of the left peak, whereas insulin-containing cells were clearly enriched in the right peak. The B-cell-enriched fraction (90% B cells, 3% A cells, 2% D cells) exhibited significant insulin secretory responses to glucose (16.7 mM), and 3-isobutyl-1-methylxanthine (0.1 mM), during a 24-h culture period, and these responses were slightly greater than those observed in the original mixed islet cell preparation (66% B cells, 14% A cells, and 4% D cells). These results indicate that flow cytometry can be applied to sort pancreatic islet cells into populations enriched in specific endocrine cell types for further study of the functions of individual cell types.

A New in Vitro Model for the Study of Pancreatic A and B Cells*

“…The actual trypsin concentration is 20-to 200-fold lower than in previously reported methods (11,(20)(21)(22)(23). Differences in enzyme activity might certainly explain in part this wide concentration range, but part seems also attributed to the design of the dissociation procedure.…”

“…Among the techniques currently available to isolate one of the islet cell types, centrifugal elutriation represents certainly the simplest method for the preparation of purified B cells (5). Cell sorting has been employed to purify islet cells by light scatter activity alone (10,11) or by their selective binding to fluorescently labeled surface antibodies (7)(8)(9). In our experience, the differences in light scatter activity are not sufficient for a complete separation of A and B cells without considerable cell loss.…”

“…Several methods have been described for the isolation and dissociation of pancreatic islets (5,11,14,15,(20)(21)(22)(23)(25)(26)(27), but quality control data are not always reported or are often unsuitable for comparison. In the present procedure the tissue is exposed to more gentle mechanical treatment and lower enzyme concentrations than in previous reports, which results in a high yield of single and viable islet cells per pancreas.…”

Interleukin 2 Activates BB/W Diabetic Rat Lymphoid Cells Cytotoxic to Islet Cells

“…The islets were dissociated into single cells by incubation in Ca 2+ /Mg 2+ -free Hanks' balanced salt solution supplemented with 0.5 mg/ml trypsin and 0.2 mg/ml EDTA (trypsin-EDTA) solution at 37°C for 10 min. Cell viability was >85%, and 80-90% of the cells were islet endocrine cells, of which -8 0 % were insulin-containing p-cells (14). WF splenic lymphocytes were isolated by Ficoll-Hypaque density gradient centrifugation.…”

Functional and morphological study of cultured pancreatic islets treated with cyclosporine

“…Insulin was extracted from the isolated islets by acidified ethanol, as follows: Islets were pelleted by centrifugation at 170 ×g for 10 min then 1 ml acid ethanol (HCl 1.5%: ethanol 75%: H 2 O 23.5%) was added and sonicated for 10 s (Rabinovitch et al, 1982). Extraction was allowed to proceed overnight at 4°C.…”