Immunomagnetic purification of beta cells from rat islets of Langerhans

Aims/hypothesis. The aim of this study was to develop immunomagnetic purifi cation by the Dynabead system to separate insulin-containing beta cells fro m a mixed rat islet cell population. Functional studies on insulin secretio n and a test of the susceptibility of Dynabead-separated beta cells to DNA damage following cytokine exposure were carried out. Methods. Dynabeads are uniform, paramagnetic particles coated with specific antibodies. Single rat islet cells were initially incubated with the beta- cell surface specific antibody (K14D10 mouse IgG) for 20-60 min. A suspensi on of Dynabeads coated with a secondary antibody (anti-mouse IgG) was added for a further 15 min, after which the Dynabead-coated cells were instantan eously pelleted by contact between the tube and a magnet (Dynal MPG). Immun ocytochemistry was used to confirm that the Dynabead-coated cells contained insulin and to quantify the efficiency of the method. Dynabead-coated and non-coated cells were stained for insulin and glucagon. Results. Dynabead immunopurification yielded 95% pure insulin-containing be ta cells, which released insulin in response to isobutylmethylxanthine and glucagon-like polypeptide 1. The insulin content of Dynabead-coated beta ce lls was significantly higher than that of non-coated cells. Successful sepa ration was achieved using as few as 30 islets as starting material. Using t he comet assay, we found that Dynabead-coated beta cells showed equal susce ptibility to cytokine-induced DNA damage as non-coated cells. Conclusion/interpretation. We conclude that Dynabead separation of beta cel ls is simple, rapid, applicable to large or small numbers of islets and can be used to study beta-cell specific function and responsiveness.