Immunomagnetic purification of beta cells from rat islets of Langerhans.

Autor: Hadjivassiliou V; Biochemistry Department, University of Sussex, Brighton, UK., Green MH, Green IC
Jazyk: angličtina
Zdroj: Diabetologia [Diabetologia] 2000 Sep; Vol. 43 (9), pp. 1170-7.
DOI: 10.1007/s001250051509
Abstrakt: Aims/hypothesis: The aim of this study was to develop immunomagnetic purification by the Dynabead system to separate insulin-containing beta cells from a mixed rat islet cell population. Functional studies on insulin secretion 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 suspension of Dynabeads coated with a secondary antibody (anti-mouse IgG) was added for a further 15 min, after which the Dynabead-coated cells were instantaneously pelleted by contact between the tube and a magnet (Dynal MPC). Immunocytochemistry 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 beta cells, which released insulin in response to isobutylmethylxanthine and glucagon-like polypeptide 1. The insulin content of Dynabead-coated beta cells was significantly higher than that of non-coated cells. Successful separation was achieved using as few as 30 islets as starting material. Using the comet assay, we found that Dynabead-coated beta cells showed equal susceptibility to cytokine-induced DNA damage as non-coated cells.
Conclusion/interpretation: We conclude that Dynabead separation of beta cells is simple, rapid, applicable to large or small numbers of islets and can be used to study beta-cell specific function and responsiveness.
Databáze: MEDLINE
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