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.
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.
Authors: Louise A Sigfrid; James M Cunningham; Neil Beeharry; L A Håkan Borg; Alma L Rosales Hernandez; Carina Carlsson; Adrian J Bone; Irene C Green Journal: J Mol Med (Berl) Date: 2004-03-09 Impact factor: 4.599