Immunomagnetic purification of beta cells from rat islets of Langerhans.
OBJECTIVE
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.


CONCLUSIONS
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.
Hadjivassiliou
V
V
Biochemistry Department, University of Sussex, Brighton, UK.
Green
M H
MH
Green
I C
IC
D000818:Animals; D016207:Cytokines / Q000494:pharmacology*; D004249:DNA Damage; D005934:Glucagon; D006801:Humans; D007074:Immunoglobulin G; D018189:Immunomagnetic Separation / Q000295:instrumentation / Q000379:methods; D007328:Insulin / Q000032:analysis / Q000378:metabolism; D000078790:Insulin Secretion; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D007515:Islets of Langerhans / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D008297:Male; D051379:Mice; D008855:Microscopy, Electron, Scanning; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016207:Cytokines; D007074:Immunoglobulin G; D007328:Insulin; D007375:Interleukin-1; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D005934:Glucagon