Magnetic bead separation of anterior pituitary cells.
The use of magnetic beads coated with anti-IgG antibodies should allow simultaneous purification and depletion of differing anterior pituitary cell types labelled with anti-hormone antibodies. This technique would be expected to give very similar results to fluorescence-activated cell sorting (FACS). Magnetic bead separation of dispersed, labelled anterior pituitary cells is cheap, easy and quick to perform (time from the end of anti-hormone antibody labelling to completion of purification is approximately 30 min) and the resulting cells are viable for at least 24 hours after purification. While the cell recovery for beads and FACS, 94% (SEM +/- 4.4) vs. 89% (SEM +/- 3.9) and purity of 88% (SEM +/- 2.2) vs. 96.7% (SEM +/- 1.7) for lactotrophs and purity of 87% (SEM +/- 1.9) vs. 98% (SEM +/- 1) for somatotrophs are similar, the results for depletion by the magnetic bead separation method are disappointing, only 30-40% of the labelled lactotrophs or somatotrophs cells bind to the beads and thus only a sub-population of cells may be purified by this method. These results are explicable on the basis of the sensitivity of the two techniques. Pituitary cells co-incubated with two specific anti-prolactin antibodies (one raised in rabbit and one in sheep) demonstrate that removal by Dynal magnetic beads (coated with rabbit IgG antibody) of those prolactin molecules bound to the rabbit anti-prolactin antibody also removed those prolactin molecules bound to a sheep anti-prolactin antibody. In contrast, co-incubating cells with the rabbit anti-prolactin antibody and a sheep anti-growth hormone antibody did not remove growth hormone labelling when the prolactin bound to the beads was removed.(ABSTRACT TRUNCATED AT 250 WORDS)
Wynick
D
D
Department of Medicine, Royal Postgraduate Medical School, London, UK.
Bloom
S R
SR
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D005260:Female; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D013006:Growth Hormone / Q000378:metabolism; D007118:Immunoassay; D008280:Magnetics; D008863:Microspheres; D010903:Pituitary Gland, Anterior / Q000166:cytology*; D011388:Prolactin / Q000276:immunology / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013861:Thiocyanates
D000906:Antibodies; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013861:Thiocyanates; D011388:Prolactin; D013006:Growth Hormone; D016650:Fluorescein-5-isothiocyanate