Purification of functional lactotrophs and somatotrophs from female rats using fluorescence-activated cell sorting.
As the secretory granules of anterior pituitary cells fuse with the cell surface, there would appear to be sufficient hormone present on the cell surface to be labelled by polyclonal hormone antibodies and thus analysed by flow cytometry. We have therefore applied fluorescence-activated cell sorting to these labelled pituitary cells. Percentage purity and depletion of other cell types was assessed by immunocytochemistry and the reverse haemolytic plaque assay (RHPA). Results demonstrate that fluorescence-activated cell sorting allows almost complete purification of functional lactotrophs and somatotrophs to 96.7 +/- 1.7 (S.E.M.)% and 98 +/- 1.0% respectively by immunocytochemistry, and to 95.8 +/- 1.1% and 97 +/- 0.8% respectively by RHPA. Depletion of other anterior pituitary cell types to less than 2% was demonstrated by both immunocytochemistry and RHPA. Fluorescence-activated cell sorting to this degree of purity was routinely possible with cell yields of 91 +/- 3.4%. To obtain such purity/depletion, it was necessary to use specific antisera of high titre, at concentrations which ensured maximal cell-surface labelling associated with maximal stimulation of hormonal secretion by the appropriate hypothalamic stimulatory factor. Separating cells on the basis of the intensity of prolactin cell-surface labelling demonstrated a low level of binding of the prolactin antibody to gonadotrophs (but not of sufficient fluorescence intensity to be sorted into the prolactin enriched population), raising the possibility of prolactin receptors on gonadotrophs. We were unable to demonstrate the presence of mammosomatotrophs in the normal female rat, since purified lactotrophs did not contain or secrete GH nor did purified somatotrophs contain or secrete prolactin.
Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London.
D
D
Wynick
R
R
Critchley
M S
MS
Venetikou
J M
JM
Burrin
S R
SR
Bloom
D000818:Animals; D002469:Cell Separation / Q000379:methods; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D013006:Growth Hormone / Q000378:metabolism; D006462:Hemolytic Plaque Technique; D010903:Pituitary Gland, Anterior / Q000166:cytology* / Q000378:metabolism; D011388:Prolactin / Q000378:metabolism; D051381:Rats
D011388:Prolactin; D013006:Growth Hormone