Preparation of thyrotroph cells from adult male rat pituitary glands by centrifugal elutriation.
To study the metabolism of thyrotrophs and dynamics of TSH secretion in vitro, it is desirable to have a highly enriched population of thyrotrophs. For that purpose, centrifugal elutriation, a recently developed cell isolation method based on the size and density of cells, was used to prepare thyrotrophs from a cell suspension of adult male rat pituitary cells. Trypsin-dispersed cells (4-8 X 10(7] were loaded into the elutriation rotor (Beckman, JE-6) operating at 2800 rpm. Twelve cell fractions were collected at variable rotor speed (2000-2800 rpm) and increasing medium flow rate (10-103 ml/min). Cell recovery was 77-98%. The viability of the cells after elutriation was 90-95% based on trypan blue exclusion. Each fraction was analyzed for TSH, GH, and PRL content and for TRH-stimulated TSH release by RIA. Thyrotrophs were found predominantly in fractions 8-11 (flow rate 38-75 ml/min) based on TSH RIA. The mean TSH concentration in these fractions was 56 +/- 13.6 (+/- SD) microU/10(3) cells compared with 7.6 +/- 3.8 microU/10(3) cells in the initial cell suspension, representing a 7- to 8-fold enrichment of the thyrotrophs. Incubation with 20 nM TRH for 3 h increased the TSH release of cells eluted in fractions 8-11 by 3- to 5-fold; there was no significant increase in TSH release in fractions 3-6. Centrifugal elutriation may be used to prepare a uniform highly enriched thyrotroph fraction with excellent recovery from a suspension of rat pituitary cells. This technique should be valuable for study of the metabolism of thyrotrophs.
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002498:Centrifugation / Q000379:methods; D013006:Growth Hormone / Q000032:analysis; D010903:Pituitary Gland, Anterior / Q000166:cytology* / Q000187:drug effects; D011388:Prolactin / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D013972:Thyrotropin / Q000378:metabolism; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology
D013973:Thyrotropin-Releasing Hormone; D011388:Prolactin; D013972:Thyrotropin; D013006:Growth Hormone