Application of a Percoll Density Gradient to Separate and Enrich Porcine Pituitary Cell Types

Abstract

The application of a Percoll density gradient cell separation procedure to the pituitary gland of neonatal, prepubertal and mature pigs is described. After enzymatic dispersion, cell viability was 90% to 98% as determined by uptake of Trypan Blue. Recovery of dissociated cells after application of the gradient ranged from 80% to 95%. The dissociated cells were separated in several fractions that were characterized immunocytochemically using different antisera. We obtained highly enriched fractions of gonadotrope (gonadotropins), somatotrope (growth hormone) and lactotrope (prolactin) cells for each age. Concentration of the cell types (purity) was higher than 60% in the following fractions: 1) gonadotropin cells, fraction 15 (1.033 g/cc of density) from mature animals; 2) growth hormone cells, fraction 3 (1.121 g/cc of density) from neonatal animals and fraction 9 (1.087 g/cc) from prepubertal animals; and 3) prolactin cells, fraction 7 (1.094 g/cc) and 10 (1.082 g/cc) from neonatal animals and fraction 14 (1.051 g/cc) from mature pigs. In thyrotrope (thyroid-stimulating hormone) and corticotrope (adrenocorticotropin) cells, enriched fractions were also obtained, although the values of purity were lower (20% to 58%). In conclusion, the proposed cell separation and enrichment technique is suitable for the isolation, purification and examination of porcine pituitary cell types and subpopulations, and offers major advantages such as simplicity, rapidity, efficiency and reproducible results.

Cortistatin mimics somatostatin by inducing a dual, dose-dependent stimulatory and inhibitory effect on growth hormone secretion in somatotropes

“…The cell number was assessed in a hemocytometer, and cell viability, as determined by the Trypan blue exclusion test, was consistently over 85%. Initial cell suspension (ICS,(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) 6 cells) was centrifuged (3000 g, 25 min) in a hyperbolic, continuous Percoll density gradient (Amersham, Buckinghamshire, UK) at 25-80% (1033-1121 g/cm 3 ) to separate the two subpopulations of pig somatotropes of low (LD; 1051-1064 g/cm 3 ) and high (HD; 1076-1098 g/cm 3 ) density, which have been characterized previously in our laboratory (Torronteras et al 1993, Castaño et al 1996, Ramírez et al 2002.…”

“…Anterior pituitaries were dispersed into single cells by the enzymatic and mechanical method described previously (Torronteras et al 1993, Castaño et al 1996, Ramírez et al 2002. For each experiment, three to four anterior pituitary glands were pooled and dispersed together, and the cell suspension obtained (initial cell suspension (ICS)) was filtered through a nylon gauze (100 µm mesh).…”

Morphological and Functional Heterogeneity of Frog Melanotrope Cells

“…lmmunostaining was performed as previously described [26] using the ABC complex method [30]. A specific antiserum to a-MSH.…”

“…The pars intermedia, which consists of a single endocrine cell type, represents a valuable model in which to investigate cellular heterogeneity [21,22], Various methods can be applied to separate subpopu lations of endocrine cells [23], Centrifugation on Percoll density gradients is a simple approach which appears to be particularly appropriate for relating density differences to morphological and functional characteristics of cells [24,25]. This kind of technique has been used successfully to separate subpopulations of various pituitary cell types [26].…”

Porcine sst1 can physically interact with other somatostatin receptors, and its expression is regulated by metabolic/inflammatory sensors

“…Pituitary glands were dispersed to obtain a suspension of monodispersed cells following an enzymatic-mechanic process as previously reported (49). Part of the dispersed cells obtained from the whole pituitary was used in a Percoll density gradient centrifugation protocol (Amersham Pharmacia Biotech, Uppsala, Sweden) to separate both somatotrope subpopulations, LD cells and HD cells, as previously reported by our group (7,49).…”