sorting of proglucagon into secretory granules are yet to be identified. Endocrine and neuroendocrine cells are characterised by the presence of a regulated secretory pathway, with specific intracellular compartments and associated proteins for the sorting
Rebecca McGirr, Leonardo Guizzetti and Savita Dhanvantari
D. Wynick, R. Critchley, M. S. Venetikou, J. M. Burrin and S. R. Bloom
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.
Journal of Endocrinology (1990) 126, 269–274
K Josefsen, J P Stenvang, H Kindmark, P-O Berggren, T Horn, T Kjær and K Buschard
Studies of individual cell types in the islets of Langerhans are complicated by the cells' functional coupling by gap junctions and paracrine interaction. Access to purified alpha and beta cells is therefore desirable. We present a simplified and optimized method for fluorescence-activated cell sorting of endocrine pancreatic rat islets. For dispersion of the islets, dispase was superior to trypsin, as the number of vital single cells was higher (1·1 ± 0·1 × 103 vs 0·6 ± 0·1 × 103/islet, P<0·05). The purity of the sorted cells was 96·7 ± 1·2% for the non-beta cells and 97·8 ± 0·6% for the beta cells (numbers in percentages of endocrine cells). In culture, isolated beta cells, non-beta cells and mixtures of beta and non-beta cells formed aggregates, but not at low temperature (4 °C) and not in medium with low serum content (2%). Finally, in pure beta cell aggregates, glucose stimulated changes in cytoplasmic free Ca2+ concentration although both glucose- and arginine-induced insulin secretion was much reduced. We conclude that alpha cells are necessary for insulin secretion but not for glucose sensing.
Journal of Endocrinology (1996) 149, 145–154
SN Lee, E Prodhomme and I Lindberg
Prohormone convertase 1 (PC1) is a serine proteinase responsible for the proteolytic processing of many precursor proteins within the regulated secretory pathway. The activity of PC1 is potentially regulated by two endogenous inhibitors, the PC1 propeptide and proSAAS. Here we have investigated the effect of proSAAS and propeptide-containing constructs on PC1 carboxy-terminal processing and activity. In AtT-20 cells, proSAAS expression inhibited both C-terminal PC1 processing and proopiomelanocortin (POMC) processing under pulse/chase conditions. SAAS CT peptide-propeptide chimeric constructs had no effect on the cleavage of PC1 and POMC under pulse/chase conditions. However, a construct containing the propeptide alone reduced C-terminal PC1 processing under pulse/chase conditions and also inhibited POMC processing. In contrast, experiments using HEK293 cells transiently expressing PC1 plus the respective constructs demonstrated significant inhibition of zymogen processing and decreased C-terminal processing of PC1 by the SAAS CT peptide portion of the chimera. Our results suggest that the PC1 propeptide expressed in trans is able to act as an endogenous inhibitor of PC1, but that SAAS CT peptide-containing/propeptide constructs cannot function as effective inhibitors of precursor maturation in the regulated pathway.
Domenico Bosco, Dominique G Rouiller and Philippe A Halban
fluorescence-activated sorting using a FACStar-Plus cell sorter (Becton–Dickinson, San Jose, CA, USA), as previously described ( van de Winkel & Pipeleers 1983 , Rouiller et al. 1990 ), resulting in a population comprising 95–98% (insulin-positive) β
Caroline Alfaia, Vincent Robert, Kevin Poissenot, Yves Levern, Daniel Guillaume, Shel-Hwa Yeo, William H Colledge and Isabelle Franceschini
population at E16.5. The discovery of higher levels of Esr1 in the ARC Kiss1 cells of female fetuses prompted us to compare the direct estradiol responsiveness of ARC Kiss1 cells in vitro specifically sorted from male and female fetuses
Kotaro Horiguchi, Tom Kouki, Ken Fujiwara, Motoshi Kikuchi and Takashi Yashiro
Anterior pituitary cells of male S100b-GFP rats were dispersed as described previously ( Horiguchi et al . 2008 ). Dispersed cells were separated into GFP-positive and GFP-negative cells by a cell sorter (MoFlo XDP: Beckman Coulter, Inc., Fullerton, CA
Nathalie Hinfray, Rafael Henrique Nóbrega, Morgane Caulier, Damien Baudiffier, Emmanuelle Maillot-Maréchal, Edith Chadili, Olivier Palluel, Jean-Marc Porcher, Rüdiger Schulz and François Brion
/total fish wet weight×100). Expression of cyp17a1 and cyp19a1 genes in sorted testicular cell fractions To generate data on the cellular localization of testicular cyp17a1 and cyp19a1 expression with an independent approach, we used testes from vasa
Wei Zhang, Xin-Hong Wang, Si-Feng Chen, Guo-Ping Zhang, Ning Lu, Ren-Ming Hu and Hui-Ming Jin
-ac-LDL) was from Molecular Probes, Inc. (Carlsbad, CA, USA). CD117 MicroBead Kit and magnetic activated cell-sorting system (MACS) were from Miltenyi Biotec (Bergisch Gladbach, Germany). Annexin-V/PI kit was from Bender MedSystems Inc. (Burlingame, CA, USA
Hyunju Chung and Seungjoon Park
was directly proportional to the number of living cells. Fluorescence-activated cell sorting (FACS) analysis Cell cycle distribution was examined by FACS analysis. 1×10 6 cells were collected and fixed with 3.7% paraformaldehyde. After