2012 ). One of the major functions of CgA is to sort proteins to the regulated secretory pathway ( Chanat et al . 1991 , Taupenot et al . 2002 , Bartolomucci et al . 2011 ). Although the role of CgA in sorting or folding of membrane proteins is yet
Kechun Tang, Teresa Pasqua, Angshuman Biswas, Sumana Mahata, Jennifer Tang, Alisa Tang, Gautam K Bandyopadhyay, Amiya P Sinha-Hikim, Nai-Wen Chi, Nicholas J G Webster, Angelo Corti and Sushil K Mahata
Susanne Granholm, Pernilla Lundberg and Ulf H Lerner
). Fluorescence-activated cell sorting (FACS) Crude bone marrow and BMM cells, obtained (by culturing bone marrow cells for 6 days in the presence of M-CSF) as described above, were washed with PBS/3% FBS and stained with antibodies (0.4 μg/10 6 cells) against
Anaies Nazarians-Armavil, Jennifer A Chalmers, Claire B Lee, Wenqing Ye and Denise D Belsham
construct were selected for by treatment with 100 μg/ml G418 (Geneticin; Gibco, Life Technologies), a neomycin analog. Following immortalization, in order to select for POMC neurons specifically, each hypothalamic culture was fluorescence-activated cell (FAC)-sorted
Diego Crespo, Moline Severino Lemos, Yu Ting Zhang, Diego Safian, Birgitta Norberg, Jan Bogerd and Rüdiger W Schulz
submitted to fluorescence-activated cell sorting (FACS) using an in Flux cell sorter (BD Bioscience). EGFP-positive and -negative cells were collected, centrifuged in PBS at 100 g for 10 min and the pellet stored at −80°C until use for gene expression
M Candolfi, G Jaita, D Pisera, L Ferrari, C Barcia, C Liu, J Yu, G Liu, M G Castro and A Seilicovich
pituitary cells from two different rat strains and also in the pituitary cell lines GH3 and AtT20. We investigated the proapoptotic effect of the vectors expressing TNF-α and FasL by fluorescence-activated cell sorting (FACS) analysis and the TUNEL method in
Catherine Roche, Alfredo J Zamora, David Taïeb, Esteban Lavaque, Ramahefarizo Rasolonjanahary, Henri Dufour, Claude Bagnis, Alain Enjalbert and Anne Barlier
determined by fluorescence-activated cell sorting (FACS), and the viral titer was calculated as previously described ( Limon et al. 1997 ). Titers around 1 × 10 8 transducing units per ml were obtained. The titers of lenti-PGK-eGFP supernatants were
D. Wynick, M. S. Venetikou, R. Critchley, J. M. Burrin and S. R. Bloom
Laser-light scatter signals generated from living cells provide useful information with regard to both cell size (forward-angle light scatter) and granularity (ninety-degree or perpendicular light scatter). By measuring angles of light scatter and fluorescence, a fluorescence-activated cell sorter is capable of analysing and sorting cells on the basis of their size, granularity and cell-surface fluorescence. Using an electronically programmable individual cell sorter we were able to analyse single, viable, dispersed anterior pituitary cells of the female rat on the basis of their laser light scatter characteristics. Two distinct populations of differing granularity were defined: 26±2·2% (mean ± s.e.m.) were more granular and 74±3·5% less granular. Acutely dispersed anterior pituitary cells were labelled with antibodies against four of the anterior pituitary hormones, and cell size and granularity were compared amongst the different hormonal cell types. Somatotrophs were the most granular cell type, gonadotrophs were the largest and corticotrophs the smallest, whilst lactotrophs were of intermediate size. Labelling was demonstrated to be dependent upon the secretory state of the cell. Hypothalamic stimulating factors increased cell-surface labelling, whilst dopamine and somatostatin decreased labelling. These changes compare favourably with published data obtained by immunocytochemistry. Using dual-colour fluorescence cell surface labelling we were unable to define a population of cells secreting both prolactin and growth hormone (mammosomatotrophs).
Journal of Endocrinology (1990) 126, 261–268
Madan L Nagpal, Yue Chen and Tu Lin
detection. Fluorescence-activated cell sorter (FACS) analysis Flow cytometry was performed with a Coulter Epics×L-MCL flow cytometer at the Flow Cytometry Core Laboratory, South Carolina Cancer Center, University of South
E. R. Norwitz, P. M. Starkey, A. López Bernal and A. C. Turnbull
Human term decidua produces prostaglandins (PGs) which have been implicated in the initiation of human parturition. Using flow cytometry to isolate pure cell populations, we have investigated the cell types responsible for decidual PG production. Cell dispersions were prepared enzymatically from decidua vera isolated from term placentae, and were incubated in Dulbecco's Modified Eagle's Medium containing 0·25% bovine serum albumin at 37 °C. PGF2α and PGE2 output were measured by radioimmunoassay of the conditioned medium. Production of PGF2α (fmol/106 cells per 3 h) exceeded that of PGE2 at 273 (108–322) 322) versus 97 (38–127) respectively (median (range)). The decidual cell dispersions were then incubated with monoclonal antibodies (anti-CD45 which labels the leukocyte common antigen or anti-human leukocyte antigen class II (HLA-DR) which is specific for macrophages in this tissue) and sorted by flow cytometry. The resultant antibody-positive and -negative cell populations were incubated and PG production was measured. Controls showed that antibody labelling and sorting did not alter PG production. PGF2α and PGE2 output by bone marrow-derived (CD45-positive) cell populations exceeded that of non-bone marrow-derived (CD45-negative) cells. Furthermore, we were able to demonstrate that the HLA-DR-positive macrophage population had the highest PGF2α and PGE2 production rates in human term decidua in vitro.
Journal of Endocrinology (1991) 131, 327–334
T Tierney and IC Robinson
The dwarf (dw/dw) rat differs from all other rodent models of GH deficiency in that its pituitary prolactin (PRL) content is normal or even increased. We have now studied this throughout postnatal development, using a combination of immunocytochemistry, RIA and fluorescence-activated cell sorting (FACS) and analysis. Compared with normal Albino Swiss (AS) rats, adult dw/dw rats showed a profound reduction in pituitary GH content accompanied by increased PRL content, significantly so in females (AS vs dw/dw; P<0.01). Somatotroph hypoplasia was evident in the adult dw/dw rats, with most GH(+ve) cells showing weak immunostaining, whereas many more strongly stained PRL cells were evident in pituitary sections from dw/dw rats. Facs analysis confirmed both somatotroph hypoplasia and relative lactotroph hyperplasia in dw/dw rats at all ages studied (9-144 days); the difference in somatotrophs increased with age whereas the difference in lactotrophs declined with age. At 9 days, the percentage of lactotrophs was 10-fold higher in dw/dw rats than in AS rats. Young dw/dw rats also had a higher proportion of mammosomatotrophs than AS rats, although this difference disappeared as the mammosomatotroph proportions increased with age in both strains. GHRH released GH from both dw/dw and as cells maintained in culture for 5 days. The sensitivity to GHRH and the amount of GH released was lower in the dw/dw cultures, mostly explained by their fewer GH cells and lower initial GH content. GHRH increased cAMP in as but not in dw/dw cultures, even when these were greatly enriched for dw/dw somatotrophs by FACS sorting prior to culture. These results suggest that GHRH-induced cAMP stimulation is required for trophic effects on GH synthesis and somatotroph proliferation, but is not required for GHRH-stimulated GH release. The inverse changes in somatotroph and lactotroph numbers suggest that the dw/dw mutation disturbs the mechanism that specifies and retains appropriate numbers of somatotrophs in their differentiated state, and results in a higher proportion of the remaining cells progressing to lactotrophs. The dw/dw phenotype is thus not confined to somatotrophs.