Clonal characterization of mouse mammary luminal epithelial and myoepithelial cells separated by fluorescence-activated cell sorting |
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Authors: | Matthew J Smalley Jenny Titley Michael J O’Hare |
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Institution: | (1) Section of Cell Biology and Experimental Pathology, Institute of Cancer Research: Royal Cancer Hospital, SM2 5NG Sutton, Surrey, United Kingdom;(2) LICR/UCL Breast Cancer Laboratory, Department of Surgery, University College London, Charles Bell House, 67-73 Riding House Street, W1P 7LD London, United Kingdom |
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Abstract: | Summary Lineage analysis in vitro of heterogeneous tissues such as mammary epithelium requires the separation of constituent cell types and their growth as
clones. The separation of virgin mouse mammary luminal epithelial and myoepithelial cells by fluorescence-activated cell-sorting,
their growth at clonal density, and the phenotyping of the clones obtained with cell-type specific markers are described in
this paper. Epithelial cells were isolated by collagenase digestion followed by trypsinization, and the luminal and myoepithelial
cells were flow-sorted with the rat monoclonal antibodies 33A10 and JB6, respectively. Sorted cells were cloned under, using
low oxygen conditions (<5% vol/vol), in medium containing cholera toxin and insulin, with an irradiated feeder layer of 3T3-L1
cells. Clones were characterized morphologically, and antigenically by multiple immunofluorescence with a panel of antibodies
to cytoskeletal antigens specific to either luminal epithelial or myoepithelial cells in situ. Whereas sorted myoepithelial cells gave a single clone type, sorted luminal cells gave three morphological clone types,
two of which grew rapidly. All myoepithelially derived clones showed a limited proliferative capacity in vitro, in contrast to their rat and human counterparts, as shown in previous studies. The present results with sorted mouse cells
have also allowed the stability of the differentiated phenotype in mouse, rat, and human mammary luminal epithelial and myoepithelial
cells in primary clonal culture to be compared. They show that the mouse mammary cells are the least stable in terms of expression
of differentiation-specific cytoskeletal markers in vitro. |
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Keywords: | epithelium murine cloning cytokeratins oxygen growth |
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