Journal: Nature Communications
Article Title: Modelling breast cancer requires identification and correction of a critical cell lineage-dependent transduction bias
doi: 10.1038/ncomms7927
Figure Lengend Snippet: ( a ) Primary cell outgrowth, derived from a reduction mammoplasty tissue (RMT) from a 24-year-old woman, cultured in MCDB170 medium, and inoculated with pLenti6/CMV-H2B-GFP lentivirus (1 μg ml −1 polybrene). ( b ) Overlay of H2b-GFP signal. ( c ) Overlay of keratin 19 (blue) and keratin 14 (red) immunofluorescence with TO-PRO-3 nuclear counterstain (white). ( d , left) Primary breast cells (passage 1), derived from RMT from a 34-year-old woman, transduced with pLenti6/CMV-ZsGreen lentivirus (+6 μg ml −1 polybrene) and immunostained as in c . ( d , right) Digital removal of red keratin 14 signal; arrowheads mark 3 of the 12 k19 + mitotic cells ( e ) Flow cytometric characterization of primary cells, derived from RMT from a 26-year-old woman (sample N135), cultured in M87 medium and inoculated with pLenti6/CMV-H2B-GFP lentivirus. GFP in transduced cells is compared with the cell expression of lineage markers associated with luminal (Muc1, c-Kit) and basal (CD10, CD49f, Thy1) cell types. ( f ) Quantification of flow cytometry data shown in e . ( g ) Transduction efficiencies of first passage of N135 cells (mixed culture) inoculated with twofold serial dilutions of 1,500 × concentrated CMV-H2B-GFP lentivirus. The fraction of GFP+ cells in the MEPs and LEPs was determined by multi-parameter flow cytometry using Muc1 and Thy1 specific antibodies. The transductional bias has been observed in every (over two dozens) primary culture tested to date. ( a – d ) Scale bars, 100 μm.
Article Snippet: The resulting divested tissue fragments (organoids) were collected by centrifugation (100 g × 2 min) and either archived in liquid nitrogen (90% FBS+10% dimethylsulphoxide) or immediately placed into culture using serum-free MCDB170 (Lonza) or M87 (M87+CT+X) minimal serum (0.25% FBS) medium , as indicated in the figure legends.
Techniques: Derivative Assay, Cell Culture, Immunofluorescence, Transduction, Expressing, Flow Cytometry
