Figure 3

Flow Cytometric Analysis of MAM-1 co-cultures. A. Density plot of MAM-1 co-culture stained for HER2/neu and α-SMA. MAM-1 co-cultures were stained with the same ready-to-use, histological grade antibodies that were used for IHC and IC to detect HER2, PAD: Z4881 and α-SMA, (1A4) in tissues. Primary antibodies were labeled with -PE and FITC conjugated secondaries as described in the methods. Quadrant analysis revealed that MAM-1 cultures consisted of 52% HER2/neu⁺, α-SMA^- tumor cells, 40% HER2/neu^-, α-SMA⁺ stromal cells, a 5% population of HER2/neu^-, α-SMA^- cells and a 3% population of HER2/neu⁺, α-SMA⁺ cells which may represent cells undergoing epithelial-mesenchymal transition. At the right, Histogram analysis of the separate FL1 (top) and FL2 (bottom) events indicates 54% of the culture as positive for HER2/neu (top) and 43% of cells as positive for α-SMA (bottom) which is consistent with the quadrant analysis. Filled peaks are specific antibody stained cells and open peaks represent background staining with the isotype control. B. Subpopulation analysis of Tumor cell fractions in MAM-1 co-cultures based on Forward scatter profiles. Dot plot analysis of a-SMA-PE stained cells versus Forward scatter indicates the presence of two distinct subpopulations of different size in the a-SMA negative fraction labeled as S (small) and L (large). Histogram analysis of these gated subpopulations (defined by the circles) for HER2/neu specific staining indicates that the small population has lower levels of HER2/neu, a mean channel fluorescence of 110 compared to the large population enriched in dividing cells, with a mean channel fluorescence of 366.