Multiparametric flow cytometry analysis of mammary epithelial and stem cell markers in matched adherent and sphere cultures of breast cancer cell lines.

<p>(<b>A</b>) <b>Experiment design.</b> Matched adherent and sphere cultures of luminal-like and basal-like/claudin-low cell lines were generated in triplicate from subconfluent, adherent parent cultures on d0, harvested and dissociated on d7 into single cell suspensions, then concurrently stained with combinations of fluorescent antibody conjugates. Panel 1: MUC1-FITC, HER2-PE and the LIVE/DEAD® red cell viability stain; Panel 2: CD49f-Pacific Blue, Aldefluor® reagent (for ALDH1 activity), CD24-PE, LIVE/DEAD® red, AC133-APC, EpCAM-PerCP Cy5.5 and CD44-APC-Cy7. (<b>B</b>) <b>Gating strategy for simultaneous detection of cell surface markers.</b> Acellular particles and dead cells were excluded based on low light scatter and LIVE/DEAD® red positivity (live cells designated ‘LDR live’). For each experiment, gates were placed based on autofluorescence of unstained adherent or sphere control samples to account for differences in the autofluorescence of cells grown in the different formats. For Aldefluor® (ALDH1 activity), gates were placed based on the fluorescence of the DEAB negative control (refer to supp methods). Population frequencies were determined for individual parameters (MUC1+, HER2+, AC133+, EpCAM+, ALDH1+, CD44+, CD24+, CD49f+). For panel 2, combination gating was performed to investigate the frequencies of stem cell populations, differentiation states and other subpopulations (see Fig. 3).