Figure 1. Phenotypic characterization of mammospheres.

A: Representative phase contrast photomicrographs of organoids derived from primary human breast tissue; ×10 objective (a) and a primary mammosphere formed in suspension after 7 days; ×10 objective (b). B: Measurement of mammosphere size; ×10 objective (a) and cell size comprising the sphere (b) using LSM Image Browser from the Carl Zeiss website. C: Immunostaining of intact mammospheres viewed using a Zeiss 510 Meta confocal laser scanning microscope, and optical sectioning done along the XZ-plane to get a Z stack of the specimen. Photomicrographs represent negative control (minus primary antibody) (a), positive immunostaining for E-Cadherin (b) and Epithelial Specific Antigen (ESA) (c), but not for CD34 (d). RT-PCR analysis shows lack of nestin transcripts in mammospheres compared to U251 glioma cell line used as positive control (e). D: Photomicrographs represent immunostaining of single cells derived from primary mammospheres showing positivity for CK18 (a) CK14 (b) and CK19 (c). Insets show confocal images of intact spheres stained for the corresponding antigen. Hoechst 33342 and propidium iodide (PI) were used as nuclear counter stains for single cell and intact spheres, respectively (blue, Hoechst 33342; green, FITC; red, PI; scale bar = 20 µm). E: Gel picture shows hTERT expression in primary mammospheres (MS) by RT-PCR using HeLa as a positive control (a). Bar graph represents quantitative real time RT-PCR for hTERT expression in mammospheres compared to HeLa using primary human diploid fibroblasts (hF) as negative control (b). F: Mammospheres show few label retaining cells in T4 spheres. (a–d) represent photomicrographs of mammospheres immunostained for BrdU at each passage. (green, FITC signal; scale bar = 20 µm).