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Fig. 1 | SpringerPlus

Fig. 1

From: Murine mammary stem/progenitor cell isolation: Different method matters?

Fig. 1

Effects of digestion method and surface marker on the purity of stem/progenitor cell isolation. a Mammary glands from two mice were divided into two equal portions with one thoracic gland and one inguinal gland from one mouse mixed with another mouse, and used for fast (1 h) and slow (15 h) digestion protocols. The experiment was repeated three times with a total of 6 C57BL/6J mice. Flow cytometry profiles showing predominance of non-epithelial lineage (CD45, CD31, TER119, BP-1) positive cells (Lin pos) in tissues digested with fast protocol versus the predominance of Lin (Lin neg) cells in tissues from the same mice digested with slow protocol. Within the Lin cells, basal (green circles) and luminal (red circles) cells are characterized by EpCAM+CD49fhi and EpCAMhiCD49flo, respectively. Fast digestion yielded much lower percentage of basal cells than slow digestion. b Representative epithelial colony formed by luminal progenitor and c non-epithelial colony formed by stromal cells on the irradiated NIH3T3 fibroblast feeder layer (2D assay). d Solid organoids formed by basal-like stem cells, and e hollow organoids formed by luminal progenitors after they were seeded in Matrigel (3D assay). f Branching-like structures formed by non-epithelial stromal cells in the 3D assay. g Inclusion of luminal progenitor in basal fraction showing hollow organoid formation in 3D assay. h Flow cytometry profiles showing Lin mammary cells isolated from regenerated glands derived from GFP transgenic C57BL/6J donor mice. Thus, epithelial cells were GFP+ (green dots) and niche stromal cells were GFP (black dots). Regenerated glands of virgin mice were pooled from ten transplants, and regenerated glands of pregnant mice (day 16) were pooled from four transplants. Isolated single cell suspension from these transplants were divided into three equal portions for flow profiling comparison using CD24/CD29, CD24/CD49f, and EpCAM/CD49f. i Flow cytometry profile showing the use of EpCAM/CD49f in regenerated pregnant glands derived from GFP FVB donor mice. j A summary table showing basal cell gate profiled by the three pairs of surface markers (CD24/CD29, CD24/CD49f, EpCAM/CD49f) contains a different number of non-epithelial stromal cells evaluated by the 2D and 3D in vitro assays as well as the GFP-marked regenerated glands (reg glands). Scale bars in b–g 500 μm

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