Extended Data Figure 6: Clonal labelling enables quantification of the number of functional MaSCs based on labelling density.
From: Identity and dynamics of mammary stem cells during branching morphogenesis
a, Cartoon depicting the method used to determine the number of MaSCs. Clonal labelling at the onset of puberty results in the confetti labelling of 1 MaSC out of N MaSCs in a TEB (red cell). Clonal analysis in the resulting subtree at the end of puberty enables us to determine the contribution of one labelled MaSC to the entire subtree that is formed after the induction of confetti tracing. The labelled fraction is calculated by dividing the clone size (red cells) by the total number of cells in the subtree (1/N). By taking the inverse, the number N of functional MaSCs can be calculated. b, Representative images of a part of a whole-mount fourth mammary gland, stained for K14 (basal marker) used to determine the identity of the labelled clones. Magnified images show a single Z-plane with basal cells (red outline), which overlap with the staining, or luminal cells (yellow outline), which do not overlap with the basal staining. Scale bars, 100 μm. c, Maximum projection of part of a whole-mount fifth mammary gland, stained for E-cadherin (luminal marker) used to determine the identity of the labelled clones. Magnified images show a single Z-plane with basal cells (yellow outline), which do not overlap with the staining, or luminal cells (red outline), which do overlap with the luminal staining. Scale bars, 1 mm (left) and 100 μm (right). d, Quantification of number of basal and luminal cells in the TEB (n = 10 TEBs, scored over three mice). Data are mean ± s.e.m. e, Representative images of TEBs at 5 weeks of age, showing a single Z-plane of the TEB stained for DAPI and K14 or E-cadherin to determine the number of luminal and basal cells per TEB. Z-stacks of complete TEBs (Z-step size, 5 μm) were used to count the total number of basal cells per TEB. Scale bars, 100 μm.