Figure 5: Quantification of DNA synthesis rate by EdU incorporation in Tis21-GFP− and Tis21-GFP+ NPCs.

Following a 30-min EdU pulse labelling (two litters), cortical cells were dissociated, analysed by flow cytometry and sorted for Tis21-GFP fluorescence. Tis21-GFP− and Tis21-GFP+ cells were then separately analysed for DNA content and EdU incorporation in another round of flow cytometry, as shown in the panels. (a, b) Granularity (side scatter, SSC; AU, arbitrary units) versus EdU fluorescence of single Tis21-GFP− (a, 8,053 cells) and Tis21-GFP+ (b, 8,029 cells) cells. Dashed lines, overtly EdU-incorporating cells quantified in (c). Plots show one of the two litters analysed. (c) Mean EdU fluorescence of the overtly EdU-incorporating single Tis21-GFP− (grey column) and Tis21-GFP+ (green column) cells. For each litter, the mean EdU fluorescence of Tis21-GFP− cells was arbitrarily set to 100, and the mean EdU fluorescence of Tis21-GFP+ cells was expressed relative to this. Data are the mean of two litters, dots indicate the two individual values. (d, e) Cell number versus DNA content of the single Tis21-GFP− (d) and Tis21-GFP+ (e) cells shown in (a) and (b), respectively. Boxes indicate the sub-populations of cells in G0/G1, early S, late S and G2+M phases, as defined by DNA content; cells in early and late S-phases were analysed for SSC versus EdU fluorescence in (g–j). (f) Mean EdU fluorescence of the overtly EdU-incorporating single Tis21-GFP− (grey columns) and Tis21-GFP+ (green columns) cells in early (g, i) and late (h, j) S-phase, as defined by DNA content (d, e) and after analysis of SSC versus EdU fluorescence (g–j). Quantification as in (c). (g–j) Granularity (SSC) versus EdU fluorescence of single Tis21-GFP− (g, h) and Tis21-GFP+ (i, j) cells in early (g, i) and late (h, j) S phase shown in (d) and (e), respectively. Dashed lines, overtly EdU-incorporating cells quantified in (f). Plots show one of the two litters analysed. AU, arbitrary units.