Fig. 6. The quantitative evaluation of in vitro neurogenesis using flow-cytometry revealed a development-dependent distribution pattern. A clear delay in the generation of strong EGFP-positive progenitors was observed in the retinoic acid untreated (right panels) versus the treated (left panels) EBs. The maximal generation of progenitors occurred between d4 and d10 after plating in the RA-treated cells. Notice the decline in the number of EGFP-positive cells accompanied by a compensatory increase in EGFP-negative cells, suggesting a transition to the differentiated neuronal population. While the population of undifferentiated D3 ES cells (A) was used for calibration of the system (estimate of the range of auto-fluorescence), a relatively homogenous EGFP expression was noticed in the undifferentiated h-nestin-EGFP cells (B). The EGFP fluorescence intensity on the horizontal axis is displayed as log scale; the vertical axis represents the percentage of gated cells. Numbers in the top-left corner indicate the differentiation stage. (B) A quantitative evaluation of the in vitro neurogenesis was obtained by averaging two to three experiments per time point of differentiation. This analysis clearly showed that the decline in the strong EGFP-positive (EGFP⁺⁺⁺ and EGFP⁺⁺) and weak EGFP-positive (EGFP⁺) cell population was accompanied by a parallel increase in EGFP-negative (EGFP^-) cells.