ImageJ was used to count the number of EdU labeled cells in a defined area of neural tube from several different embryos at each of three concentrations. In each case a cell count from the middle region of one half of the neural tube was selected using the marquee tool. White lines have been placed on Fig. 3A, indicating the level at which cells were counted. The selected area was cropped to include only neural tube, adjusted to a black and white image, and then the ImageJ threshold tool was applied to select cells labeled with EdU and TO-PRO-3 (these appear light gray in the image and are easily distinguished from the dark TO-PRO-3 labeled cells). ImageJ was used to count the cells and the area was measured. The cell count was normalized to give an average number of cells per unit of area. 11, 12 and 11 individual sections, from 3, 5 and 2 individual embryos, for 500 μM, 1 mM and 2 mM concentrations were counted, respectively (see Table 1). The Mixed Procedure applied uses an analysis of variance to compare EdU across the 3 dose levels. Terms in the model include the fixed effect of dose and the random effect of embryos within each dose.