based on the camera's capture of a discernable image above background (black) level and by overlay of DAPI staining and/or bright field images from the analogous field. Alexa 488 stained fluorescent images were converted to 8-bit color format and analyzed with Image J. Calibration was set based on 256 levels of green scale, 2nd degree polynomial function. Cells were selected for analysis using a numbered grid overlay to select fields. Cells with a distinguishable, stained nuclear boundary were circled using the freehand circle tool. Each cell within the field chosen for analysis was marked with a numeric label to avoid double counting. The staining intensity of marks in a region of neurosphere was defined by the intensity of stained cells and the majority proportion of the intensity-ranked cells in the region, as defined in the following. The staining intensity of cells (e.g. Figure 1 and 2) were defined by Absent (0), Weak (+) = <0.08, Moderate (++) = 0.08-0.089, High (+++) = 0.09-0.099, Intense (++++) = >0.1) (see Figure 2, bottom panel). The intensity of the marks per region of neurosphere was defined by the ≥60% of the cells with the above rank. Based on the rank values, Mann-Whitney U
