Since its inception in 1992 [Reynolds and Weiss, Science 255:1707-10, 1992], the neurosphere assay (NSA) has proven an exceptionally useful tool in detecting neural stem cells (NSCs) in both the developing and adult mammalian brain. To date, over 1,300 manuscripts have been published employing the assay, attesting to the robustness of the assay, and its ease of use. However, a brief survey of the literature demonstrates that the number of primary neurospheres generated from essentially the same anatomical region (i.e., the periventricular region of the rostral lateral ventricle) ranges between 150 and 936 [Gritti et al., J Neurosci 22:437-445, 2002; Tropepe et al., J Neurosci 17:7850-59, 1997; Doetsch et al., Cell 97:703-16, 1999; Enwere et al., J Neurosci 24:8354-65, 2004]. Indeed, in our hands we typically generate approximately 1,800 primary spheres when harvesting tissue from the same region.