Serum-based media have been the mainstay of microglial isolation techniques over the years but these undefined culture conditions may unpredictably alter their characteristics. Building on recent refinements of the work of Brewer and colleagues 14 we have developed a fully-defined, serum-free Neuro-Glial Differentiation media (referred to as NGD), the component concentrations of which were adjusted to match those of human cerebrospinal fluid (Supplementary Table 1). Compared to the complexity of other published culture conditions, NGD constitutes a single defined serum-free medium allowing culture of multiple neural and glial cell types, as well as other cells of interest. Neural differentiation can be initiated in NGD by addition of a small molecule inhibitor of TGFβ signaling (dorsomorphin) 15. Neural progenitors can be expanded by addition of FGF2 to the NGD base and differentiation into neurons and glia can be subsequently triggered by FGF2 removal.
