Cortical neurons endogenously synthesize cholesterol. Primary neurons and astrocytes were incubated for 3, 6, and 9 days in defined cholesterol-free medium. Levels of cholesterol, 7-DHC, desmosterol and lanosterol levels were measured by LC-MS/MS. Panels on the left show neuronal sterols (A, C, E, G), while panels on the right denote astrocytic sterol levels (B, D, F, H). Sterol levels were normalized to the total cell count at the end point of each experiment. Sterol values correspond to the mean ± SEM of 8–12 replicates. Statistical significance is denoted by asterisks (*p < 0.05; ***p <0.001; ****p < 0.0001). Note that all sterols accumulate in neurons in a time-dependent manner and that a similar process is not observed in astrocytes.