Nissl staining of neural stem cells (NSCs) and cell culture medium concentration of GHB as a function of cell line genotype.

To confirm that NSCs retained features of neural cells, Nissl stain (red) was initially undertaken, and co-localization with a nuclear stain (blue) was performed to confirm that cells were of neuronal origin (A), although the Nissl stain does not differentiate mature neurons from progenitors. Scale bar = 50 μm (white bar). GHB concentration in cell culture medium is also displayed as a function of donor cell line (B). Media were collected for 24 h following addition of fresh medium and quantified for GHB content employing isotope-dilution gas chromatography-mass spectrometry [29]. GHB content was normalized to the recovery of ²H₆-GHB, the latter added to each medium sample as internal standard stable isotope. The lower limit of quantification for this assay was 30 μM; all wild-type samples were below the limit of quantification (BLQ). Error bars display mean and SD. Abbreviations: WT, wild-type; MT, mutant. Statistical analysis employed one-way ANOVA (F (3, 42) = 378.9; p < 0.0001).