Ogy in cryoform (Tissue-Tek O.C.T. Compound, Sakura Finetek, Netherlands) and frozen in liquid nitrogen-cooled CASIN custom synthesis 2methylbutane (Sigma-Aldrich, Germany); or directly frozen in liquid nitrogen and kept at 280uC until analysis.In situ Cell Death DetectionTo detect typical features of apoptosis (fragmented nuclei, apoptotic bodies), nuclear DNA was stained using the blue fluorescent 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, USA). Aggregate cryosections (16 mm) were incubated with DAPI for 10 min at room temperature. In situ detection of cell death was performed using terminal deoxynucleotidyl transferase (TdT)mediated dUTP nick end labeling (TUNEL) on 16 mm cryosections of aggregates. TUNEL staining was performed according to supplier recommendations using the In Situ Cell Death Detection Kit with BIBS39 manufacturer Fluorescein (Roche Applied Science, Switzerland) resulting in green fluorescence in dying cells.Treatment ProtocolCultures were treated with 1 mM glutaric acid (GA; SigmaAldrich, Germany) or 3-hydroxyglutaric acid (3-OHGA; Ernesto Brunet-Romero, Madrid, Spain) buffered in 25 mM HEPES with pH adjusted to 7.5. Cultures were exposed to one of the two metabolites 6 times every 12 hours at two different developmental stages starting from DIV 5 in protocol A or from DIV 11 in protocol B (Figure 1). Aggregates were harvested 5 hours after the last treatment at DIV 8 in protocol A and at DIV 14 in protocol B.Western Blot AnalysisAggregates were homogenized in 150 mM sodium chloride, 50 25837696 mM Tris-HCl, pH 8, 1 NP-40 (Sigma-Aldrich, Germany) 
and Protease Inhibitor Cocktail – Complete Mini (Roche Applied Science, Switzerland) and sonicated for 5 seconds. Lysates were ?cleared by centrifugation at 129000 rpm for 30 min at 4C. After dilution, protein content was measured by bicinchoninic acid assay (Thermo Scientific, USA) and diluted with NuPAGEH LDS Sample Buffer (Life Technologies, USA) to a final concentration of 1.2 mg/ml. Samples were heated at 70uC for 10 min and resolvedImmunohistochemistryImmunohistochemical staining was carried out on 16 mm aggregate cryosections using antibodies against different markers of brain cell types: phosphorylated medium weight neurofilament (p-NFM; clone NN18, Sigma-Aldrich, USA) for neurons [16], glialBrain Cell Damage in Glutaric Aciduria Type IFigure 1. Treatment protocols. Cultures of aggregates were exposed to 1 mM GA and 3-OHGA at two time points representing different developmental stages of brain cell maturation (Protocols A and B). Metabolites were added 6 times every 12 hours (indicated by arrows) starting on DIV 5 in protocol A and on DIV 11 in protocol B (treatment days are indicated by black boxes) 12 hours after the change of the medium. Aggregates were harvested 5 hours after the last treatment at DIV 8 in protocol A and at DIV 14 in protocol B. doi:10.1371/journal.pone.0053735.gon NuPAGEH 4?2 Bis ris Gel (for p-NFM) or NuPAGEH 12 Bis ris Gel (for GFAP, MBP, actin and caspase-3) using NuPAGEH MOPS SDS Running Buffer (Life Technologies, USA) at a constant voltage (200 V, 60 min). Proteins were transferred onto Immobilon-FL PVDF, 0.45 mm membranes (Millipore, USA). Membranes were blocked with 5 non-fat dry milk in TBS-Tween (20 mM Trizma base, 137 mM NaCl, 0.05 Tween, pH 7.6) for 1 h at room temperature. After blocking, the membranes were incubated overnight with different primary antibodies against GFAP, MBP, p-NFM, Actin (I-19) (Santa Cruz Biotechnology, USA) or full-length (35 kDa) and large fragment.Ogy in cryoform (Tissue-Tek O.C.T. Compound, Sakura Finetek, Netherlands) and frozen in liquid nitrogen-cooled 2methylbutane (Sigma-Aldrich, Germany); or directly frozen in liquid nitrogen and kept at 280uC until analysis.In situ Cell Death DetectionTo detect typical features of apoptosis (fragmented nuclei, apoptotic bodies), nuclear DNA was stained using the blue fluorescent 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, USA). Aggregate cryosections (16 mm) were incubated with DAPI for 10 min at room temperature. In situ detection of cell death was performed using terminal deoxynucleotidyl transferase (TdT)mediated dUTP nick end labeling (TUNEL) on 16 mm cryosections of aggregates. TUNEL staining was performed according to supplier recommendations using the In Situ Cell Death Detection Kit with Fluorescein (Roche Applied Science, Switzerland) resulting in green fluorescence in dying cells.Treatment ProtocolCultures were treated with 1 mM glutaric acid (GA; SigmaAldrich, Germany) or 3-hydroxyglutaric acid (3-OHGA; Ernesto Brunet-Romero, Madrid, Spain) buffered in 25 mM HEPES with pH adjusted to 7.5. Cultures were exposed to one of the two metabolites 6 times every 12 hours at two different developmental stages starting from DIV 5 in protocol A or from DIV 11 in protocol B (Figure 1). Aggregates were harvested 5 hours after the last treatment at DIV 8 in protocol A and at DIV 14 in protocol B.Western Blot AnalysisAggregates were homogenized in 150 mM sodium chloride, 50 25837696 mM Tris-HCl, pH 8, 1 NP-40 (Sigma-Aldrich, Germany) and Protease Inhibitor Cocktail – Complete Mini (Roche Applied Science, Switzerland) and sonicated for 5 seconds. Lysates were ?cleared by centrifugation at 129000 rpm for 30 min at 4C. After dilution, protein content was measured by bicinchoninic acid assay (Thermo Scientific, USA) and diluted with NuPAGEH LDS Sample Buffer (Life Technologies, USA) to a final concentration of 1.2 mg/ml. Samples were heated at 70uC for 10 min and resolvedImmunohistochemistryImmunohistochemical staining was carried out on 16 mm aggregate cryosections using antibodies against different markers of brain cell types: phosphorylated medium weight neurofilament (p-NFM; clone NN18, Sigma-Aldrich, USA) for neurons [16], glialBrain Cell Damage in Glutaric Aciduria Type IFigure 1. Treatment protocols. Cultures of aggregates were exposed to 1 mM GA and 3-OHGA at two time points representing 
different developmental stages of brain cell maturation (Protocols A and B). Metabolites were added 6 times every 12 hours (indicated by arrows) starting on DIV 5 in protocol A and on DIV 11 in protocol B (treatment days are indicated by black boxes) 12 hours after the change of the medium. Aggregates were harvested 5 hours after the last treatment at DIV 8 in protocol A and at DIV 14 in protocol B. doi:10.1371/journal.pone.0053735.gon NuPAGEH 4?2 Bis ris Gel (for p-NFM) or NuPAGEH 12 Bis ris Gel (for GFAP, MBP, actin and caspase-3) using NuPAGEH MOPS SDS Running Buffer (Life Technologies, USA) at a constant voltage (200 V, 60 min). Proteins were transferred onto Immobilon-FL PVDF, 0.45 mm membranes (Millipore, USA). Membranes were blocked with 5 non-fat dry milk in TBS-Tween (20 mM Trizma base, 137 mM NaCl, 0.05 Tween, pH 7.6) for 1 h at room temperature. After blocking, the membranes were incubated overnight with different primary antibodies against GFAP, MBP, p-NFM, Actin (I-19) (Santa Cruz Biotechnology, USA) or full-length (35 kDa) and large fragment.
