Ipped with a UC30 digital camera.Rat 3D Organotypic Brain Cell Cultures in AggregatesPregnant Sprague-Dawley rats (Harlan; Netherlands) were sacrificed on day 15 of gestation. Fetal whole brains were extracted, pooled and mechanically dissociated. 3.66107 cells were grown in 8 ml of a serum-free, chemically-defined medium with 25 mM glucose and maintained under constant gyratory agitation at 37uC, in an atmosphere of 10 CO2 and 90 humidified air to form reaggregated 3D primary brain cell cultures as previously described [14,15]. Media were replenished every three days from day-in-vitro (DIV) 5, by exchanging 5 ml of medium per culture. On the day of harvest aggregate pellets were washed three times with ice-cold PBS and either embedded for histology 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 Daclatasvir (dihydrochloride) 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 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: Conduritol B epoxide chemical information 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.Ipped with a UC30 digital camera.Rat 3D Organotypic Brain Cell Cultures in AggregatesPregnant Sprague-Dawley rats (Harlan; Netherlands) were sacrificed on day 15 of gestation. Fetal whole brains were extracted, pooled and mechanically dissociated. 3.66107 cells were grown in 8 ml of a serum-free, chemically-defined medium with 25 mM glucose and maintained under constant gyratory agitation at 37uC, in an atmosphere of 10 CO2 and 90 humidified air to form reaggregated 3D primary brain cell cultures as previously described [14,15]. Media were replenished every three days from day-in-vitro (DIV) 5, by exchanging 5 ml of medium per culture. On the day of harvest aggregate pellets were washed three times with ice-cold PBS and either embedded for histology 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 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.