Fusion fx

The conditioned medium or supernatant of the control and BF-treated cortical organoids from both the 24 and 72 h treatments were stored and used for the proteome profiler antibody array. The relative expression levels of 105 soluble human proteins and cytokines were determined using the Human XL Cytokine Array Kit from R&D Systems. The cytokine array was performed following the manufacturer’s guidelines. In brief, the membranes were blocked for 1 h on a rocking platform using the provided blocking buffer and then the samples were prepared by diluting the desired quantity to a final volume of 1.5 mL with the distinct array buffer (array buffer 6). The sample mixtures were pipetted onto the blocked membranes and were incubated overnight at 4 °C on a rocking platform. The membranes were then washed three times with washing buffer for 10 min each at RT. Then, the membranes were incubated with the detection antibody cocktail for 1 h at RT and then washed three times thoroughly. Afterward, Streptavidin-HRP was added onto the membranes, which were incubated for 30 min at RT. The ECL detection reagent (Cytiva, Freiburg, Germany) was used to visualize the spots on the membrane and then detected in a Fusion FX instrument (PeqLab, Erlangen, Germany).

Protein lysates were separated by SDS PAGE (6–10%). Gels were either stained with Coomassie brilliant blue or - for IB - proteins were transferred to nitrocellulose membranes. The membranes were blocked with 5% milk powder in PBS with 0.1% Tween-20 and subsequently incubated with primary antibodies followed by incubation with HRP-conjugated secondary antibodies. Detection was carried out using a Fusion FX chemiluminescence system (PEQLAB, Erlangen, Germany). Quantification of protein bands was performed using QuantiScan version 1.5 software (Biosoft, Cambridge, UK).

Citrate synthase activity assays to assess tissue mitochondrial function were performed as described previously^{33 (link)}. Protein lysate (8 μg) was used for the citrate synthase activity assay following the manufacturer’s protocol (Sigma CS070). Tissue samples were homogenized in tissue lysis buffer provided with the kits to obtain protein lysates. After centrifugation and removal of the lipid layer, protein concentration was determined using the BCA Protein Assay (Thermo Fisher Scientific Inc.).
For gene expression assays, total RNA was extracted using Trizol (Invitrogen), treated with DNase (ThermoScientific) and reverse transcribed to cDNA (AppliedBiosystems) according to manufacturer’s instructions. Gene expression, normalized to 36B4, was analyzed by quantitative real-time RT-PCR (Sybr Green, 384-well plates) using the QuantStudio 6 PCR System (ThermoFisher). Primer sequences are available upon request.
To perform immunoblot analysis, homogenized tissue was lysed in protein lysis buffer (Sigma) containing protease and phosphatase inhibitors. Standard western blotting was performed using rabbit polyclonal antibodies to Ucp1 (1:500; Abcam; ab23841) and GAPDH (1:3000; CellSignaling; #2118). HRP linked Goat anti-Rabbit (1:3000; Biorad; #170–6515) was used as secondary antibody. Proteins were detected by chemiluminescence (Roche) and images were acquired using FusionFx (Peqlab).