Neurobasal a

Astrocytes were seeded into 6-well plates at a density of 100,000 cells. At 90% confluency, cells were washed 3 times with Neurobasal A (GIBCO), then cultured overnight in serum-free media (Neurobasal A, Glutamax and Penicillin/Streptomycin). Astrocytes were treated for 24 hours with vehicle, thapsigargin or both thapsigargin and GSK2606414. Media was collected and centrifuged for 5 minutes at 4,000 x g to remove any dead cells. Proteins were precipitated using trichloroacetic acid. Precipitates were washed with acetone, resuspended in SDS-PAGE buffer and separated on a 4%–12% gel. The precipitation procedure was also performed on serum-free media alone, as an additional control. Proteins were analyzed by LC/MS. A protein was identified if it received ≥ 99% confidence with ≥ 3 unique peptides at 95% confidence in the vehicle- or thapsigargin-treated condition.

All DMG cell lines used in the study (except for SF7761) were cultured in ultra-low attachment flasks in culture medium with 1:1 ratio of DMEM/F-12 (Invitrogen, 11330-032) and Neurobasal A (Invitrogen, 10888-022) and ten percent each of HEPES Buffer Solution 1 M (Thermo Fisher, 15630080), Sodium Pyruvate solution 100 nM (Life Technologies, 11360070), MEM non-essential amino acids solution 10 mM (Thermo Fisher, 11140050), Glutamax-I Supplement (Thermo Fisher, 35050061), and Penicillin/Streptomycin solution (Life Technologies, 15140122). The media was supplemented with B27 Minus Vitamin A (Invitrogen, 12587-010), epidermal growth factor (EGF; StemCell Tech. Inc., 78006), fibroblast growth factor (FGF; GF003, StemCell Tech., Inc., 78003) and heparin solution, 0.2% (StemCell Tech. Inc., 07980), as well as PDGF-AA (Shenandoah Biotech, 100-16) and PDGF-BB (Shenandoah Biotech, 100-18). SF7761 cell line was cultured in medium with Neurobasal A (Invitrogen, 10888-022) and N-2 Supplement (Invitrogen, 17502), further supplemented with B27 Minus Vitamin A (Invitrogen, 12587-010), epidermal growth factor (EGF; StemCell Tech. Inc., 78006), fibroblast growth factor (FGF; GF003, StemCell Tech. Inc., 78003) and heparin solution, 0.2% (StemCell Tech. Inc., 07980). Cells were dissociated using Accutase (StemCell Tech. Inc., 07922) and passaged every 3 or 4 days.

Hippocampal neuronal cultures were prepared from one to four days old Wistar rats (Charles River, USA) as described [9] (link). Briefly, newborn rats were sacrificed by decapitation in accordance with the guidelines of the State of Bavaria. Hippocampi were removed from the brain in ice cold Hank's salt solution, and the dentate gyrus was cut away. After digestion with trypsin (5 mg/ml) cells were mechanically triturated and plated in MEM medium, supplemented with 10% fetal calf serum and 2% B27 Supplement (all from Invitrogen, Taufkirchen, Germany). Neurons were transfected on DIV3 with synapto-pHluorin [4] (link) under control of a synapsin promoter or with VGLUT1-pHluorin [10] (link), [11] (link) (kindly provided by Susan Voglmaier, Department of Psychiatry at University of California, San Francisco) under control of a beta-actin-promotor with CMV-enhancer, using a modified calcium phosphate method [12] (link). In brief, the culture medium was removed and replaced with Neurobasal A (Invitrogen, Taufkirchen, Germany). The calcium phosphate/DNA precipitate was allowed to form in BBS buffer (pH 7.05) for 30 minutes, then cells were transfected with the precipitate in Neurobasal A and incubated for 30 minutes prior to washing with HBSS (Invitrogen, Taufkirchen, Germany). Experiments were performed between 25 and 35 days in vitro.

The amygdala was dissected from an embryo in dams on an embryonic day 18 (E18), then the tissues were treated for 15 min at 37 °C by 0.05% of trypsin EDTA, then replaced by soybean trypsin inhibitor (Sigma) to stop the reaction for 5 min at 37 °C. The tissues were then dissociated in supplemented Neurobasal A (Invitrogen) solution and then resuspended in culture media, including Neurobasal A, B27, 1× GlutaMAX, and 100 U/ml Pen/Strep (from Invitrogen), the isolated amygdala neurons were maintained at 37 °C and 5% CO₂ for further biomedical analysis^{61 (link)}.

Ten-week-old offspring were used for the preparation of primary amygdala neurons. Amygdala tissues were dissected from rat offspring that were humanely sacrificed through cervical dislocation. The tissues were treated with 0.05% trypsin EDTA for 15 min at 37 °C. Trypsin EDTA was replaced with soybean trypsin inhibitor (Sigma) for 5 min at 37 °C to stop the reaction. This was then replaced with supplemented Neurobasal A (Invitrogen) followed by mechanical dissociation. The cells were then resuspended in culture media, including Neurobasal A, B27, 1×GlutaMAX and 100 U/ml Pen/Strep (from Invitrogen), and then, the cells were incubated at 37 °C, 5% CO₂ [28 (link)]. The isolated amygdala neurons were used for the analysis of DNA methylation, epigenetic changes by chromatin immunosuppression (ChIP) assay on the ERβ promoter, and in vitro fatty acid lipid uptake.