Fibroblasts were transduced with lentiviruses containing transgenes for miR-9/9*-124, NEUROD2, ASCL1 and MYT1L as previously described.9 (link) Fibroblasts were plated at a density of 10–15 k cm−2 in fibroblast medium (Dulbecco's Modified Eagle Medium; Invitrogen) containing 10% fetal bovine serum (FBS; Gemini Bio-Products), nonessential amino acids, glutamate and penicillin/streptomycin (Invitrogen) on plates coated with 0.1% gelatin (Millipore, Billerica, MA, USA). Fibroblast medium contained DMEM (Invitrogen) with 10% FBS (Gemini Bio-Products), 1% Pen-Strep (Invitrogen, Grand Island, NY, USA), 1% non-essential amino acids (Invitrogen) and 1% 200 mM L-glutamine (Invitrogen). Infection with the four lentiviruses occurred 24 h after plating using 8 μg ml−1 polybrene (Sigma) with plates centrifuged at 1000 g for 20 min to enhance the lentivirus infection efficiency. The next day, medium was changed to fibroblast medium containing 1 mM valproic acid (Sigma), and 3 days later, the medium was changed to Neuronal Medium (ScienCell, Carlsbad, CA, USA) containing 1 mM valproic acid along with selection antibiotics Geneticin, Blasticidin and Puromycin (all Invitrogen). Selection antibiotics were removed after 6–7 days, and the cells were maintained in Neuronal Medium (ScienCell) with 1 mM valproic acid for 1–3 days until passaging with Accutase (Sigma) onto a 384-well BIND biosensor.
Valproic acid
Manufactured by Merck Group
Sourced in United States, Germany, China, Italy, Sao Tome and Principe, United Kingdom, France
Valproic acid is a chemical compound used in various laboratory applications. It is a clear, colorless liquid with a characteristic odor. Valproic acid is commonly used as a standard reference material in analytical chemistry and as a reagent in biochemical and pharmaceutical research. Its core function is to serve as a reference or control substance in scientific experiments and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Trichostatin a, by Merck Group (13 mentions)
Dmem f12, by Thermo Fisher Scientific (13 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (12 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (11 mentions)
Matrigel, by BD (8 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Glutamax, by Thermo Fisher Scientific (7 mentions)
Sodium butyrate, by Merck Group (6 mentions)
Carbamazepine, by Merck Group (6 mentions)
Insulin, by Merck Group (5 mentions)
Clobazam, by Merck Group (5 mentions)
Topiramate, by Merck Group (5 mentions)
Phenobarbital, by Merck Group (5 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (5 mentions)
N2 supplement, by Thermo Fisher Scientific (5 mentions)
Forskolin, by Merck Group (4 mentions)
Pentylenetetrazole, by Merck Group (4 mentions)
Polybrene, by Merck Group (4 mentions)
Matrigel, by Corning (4 mentions)
183 protocols using valproic acid
1
Reprogramming Fibroblasts into Neurons
2
Cell lines and drug treatments
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A375, BCPAP, TPC1, MDA-MB231 were cultured in DMEM, H1299 and PC3 were cultured in RPMI and HCT-116 were cultured in IMDM; all cell lines were grown at 37 °C/5% CO2 in medium added with 10% fetal bovine serum and 1% penicillin – streptomycin. All cell lines were routinely tested for Mycoplasma contamination and authenticated by SNP profiling at Multiplexion GmbH (Heidelberg, Germany), last authentication was performed in January 2019. All cell lines were treated for 24–48 – 72 h (depending on the assay performed) with different concentrations of the following drugs: Tubacin, SAHA, Valproic acid (Sigma-Aldrich, St. Louis, Missouri, USA), TMP-269, PCI-34051, 4SC-202 (Selleckchem, Munich, Germany) or the respective control. All drugs were resuspended in DMSO (Sigma-Aldrich, St. Louis, Missouri, USA) except for Valproic acid which were reconstituted in water. For proliferation assays, treated cells were counted by trypan blue exclusion with Countess® Automated Cell Counter (Thermo Scientific, Waltham, Massachusetts, USA).
3
Valproic Acid Induced Autism Model
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To induce autism-like pup model, valproic acid (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) was dissolved in saline at 0.1-mL/kg concentration, and 400-mg/kg valproic acid was administered subcutaneously to mice on day 12 of pregnancy, as the previous mentioned method [19 ].
4
Anticonvulsant Compound Preparation Protocol
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All compounds were prepared in 0.5% methylcellulose (Sigma), except for valproic acid, which was prepared using saline (0.9% NaCl). Carbamazepine, clonazepam, celecoxib, diclofenac, dexamethasone, ethosuximide, ibuprofen, minocycline, phenobarbital, phenytoin, prednisone, ezogabine, and valproic acid were obtained from Sigma (Sigma). Gabapentin, levetiracetam, tiagabine, and topiramate were obtained from TCI America. Lacosamide was obtained from Axon Medchem, and lamotrigine was obtained from AK Scientific. Doses for each compound are shown in Tables 1 , 2 , 3 . All compounds were administered as suspensions, with the exceptions of ethosuximide, ezogabine, Gabapentin, levetiracetam, tiagabine, and valproic acid.
5
Efficient Conversion of Naive hPSCs to Trophoblast Cells
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The conversion from naive hPSCs to trophoblast cells11 ,13 (link) was performed as follows. Cell culture plates were coated with 5 μg ml−1 collagen IV (Corning, 354233) overnight at 37 °C. Naive colonies were dissociated to single cells with TrypLE (Thermo Fisher, 12605010; 10 min at 37 °C), followed by filtering through a 40-μm cell strainer. After washing the plates once with PBS, the naive hPSCs were seeded onto the collagen IV-coated plates in filter-sterilized trophoblast stem cell medium13 (link),62 (link) comprising DMEM/F12 medium (Gibco, 11320033) supplemented with 0.3% BSA (Sigma-Aldrich, A3059), 0.2% FBS (Thermo Fisher, 10270106), 0.5% penicillin–streptomycin (Gibco, 15140122), 1% insulin-transferrin-selenium-ethanolamine supplement (ITS-X) (Thermo Fisher, 51500056), 8.5 μM l -ascorbic acid (Sigma-Aldrich, A4403), 0.5 μM A83-01 (PeproTech, 9094360), 1 μM SB431542 (Axon Medchem, 301836-41-9), 50 ng ml−1 human epidermal growth factor (Miltenyi Biotec, 130-097-749), 2 μM CHIR99021 (Axon Medchem, HY-10182), 0.8 mM valproic acid (Merck, V0033000) and 0.1 mM β-mercaptoethanol (Gibco, 31350010). The medium was changed daily and supplemented with 5 μM Y-27632 (Tocris) and 1 μM UNC1999 or an equivalent volume of DMSO.
6
Angiogenic Factors and Epigenetic Modulators
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Recombinant VEGF-A and VEGF-C were purchased from ReliaTech (Wolfenbüttel, Germany). Sodium Butyrate (NaB), Trichostatin A (TSA) and Valproic Acid (VPA) were obtained from Merck Millipore (Billerica, MA, USA).
7
HEK293-F Co-expression of MHC I and TAPBPR
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HEK293-F cells were grown in FreeStyle 293 Expression Medium (Gibco) at 37 °C, 5% CO2 and shaken at 120 rpm. For co-expression of the MHC IFos and TAPBPRJun, 5 mL expression medium was supplemented with 75 µg β2m-HLA-A*68:02Fos and 75 µg TAPBPRJun DNA (equimolar ratio) and mixed with a 4-fold molar excess of linear PEI (Polysciences) diluted in 5 mL expression medium. DNA used for transfection was purified using the PureYield Plasmid Midiprep System (Promega). After incubation for 30 min, the transfection mixture was added to 50 mL of HEK293-F cells at a density of 16×106 cells/mL. Cells were diluted to 3×106 cells/mL 3 hr post transfection. Protein expression was performed in the presence of 3.5 mM valproic acid (Merck Millipore) and 10 µM kifunensine (Biomol). The culture supernatant containing the secreted proteins was harvested after five days. The HsUGGT1D1316N construct was transfected into HEK293-F cells at a density of 1.6×106 cells/mL. The cell suspension was diluted to 0.8×106 cells/mL 3 hr post transfection and was further cultured for five days.
8
Epigenetic Regulation of Viral Gene Expression
9
MEF Reprogramming to iPSCs
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For reprogramming 6,5×103 MEFs per cm2 were seeded on gelatin-coated dishes one day prior to transduction. MEFs were transduced virally using a multiplicity of infection of 4–7. Medium was changed to MEF medium 8–16 h p.t. Cells were further cultured in MEF medium until day 4 p.t. and in iPSC medium hereafter. Medium was exchanged every other day supplemented with 2 mM valproic acid (Merck, Darmstadt, Germany) from day 2 onwards, 25 µg/ml vitamin C (Sigma-Aldrich) from day 2–8 and “3i” cocktail from day 8 onwards. To harvest the cells, dishes were washed once in PBS, pre-treated with 1 mg/ml Dispase (Roche, Penzberg, Germany) for 7 min at 37°C, washed and dissociated in 0.25% trypsin-EDTA (Sigma-Aldrich) for 5 min at 37°C.
10
Establishing PS1-iPSCs from Fibroblasts
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PS1-iPSCs were established from the PS1 patient’s skin fibroblasts (Coriell Institute) as previously described (Okita et al., 2007 (link)), with slight modifications. In brief, PS1 fibroblasts were seeded at 3 × 105 cells in 60 mm2 dishes coated with gelatin. On day 1, the VSV-G pseudotyped retroviral vector system carrying OCT4, SOX2, KLF4, and c-Myc was added to fibroblast cultures. On day 2, cells were subjected to the same transduction procedures and harvested 24 h later. Transduced cells were replated on mouse embryonic fibroblast (MEF) layers in 100 mm2 dishes containing the fibroblast medium. On the next day, the medium was changed to complete ES medium with 0.5 mM valproic acid (Sigma-Aldrich), and thereafter changed every other day. After 20 d, ES-like colonies appeared and were picked up to be reseeded on new MEF feeder cells. Cloned ES-like colonies were subjected to further analysis. Normal iPSC line (HPS0063) was obtained from the RIKEN Bioresource Center (Takahashi et al., 2007b (link)).
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