Cortices from postnatal day 1–3 CD1, C57BL/6 or il1r1 null mutant mouse pups were dissected, pooled, and cells dissociated by exposure to trypsin (0.025%, 15 min, 37 °C). Cells, diluted 2 hemispheres/10 ml of glial plating medium/plate, were plated into multi-well culture plates (Falcon Primaria; BD Biosciences, Lincoln Park, NJ) as described (Hamby et al. 2006 (link); Uliasz et al. 2012 (link)). DNA extracted from extraneous brain was used to confirm genotype. Once confluent, astrocyte monolayers were treated with 8 µM β-D-arabinofuranoside (Sigma, St. Louis, MO) once for 4–6 days to reduce the number of microglia. Cells were then placed in maintenance media that was replaced once per week until experimentation. Purified astrocyte cultures were generated by removing residual microglia by treating monolayers with 50–75 mM L-leucine methyl ester for 30–90 min, one day prior to experimentation (Hamby et al. 2006 (link); Uliasz et al. 2012 (link)). Cultures were maintained at 37°C in a humidified 6.0% CO2, 21% O2 -containing incubator and were used for experimentation at ≤35 days in vitro.
β d arabinofuranoside
Manufactured by Merck Group
Sourced in United States, Hungary
β-d-arabinofuranoside is a laboratory reagent used in various biochemical and molecular biology applications. It functions as a monosaccharide, specifically a pentose sugar, that can be utilized in various experimental procedures. The core function of this compound is to serve as a chemical building block or substrate for further analysis and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Falcon primaria, by BD (1 mentions)
Polylysine, by Merck Group (1 mentions)
Sprague dawley rat, by Charles River Laboratories (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Zearalenone, by Merck Group (1 mentions)
3 3 5 triiodo l thyronine, by Merck Group (1 mentions)
L thyroxine, by Merck Group (1 mentions)
Heat inactivated fetal bovine serum, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
17β estradiol, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Coomassie brilliant blue r 250, by Thermo Fisher Scientific (1 mentions)
6 protocols using β d arabinofuranoside
1
Primary Astrocyte Culture Protocol
2
Preparation and Transfection of Primary Hippocampal Neurons
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Primary hippocampal neurons were prepared from embryonic day18 Sprague–Dawley rats (Charles River) as previously described (Kaech and Banker, 2006 (link)). Neurons were maintained in neurobasal medium with B27 supplement and GlutaMAX (all Thermo Fisher Scientific) on poly-lysine (Sigma-Aldrich)–coated 18-mm-diameter #1.0 glass coverslips (Menzel) or µGrid glass-bottom Petri dishes (ZellKontakt GmbH) at 37°C in a CO2-controlled humidified incubator. After 3 d, the cytostatic cytosine β-d -arabinofuranoside (Sigma-Aldrich) was added at a final concentration of 5 µM. Neurons were transiently transfected with protein fusion constructs between DIV2 and DIV8 using Lipofectamine 2000 (Thermo Fisher Scientific). The GPI-GFP protein fusion construct was a gift from the Helenius laboratory (ETH Zurich, Zurich, Switzerland; Keller et al., 2001 (link)). The L-YFP-GT-mHoneydew-46 construct was based on L-YFP-GT46, a gift from P. Keller (Max Planck Institute for Cell Biology and Genetics, Dresden, Germany; Keller et al., 2001 (link)), and modified by inserting mHoneydew between the transmembrane domain and the CD46 domain to increase the size of the cytosolic part of the fusion protein.
3
Embryonic Mouse Hippocampal Neuron Culture
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Hippocampal neurons from 17- to 19-day-old embryonic mice were cultured (5 × 105 cells per square centimeter) on plates coated with poly-L-lysine (Catalog number P1399, Sigma, USA) as described previously [17 (link)]. The neurons were then maintained in neurobasal medium (Catalog number 21103-049, Gibco, USA) supplemented with B27 (Catalog number 17504-044, Gibco) and 0.5 mM L-glutamine (Catalog number G3126, Sigma). Approximately 1/3 to 1/2 of the culture medium was changed every 3-4 days. Ten micromolar cytosine β-D-arabinofuranoside (Catalog number C1768, Sigma) was added to the culture medium at 3 days in vitro (DIV3) to inhibit the growth of gliocytes. The cultured neurons were stained at DIV7 with a neuron-specific marker, microtubule-associated protein 2 (MAP2) (Catalog number 11267, Abcam, USA), to evaluate the purity of the cultured neurons. Only the cultured cells whose purity was higher than 98% were used for the following experiment.
4
Conditional Knockout of Hippocampal GABA Receptors
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Mouse hippocampal neurons were isolated and cultured from floxed-GABABR1 mice as described previously.27 (link),46 (link) Cells were cultured in poly-d -lysine-coated (0.1 mg/mL) plastic dishes and/or glass coverslips in a neuron maintenance medium27 (link),46 (link) for 3 days and supplemented with cytosine β-d -arabinofuranoside (3 μmol/L; Sigma, St. Louis, MO, USA) for an additional 3–4 days. These neurons were then infected with adenoviruses (8 pfu/cell) carrying a cDNA encoding Cre recombinase (Ad-Cre; Microbix Inc., Toronto, Canada) or empty viral vector (Ad-Cont Microbix Inc., Toronto, Canada) as described previously.27 (link),46 (link)
5
Thyroid Receptor Antibody Protocol
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TRα and TRβ primary antibodies were purchased from Abnova (Budapest, Hungary). Secondary antibodies were from Vector Laboratories. Protease inhibitor cocktail, β-d -arabinofuranoside, 17β-estradiol, 3,3′,5-triiodo-l -thyronine, l -thyroxine, and zearalenone were purchased from Sigma-Aldrich (Budapest, Hungary). Culture media and TRI reagent were from Invitrogen (Carlsbad, CA, USA). Penicillin/streptomycin and heat-inactivated fetal bovine serum were purchased from GIBCO (Budapest, Hungary). Bicinchoninic acid (BCA) kit from Pierce (Rockford, IL, USA). Immobilon Western chemiluminescent HRP substrate was from Merck Millipore (Darmstadt, Germany).
6
Pericyte Migration Inhibitor Assay
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A confluent monolayer of pericytes was scratched with a P200 pipette tip. Cells were washed two times in warm PBS. Cells were then given fresh media with or without mitotic inhibitors cytosine β-d -arabinofuranoside (AraC, 1 µM; Sigma), 5-fluoro-2’-deoxyuridine (FdUR, 1 µM; Sigma), uridine (Urd, 1 µM; Sigma). Pericytes were treated with vehicle or sunitinib, wortmannin, U0126 or TPCA-1 for 2–4 h (see Table 1 for details), then treated with vehicle or PDGF-BB and left to migrate into the scratch for a further 48 h. Cells were then fixed in 4% PFA for 15–20 min and washed in PBS-T. Cells were stained with Coomassie Brilliant Blue R250 (0.25% w/v in 10% glacial acetic acid, 45% methanol; Gibco, CA, USA) for 30 min. The stain was then removed and cells were allowed to air dry. Wells were imaged at 4× magnification using brightfield imaging on the ImageXpress Micro XLS microscope.
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