Cytosine β d arabinofuranoside

Manufactured by Merck Group

Sourced in United States, United Kingdom, Germany, Italy

Cytosine β-D-arabinofuranoside is a synthetic pyrimidine nucleoside that acts as a cytostatic agent. It is a structural analog of the naturally occurring nucleoside cytidine.

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Lab products found in correlation

88 protocols using cytosine β d arabinofuranoside

Isolation and Culture of Mouse Primary Cortical Neurons

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Mouse primary cortical neurons were prepared from ICR mice (SLC, Inc., Shizuoka, Japan) on the 16th day of gestation as described [44 (link)]. The cortex was separated, and the meninges were removed. The tissues were cut into small pieces and then dissociated using a papain dissociation system (Worthington Biochemical, Freehold, NJ, USA). The resulting cell suspension was filtered through a cell strainer (40 μm, Falcon, Tewlsbury, MA, USA) and plated on polyethylenimine-coated dishes with neurobasal medium and B27 supplement. After 2 days of culture at 37 °C in 5% CO₂ and 95% air, cytosine β-d-arabinofuranoside (Sigma) was added to inhibit glial proliferation (final concentration, 1 μM), and the medium was changed completely after 2 days to remove the cytosine β-d-arabinofuranoside.

Oguro A., Fujita K., Ishihara Y., Yamamoto M, & Yamazaki T. (2021). DHA and Its Metabolites Have a Protective Role against Methylmercury-Induced Neurotoxicity in Mouse Primary Neuron and SH-SY5Y Cells. International Journal of Molecular Sciences, 22(6), 3213.

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GABA Receptor Subunit Detection

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RA was purchased from Sigma-Aldrich Co (St. Louis, MO, USA). Muscimol (Tocris Bioscience, Bristol, UK) and dimethyl sulfoxide (Amresco Solon, Ohio, USA) was purchased respectively. Sodium pentobarbital (100 mg/2 ml) and diazepam (10 mg/2 ml) were respectively purchased through Hanlim Pharm. Co., Ltd. and Samjin Pharm (Seoul, Korea). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle medium, neurobasal A medium, Trypsin-EDTA and Penicilline-Streptomycin was purchased from GIBCO (Grand island, NY, USA). N-(ethoxycarbonyl methyl)-6-methyl quinolinium bromide (MQAE) and cytosine beta-D-arabinofuranoside was purchased from Sigma-Aldrich Co. Specific polyclonal antibodies on the GABA_A receptors subunits of the GAD_65/67 extracted from rabbits and anti-rabbit immunoglobulin G-horseradish peroxidase was purchased from Abcam Inc. in Cambridge, UK. Chemiluminescent HRP substrate was purchased from Millipore Corporation (Billerica, MA, USA).

Kwon Y.O., Hong J.T, & Oh K.W. (2016). Rosmarinic Acid Potentiates Pentobarbital-Induced Sleep Behaviors and Non-Rapid Eye Movement (NREM) Sleep through the Activation of GABAA-ergic Systems. Biomolecules & Therapeutics, 25(2), 105-111.

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Cell Culture Reagent Preparation

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Prior to addition to cell culture media, Cytosine Beta-D-Arabinofuranoside (Sigma-Aldrich, St. Louis, MO) and 2-Deoxy-D-glucose (Sigma-Aldrich) were dissolved in H₂O, CCCP (Sigma-Aldrich), Disulfiram (Sigma-Aldrich), Etacrynic acid (Sigma-Aldrich), and Torin1 (Cell Signaling Technology, Danvers, Massachusetts) were dissolved in DMSO, and IFNg (Peprotech, Rocky Hill, NJ) was dissolved in culture media to 1000x the desired final concentration. Final DMSO (or other diluent) concentration was always 0.1%.

Morinishi L., Kochanowski K., Levine R.L., Wu L.F, & Altschuler S.J. (2020). Loss of TET2 affects proliferation and drug sensitivity through altered dynamics of cell-state transitions. Cell systems, 11(1), 86-94.e5.

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Culturing Hippocampal Neurons and Glia

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Cultures of hippocampal neurons and glial cells were prepared from E18 WT and Fmr1 KO embryos. Pregnant mice were killed by decapitation after deep anesthesia with isoflurane and the uterine horn dissected. Hippocampi were subsequently dissected from the embryos in ice-cold dissection solution and then dissociated in trypsin (Sigma, 0.25%). Briefly, cells were plated at a density of 100 to 200 × 10³ cells per milliliter on poly-L-lysine (Sigma) precoated coverslips and kept at 37 °C in 5% CO₂. The original plating Neurobasal culture medium (Gibco) supplemented with B27 (Gibco, 2%) and complemented with 5% fetal bovine serum was replaced with a serum free medium on day in vitro (DIV) 2. Cytosine B-D-arabinofuranoside (Sigma, 5 μM) was added on DIV 4. All the experiments were performed at DIV 12/15.

Aloisi E., Le Corf K., Dupuis J., Zhang P., Ginger M., Labrousse V., Spatuzza M., Georg Haberl M., Costa L., Shigemoto R., Tappe-Theodor A., Drago F., Vincenzo Piazza P., Mulle C., Groc L., Ciranna L., Catania M.V, & Frick A. (2017). Altered surface mGluR5 dynamics provoke synaptic NMDAR dysfunction and cognitive defects in Fmr1 knockout mice. Nature Communications, 8, 1103.

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Isolation of Primary Cortical Neurons from Mice

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Neonatal Sprague-Dawley mice were used for the isolation of primary cortical neuronal (PCN) cells. Briefly, the cerebral cortices were digested (0.25% trypsin at 37°C) for 1 hour and then incubated in wells precoated with 0.1 mg/ml poly-l-lysine. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 10 mmol/L cytosine-b-d-arabinofuranoside (catalog no. C1768, Sigma). Three hours later, the culture solution was replaced by Neurobasal-A medium (catalog no. 10888022, Thermo Fisher Scientific) containing 2% B27 NeuroMix, 10 mM cytosine-b-d-arabinofuranoside, 100 U/ml penicillin, and 100 U/ml streptomycin.

Tian Y.S., Zhong D., Liu Q.Q., Zhao X.L., Sun H.X., Jin J., Wang H.N, & Li G.Z. (2019). Upregulation of miR-216a exerts neuroprotective effects against ischemic injury through negatively regulating JAK2/STAT3-involved apoptosis and inflammatory pathways. Journal of neurosurgery, 130(3).

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Embryonic Hippocampal Neuron Culture

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Primary cultures of embryonic hippocampal neurons were prepared from CD1 mice on embryonic day 17–18. Pregnant animals were sacrificed by cervical dislocation. Embryonic hippocampi were isolated aseptically and hippocampal tissue was digested and triturated according to Czöndör et al.^{44 (link)}. Cells were seeded onto poly-l-lysine-laminin (Sigma-Aldrich) -coated glass coverslips in 24-well plates at 6.3–6.8 × 10⁴ or 6-well plates at 4.7–5 × 10⁴ cells/cm² density and cultivated at 37 °C in 5% CO₂ atmosphere. Neurobasal medium (Invitrogen) containing 2% B27 supplement (Invitrogen), 0.5 mM Glutamax (Gibco) and 5% FCS (Invitrogen) was used for plating and for a complete medium change on the first day after plating (DIV1). On the 5th, 9th and 12th day after plating, third of the culture medium was changed to FCS-free fresh medium. To inhibit glial cell division, 10 µM cytosine β-d-arabinofuranoside (Sigma-Adrich) was added to the cultures between DIV4 to 6. To achieve a complete blockade of firing, cell cultures were treated with 1 µM tetrodotoxin (TTX, Tocris) between DIV12–14.

Rátkai A., Tárnok K., Aouad H.E., Micska B., Schlett K, & Szücs A. (2021). Homeostatic plasticity and burst activity are mediated by hyperpolarization-activated cation currents and T-type calcium channels in neuronal cultures. Scientific Reports, 11, 3236.

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Isolation and Culture of Embryonic Mouse Hypothalamus

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Wild-type mice at embryonic days 15-17 were used. The protocol used was described in [43] (link). Embryos were quickly removed after pregnant mice dislocation. Embryo brains were placed on the dorsal face to remove the hypothalamus using forceps. Hypothalamus were placed in sterile Hank's solution and rinsed twice. Cell dissociation was done at 37 °C for 20 min with 0.25 mg/ml trypsin (Microvet). After that, 300 μl FBS was used to stop the enzyme digestion and 0.28 mg/ml deoxyribonuclease I from bovine pancreas (Sigma-Aldrich) was added. Cells were mechanically dissociated and plated (about 60,000 cells) on 12-mm-diameter glass coverslips treated previously with poly-L-lysine (Sigma-Aldrich) and laid over 24-well plates. Cells were placed at 37 °C in a 95% air and 5% CO 2 atmosphere with DMEM (Microvet)/F12 1:1 medium supplemented with B27 supplement (1:50; Gibco), 10% FBS, 0.25% glucose, 2 mM glutamine (Gibco), 3.3 μg/ml insulin (Novo Nordisk Pharmaceutical Industries, Inc.), 40 μg/ml gentamicin sulfate salt (Richet), and 1% vitamin solution (Microvet). After 4 days in culture, half of the medium was replaced by fresh medium containing cytosine β-D-arabinofuranoside (Sigma-Aldrich) to reach a final concentration of 5 μM.

Mustafá E.R., Cordisco Gonzalez S, & Raingo J. (2020). Ghrelin Selectively Inhibits Ca_V3.3 Subtype of Low-Voltage-Gated Calcium Channels. Molecular neurobiology, 57(2).

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Primary Neuron-Glia Co-Cultures from Rodents

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All procedures involving animals were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of California, Davis, Institutional Animal Care and Use Committee. Timed-pregnant Sprague Dawley rats were purchased from Charles River Laboratories (Hollister, CA, USA). Timed-pregnant C57BL/6J wild-type mice were purchased from Jackson Labs (Bar Harbor, ME, USA). All animals were housed in clear plastic cages containing corn cob bedding in a room with a constant temperature (22 ± 2 °C) and a 12 h light-dark cycle. Food and water were provided ad libitum.
Primary hippocampal or cortical neuron-glia co-cultures were prepared from postnatal day 0 mouse or rat pups, respectively, as previously described (Sethi et al., 2017 (link)). Dissociated cells were plated on glass coverslips (BellCo, Vineland, NJ, USA) pre-coated with 500 μg/ml poly-L-lysine (Sigma-Aldrich, St. Louis, MO, USA) and maintained at 37 °C in NeuralQ Basal Medium (MTI-GlobalStem, Gaithersburg, MD, USA) supplemented with 2% GS21 (MTI-GlobalStem) and GlutaMAX (ThermoScientific, Waltham, MA, USA) under 5% CO2. Neurons were plated at 83,000 cells/cm² in 24-well tissue culture plates for analyses of dendritic morphology. At 4 days in vitro (DIV), cultures were treated with cytosine β-D-arabinofuranoside (Sigma-Aldrich) at 2.5 μM to limit glial growth.

Schmuck M.R., Keil K.P., Sethi S., Morgan R.K, & Lein P.J. (2020). Automated high content image analysis of dendritic arborization in primary mouse hippocampal and rat cortical neurons in culture. Journal of neuroscience methods, 341, 108793.

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Isolation and Culture of Primary Spinal Cord Neurons

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Primary spinal cord neurons were obtained from Sprague-Dawley (SD) rat embryonic day 15 (E15) pups according to a previously established protocol (Jiang et al., 2006 (link)). In brief, E15 rat spinal cords were isolated and placed in L15 medium (Wu et al., 2017 (link)). Meninges were carefully removed and spinal cords were cut into small pieces, dissociated by incubation in 0.05% trypsin/EDTA 15 min at 37°C and triturated every 5 min. The dissociated cells were washed with and triturated in 10% heat-inactivated fetal bovine serum (FBS), 5% heat-inactivate horse serum (HS), 2 mM glutamine-DMEM (all from Gibco, Grand Island, NY) and cultured in 10 cm plates for 30 min at 37°C to eliminate glial cells and fibroblasts. Neurons were plated on poly-L-lysine coated 48 well plates and incubated in a humidified atmosphere containing 5% CO₂ at 37°C with 10% FBS +5% HS+2 mM glutamine DMEM (all from Gibco). After 16 hr, medium was replaced with serum free neurobasal medium with 2% B27 (Gibco), 1% N2 (Gibco) and 2 mM glutamine (Gibco). On day 3, 5 µM cytosine-β-D-arabinofuranoside (Sigma-Aldrich, Saint Louis, MO) was added for 24 hr to inhibit glia cell proliferation. Using this culture protocol, a purity of >89% spinal cord neurons was obtained by 7 days in vitro (DIV). All experiments were performed between 7 – 10 DIV.

Wang Y., Wu W., Wu X., Sun Y., Zhang Y.P., Deng L.X., Walker M.J., Qu W., Chen C., Liu N.K., Han Q., Dai H., Shields L.B., Shields C.B., Sengelaub D.R., Jones K.J., Smith G.M, & Xu X.M. (2018). Remodeling of lumbar motor circuitry remote to a thoracic spinal cord injury promotes locomotor recovery. eLife, 7, e39016.

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Characterizing Cell Culture Reagents

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Culture media (Dulbecco’s modification of Eagle’s medium [DMEM], Ham’s F-12 50/50 mix), and TRI reagent were from Invitrogen (Carlsbad, CA, USA). Penicillin/streptomycin and heat-inactivated fetal bovine serum were purchased from GIBCO (Budapest, Hungary). Cytosine β-D-arabinofuranoside (CAS: 147-94-4), 17β-estradiol (MDL number MFCD00133134; CAS: 50-28-2), 3,3′,5-triiodo-l-thyronine (purity: 96%; CAS: 6893-02-3), zearalenone (purity: ≥99%; CAS: 17924-92-4), bisphenol A (purity: ≥99%; CAS: 80-05-7), and sodium (meta)arsenite (purity: ≥90%; CAS: 7784-46-5) were purchased from Sigma-Aldrich (Budapest, Hungary). Bicinchoninic acid (BCA) kit from Pierce (Rockford, IL 61111, USA).

Jocsak G., Ioja E., Kiss D.S., Toth I., Barany Z., Bartha T., Frenyo L.V, & Zsarnovszky A. (2019). Endocrine Disruptors Induced Distinct Expression of Thyroid and Estrogen Receptors in Rat versus Mouse Primary Cerebellar Cell Cultures. Brain Sciences, 9(12), 359.

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