Cytosine arabinoside

Manufactured by Merck Group

Sourced in United States, Germany, Italy

Cytosine arabinoside is a laboratory reagent used for research purposes. It is a synthetic nucleoside analog that inhibits DNA synthesis and can be used to study cellular processes and mechanisms.

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Close spelling variants of this product

Cytosine (40 citations) Cytosine arabinoside (ARAC; (39 citations) Cytosine β-d-arabinoside (5 citations) Cytosine-D-arabinoside (3 citations) Cytosine-β-D arabinoside (Ara-C) (2 citations)

120 protocols using cytosine arabinoside

Extracellular Vesicle Impacts on DRG Neurons

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DRGs were obtained from SD rat embryos (14.5 days). Briefly, DRGs were isolated and cultured on coverslips coated with poly-lysine in neurobasal medium (Gibco) with 2% B27, 1% mM l-glutamine, 50 ng/ml human nerve growth factor (NGF, R&D), and 1% penicillin-streptomycin. Besides, 10 mM cytosine arabinoside (Sigma) was added to remove non-neuronal cells. Then, low (1 × 10⁸ particles/ml), medium (2 × 10⁸ particles/ml), and high (4 × 10⁸ particles/ml) dose of EVs and PBS (control) were added to neurons daily during 5 days.
Moreover, DRG sensory neurons were primarily cultured as previously described [31 (link)]. Briefly, DRGs were collected from neonatal rats (1 day), then digested sequentially in 0.1% collagenase type I (Sigma) for 30 min and 0.25% trypsin (Gibco) for 20 min; then, single cells were collected and purified by centrifugation in PBS solution with 15% Bovine Serum Albumin (Sigma) before 10 mM cytosine arabinoside was added to remove non-neuronal cells. The obtained sensory neurons were cultured on the coated plates in medium the same as DRG explant culture medium, with treatment of medium-dose EVs (2 × 10⁸ particles/ml) for 12 h.
After the immunofluorescence staining of β-tubulin III (TUJ1, Abcam, 1:500), the axonal area of each DRG explant and the neurite length of single sensory neuron were measured with Image J software.

Cong M., Shen M., Wu X., Li Y., Wang L., He Q., Shi H, & Ding F. (2021). Improvement of sensory neuron growth and survival via negatively regulating PTEN by miR-21-5p-contained small extracellular vesicles from skin precursor-derived Schwann cells. Stem Cell Research & Therapy, 12, 80.

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Cerebellar Granule Cell Culture

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Cerebellar granule cells were obtained from 6–8 days old Sprague–Dawley rats, as described previously^{19 (link)}. They were plated at a density of 1.5–2.5 10⁶ cells/well coated with 20 μg/ml Poly-l-Lysine (Sigma-Aldrich, St. Louis, MO, USA).
The neurons were kept at 37 °C in a humidified 95% air/5% CO₂ atmosphere and grown in 90% basal medium Eagle, 10% fetal calf serum (Sigma-Aldrich, St. Louis, MO, USA), 25 mM KCl, 2 mM glutamine and 100 µg/ml gentamicin.
At 18–24 h from plating, 10 μM cytosine arabinoside (Sigma-Aldrich, St. Louis, MO, USA) was added to the culture medium to prevent glial cell growth; at 48 h the medium was refreshed and 10 μM cytosine arabinoside was renewed. The cells were studied from the sixth to the tenth day in vitro.

Cozzolino M., Bazzurro V., Gatta E., Bianchini P., Angeli E., Robello M, & Diaspro A. (2020). Precise 3D modulation of electro-optical parameters during neurotransmitter uncaging experiments with neurons in vitro. Scientific Reports, 10, 13380.

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Cerebellar Granule Cell Culture Protocol

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Cerebellar granule cells were obtained from 6-8 days old Sprague-Dawley rats (male and female), as described previously [19] (link). They were plated at a density of 1.5-2.5 × 10 6 cells per well coated with 20 μg/mL poly-L-lysine (Sigma-Aldrich, St. Louis, MO, USA).
Neurons were kept at 37°C in a humidified 95% air/ 5% CO 2 atmosphere and grown in 90% basal medium Eagle, 10% fetal calf serum (Sigma-Aldrich, St. Louis, MO, USA), 25 mM KCl, 2 mM glutamine, and 100 µg/mL gentamicin.
At 18-24 h from plating, 10 μM cytosine arabinoside (Sigma-Aldrich, St. Louis, MO, USA) was added to the culture medium to prevent glial cell growth; at 48 h, the medium was refreshed, and 10 μM cytosine arabinoside was renewed. The cells were studied from the sixth to the tenth day in vitro.

Gatta E., Bazzurro V., Angeli E., Salis A., Damonte G., Cupello A., Robello M, & Diaspro A. (2022). Electrophysiological study of the effects of side products of RuBi-GABA uncaging on GABA_A receptors in cerebellar granule cells. Biomolecular concepts, 13(1).

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Isolation and Culture of Primary Hippocampal Neurons

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Primary hippocampal neurons were isolated from newborn hippocampi with accutase (#A11105-01, Life Technologies, CA). After filtering through a 40 µm strainer (Falcom, United States), 4 × 10^⁵ neurons were seeded in poly-d-lysine (Sigma-Aldrich, MO)-coated dishes and cultured with DMEM medium containing 10% FBS and 10% horse serum (Solarbio Life Sciences, Beijing, China). Six hours later, neurobasal medium (#10888022, Life Technologies, CA) containing B27 supplement (#17504, Life Technologies, CA), 0.5 mM glutamine (#35050, Life Technologies, CA), and cytosine arabinoside (1 ug/mL, # C3350000, Sigma-Aldrich, MO) was added. Culture medium was changed every 2 days. Experiments were performed on day seven. The primary hippocampal neurons were positively stained with NeuN (#ab104224, Abcam, UK), a neuron-specific marker (Supplementary Figure 1C).

Hu Y., Zhou Y., Yang Y., Tang H., Si Y., Chen Z., Shi Y, & Fang H. (2022). Metformin Protects Against Diabetes-Induced Cognitive Dysfunction by Inhibiting Mitochondrial Fission Protein DRP1. Frontiers in Pharmacology, 13, 832707.

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Culturing Embryonic Hippocampal Neurons

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All animal studies conformed to the guidelines outlined in the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and were approved by the University of Pacific Institutional Animal Care and Use Committee. Hippocampal neurons were isolated from 19 days gestation embryos (Simonsen, Gilroy, CA, United States) as described previously (Halliwell et al., 2002 (link)) and plated on poly-D-lysine-coated 24-well plates in Neurobasal medium supplemented with B27 (1%, v/v) (Invitrogen, Carlsbad, CA, United States), Penicillin/Streptomycin (1%, v/v), and 0.5 mM L-glutamine (Sigma-Aldrich). Cells were placed in an incubator (Isotemp, Fisher Scientific, Inc., Santa Clara, CA, United States) at 37°C, 100% humidity and 5% CO₂. Non-neuronal cell division was halted by the addition of 10 μM cytosine arabinoside (Sigma-Aldrich) after 2 days in culture. Fresh media was added every 3 days thereafter.

Khansari P.S, & Halliwell R.F. (2019). Mechanisms Underlying Neuroprotection by the NSAID Mefenamic Acid in an Experimental Model of Stroke. Frontiers in Neuroscience, 13, 64.

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Neuronal Culture Reagents and Protocols

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Trypsin, penicillin, streptomycin, heat-inactivated fetal bovine serum, horse serum, and soybean Trypsin inhibitor were obtained from Atlanta Biologicals (Norcross, GA, USA). Minimum essential medium (MEM), deoxyribonuclease (DNase), poly-L-lysine, poly-D-lysine hydrobromide, cytosine arabinoside, NMDA, protease inhibitor cocktail, MK-801, and ifenprodil were purchased from Sigma (St. Louis, MO, USA). Pluronic acid and Fluo-3 AM were purchased from Molecular Probes (Eugene, OR, USA). TCN-201, IPA-3, and NSC23766 were purchased from Tocris (Bristol, UK). Pierce ECL kits (Thermo Fisher Scientific, Rockford, IL, USA), Neurobasal, and B-27 supplements were purchased from Invitrogen Corporation (Carlsbad, CA, USA); the p-PAK1 antibody (Thr 212) from Santa Cruz Biotechnology (Dallas, TX, USA); and the PAK1 antibody anti-rabbit IgG HRP-linked antibody from Cell Signaling Technology (Danvers, MA, USA). Brevetoxin-2 (PbTx-2) was isolated and purified from Karinia breve cultures at the Center for Marine Sciences at the University of North Carolina (Wilmington, NC, USA). QNZ-46 was a gift from SF Traynelis, Department of Pharmacology, Emory University, Atlanta, GA. The GluN2D subtype of NMDA receptor knockout mice was obtained from Daniel T. Monaghan, Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska.

Mehrotra S., Pierce M.L., Dravid S.M, & Murray T.F. (2022). Stimulation of Neurite Outgrowth in Cerebrocortical Neurons by Sodium Channel Activator Brevetoxin-2 Requires Both N-Methyl-D-aspartate Receptor 2B (GluN2B) and p21 Protein (Cdc42/Rac)-Activated Kinase 1 (PAK1). Marine Drugs, 20(9), 559.

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Preparation of Near-Pure Neuronal Cultures

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Primary near-pure neuronal cultures were prepared from embryonic mice at 13–14 days as described previously [18 (link)]. In brief, triturated cortical cells were seeded onto a poly-D-lysine coated plate (SPL Life Sciences, Gyeonggi-do, South Korea) at seven hemispheres per 24-well plate or nine hemispheres per 6-well plate, respectively. Growth medium consisted of glutamine-free Dulbecco’s modified Eagle medium (DMEM, GibcoBRL, Grand Island, NY, USA) with 25 mM glucose, 44 mM sodium bicarbonate, 2 mM glutamine, 5 % fetal bovine serum, and 5 % horse serum. For pure neuronal cell culture, 10 μM cytosine arabinoside (Sigma, St. Louis, MO, USA) was added at days in vitro (DIV) 3. Cultures were incubated at 37 °C in a humidified 5 % CO₂ atmosphere. This procedure routinely produced near-pure cortical neuronal cultures through DIV1 to DIV7 consisting of > 96 % neurons, < 1 % astrocytes, and < 0.5 % microglia. All experiments were performed at DIV 6–7.
The present study was conducted in accordance with the guidelines for care and use of mice in research and under protocols approved by the Animal Care and Use Committee of The Sejong University.

Eom J.W., Lee J.M., Koh J.Y, & Kim Y.H. (2016). AMP-activated protein kinase contributes to zinc-induced neuronal death via activation by LKB1 and induction of Bim in mouse cortical cultures. Molecular Brain, 9, 14.

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Neurochemical Reagents for Cellular Studies

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Bay K8644, bicuculline methiodide, caffeine, isradipine, riluzole, CNQX, dimethyl sulfoxide (DMSO), cytosine arabinoside, poly-D-lysine and bulk chemicals were purchased from Sigma-Aldrich (Vienna, Austria). Tetrodotoxin was obtained from Latoxan (Valence, France).

Stiglbauer V., Hotka M., Ruiß M., Hilber K., Boehm S, & Kubista H. (2017). Cav1.3 channels play a crucial role in the formation of paroxysmal depolarization shifts in cultured hippocampal neurons. Epilepsia, 58(5), 858-871.

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Recovery and Amplification of Recombinant VSV

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Recovery of recombinant VSV was performed as described (Whelan et al., 1995 (link)). Briefly, BSRT7/5 cells (Buchholz et al., 1999 (link)) were inoculated with vaccinia virus vTF7-3 (Fuerst et al., 1986 (link)) and subsequently transfected with T7-expression plasmids encoding VSV N, P, L, and G, and an antigenomic copy of the viral genome. Cell culture supernatants were collected at 56-72 h, clarified by centrifugation (5 min at 1,000 x g), and filtered through a 0.22 μm filter. Virus was plaque-purified on Vero CCL81 cells in the presence of 25 μg/mL of cytosine arabinoside (Sigma-Aldrich), and plaques in agarose plugs were amplified on Vero CCL81 cells. Viral stocks were amplified on MA104 cells at an MOI of 0.01 in Medium 199 containing 2% FBS and 20 mM HEPES pH 7.7 at 34°C. Viral supernatants were harvested upon extensive cytopathic effect and clarified of cell debris by centrifugation at 1,000 x g for 5 min. Aliquots were maintained at −80°C. Construction and use of VSV-SARS-CoV-2 was approved by the Washington University School of Medicine Institutional Biosafety Committee at Biosafety level 2.

Case J.B., Rothlauf P.W., Chen R.E., Liu Z., Zhao H., Kim A.S., Bloyet L.M., Zeng Q., Tahan S., Droit L., Ilagan M.X., Tartell M.A., Amarasinghe G., Henderson J.P., Miersch S., Ustav M., Sidhu S., Virgin H.W., Wang D., Ding S., Corti D., Theel E.S., Fremont D.H., Diamond M.S, & Whelan S.P. (2020). Neutralizing Antibody and Soluble ACE2 Inhibition of a Replication-Competent VSV-SARS-CoV-2 and a Clinical Isolate of SARS-CoV-2. Cell Host & Microbe, 28(3), 475-485.e5.

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Quantifying Cell Migration and Invasion

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In wound scratch assay, 1.7 × 10⁵ cells were used in a 24-well plate. Quiescent cells were wounded [16 (link)] and left un-stimulated or stimulated for 28 h, in the absence or presence of the indicated compounds. To avoid cell proliferation, cytosine arabinoside (Sigma-Aldrich) at 50 μM (final concentration) was included in the cell medium. Different fields were analyzed using DMIRB inverted microscope (Leica) equipped with N-Plan 10× objective (Leica). Phase-contrast images, representative from three different experiments, were captured using a DFC 450C camera (Leica) and acquired using Leica Suite Software (Leica). The wound width was calculated using Image J Software and expressed as % of the decrease in the wound area. Migration and invasion assays were performed by using collagen- or Matrigel- pre-coated Transwells with 8 μm polycarbonate membrane (Corning; Corning, NY, USA), respectively [16 (link)]. The indicated compounds were added and cytosine arabinoside was included in cell medium. Cells were allowed to migrate or invade for seven or 18 h for LNCaP cells respectively and for nine h or 24 h for DuCaP and 22Rv1, respectively. Migrating or invading cells were then scored [16 (link)].

Di Donato M., Giovannelli P., Barone M.V., Auricchio F., Castoria G, & Migliaccio A. (2021). A Small Peptide Targeting the Ligand-Induced Androgen Receptor/Filamin a Interaction Inhibits the Invasive Phenotype of Prostate Cancer Cells. Cells, 11(1), 14.

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