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Aphidicolin

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Aphidicolin is a specific inhibitor of eukaryotic DNA synthesis. It acts by inhibiting DNA polymerase α and δ, which are essential enzymes for DNA replication in eukaryotic cells.

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Lab products found in correlation

Lipofectamine rnaimax, by Thermo Fisher Scientific (4 mentions) Collagenase, by Worthington (3 mentions) Trypsin, by Worthington (2 mentions) Nerve growth factor (ngf), by Alomone (2 mentions) Hydroxyurea, by Merck Group (2 mentions) On targetplus smart, by Thermo Fisher Scientific (2 mentions) Laminin, by Merck Group (1 mentions) Poly l lysine (pll), by Merck Group (1 mentions) Trypsin, by Merck Group (1 mentions) Hibernate a, by Transnetyx (1 mentions) Neurobasal a, by Thermo Fisher Scientific (1 mentions) Mouse monoclonal anti tubulin antibody, by Merck Group (1 mentions) Dnase 1, by Thermo Fisher Scientific (1 mentions) Mouse anti map2 antibody, by Abcam (1 mentions) Papain, by Merck Group (1 mentions) Dispase 2, by Roche (1 mentions) Nerve growth factor 2.5s, by Alomone (1 mentions) Kt5720, by Bio-Techne (1 mentions) Il 1β, by Fujifilm (1 mentions) Zd7288, by Cayman Chemical (1 mentions)

8 protocols using aphidicolin

1

Synchronizing Cells with Aphidicolin

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For experiments requiring synchronization with aphidicolin (AG Scientific, San Diego, CA, USA), the cells were prepared as described previously 19 (link),20 (link). Briefly, the cells were grown to confluence to arrest them in G0 and replated at a lower density to allow the cells to re-enter the cell cycle in the presence of aphidicolin (2 μg mL−1); the cultures were incubated for 24 h to accumulate the cycling cells at the beginning of the S phase 19 (link)–21 (link). The DNA synthesis inhibitor was removed from the cells by washing the cultures 3 times with warm Hanks’ balanced salt solution (HBSS, HyClone) and adding pre-warmed fresh culture medium. The cells were exposed to UVC at 45 min, 3 h and 5 h after release. For synchronization without the inhibitor, the cells were grown to confluence arrest, replated at a lower density to allow them to re-enter the cell cycle, and treated 15, 18, 21 and 24 h after replating.
Chastain P.D., Brylawski B.P., Zhou Y.C., Rao S., Chu H., Ibrahim J.G., Kaufmann W.K, & Cordeiro-Stone M. (2014). DNA Damage Checkpoint Responses in the S Phase of Synchronized Diploid Human Fibroblasts. Photochemistry and Photobiology, 91(1), 109-116.
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2

Culturing Newborn DRG Neurons

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DRGs were obtained from newborn (postnatal days 0–2) WT and Gdap1 knockout (Gdap1−/−) mice according to (45 (link)). Ganglia were digested in collagenase (Worthington, USA) and trypsin (Sigma-Aldrich, St. Louis, MO), dissociated by trituration and plated in DMEM-F12 containing 50 ng/ml NGF (Tebu-Bio, Le-Perray-en-Yvelines, France), 10% FBS and 5 ng/ml of aphidicolin (A.G. Scientific, San Diego, CA). DRG neurons were seeded on poly-l-lysine and laminin (1 μg/ml)-coated (Sigma-Aldrich, St. Louis, MO) plates and used for experimentation between 2 and 4 DIV.
Nuevo-Tapioles C., Santacatterina F., Sánchez-Garrido B., de Arenas C.N., Robledo-Bérgamo A., Martínez-Valero P., Cantarero L., Pardo B., Hoenicka J., Murphy M.P., Satrústegui J., Palau F, & Cuezva J.M. (2021). Effective therapeutic strategies in a preclinical mouse model of Charcot–Marie–Tooth disease. Human Molecular Genetics, 30(24), 2441-2455.
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3

Isolation and Culture of Dopaminergic Neurons

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α-Endosulfan was purchased from Accustandard (New Haven, CT). Hibernate A and Hibernate A- Calcium were purchased from BrainBits (Springfield, IL). B27, DNase1, and Neurobasal A were purchased from Life Technologies (Carlsbad, CA). Papain was obtained from Sigma (St. Louis, MO). Dispase II was purchased from Roche (Nutley, NJ). The BCA protein assay kit was obtained from Pierce (Rockford, IL). Aphidicolin was purchased from A.G. Scientific (San Diego, CA). Monoclonal anti-rat dopamine transporter and polyclonal anti-rabbit tyrosine hydroxylase were purchased from EMD Millipore (Billerica, MA). Monoclonal mouse-anti tubulin antibody was purchased from Sigma (St. Louis, MO). Mouse anti-GABAA 2α receptor subunit was purchased from Synaptic Systems (Germany) and mouse anti-MAP2 antibody was purchased from Abcam (San Francisco, CA). Secondary antibodies conjugated to fluorescent tags were obtained from Life Technologies (Grand Island, NY). SuperSignal West Dura Extended duration substrate and stripping buffer were obtained from Pierce.
Wilson W.W., Shapiro L.P., Bradner J.M, & Caudle W.M. (2014). Developmental exposure to the organochlorine insecticide endosulfan damages the nigrostriatal dopamine system in male offspring. Neurotoxicology, 0, 279-287.
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4

Screening of Neuroprotective Compounds

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Compounds used in the study are as follows: Acycloguanosine, FUDR, Uridine, SP600125, GNE-3511, GSK-J4, L-glutamic acid, and Ivabradine (Millipore Sigma); Forskolin, LY 294002, 666–15, SQ 22536, KT 5720, tetraethylammonium chloride, cesium chloride, OG-L002, S2101, tetrotdotoxin, and ESI-09 (Tocris); 1,9-dideoxy-Forskolin, ZD 7288 and 8-bromo-cyclic AMP (Cayman Chemicals); nerve growth factor 2.5S (Alomone Labs); Primocin (Invivogen); aphidicolin (AG Scientific); IL-1β (Shenandoah Biotechnology); WAY-150138 was kindly provided by Pfizer, Dr. Jay Brown at the University of Virginia, and Dr. Lynn Enquist at Princeton University. Compound concentrations were used based on previously published IC50s and assessed for neuronal toxicity using the cell body and axon health and degeneration index (Supplementary file 1 Table 1 and 2). All compounds used had an average score ≤1. Untreated controls are quantified as ‘Mock’ treatments for all experiments.
Cuddy S.R., Schinlever A.R., Dochnal S., Seegren P.V., Suzich J., Kundu P., Downs T.K., Farah M., Desai B.N., Boutell C, & Cliffe A.R. (2020). Neuronal hyperexcitability is a DLK-dependent trigger of herpes simplex virus reactivation that can be induced by IL-1. eLife, 9, e58037.
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5

Isolation and Culture of Newborn Mouse Ganglia

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Ganglia were dissected from newborn mice (postnatal day 0–1), digested in collagenase and trypsin (Worthington, Lakewood, NJ), dissociated by trituration and plated on dishes previously coated with polylysine (250 μg/mL)-laminin (10 μg/mL) in DMEM-F12 (all three from Life Technologies, Thermo Fisher Scientific) containing 10 ng/mL NGF (Alomone labs, Jerusalem, Israel), 5 % horse serum, and 5 ng/mL of aphidicolin (A.G. Scientific, San Diego, CA) for 3 days.
Cabrera J.R., Viejo-Borbolla A., Alcamí A, & Wandosell F. (2016). Secreted herpes simplex virus-2 glycoprotein G alters thermal pain sensitivity by modifying NGF effects on TRPV1. Journal of Neuroinflammation, 13(1), 210.
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6

Culturing Sympathetic Neurons from Newborn Mice

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Mouse sympathetic neurons from SCGs were cultured as previously described [65 (link)]. Ganglia were dissected from newborn mice (postnatal day 0–1), digested in collagenase and trypsin (Worthington, Lakewood, NJ), dissociated by trituration and plated on dishes previously coated with rat tail collagen I (BD Biosciences) in DMEM containing 50 ng/mL NGF (Alomone labs), 10% fetal bovine serum and 5ng/mL of aphidicolin (A.G. Scientific, San Diego, CA) for 5–7 days. For retrograde transport analysis SCG neurons were cultured in polylysine (100 μg/mL)—laminin (10 μg/mL) using microfluidic devices AXIS Axon Isolation Devices, 450um from Millipore (Billerica, MA)
Cabrera J.R., Viejo-Borbolla A., Martinez-Martín N., Blanco S., Wandosell F, & Alcamí A. (2015). Secreted Herpes Simplex Virus-2 Glycoprotein G Modifies NGF-TrkA Signaling to Attract Free Nerve Endings to the Site of Infection. PLoS Pathogens, 11(1), e1004571.
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7

HUES6 Cell siRNA Knockdown

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HUES6 cells were transfected using Lipofectamine RNAiMAX (ThermoFisher) transfection reagent according to the manufacturer’s recommendations. ON-TARGETplus SMART pool of four siRNAs from Dharmacon (ThermoFisher) were used for siRNA knockdown: TRF1 (catalogue #L-010542-02-0005); BLM: (catalogue #L-007287-00-0005); WRN: (catalogue #L-010378-00-0005); Exo1: (catalogue #L-013120-00-0005); DNA2: (catalogue #L-026431-01-0005); SMARCAL1: (catalogue # L-013058-00-0005); XRCC3: (catalogue # L-012067-00-0005) or control non-targeting pool (catalogue # D-001810-10-20). The cells were collected 72 h or 96 h post treatment after two serial transfections and processed for protein, RNA and DNA analyses.
Cells were treated with 0.2mM or 3mM hydroxyurea (Sigma), 5μM aphidicolin (AG Scientific) or 0.5μM RHPS4 for 24h, and collected for DNA analysis.
Rivera T., Haggblom C., Cosconati S, & Karlseder J. (2016). A balance between elongation and trimming regulates telomere stability in stem cells. Nature structural & molecular biology, 24(1), 30-39.
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8

HUES6 Cell siRNA Knockdown

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HUES6 cells were transfected using Lipofectamine RNAiMAX (ThermoFisher) transfection reagent according to the manufacturer’s recommendations. ON-TARGETplus SMART pool of four siRNAs from Dharmacon (ThermoFisher) were used for siRNA knockdown: TRF1 (catalogue #L-010542-02-0005); BLM: (catalogue #L-007287-00-0005); WRN: (catalogue #L-010378-00-0005); Exo1: (catalogue #L-013120-00-0005); DNA2: (catalogue #L-026431-01-0005); SMARCAL1: (catalogue # L-013058-00-0005); XRCC3: (catalogue # L-012067-00-0005) or control non-targeting pool (catalogue # D-001810-10-20). The cells were collected 72 h or 96 h post treatment after two serial transfections and processed for protein, RNA and DNA analyses.
Cells were treated with 0.2mM or 3mM hydroxyurea (Sigma), 5μM aphidicolin (AG Scientific) or 0.5μM RHPS4 for 24h, and collected for DNA analysis.
Rivera T., Haggblom C., Cosconati S, & Karlseder J. (2016). A balance between elongation and trimming regulates telomere stability in stem cells. Nature structural & molecular biology, 24(1), 30-39.
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