All animal procedures in the study were carried out following the guidelines for the Welfare and Use of Laboratory Animals and approved by the Laboratory Animal Welfare & Ethics Committee of Fujian Medical University. DRGs obtained from the spinal cords of day-15 rat embryos (Fujian Medical University) were isolated as described elsewhere [22 ]. DRGs were cultured in Basic Medium (BM), which was formulated using Neurobasal medium supplemented with B-27 (2 %, Sigma–Aldrich), l -glutamine (0.4 mM, Invitrogen), glucose (2.5 mg/mL, Sigma–Aldrich), 2.5S nerve growth factor (10 ng/mL, NGF, Gibco) and fetal bovine serum (1 %, FBS, HyClone). Cytosine 1-β-d -arabinofuranoside (5 μM, Ara-C, Sigma–Aldrich), 5-fluoro-2′-deoxyuridine (5-FdU) (20 μM, Sigma–Aldrich) and uridine (20 μM, Sigma–Aldrich) were added into the BM in the first 2 days. Starting from day 3, BM containing 20 μM 5-FdU and 20 μM uridine was used for long-term culture.
Cytosine 1 β d arabinofuranoside
Manufactured by Merck Group
Sourced in Sao Tome and Principe, United States
Cytosine-1-β-D-arabinofuranoside is a laboratory compound used in scientific research. It is a synthetic nucleoside analog that functions as a cytosine derivative. The core function of this product is to serve as a research tool for investigating biological processes and pathways.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (3 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (3 mentions)
Seahorse xf extracellular flux analyser, by Agilent Technologies (2 mentions)
Seahorse xf glycolysis stress test buffer, by Thermo Fisher Scientific (2 mentions)
Poly ornithine, by Merck Group (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Nerve growth factor (ngf), by Thermo Fisher Scientific (1 mentions)
Uridine, by Merck Group (1 mentions)
Ara c, by Merck Group (1 mentions)
5 fluoro 2 deoxyuridine 5 fdu, by Merck Group (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Cck 8 kit, by Beyotime (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Phospho p70 s6 kinase t389, by Cell Signaling Technology (1 mentions)
Phospho 4e bp1 thr37 46, by Cell Signaling Technology (1 mentions)
S6 ribosomal protein, by Cell Signaling Technology (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
2 7 dichlorofluorescein diacetate dcfh da, by Thermo Fisher Scientific (1 mentions)
Dihydroethidium, by Merck Group (1 mentions)
P70 s6 kinase, by Cell Signaling Technology (1 mentions)
8 protocols using cytosine 1 β d arabinofuranoside
1
Cultured Rat DRG Isolation Protocol
2
Measuring Glycolysis Through Proton Flux
3
Measuring Glycolysis via Seahorse Analysis
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To measure glycolysis through proton production [36 (link)], a Seahorse extracellular flux (XF) analyser (Seahorse Biosciences) was used as per the manufacturer’s protocol. Briefly, non-purified granule cells were prepared as described above and were grown in DMEM supplemented with 0.5-mM L-glutamine and 10% fetal calf serum (Gibco) on 96-well plates pre-treated with poly-ornithine (Sigma). The day after splitting, cells were treated with 0.01-mM cytosine-1-β-D-arabinofuranoside (Sigma) and cultured for 7 days. On the day of the assay, cells were treated with vehicle or 25-μM arsenite for 4 h before being transferred to minimum medium (Seahorse XF glycolysis stress test buffer supplemented with 2-mM L-glutamine Gibco, pH 7.4) for 1 h. To quantify glycolysis levels, the glycolysis stress test assay is performed by first injecting glucose (to feed glycolysis), then oligomycin (to drive glycolysis), and finally (to inhibit glycolysis). Glycolysis is determined through measurements of the extracellular acidification rate (ECAR) of the surrounding media in which glucose, oligomycin, and 2-deoxyglucose (2-DG) are sequentially added. The ECAR values were normalised to the protein level per well, as determined by BCA assay at the end of the run.
4
Mangiferin Modulates Cellular Metabolism
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Mangiferin, dihydroethidium (DHE), DMSO, and cytosine 1-β-D -arabinofuranoside were purchased from Sigma-Aldrich (St. Louis, MO, United States). Antibodies against LC3, p62, and anti-GAPDH were purchased from Abcam (Cambridge, MA, United States). Antibodies against AMPK, p-AMPK, ACC, p-ACC (Ser79), mTOR, phospho-mTOR (Ser2448), Akt, phospho-Akt (Ser473), phospho-Akt (Thr308), cleaved caspase 3, PARP, p70 S6 kinase, phospho-p70 S6 kinase (T389), S6 ribosomal protein, phospho-S6, 4EBP1, and phospho-4EBP1 (Thr37/46) were purchased from Cell Signaling Technology (Boston, MA, United States). tf-LC3 plasmid (21074) was purchased from Addgene. CCK-8 Kit, JC-1, and MAO ELISA Kit were purchased from Beyotime (Shanghai, China). 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was purchased from Molecular Probes (New York, NY, United States). Mangiferin was dissolved in DMSO.
5
Neuroprotective Effects of Albumin on Excitotoxicity
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Hippocampal neurons were dissected from rat embryos (18th day of gestation; Shanghai Super-B&K Laboratory Animal Corporation, Ltd., Shanghai, China) via treatment with 10 U/l papain solution (Sigma-Aldrich). Primary neurons (2×105/ml) were seeded on poly-L-lysine-coated dishes and cultured in minimum essential medium supplemented with 5% fetal bovine serum (both purchased from Signa-Aldrich) and 5% horse serum (Thermo Fisher Scientific, Inc.) under 5% CO2 conditions on the first day. On the second day, cells were cultured in neurobasal medium with B27 (Thermo Fisher Scientific, Inc.), containing 0.025 mM glutamate; whereas on the third day, cells were treated with 5 µM cytosine-1-β-D-arabinofuranoside (Sigma-Aldrich)to inhibit the growth of gliocytes. After two days, half of the neurobasal medium was replaced by fresh medium. Cells were subsequently treated with saline, 10 µM KA, 10 µM KA + 10 µM albumin, 10 µM KA + 50 µM albumin and 10 µM KA + 100 µM albumin at 37°C and 5% CO2 for 24 h. Staining of Evans blue (EB) and immunofluorescent staining of NeuN were performed after cell were washed with PBS.
6
Dynamin-amphiphysin SH3 domain interaction
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Bovine serum albumin, poly-L -lysine, cytosine-1-β-D -arabinofuranoside, and papain were obtained from Sigma (St. Louis, MO, USA); Dulbecco’s modified Eagle’s medium, fetal calf serum, gentamicin and penicillin/streptomicin from Gibco (Carlsbad, CA, USA); tetrodotoxin from Alomone Labs (Jerusalem, Israel); the monoclonal antibody against dynamin (it binds dynamin 1 and 2) from BD Biosciences (San Jose, CA, USA). The GST fusion protein GST-Dyn829-842, which contains the amino-acids 829 to 842 of human dynamin (PPQVPSRPNRAPPG) was obtained as described before (Gonzalez-Gutierrez et al., 2007 (link)). As a negative control of GST-Dyn829-842, we used a GST fusion protein containing mutations at positions 835 (arginine to aspartic acid) and 836 (proline to alanine), which reportedly does not disrupt the binding to the amphiphysin SH3 domain (Grabs et al., 1997 (link)).
7
Primary Hippocampal Neuron Culture from E18 Mice
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Primary hippocampal neurons were prepared from E18 mice, as previously described,113 (link) with some modifications. Briefly, bilateral hippocampi were collected and digested with 0.22 μm PES membrane-filtered custom digestion solution containing 5 mg/mL deoxyribonuclease I (Sigma, D4527/10KU), 1.5 mM CaCl2, 0.75 mM EDTA, 200 units of papain (Worthington, LS003127), and 2.5 mM L-cysteine (Sigma, C7352) for 5 min at 37°C. The cells were plated on autoclaved, nitric acid-washed, poly-L-lysine (Sigma) coated glass cover slips (Electron Microscopy Sciences) in Neuronal Plating Medium for 2-4 hours. They were maintained in modified Neurobasal/B27 Medium, which also had 1 mM sodium pyruvate (Gibco, 11360070) and 100 U/mL penicillin-streptomycin (Gibco, 15070063) in addition to the B27 and GlutaMAX-I supplements. On days in vitro (DIV) 0, 4 μM cytosine-1-β-D-arabinofuranoside (Sigma, 251010) was added to limit glial proliferation.
8
Culturing Mouse and Rat Cardiomyocytes
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The HL-1 mouse cardiomyocytes, a generous gift from Dr. William Claycomb at the Louisiana State University Medical Center, were cultured in Claycomb media (Sigma-Aldrich, #51800) supplemented with 10% heat-inactivated newborn fetal bovine serum, 100 μg/ml penicillin-streptomycin, 2 mM L-glutamine, and 0.1 mM norepinephrine as described previously [10 (link)].
Rat neonatal cardiomyocytes were obtained by enzymatic dissociation of cardiac ventricles from 1-2 day old Sprague-Dawley rat neonates as previously described [45 (link)]. Non-myocytes were removed through two rounds of pre-plating and cytosine 1-β-D-arabinofuranoside (Sigma-Aldrich, #C1768) was added to inhibit the growth of contaminating non-myocytes. The enriched cardiomyocytes were cultured in Dulbecco's modified Eagle’s media with 10% bovine calf serum and 10% horse serum.
Rat neonatal cardiomyocytes were obtained by enzymatic dissociation of cardiac ventricles from 1-2 day old Sprague-Dawley rat neonates as previously described [45 (link)]. Non-myocytes were removed through two rounds of pre-plating and cytosine 1-β-D-arabinofuranoside (Sigma-Aldrich, #C1768) was added to inhibit the growth of contaminating non-myocytes. The enriched cardiomyocytes were cultured in Dulbecco's modified Eagle’s media with 10% bovine calf serum and 10% horse serum
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