Mixtures of Ac, Bu, and Cl sodium salts have been prepared using sodium acetate (>98%, Fisher Chemical, Hampton, NH, USA), sodium butyrate (>98%, Alfa Aesar, Haverhill, MA, USA), and sodium chloride (>99.9%, VWR Chemicals, Radnor, PA, USA). Electrode compartment solution has been prepared with sodium sulfate (>99.9% VWR Chemicals, Radnor, PA, USA).
Sodium butyrate
Manufactured by Thermo Fisher Scientific
Sourced in United States
Sodium butyrate is a chemical compound that is commonly used in laboratory settings. It is a salt of butyric acid and serves as a source of this short-chain fatty acid. Sodium butyrate is often utilized in cell culture and biochemical applications, though its precise functions may vary depending on the specific research or experimental requirements.
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Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (4 mentions)
Axiovert microscope, by Zeiss (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by PanEco (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Varioskan lux, by Thermo Fisher Scientific (2 mentions)
Polyethylenimine (pei), by Polysciences (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Sodium chloride (nacl), by Avantor (1 mentions)
Sodium sulfate, by Avantor (1 mentions)
Sodium acetate, by Thermo Fisher Scientific (1 mentions)
Midiprep kits, by Macherey-Nagel (1 mentions)
Hygromycin, by Merck Group (1 mentions)
Salbutamol, by Merck Group (1 mentions)
Phentolamine, by Merck Group (1 mentions)
Atenolol, by Merck Group (1 mentions)
Prazosin, by Merck Group (1 mentions)
Ici 118 551, by Merck Group (1 mentions)
Probenecid, by Merck Group (1 mentions)
125i cyp, by PerkinElmer (1 mentions)
9 protocols using sodium butyrate
1
Sodium Salt Mixture Preparation
2
KSHV Recombinant Virus Production in 293T Cells
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KSHV BAC DNA was purified from GS1783 E. coli cells using Midiprep kits (Macherey-Nagel). BACΔLANA, BACLANA-R, or BACLANA-R mutants were transfected into 293T cells with Lipofectamine 3000 (Thermo Fisher Scientific) following the manufacturer’s protocol. Clonal selection was obtained using 0.2 mg/mL hygromycin (EMD Millipore). To assess the ability of the recombinant virus to produce virions, lytic reactivation was induced shortly after establishment of 293T stable cell lines containing BACΔLANA, BACLANA-R, or BACLANA-R LANA. Reactivation was induced overnight using TPA (20 ng/mL; Sigma-Aldrich) and sodium butyrate (32 mM; Alfa Aesar). At 5 d after reactivation, supernatant was harvested, filtered through a 0.45-µm filter (Celltreat), and used to infect 293T cells by spinoculation at 1,500 rpm for 1 h at room temperature. Cells were imaged using a Zeiss Axiovert microscope to assess for GFP expression from the recombinant BAC16.
3
Membrane Preparation from Transfected Cells
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Reagents were purchased from the following commercial suppliers: A-61603, xamoterol, procaterol and fenoterol from Tocris (Ellisville, MO); norepinephrine, prazosin, phentolamine, ICI 118551, atenolol, salbutamol, propranolol, probenecid, BSA and glucose from Sigma-Aldrich (St. Louis, MO); isoproterenol and oxymetazoline from MP Biomedicals (Irvine, CA), sodium butyrate from Alfa Aesar (Ward Hill, MA); HEPES, HBSS, 3-isobutyl-1-methylxanthine (IBMX) from Axxora (San Diego, CA) and Fluo3-AM from Invitrogen (Carlsbad, CA). [3H]-prazosin and [125I]-CYP were purchased from Perkin Elmer (Boston, MA). Crude membranes were prepared from transduced or transfected HEK-293/EBNA cells as published [28 (link)] with one modification. Cells were resuspended in the lysis buffer without sucrose and broken using a Polytron homogenizer (3 × 30 s pulses).
4
Lentivirus Production and Cell Transduction
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The HEK293FT cells were co-transfected using PEI (polyethyleneimine, linear, 25 kDa, Polysciences) with a mixture of three plasmids in serum-free DMEM without antibiotics. Virus-containing supernatants were harvested after 24, 48 and 72 h in complete DMEM with 10% FBS, 3 mM Sodium butyrate, 1% non-essential amino-acids (ThermoFisher, USA), 2 mM GlutaMAX (ThermoFisher, USA) and 1% sodium pyruvate (PanEco, Russia), pooled, filtered through a 0.45 µm filter and concentrated using a PEG6000/NaCl solution. The lentivirus titers were determined by counting fluorescently tagged cells according to the protocol at https://www.addgene.org/protocols/fluorescence-titering-assay/ . CT-26 cells were transduced with an iRFP720 vector and were cloned in 96-well plates containing feeder of mouse peritoneal macrophages to generate individual single colonies that were analyzed further with aid of VarioSkanLUX (ThermoFischer, USA).
5
Oral Sodium Butyrate Mitigates Morphine Tolerance
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Sodium butyrate (ThermoFisher Scientific, Waltham, MA) was prepared in saline at concentrations of 0.125, 0.250, 0.500, and 1.000 M and administered twice daily by oral gavage. For the dose-response experiment in Figure 3A , mice injected with ramping doses of morphine (as described above) were orally administered saline or different concentrations of Sodium butyrate (0.125, 0.250, 0.500, or 1.000 M) for four days. Antinociception was measured on day 5 using the warm-water tail-withdrawal test described below. In all subsequent experiments, 0.250 M Sodium butyrate or its vehicle, saline, was administered twice daily through oral gavage. The number of treatments with Sodium butyrate was contingent on the duration of exposure to morphine, such that mice subcutaneously implanted with pellets were administered 0.250 M Sodium butyrate for six days (Figures 3B , 5 , 6B , 7 , and S2), and mice injected with ramping doses of morphine received 0.250 M Sodium butyrate for four days (Figures 3A , 4B , and 6B ).
6
HDAC Inhibitor Treatment of HAA1 Cells
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HAA1 cells were plated onto 60 mm plates at a density of 8.0 × 105 in RPMI 1640 medium supplemented with 10% FBS. Then, 24 h after plating, cells were either left untreated or treated with sodium butyrate (3 mM, Thermo) or trichostatin A (100–300 nM, Sigma-Aldrich, Saint Louis, MO, USA). Cells were maintained in the presence of the HDACi for a defined number of days, at which time the media was removed and cells harvested directly on the plate with TRI® reagent (Sigma-Aldrich, St. Louis, MO, USA). Experiments with HDAC isoform-specific inhibitors and suberoylanilide hydroxamic acid (SAHA) were performed in a similar fashion, except that HAA1 cells were grown in triplicate in 6-well plates (Corning) and either left untreated or treated for 4–6 days with sodium butyrate, trichostatin A, SAHA (5 µM, #SML0061, Sigma-Aldrich, St. Louis, MO), CI994 (50 µM, #S2818, SelleckChem, Houston, TX, USA), PC-34051 (50 µM, #S2012, SelleckChem) or RGFP996 (50 µM, #S7229, SelleckChem, Houston, TX, USA). RNA from untreated and treated cells was prepared and analyzed as described below.
7
Versatile Cell Line Maintenance and Biochemical Assays
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Madin Darby bovine kidney (MDBK) cells, HEK293T cells, and HeLa cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and penicillin (100 U/ml)-streptomycin (0.1 mg/ml) at 37°C in a 5% CO2 incubator. The antibodies used in this study were obtained from the following manufacturers: rabbit polyclonal antibodies to anti-Flag (GTX115043), anti-HA (GTX115044), anti-Myc (GTX115046), and anti-GAPDH (GTX100118) were purchased from GeneTex, Inc. (United States). Antibody against TRAF6 (A5724) and Anti-Flag magnetic beads (B26102) were purchased from Bimake. Anti-HA magnetic beads (HY-K0201), MG132, and chloroquine were purchased from MedChemExpress (MCE). The rabbit monoclonal antibodies against Ubiquitin (ab134953), K48-Ubiquitin (ab140601), and K63-Ubiquitin (ab179434) were purchased from Abcam. Horseradish peroxidase (HRP)-conjugated secondary antibody was purchased from ZSGB-BIO. The dual-luciferase reporter assay system was obtained from Promega. Sodium butyrate and Lipofectamine 2000 were purchased from Thermo Fisher Scientific, Inc.
8
Efficient Lentivirus Production and Transduction
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The HEK293FT cells were co-transfected using PEI (polyethyleneimine, linear, 25kDa, Polysciences) with a mixture of three plasmids in serum-free DMEM without antibiotics. Virus-containing supernatants were harvested after 24, 48 and 72h in complete DMEM with 10% FBS, 3 mM Sodium butyrate, 1% non-essential amino-acids (ThermoFisher, USA), 2 mM GlutaMAX (ThermoFisher, USA) and 1% sodium pyruvate (PanEco, Russia), pooled, filtered through a 0.45 µm filter and concentrated using a PEG6000/NaCl solution. The lentivirus titers were determined by counting fluorescently tagged cells according to the protocol at https://www.addgene.org/protocols/fluorescence-titering-assay/. CT-26 cells were transduced with an iRFP720 vector and were cloned in 96-well plates containing feeder of mouse peritoneal macrophages to generate individual single colonies that were analyzed further with aid of VarioSkanLUX (ThermoFischer, USA).
9
Cytotoxicity Evaluation of Butyric Acid Derivatives
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Cell culture. Human colorectal carcinoma cell line, HCT116, was obtained from the National Centre for Cell Sciences, Pune. Cells were cultured in RPMI1640 media (Sigma-Aldrich, Darmstadt, Germany) supplemented with 10% Fetal Bovine Serum (FBS) and antibioticantimycotic solution (1 ml/100 ml) in a humidified atmosphere at 37˚C with 5% CO 2 (19) (link).
Determination of IC 50 value of butyric acid derivatives in HCT116 cells. IC 50 values of sodium butyrate (Himedia), indole-3-butyric acid (Fischer Scientific, Mumbai, India), tributyrin (Himedia), 2-amino-nbutyric acid (Sisco Research Laboratories Pvt. Ltd., Mumbai, India) and nicotinate (S D Fine-Chem Limited, Maharashtra, India) were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cells were treated with increasing concentrations (1-10 mM) of butyric acid derivatives, nicotinate (GPR109A receptor ligand) and 5-fluorouracil (Himedia) (Positive control) for 24, 48 and 72 h (15, (link)20) (link). Stock solutions of indole-3butyric acid and tributyrin were prepared in dimethyl sulfoxide (DMSO) and that of other compounds were prepared in phosphate buffered saline (PBS).
Determination of IC 50 value of butyric acid derivatives in HCT116 cells. IC 50 values of sodium butyrate (Himedia), indole-3-butyric acid (Fischer Scientific, Mumbai, India), tributyrin (Himedia), 2-amino-nbutyric acid (Sisco Research Laboratories Pvt. Ltd., Mumbai, India) and nicotinate (S D Fine-Chem Limited, Maharashtra, India) were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cells were treated with increasing concentrations (1-10 mM) of butyric acid derivatives, nicotinate (GPR109A receptor ligand) and 5-fluorouracil (Himedia) (Positive control) for 24, 48 and 72 h (15, (link)20) (link). Stock solutions of indole-3butyric acid and tributyrin were prepared in dimethyl sulfoxide (DMSO) and that of other compounds were prepared in phosphate buffered saline (PBS).
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