mpkCCD cells (a contribution from Alain Vandewalle, Paris) were cultured in modified DM medium as previously described41 (link),42 (link) and were seeded on semipermeable filters (Transwell 0.4-μm pore size, 4.67 cm2; Corning Costar). The cells were cultured for 5 days, following which they were serum-starved and hormone-deprived for 12 h. The culture medium was changed daily. Tolvaptan (LKT Laboratories) (10–200 μM), L-sulforaphane (Sigma-Aldrich) (10 μM), ML385 (Selleck) (50 μM), dDAVP (Sigma-Aldrich) (1 nM), GSK2606414 (Sigma-Aldrich) (5 μM), thapsigargin (Sigma-Aldrich) (1 μM), and bardoxolone methyl (Cayman Chemical) (1–50 nM), and mozavaptan (Cayman Chemical) (100 μM) were applied to the basolateral side of the mpkCCD cells. The H9C2 cells were cultured in DMEM (Nacalai Tesque) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. On reaching 70–80% confluence, cells were exposed to Tolvaptan (200 μM), L-sulforaphane (10 μM), and dDAVP (Sigma-Aldrich) (1 nM). The renal proximal tubule-derived HK-2 cells were cultured in modified DM medium with 10% fetal calf serum, 100 U/ml penicillin, and 0.1 mg/mL streptomycin. HK2 cells were treated with Tolvaptan (200 μM), L-sulforaphane (10 μM), and dDAVP (Sigma-Aldrich) (1 nM) at 90–95% confluence. All reagents were solved with dimethyl sulfoxide (DMSO).
Tolvaptan
Manufactured by Merck Group
Sourced in United States
Tolvaptan is a lab equipment product manufactured by Merck Group. It is a selective vasopressin V2-receptor antagonist.
Automatically generated - may contain errors
Lab products found in correlation
Ddavp, by Merck Group (3 mentions)
Forskolin, by Merck Group (2 mentions)
8 br camp, by Merck Group (2 mentions)
P erk1 2, by Cell Signaling Technology (2 mentions)
Ecl reagent, by PerkinElmer (2 mentions)
Opc31260, by Merck Group (2 mentions)
Opc41061, by Merck Group (2 mentions)
Erk1 2, by Cell Signaling Technology (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Cyclophosphamide monohydrate, by Merck Group (1 mentions)
Arginine vasopressin, by Merck Group (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Transwell membrane, by Corning (1 mentions)
Berberine chloride, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
2 deoxy d glucose 2 dg, by Merck Group (1 mentions)
Tolvaptan, by LKT Laboratories (1 mentions)
Gsk2606414, by Merck Group (1 mentions)
Ml385, by Selleck Chemicals (1 mentions)
L sulforaphane, by Merck Group (1 mentions)
9 protocols using tolvaptan
1
Pharmacological Modulation of Renal Cell Lines
2
Cyclophosphamide and Tolvaptan Treatment Protocol
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Cyclophosphamide monohydrate (218707) and tolvaptan (T7455) were obtained from Sigma‐Aldrich and administered as a freshly prepared solution in saline.
3
Microtubule-based Tissue Engineering Protocol
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At 2 days, microtubules were harvested from PDMS scaffolds, and transferred onto polyester Transwell membranes (0.4 μm pore size; Cat#CC3460, Corning-Costar, MA, USA) containing 200 μl of 2.4 mg/ml rat tail collagen type I. Following collagen polymerization (45 min at 37 °C), 1.5 ml of culture medium, composed of MEM supplemented with 10% FBS (Invitrogen), 1% l -glutamine and 1% Pen Strep, was added. Samples were cultured for 7 days in the presence of the following agents at doses that were selected on the basis of previous studies: 10 μM forskolin [30 (link)] (Cat#F6886, Sigma-Aldrich), then for another 7 days with 102 μM Octreotide acetate (OCTR) [31 (link)] (Cat#0239950, Toronto Research Chemicals, Brisbane Rd., North York, Canada), 102 μM Pasireotide diaspartate (PAS) [31 (link)] (kind gift from Novartis Farma S.p.A., Varese, Italy), 104 μM 2-Deoxy-d -glucose (2DG) [32 ] (Cat#D6134, Sigma-Aldrich), 10 μg/ml berberine chloride [33 (link)] (Cat#B3251, Sigma-Aldrich), 0.5 μM Rapamycin [34 (link)] (Cat#R0395, Sigma-Aldrich), or 10−2 μM Arginine Vasopressin (AVP) (Cat#V9879, Sigma-Aldrich) alone or in combination with 10−2 μM Tolvaptan [35 (link)] (Cat#T7455, Sigma-Aldrich). For OCTR and PAS dosing, we based this on data from Masyuk et al. [31 (link)] to optimize the best dosage for our system. Control samples were maintained in culture medium alone for 14 days.
4
Vasopressin Receptor Signaling Modulators
5
Signaling Pathways in Mouse Cell Lines
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Cell culture media, reagents, and foetal bovine serum (FBS) were from Euroclone. Restriction enzymes from New England Biolabs. Coelenterazine and luciferin (Na salt) from Biotium Inc. or Nanolight Technology; VisiGlo HRP Chemiluminescent Substrate from VWR; mouse monoclonal antibody conjugated to peroxidase (anti-cmyc-peroxidase) from Roche; [Arg8]Vasopressin, Tolvaptan, Forskolin and all other biochemicals were purchased from Sigma-Aldrich. Pertussis toxin was from List Biologicals or Sigma-Aldrich. All other reagents were from Sigma-Aldrich, Merck or Fisher Scientific. The clonal fibroblast line isolated from mouse embryos carrying the targeted ablation of both β-arrestin 1 and β-arrestin 2 (MEF β-arr 1/2 KO cells)23 (link) was made available to us by Prof. Robert J. Lefwokitz (Duke University, Durham, NC, USA). The clonal 2B2 cell line derived from mice carrying the targeted ablation of Gnas exon 224 (link) was supplied by Dr M. Bastepe (Harvard Medical School, Boston, MA, U.S.A.).
6
Vasopressin Receptor Signaling Modulators
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Tolvaptan (OPC41061, 7-chloro-5-hydroxy-1-[2-methyl-4-(2-methylbenzoylamino) benzoyl]-2,3,4,5- tetrahydro-1H-1-benzazepine), OPC31260 (Mozavaptan, 5-(Dimethylamino)-1-[4-(2-methylbenzamido)benzoyl]-2,3,4,5-tetrahydro-1H-benzazepine hydrochloride), 8-Br-cAMP, and dDAVP (1-desamino-8-d-arginine vasopressin) were from Sigma Millipore (St. Louis, MO). Primary antibodies were purchased for phosphorylated cAMP response element binding protein (pCREB) (#9198S), CREB (#9197S), Ki-67 (#9449S), phosphorylated extracellular signal regulated kinase (pERK1/2) (#9101S), and ERK1/2 (#SC-94) from Cell Signaling Technology (Danvers, CA, USA); vascular endothelial growth factor (VEGF) (#SC-7269) and GAPDH (#SC-47724) from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and V2R (#V5514) from Sigma Millipore (St. Louis, MO, USA) and CD31 (#ab28364) from Abcam (Cambridge, MA, USA). Secondary antibodies were from Dako (Carpinteria, CA) and ECL reagent was from Perkin Elmer (Waltham, MA, USA).
7
Hepatocyte Spheroid Drug Screening
8
Metformin-Conjugated Micelle Formulation
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metformin hydrochloride (Sigma-Aldrich) was conjugated to DSPE-PEG2000-NHS (Avanti Polar Lipids, Alabaster, AL) by adding a 5× molar excess of metformin to the lipid dissolved in 10 mM aqueous sodium carbonate buffer (pH 8.5). After reaction at room temperature for 24 h, the mixture was purified by reverse-phase high performance liquid chromatography (HPLC) (Prominence, Shimadzu, Columbia, MD) on a C4 column (Phenomenex, Torrance, CA), and the molar mass of DSPE-PEG(2000)-metformin was characterized by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF) mass spectral analysis (Autoflex Speed, Bruker, Billerica, MA).
The micelle formulation of the drug combination of 5-aza-2′-deoxycytidine (Sigma-Aldrich), metformin, and tolvaptan (Sigma-Aldrich) was synthesized via self-assembly using thin film hydration. Aza, tolvaptan, and DSPE-PEG(2000)-metformin amphiphiles were dissolved in methanol or chloroform, then the organic solvent was evaporated under a steady stream of nitrogen. The resulting film was dried under vacuum overnight, hydrated at 80 °C for 30 min with either MilliQ water or PBS, vortexed and sonicated as needed to obtain a clear solution, and allowed to cool to room temperature.
The micelle formulation of the drug combination of 5-aza-2′-deoxycytidine (Sigma-Aldrich), metformin, and tolvaptan (Sigma-Aldrich) was synthesized via self-assembly using thin film hydration. Aza, tolvaptan, and DSPE-PEG(2000)-metformin amphiphiles were dissolved in methanol or chloroform, then the organic solvent was evaporated under a steady stream of nitrogen. The resulting film was dried under vacuum overnight, hydrated at 80 °C for 30 min with either MilliQ water or PBS, vortexed and sonicated as needed to obtain a clear solution, and allowed to cool to room temperature.
9
Tolvaptan Treatment for Zebrafish Embryos
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