Podocytes were cultured at 37°C in a water-saturated atmosphere with 5% CO2 for 5 days before initiation of experiments. Podocytes were separately exposed to Dkk1 (R&D Systems) or NS-398 (Sigma Chemical Co., St Louis, MO) in serum-free DMEM containing peptidase inhibitors (1 μM phosphoramidon, 4 μg/mL bacitracin and 1 μM captopril; Sigma) for 15 minutes at 37°C in a water-saturated atmosphere with 5% CO2. The cells were then treated with IL-17 and continuously stimulated with TWS119 (Cayman Chemical, Ann Arbor, MI) or Wnt-3a (R&D Systems) for 8, 12, or 24 hours in DMEM at 37°C. Podocytes were treated with different concentrations of IL-17.
Tws119
Manufactured by Cayman Chemical
TWS119 is a solid-phase extraction (SPE) column manufactured by Cayman Chemical. It is designed to facilitate the isolation and purification of analytes from complex matrices. The TWS119 column provides a consistent and reliable platform for sample preparation prior to analytical methods such as chromatography or mass spectrometry.
Automatically generated - may contain errors
Lab products found in correlation
Phosphoramidon, by Merck Group (2 mentions)
Bacitracin, by Merck Group (2 mentions)
Ns 398, by Merck Group (2 mentions)
Captopril, by Merck Group (2 mentions)
Wnt3a, by R&D Systems (2 mentions)
Bromocriptine, by Cayman Chemical (1 mentions)
Nimesulide, by Cayman Chemical (1 mentions)
Mesalamine, by Cayman Chemical (1 mentions)
Lithium chloride (licl), by Merck Group (1 mentions)
Bortezomib btz, by Merck Group (1 mentions)
Lithium acetate, by Merck Group (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Easysep human cd8 t cell enrichment kit, by STEMCELL (1 mentions)
Sb216763, by Merck Group (1 mentions)
Anti cd3 cd28 beads, by Thermo Fisher Scientific (1 mentions)
Recombinant human wnt3a, by R&D Systems (1 mentions)
Rapamycin, by LC Laboratories (1 mentions)
Bortezomib, by Santa Cruz Biotechnology (1 mentions)
Sp600125, by Bio-Techne (1 mentions)
Sb202190, by Bio-Techne (1 mentions)
6 protocols using tws119
1
Podocyte Response to IL-17 and Wnt Signaling
2
Screening of Small Molecule Library
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The 1040 compounds used for the small molecule screen were from the Prestwick Chemical Library (Illkirch, France). The following compounds were obtained from Cayman Chemical (Ann Arbor, MI): TWS119 (#10011251), ipsapirone (#22075), bromocriptine (#14598), urapidil (#29004), trimipramine (#15921), dichlorphenamide (#23658), nimesulide (#70640), and mesalamine (#70265). Lithium chloride (LiCl) was obtained from Sigma (St. Louis, MO, #62476), lithium acetate was obtained from Aldrich Chemical Company (Milwaukee WI, #21,319–5), and bortezomib (BTZ) was obtained from EMD Millipore (#179324-69-7).
3
Generating Tscm Cells via Wnt Signaling
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To generate the Tscm cells in vitro, Wnt signaling was activated in T cells by GSK-3β blockade with the inhibitor TWS119. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of RCC patients by Ficoll density gradient centrifugation. CD8+ and naïve CD8+ T cells were purified from PBMCs using the EasySepTM human CD8+ T cell enrichment kit (STEMCELL, 19053) and the EasySepTM human naïve CD8+ T cell enrichment kit (STEMCELL, 19158), respectively, according to the manufacturer’s instructions. PBMCs, CD8+, or naïve CD8+ T cells were stimulated by ɑ-CD3/CD28 beads (Invitrogen, 11452D) at a bead-to-cell ratio of 1:1 and 300 IU/mL IL-2 (PeproTech, AF-200-02) in the presence of 5 μM TWS119 (Cayman Chemical, 10011251) for 7 days. Culture medium (10% RPMI1640) containing TWS119 and IL-2 were supplemented on day 3 and 5.
4
T cell Expansion and Pathway Modulation
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T cells were cultured in RPMI-1640 supplemented with 8% heat inactivated, pooled human serum or 10% foetal calf serum, 50 IU/ml penicillin, 50 μg/ml streptomycin, 4 mM l -glutamine, 1% (v/v) non-essential amino acids and 50 μM 2-mercaptoethanol. Sorted TN cells were primed with anti-CD3/CD28 beads (Invitrogen) or OKT3/anti-CD28 antibody (in house, derived from hybridoma cells) and IL-2 (Proleukin®, Roche Pharma AG). Pathway interfering drugs were TWS119 (Cayman Chemical), rapamycin (LC Laboratories), PP242 (Chemdea), KU-0063794 (Chemdea), Indirubin-3-monoxime (Sigma-Aldrich), SB216763 (Sigma-Aldrich) and recombinant human Wnt3A (R&D Systems).
5
Substance P Regulation in Cultured DRG Cells
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The isolated DRG cells from adult Wistar rats (6–9 weeks of age) were cultured at 37°C in a water-saturated atmosphere with 5% CO2 for 5 days before the initiation of experiments [3 (link)]. Next, the DRG cells (5–6 DRGs/35 mm dish) were separately exposed to Dkk1 (R&D Systems, Minneapolis, MN 55413, USA) or NS-398 (Sigma Chemical Co., St Louis, MO, USA) in serum-free DMEM containing peptidase inhibitors (1 μM phosphoramidon, 4 μg/ml bacitracin and 1 μM captopril; Sigma) for 15 min at 37°C in a water-saturated atmosphere with 5% CO2. Thereafter, the cells pretreated with various drugs were continuously stimulated with TWS119 (Cayman Chemical, Ann Arbor, MI) or Wnt-3a (R&D Systems) for 1, 6, 24 or 48 h in DMEM at 37°C. The DRG cells treated by peptidase inhibitors alone were used as a control. The substance P content in the cultured rat DRG cells was measured using a highly sensitive radioimmunoassay as previously described [13 (link)].
6
Modulation of Cellular Pathways
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