Primed-hiPSCs of both patients were maintained on irradiated mouse embryonic fibroblasts (MEFs) in DMEM/F12 GlutaMAX supplemented with 20% KSR (thermofisher), 1X non-essential amino acids, 50 µM β-mercaptoethanol (thermofisher), and 10 ng/ml of bFGF (thermofisher). Media were replaced every day. Cells were passaged as clumps every 4 to 6 days using trypsin and 10 µM of ROCK inhibitor (TEBU-Bio). Primed-like hiPSCs were converted into a naive state by cultivating them on MEFs feeders cells in knock-out DMEM medium (thermofisher) supplemented with 20% KSR (thermofisher), 2 mM L-Glutamine (thermofisher), 0.1 mM MEM NEAA (thermofisher) and 50 µM β-mercaptoethanol (thermofisher) and containing basic fibroblast growth factor (b-FGF, thermofisher), human leukemia inhibitory factor (LIF, Peprotech), transforming growth factor-β1 (TGFβ, TEBU-Bio), and four small inhibitory molecules (4i), 3 μM CHIR99021 (R&D systems), 1 μM PD0325901 (R&D systems), 5 μM SB203580 (R&D systems), and 5 μM SP600125 (R&D systems)27 (link). Cells were maintained in 4i hiPSC medium for two weeks replacing daily with fresh medium. The growing naive-hiPSCs colonies were picked up and passaged every 2–3 days as single cells with accutase. For each passage, 10 μM of ROCK inhibitor (TEBU-Bio) were used.
Sp600125
Manufactured by R&D Systems
Sourced in Canada, Macao, United States
SP600125 is a potent, selective and cell-permeable c-Jun N-terminal kinase (JNK) inhibitor. It acts by inhibiting the enzymatic activity of JNK, a protein kinase involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Sb203580, by R&D Systems (4 mentions)
U0126, by R&D Systems (2 mentions)
Ly294002, by R&D Systems (2 mentions)
Rock inhibitor, by Tebu-Bio (1 mentions)
Knockout dmem medium, by Thermo Fisher Scientific (1 mentions)
Mem neaa, by Thermo Fisher Scientific (1 mentions)
Pd0325901, by R&D Systems (1 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Dmem f 12 glutamax, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Chir99021, by R&D Systems (1 mentions)
Lps escherichia coli o26 b6, by Merck Group (1 mentions)
Pd98059, by R&D Systems (1 mentions)
Gallein, by R&D Systems (1 mentions)
Forskolin, by R&D Systems (1 mentions)
Nifedipine, by R&D Systems (1 mentions)
ω conotoxin mviic, by Bio-Techne (1 mentions)
Phorbol myristate acetate (pma), by Bio-Techne (1 mentions)
6 protocols using sp600125
1
Primed-hiPSCs to Naive-hiPSCs Conversion
2
Microglial Inflammatory Signaling Modulation
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BV-2 microglial cells were stimulated for 12 h with 166 µg/ml RAGE-blocking antibody (Abcam, Cambridge, UK), 1.0 µg/ml C225 (inhibitor of TLR4), 20 µM PD98059 [inhibitor of extracellular signaling kinase (ERK)], 7 µM SB203580 (inhibitor of p38 MAP kinase), 10 µM SP600125 (inhibitor of JNK) or 50 µM PDTC (inhibitor of p65 NF-κB) (all from R&D Systems, ON, Canada) for 1 h before addition of 0.1 µM recombinant S100A8/A9 proteins or 1 µg/ml LPS (Escherichia coli O26:B6; Sigma-Aldrich, St. Louis, MO, USA) in the presence of 25 µg/ml polymyxin B (R&D Systems), and the culture supernatants were harvested. Levels of IL-6 and TNF-α in 100 µl medium were measured by commercial ELISA kits (Boster Biological Technology, Wuhan, China) according to the manufacturer's instructions.
3
Pharmacological Modulation of Ion Currents
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All drugs were purchased from Sigma (MO, USA) unless otherwise indicated. Stock solutions of 4-aminopyridine (4-AP), pertussis toxin (PTX), cholera toxin (CTX), PMA (Tocris Bioscience, Ellisville, MO) and ω-conotoxin MVIIC (Tocris Bioscience, Ellisville, MO) were prepared in distilled deionized water. Z941 was a kind gift from Dr. Terrance P. Snutch (University of British Columbia, Vancouver, British Columbia, Canada). Stock solutions of cholecystokinin-8 (Tocris Bioscience, Ellisville, MO), LY294002, CCK-4, nifedipine, forskolin, gallein, wortmannin, KT5720 (RD system), devazepide, LY225910, GW5823, BC264, SP600125, SB203580, anisomycin, PP2, PP3, Akt inhibitor III, U0126, and GF109203X were prepared in dimethylsulfoxide (DMSO). The final concentration of DMSO in the bath solution was estimated to be less than 0.01%, and this compound had no functional effects on IA (not shown).
4
Intrathecal Drug Administration for Pain Relief
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The drugs used in this study were prepared as follows. Morphine hydrochloride (10 μg/5 μL, Shenyang First Pharmaceutical Factory, China) and PDGFRβ inhibitor imatinib (10 μg/10 μL, LC Laboratories, USA)15 (link) were diluted in saline (Northeast Pharmaceutical Group, China), respectively. Specific JNK MAPK inhibitor SP600125 (50 μg/10 μL, MedChem Express, China)8 (link) and selective MOR antagonist naloxone (10 μg/10 μL, Selleckchem, USA)17 (link) were dissolved in 20% dimethyl sulfoxide (DMSO, Sigma, USA), respectively. PDGF-BB (10 pmol/10 μL, R&D Systems, USA) was dissolved in PBS with 0.1% BSA.15 (link) imatinib (10 μg), SP600125 (50 μg), or naloxone (10 μg) was intrathecally injected 30 minutes before morphine administration, respectively. PDGF-BB (10 pmol) was intrathecally injected alone, then 10 μL of saline was used to flush the catheter after intrathecal administration of drugs.
5
Inhibition of MMP-2/MMP-9 and JNK
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MMP-2/MMP-9 inhibitor V was purchased from Calbiochem (San Diego, CA; Cat. No. 444274). JNK inhibitor SP600125 was purchased from R&D Systems (Minneapolis, MN; Cat. No. 1496).
6
Modulation of BMDC by Galectin-9
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BMDC were generated from wild-type Balb/c or C57Bl/6 mice as previously described (GM-CSF was obtained from PeproTech, recombinant mouse Flt3L was a kind gift from Dr. Iglesia at the Dutch Cancer Institute and was made in-house) [15] . Some BMDC cultures were exposed to inhibitors for the mitogen-activated protein kinases (MAPK) p38 (SB203580, 10 μ M ), c-JUN N-terminal kinase (JNK; SP600125, 25 μ M ), extracellular signal-regulated kinase (ERK1/2; U0126, 10 μ M ), or phosphatidylinositol-3-kinase (PI3K; LY294002, 5 μ M ) 1 h prior to exposure to recombinant mouse galectin-9 for 24 h on day 7 of culture (1-5 μg/mL, R&D Systems). All inhibitors were purchased from Cell Signaling Technology.
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