The acute spinal cord slice culture was performed following the procedures described previously (Liu et al., 2017 (link)). Briefly, normal 4~6-week-old C57BL/6 mice were anesthetized with 10% urethane (1.5 g/kg, Sigma), then were plunged into 75% alcohol solution for 30 s. The spines were isolated quickly and put into 4°C precooled slice cutting buffer (2.5% 1 M HEPES in EBSS, Sigma). Under aseptic conditions, the transverse lumbar spinal cord slices were cut at 400 μm thickness with a vibratome (MA752, Campden) and then transferred onto 0.4 μm culture inserts (PIHP03050, Millipore) which were pre-equilibrated with 1 ml of culture medium. All culture media contained 50% MEM (Gibco), 25% EBSS (Sigma), 25% horse serum (Gibco), 6.5 mg/ml glucose (Sigma), 1% penicillin-streptomycin and 0.05% DMSO (Sigma). CSF1 group contained 1 μg/ml CSF1 (576406, BioLegend); SB203580 (Sigma) group contained 1 or 10 μM SB203580 (a selective inhibitor of p38 MAPK, Tocris), SB203580 + CSF1 group was cultured with 1 or 10 μM SB203580 1 h prior to CSF1. The spinal cord slices were cultured in humid conditions and 5% CO2 at 37°C. At 6 hours after CSF1 or vehicle (0.02% DMSO), the spinal cord slices were collected for immunofluorescence or WB, and the culture media were harvested for WB to show BDNF release.
Sb203580
Manufactured by Bio-Techne
Sourced in United Kingdom, United States
SB203580 is a selective p38 MAPK inhibitor used in cell biology research. It inhibits the p38 mitogen-activated protein kinase (MAPK) signaling pathway by targeting the p38 MAPK enzyme. The product is commonly used in studies investigating cellular stress responses, inflammation, and other biological processes involving the p38 MAPK pathway.
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Lab products found in correlation
Sp600125, by Bio-Techne (32 mentions)
Pd98059, by Bio-Techne (20 mentions)
U0126, by Bio-Techne (5 mentions)
Ly294002, by Bio-Techne (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Gf109203x, by Bio-Techne (4 mentions)
Fr180204, by Bio-Techne (3 mentions)
Total p38, by Cell Signaling Technology (3 mentions)
Total erk, by Cell Signaling Technology (3 mentions)
Xav939, by Merck Group (3 mentions)
Staurosporine, by Enzo Life Sciences (3 mentions)
Actinomycin d, by Merck Group (2 mentions)
Indomethacin, by Merck Group (2 mentions)
Chir99021, by Axon Medchem (2 mentions)
Nsc23766, by Bio-Techne (2 mentions)
U0126, by Cell Signaling Technology (2 mentions)
Bay 876, by Selleck Chemicals (2 mentions)
Mcc950, by Selleck Chemicals (2 mentions)
N acetyl l cysteine nac, by Merck Group (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
93 protocols using sb203580
1
Acute Spinal Cord Slice Culture Assay
2
Induction and Isolation of Murine Peritoneal Macrophages
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Three days before the extraction of peritoneal macrophages, 8-week-old C57BL/6J mice were injected intraperitoneally with 2 mL of 3% thioglycolate medium (BD Biosciences, Sparks, MD). Peritoneal macrophages were isolated by washing the peritoneal cavity with endotoxin-free phosphate-buffered saline (PBS; HyClone, Logen, UT) and cultured at 37°C in 5% CO2 in RPMI 1640 media supplemented with 10% fetal bovine serum (HyClone). The cells were seeded in 6-well culture plates (2 × 106/well) in 1 mL of fresh culture medium and washed thrice with PBS before stimulation. Macrophages were stimulated with LPS (1 μg/mL) and BLP (1 μg/mL) alone or in combination or stimulated with E. coli (DH5α or JE5505, 1 × 107 CFU/well, multiplicity of infection [MOI] of 5:1). After 1 h of infection, extracellular E. coli was eliminated via washing with fresh medium containing 10 mg/mL ovalbumin and 20 U/mL interferon gamma (58 (link)). In the groups treated with the ERK inhibitor FR180204 (Tocris, Bristol, UK) or p38 inhibitor SB203580 (Tocris), macrophages were incubated in culture media supplemented with 1 μM FR180204 for 2 h or 3 μM SB203580 for 30 min before stimulation. In the groups treated with the NF-κB inhibitor celastrol (InvivoGen, San Diego, CA), macrophages were incubated in culture media supplemented with 1 μM celastrol in combination with stimulation.
3
Glutamine Starvation Effects on P815-HTR Cells
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P815-HTR cells were seeded in 6-well plates in complete DMEM for 12 h. The cells were then rinsed twice with GLN-free medium, fed with GLN-free media supplemented with or without 4 mM GLN in combinations of 10 µM SB203580 (Tocris), 10 µg/ml actinomycin D (Sigma) or 100 µM 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB, Calbiochem) in. The SB203580 reagent was added from a 10 mM stock in DMSO, and actinomycin D was added from a 10 mg/ml stock in absolute ethanol. The DRB was added from a 100 mM stock in DMSO. The control cultures were treated with the given carrier solution without the added drug. At the indicated times after treatment, the cultures were harvested and total RNA was isolated as described for Northern blotting.
4
Antagonists Regulate Nociceptive Sensitization
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Pharmacological assays with antagonists and inhibitors were performed to investigate possible mechanisms that are relevant to nociceptive sensitization in the mouse model of incisional pain. Treatments included: anti-TNF-α monoclonal antibody Infliximab (10 mg/kg, intraperitoneally) [61 (link)], non-selective COX inhibitor indomethacin (10 mg/kg, orally) [62 (link)], NF-kB antagonist PDTC (25–100 mg/kg, intraperitoneally) [63 (link)], p38 antagonist SB203580 (2.5–20 mg/kg, intraperitoneally) [64 (link)], ERK antagonist PD98059 (0.3–3 mg/kg, subcutaneously) [65 (link)], and JNK antagonist SP600125 (25–100 mg/kg, subcutaneously) [66 (link)]. Infliximab, indomethacin, and PDTC were purchased from Sigma (Saint Louis, MA, USA). SB203580, PD98059, and SP600125 were purchased from Tocris Bioscience (Bristol, UK). Treatments were made one hour before plantar incision and followed by nociceptive threshold evaluation and Real-Time PCR for detecting Nav1.8 and Nav1.9 transcription in the DRG, as described in previous sections.
5
Modulation of CXCR2 and GRK2 Expression
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The dHL-60 cells were placed into 1.5 ml microfuge tubes at a density of 1.5×106 cells/ml in Opti-MEM. The cells were then pre-treated with the p38 inhibitor, SB203580 and the ERK inhibitor, PD98059 (both from Tocris Bioscience, Ellisville, MO, USA), at a concentration of 10 µM for each for 1 h at 37°C. Following incubation, the cells were stimulated with rhMFG-E8 or PBS for 2 h followed by the assessment of CXCR2 and GRK2 expression by flow cytometry and western blot analysis.
6
Naive Conversion of Primed hESCs
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Three primed hESC lines were subjected to naive conversion on MEFs in low oxygen conditions in either: (i) naive human stem cell medium (NHSM) composed of basal hESC media supplemented with AlbumaxI, N2 supplement (Invitrogen), PD0325901 (1 μM, Cayman) and CHIR99021 (3 μM, Axon Medchem; known together as 2i), leukaemia inhibitory factor (LIF, 1000U, Sigma), bFGF, (8 ng ml−1), SP600125 (10 μM, Tocris), SB203580 (10 μM, Tocris) and TGFβ (1 ng ml−1, Peprotech)10 (link); (ii) RT consisting of high glucose DMEM-F12 (Invitrogen) further supplemented with histone deacetylase inhibitors, sodium butyrate (0.1 mM) and SAHA (50 nM) for first three passages followed by 2i and bFGF (10 ng ml−1)11 (link) or (iii) our novel medium naive conversion medium (NCM) containing hESC media with added 2i, LIF, forskolin (10 μM, Sigma), ascorbic acid (50 ng ml−1, Sigma) and bFGF(12 ng ml−1)8 (link). Upon the appearance of domed-shaped colonies, the culture was passaged using 0.05% trypsin and once the culture was well established, cells were split at a ratio of 1:4 to 1:8 every 3 days. Single-cell clonogenicity was determined from the number of single-cells plated (upon dissociating primed and naive hESCs using trypsin without ROCKi) and the subsequent number of colonies formed. Doubling time was calculated using the formula:
7
Cell Line-Specific Compound Treatments
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Geneticin (G418, Life Technologies) was used at a final concentration of 1.2 mg/ml for MCF7 cells stably expressing fluorescent histone H2B. ERRβ agonists DY131 and GSK4716, and the p38 inhibitor SB203580 (Tocris Bioscience, Ellisville, MO), were dissolved in dimethyl sulfoxide (DMSO; Sigma Aldrich, St. Louis, MO) at a concentration of 10 mM, stored at −20°C, and used at the indicated concentrations. The microtubule inhibitor nocodazole (Sigma Aldrich), Smoothened inhibitors vismodegib and cyclopamine (kind gifts from Dr. Insoo Bae), paclitaxel (generously provided by Dr. Robert Clarke), flavopiridol (kind gift from Dr. Christopher Albanese), and the ATM inhibitor KU-55933 (generously provided by Dr. Gil Palchik) were also prepared as concentrated stocks in DMSO, stored at −20°C or 4°C (nocodazole), and used at the indicated concentrations. Doxorubicin (kind gift from Dr. Robert Clarke), mitoxantrone (generously provided by Dr. Rabindra Roy), and arsenic trioxide (ATO, [54 (link)]) were prepared as concentrated stocks in molecular biology-grade water, stored at −20°C, and used at the indicated concentrations. Human ERRγ purified protein (transcript variant 2), was purchased from Origene (Rockville, MD).
8
Cell Proliferation and Signaling Assays
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Dulbecco's modified Eagle's medium (DMEM) and FBS were purchased from Gibco‐BRL (by Thermo Fisher Scientific Inc.; Waltham, MA, USA). MG‐132 was a product of Enzo Life Sciences (Exeter, UK). Cycloheximide (CHX) and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) were obtained from Sigma‐Aldrich (St. Louis, MO, USA). SP600125, U0126, and SB203580 were purchased from Tocris Bioscience (Bristol, UK). Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix was supplied by CORNING Inc. (Corning, NY, USA).
9
Lipid-Induced Actin Remodeling Assay
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LPC (18:0) was obtained from Avanti Polar Lipids (Alabaster, AL, USA), and 3 mM stock solution in phosphate-buffered saline (PBS) was prepared with sonication. NSC23766, SB203580, and Y27632 were from Tocris Bioscience (Ellisville, MO, USA). Cytofix, Cytofix/Cytoperm, and FITC anti-CD63 Ab were from BD Bioscience (San Diego, CA, USA). FITC-conjugated phalloidin and CB were from Sigma-Aldrich Chemical (St. Louis, MO, USA). Fluo-3 AM was from Molecular Probes (San Diego, CA, USA). Rho activation assay kit was from Upstate (Billerica, MA, USA). Tat-C3 (a fusion protein between Tat-peptide and C3 toxin) was purified from bacteria containing the Tat-C3 cDNA plasmid as previously described [28 (link)]. Anti-G2A Ab (N-20) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
10
Mast Cell Activation Assay
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Peptidoglycan (PGN) from S. aureus, mouse anti-human immunoglobulin E antibody (ε-chain specific) (anti-IgE) were purchased from Sigma. Human Myeloma IgE was from Merck. Pam3CSK4 was purchased from Invivogen. SP600125, SB203580, PD98059, ciclosporin and Bay11-7821 were from Tocris. Wortmannin was from Cayman. FITC-conjugated anti-human FcεRI antibody and FITC-conjugated mouse IgG2b isotype control were purchased from eBioscience. When chemicals were dissolved in DMSO, the final concentration of DMSO did not alter the normal response of LAD2 cells.
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