Chondrocytes were digested from joints of 2‐3‐week‐old male SD rats.20 Briefly, rats were euthanized and immersed into 75% alcohol for 5 minutes. Articular cartilage was resected, digested with 0.25% Trypsin‐EDTA for 30 minutes and collagenase II (Gibco) for 4 hours at 37°C and filtrated through the 200‐mesh strainer. For culture, chondrocytes were seeded at density of 1.8 × 104 cells/cm2 in high‐glucose DMEM with 1% penicillin/streptomycin (Solarbio) and 10% FBS (Gibco) in a 37°C and 5% CO2 incubator. The phenotype at P1 was identified by typical morphology and immunostaining of type 2 collagen (COL2A1; Abcam) using Inverted Ti‐E fluorescence microscope (Nikon). For experiments, unless otherwise mentioned, chondrocytes (passage 3 to 5) were seeded at density of 3 × 104 cells/cm2 and incubated with amurensin H for 2 hours, then, stimulated by 10 ng/mL human IL‐1β (PeproTech) in DMEM with 2.5% FBS. Besides, BAY61‐3606 (Syk inhibitor, Sigma) or TAK‐242 (TLR4 inhibitor, Solarbio) were used to confirm TLR4 signalling.
Bay61 3606
Manufactured by Merck Group
Sourced in United States, United Kingdom
BAY61-3606 is a laboratory product manufactured by Merck Group. It is a chemical compound used for research purposes. The core function of BAY61-3606 is to inhibit the activity of a specific enzyme. No further details on the intended use or applications of this product can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Human α thrombin, by Enzyme Research (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Piceatannol, by Merck Group (2 mentions)
Bay11 7082, by Merck Group (2 mentions)
U73122, by Merck Group (2 mentions)
Sp600125, by Santa Cruz Biotechnology (2 mentions)
Ly294002, by Santa Cruz Biotechnology (2 mentions)
Piceatannol, by Bio-Techne (2 mentions)
Anti cd18 clone game 46, by BD (2 mentions)
Col2a1, by Abcam (1 mentions)
Ti e inverted fluorescence microscope, by Nikon (1 mentions)
Human il 1β, by Thermo Fisher Scientific (1 mentions)
Tak 242, by Solarbio (1 mentions)
Collagenase 2, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Solarbio (1 mentions)
L6529, by Merck Group (1 mentions)
Aicar, by Cell Signaling Technology (1 mentions)
M0199, by Merck Group (1 mentions)
26 protocols using bay61 3606
1
Isolation and Culture of Rat Chondrocytes
2
Modulating Thymocyte Selection with Zap-70 and Syk Inhibitors
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Zap-70(AS) Inhibitor compounds 3-MB-PP1 and HXJ42 have been described
previously12 (link),34 (link). Syk inhibitor BAY61–3606 was purchased from Sigma.
For positive selection experiments on thymic slices, 2.5 µM 3-MB-PP1 was used and
for negative selection experiments on thymic slices, 5 µM 3MBPP1, 1 µM
HXJ42, or 1 µM BAY61–3606 inhibitor were used unless otherwise
indicated.
previously12 (link),34 (link). Syk inhibitor BAY61–3606 was purchased from Sigma.
For positive selection experiments on thymic slices, 2.5 µM 3-MB-PP1 was used and
for negative selection experiments on thymic slices, 5 µM 3MBPP1, 1 µM
HXJ42, or 1 µM BAY61–3606 inhibitor were used unless otherwise
indicated.
3
Isolation and Culture of Postnatal Mouse Microglia
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Cerebral cortices and hippocampi of postnatal day 1 ICR mouse pups were obtained by removing meninges in Hank's balanced salt solution (HBSS) from Welgene (Gyeongsan, Korea). The tissues were triturated with pipette and glass pipette in Dulbecco's modified Eagle's medium (HyClone, Massachusetts, USA) supplemented with 10% fetal bovine serum (FBS, HyClone) and 1% penicillin/streptomycin (Sigma, St. Louis, USA). Cells from a brain in 20 ml of culture medium were plated on a poly‐D‐Lysine (PDL)‐coated T‐75 flask. After 24‐h incubation at 37°C in a 5% CO2 incubator, the culture medium was fully changed to remove floating debris. On DIV10‐12, microglia were isolated from the mixed glial culture by tapping the flask and plated on PDL‐coated cell culture plates. The next day, the microglia were treated with LPS (10 ng/ml, L6529, Sigma) and Aβ42 (4 μM, Bachem, Bubendorf, Swiss). Bay61‐3606 (10 μM, B9685, Sigma), AICAR (1 mM, Cell Signaling, Massachusetts, USA), Mdivi‐1 (25 μM, M0199, Sigma), or FA (100 or 200 μM) was treated after LPS treatment.
4
Osteoclastogenesis Signaling Pathway Modulation
5
Blocking Innate Immune Receptors and Inflammatory Mediators
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Purified anti-human TLR2, TLR4 blocking antibodies and their matching isotype controls were obtained from BioLegend, Inc., USA. Bay 61–3606 and Phorbol 12-myristate 13-acetate (PMA), ticagrelor and Cell Detection ELISA PLUS kit were from Sigma-Aldrich, Australia. ML-171, aspirin, RGDS, cathepsin G inhibitor I, neutrophil elastase inhibitor (1-(3-methylbenzoyl)-1H-indazole-3-carbonitrile), myeloperoxidase inhibitor 1 (4-Aminobenzoic acid hydrazide), losartan were obtained from Cayman Chemical, USA. Abciximab (ReoPro) and low molecular weight heparin (Clexane enoxaparin sodium) were from Eli Lilly and Sanofi Aventis Australia Pty Ltd., respectively. DNAse I solution was purchased from STEMCELL Technologies Australia Pty Ltd. Collagen and thrombin were from Chrono-log Corporation, USA. Human PMN Elastase ELISA kit was obtained from Abcam Biotechnology, Cambridge, UK.
6
Syk Signaling Pathway Activation Analysis
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SYK inhibitor, BAY 61–3606, Phorbol 12-myristate 13-acetate (PMA), Fluorescein isothiocyanate isomer 1 and LPS were obtained from Sigma. The following commercial antibodies were used. Anti-Syk (sc-1240), anti-IKKα/β (sc-7607), anti-JNK (sc-7345), anti-p38 (sc-728), and anti-β-actin (sc-69879) were purchased from Santa Cruz. Anti-p-Syk (2711), anti-p-IKKα/β (9246), anti-p-JNK (9251), anti-p-p38 (9211) were purchased from Cell Signaling. Anti-CLEC9A (ab79661) were purchased from Abcam. Goat F(ab')2 Anti-Human IgG-PE (2042–09) was obtained from Southern Biotech. Goat F(ab')2 Anti-Rabbit IgG-Alexa Fluor 594 (A11072) was obtained from Invitrogen.
7
High Glucose-Induced EMT in Renal Epithelial Cells
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FM was purchased from Extrasynthese (Z.I Lyon Nord, Genay, France; purity ≧99.0%). BAY61-3606 was purchased from Sigma Chemical Co.(St. Louis, MO, USA; purity ≥98.0%). Human proximal tubular epithelial cells (HK-2; China Center for Type Culture Collection, CCTCC) were maintained in MEM (5.5 mM D-glucose) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 μg/mL streptomycin and 100 U/ml penicillin at 37 °C with 5% CO2. To induce EMT, HK-2 cells were cultured for 48 h in high-glucose medium (60 mM D-glucose was selected based on preliminary experiments38 and previously published findings39 (link)). High mannitol as an osmotic control exerted minimal or no effects on EMT factors and TGF-β139 (link),40 (link). Thus, no osmotic control was required. Upon reaching 60–70% confluence, HK-2 cells were cultured in serum-free medium for 24 h prior to experimentation. All primary antibodies were obtained from Abcam (Cambridge, MA, USA).
8
Investigating Immune Cell Signaling Pathways
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The antibodies for CARD9, CD206, CD68, TGF-β, IL-13, IL-4, IL-18, BCL10, MALT1, GAPDH, β-actin and IgG were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), the phospho-Syk antibody sampler kit, NF-κB pathway sampler kit were from Cell Signaling Technology (Beverly, MA, USA); the antibody for F4/80 was from Abcam (Cambridge, MA, USA); and ChemMate TM EnVision System/DAB Detection Kits were from Dako (Glostrup, Denmark). Antibodies for PerCP/Cy5.5-conjugated CD45.2, phycoerythrin (PE)-conjugated F4/80, fluorescein isothiocyanate (FITC)-conjugated F4/80, FITC-conjugated CD206, PE-conjugated CD3, FITC-conjugated CD4, FITC-conjugated CD8 and isotype control were from Biolegend (San Diego, CA, USA). FITC-conjugated IL-10, FITC-conjugated IL-1α and PE-conjugated IL-12 were from eBioscience (San Diego, CA, USA). Piceatannol and BAY61-3606 were from Sigma (St. Louis, MO, USA). Bio-Plex Cytokine Assay Kits were from Bio-Rad Laboratories (Hercules, CA, USA). Neutralizing anti-VEGF Ab and anti-MIG Ab were from R&D Systems (Minneapolis, MN, USA). Neutralizing anti-IL-15 Ab was from Abcam.
9
Analyzing Platelet Signaling Cascades
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Anti-FAK, anti-Pyk2, the anti-phosphotyrosine antibody, 4G10, and HRP-conjugated goat anti-mouse and mouse anti-rabbit light chain specific IgGs were all obtained from Millipore (Lake Placid, NJ, USA); normal rabbit and mouse IgGs and RGD peptide were from Santa Cruz (CA, USA), while anti-PLCγ2 and anti-Hic-5 were from Cell Signaling Technology, Inc. (Boston, MA, USA). Anti-Rac1 was from Tebu-Bio (Peterborough, UK). Cross-linked collagen related peptide (CRP) was purchased from Prof. Richard Farndale (Dept of Biochemistry, Cambridge University, UK). The pharmacological inhibitors, PF-573228 (hereafter referred to as PF-228), PP2, Wortmannin, EHT-1864, U73122, GF109302× and the Ca2+ chelator, BAPTA, were from Tocris Bioscience (R&D Systems Europe, UK). Tyrphostin A9 was from Calbiochem. ML171 (2-acetylphenothiazine), and BAY61-3606 (hereafter referred to as BAY) were purchased from Sigma Aldrich (St. Louis, MO, USA).
10
Inhibition of Syk by BAY-61-3606 in Aβ-induced N9 cells
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Highly selective Syk inhibitor BAY-61-3606 (B9685, Sigma, St. Louis, MO)54 , dissolved in 80% DMSO, was applied to N9 cells at different concentrations, ranging from 0.75 to 5 µM for 2 h, followed by the addition of aggregated human Aβ42 peptide at a concentration of 750 nM (ab120301, Abcam, Cambridge, UK). Briefly, Aβ peptide was diluted in DMEM to a concentration of 7.5 mM and incubated for 1 h at 37 °C to induce aggregation. Inhibition of Syk and downstream signaling molecules were assessed using western blotting.
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