Chitosan (molecular weight, 5 kDa; deacetylation degree, 90%), and Chitosanase were purchased from Sigma-Aldrich (USA). Sodium hyaluronate (molecular weight, 35 kDa) was purchased from Freda Biochem Co., Ltd. (Shandong, China). Deoxyribonuclease I (DNase I), SYBR Prime Ex Taq II were obtained from Invitrogen (USA). The streptavidin-biotin-peroxidase complex (SABC) kit and the primary antibodies against matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were purchased from Boster (Wuhan, China). Collagenase II, trypsinase, antibiotics, Dulbecco’s modified Eagle’s medium (DMEM)/F12, and fetal bovine serum (FBS) were obtained from Gibco. All reagents used in this study were of analytical grade.
Trypsinase
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
Trypsinase is a laboratory enzyme used in cell culture applications. It functions to dissociate adherent cells from cell culture surfaces, facilitating cell passaging and subculturing.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Collagenase 2, by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Chloroform, by Sinopharm (3 mentions)
Lecithin, by Sinopharm (3 mentions)
Penicillin g, by Thermo Fisher Scientific (3 mentions)
Dulbecco s modified eagle s medium dmem f12, by Thermo Fisher Scientific (2 mentions)
Chitosan, by Merck Group (2 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Thermo Fisher Scientific (2 mentions)
4 oh tamoxifen, by Merck Group (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Sodium tripolyphosphate, by Merck Group (2 mentions)
Mtt formazan, by Merck Group (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Stearic acid, by Sinopharm (2 mentions)
Chitosanase, by Merck Group (1 mentions)
Streptavidin biotin peroxidase complex sabc kit, by Boster Bio (1 mentions)
20 protocols using trypsinase
1
Chondrocyte Isolation and Culture Protocol
2
Synthesis and Evaluation of Novel Triazole Derivatives
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An AdvanceIII HD-400 NMR spectrometer (Bruker, Fällanden, Switzerland), Tensor27 IR spectrometer (Bruker), Vario ELIII elemental analyzer (Elementar Analysensysteme GmbH, Langenselbold, Germany), and SGW X-4 micro-melting-point apparatus (Shanghai Shen Guang Instrument Co. Ltd., Shanghai, China) were used in all procedures. Para-hydroxybenzaldehyde (analytical purity), cyanuric chloride (analytical purity), 1,4-dioxane (analytical purity), triethylamine, 10% anhydrous Na2CO3 (analytical purity), ethyl acetate (analytical purity), glacial acetic acid (analytical purity), 4-amino-1,2,4-triazole, trichloromethane (analytical purity), ethanol (analytical purity), sodium carboxymethyl cellulose, and silica gel GF254 (analytical purity) were purchased from SinoPharm (Shanghai, China). The human lung adenocarcinoma cell line A549 and human hepatoma cell line Bel7402 were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). RPMI-1640 culture medium, trypsinase, and fetal calf serum were obtained from Gibco (Carlsbad, CA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Invitrogen (Carlsbad, CA, USA).
3
Clinicopathological Study of Cholangiocarcinoma
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CCA (n=22) and normal tissues (n=14) were obtained by surgical operations from Second Affiliated Hospital of Nanjing Medical University from June 2014 to July 2018. This study was approved by Second Affiliated Hospital of Nanjing Medical University Review Board. All patients provided signed informed consent. All tissues were resected and put in liquid nitrogen rapidly. All patients were followed up by interview, telephone call and network communication. Overall survival (OS) was defined as the interval between surgery and death or the interval between surgery and the last observation for surviving patients. All the clinical experiments were approved by the research ethics committee of Nanjing Medical University (Nanjing, Jiangsu, PRC).
The CCA cell lines (HCCC-9810 and RBE) and HIBEpiC were obtained from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (ICBC, Shanghai, China). HCCC-9810 and RBE cell lines were cultured in RMPI-1640, while HIBEpiC in DMEM (Gibco, ThermoFisher, USA), all containing 10% fetal bovine serum (FBS, ScienCell, Carlsbad, CA, USA), 1% streptomycin (Hyclone), and 1% penicillin (Hyclone) and cells were cultured in cell incubator (37℃, 5% CO2). When the cell confluence reached 80-90%, the cells were digested by trypsinase (Gibco, ThermoFisher, USA).
The CCA cell lines (HCCC-9810 and RBE) and HIBEpiC were obtained from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (ICBC, Shanghai, China). HCCC-9810 and RBE cell lines were cultured in RMPI-1640, while HIBEpiC in DMEM (Gibco, ThermoFisher, USA), all containing 10% fetal bovine serum (FBS, ScienCell, Carlsbad, CA, USA), 1% streptomycin (Hyclone), and 1% penicillin (Hyclone) and cells were cultured in cell incubator (37℃, 5% CO2). When the cell confluence reached 80-90%, the cells were digested by trypsinase (Gibco, ThermoFisher, USA).
4
Isolation and Treatment of Primary Mouse Chondrocytes
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Primary chondrocytes were isolated from knee joint cartilage of 3-day-old mice. Dissected tissues with cartilage were first digested with 0.25% trypsinase (Gibco/Life Technologies, Carlsbad, CA, USA) at 37°C for 15 min to remove muscles, ligaments, and bone tissue. Chondrocytes were isolated from knee joints by additional digestion with 0.1% collagenase II (Gibco/Life Technologies) overnight at 37°C in a CO2 incubator. Cells were seeded in 12-well plates at a density of 2 × 105 cells/well and cultured in Dulbecco’s Modified Eagle’s Medium/F12 (1:1) supplemented with penicillin/streptomycin (Gibco/Life Technologies) and 10% fetal bovine serum until they reached sub-confluence. On day 3 of culturing, primary chondrocytes were treated with 1 μM 4OH-tamoxifen (Sigma-Aldrich) for 48 h. For MEK inhibitor treatment, chondrocytes were incubated in 250 nM U0126 or 10 nM PD98059 (both from Merck, Kenilworth, NJ, USA) for 24 h after treatment with and in the presence of 4OH-tamoxifen. For FGF18 treatment, 50 ng/ml FGF18 (PeproTech, Rocky Hill, NJ, USA) were added to cultures 5 and 30 min after 4OH-tamoxifen incubation.
5
Anti-Inflammatory Effects of IMT
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IMT was purchased from Shanghai Bomaide Biotech. Co., Ltd. (Shanghai, China); TNF-α, IL-6, and IL-1β enzyme-linked immunosorbent assay (ELISA) kits were purchased from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA); dimethyl benzene was purchased from the Sino Pharm. (Chengdu, China); Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and trypsinase were from Gibco; Thermo Fisher Scientific, Inc.; the Cell Counting Kit-8, bicinchoninic acid (BCA) protein assay reagent and goat-anti-rabbit/rat horseradish-peroxidase (HRP)-conjugated secondary antibodies (cat. nos. A0208 and A0192) were purchased from Beyotime Institute of Biotechnology (Haimen, China); lipopolysaccharide (LPS), dimethyl sulfoxide (DMSO) and Evans blue were purchased from Sigma; Merck Millipore (Darmstadt, Germany); COX-2 (cat. no. sc-19999), p65 (cat. no. sc-56735), IκB (cat. no. sc-945) and Histone H1 (cat. no. sc-8030) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); anti-β-actin antibody (cat. no. ab8226) and anti-iNOS antibody (cat. no. ab15323) were purchased from Abcam (Cambridge, MA, USA).
6
Isolation and Culture of Primary Chondrocytes
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Primary chondrocytes were isolated from the condylar cartilage of 4 week-old mice. Dissected tissues with cartilage were first digested with 0.25% trypsinase (Gibco/Life Technologies, Carlsbad, CA, USA) at 37 °C for 15 min, and then remove muscles, ligaments, and bone tissue. Chondrocytes were isolated from TMJ tissues by additional digestion with 0.1% collagenase II (Gibco/Life Technologies) overnight at 37 °C in a CO2 incubator. Cells were seeded in 12-well plates at a density of 2 × 105 cells/well and cultured in Dulbecco’s Modified Eagle’s Medium/F12 (1:1) supplemented with penicillin/streptomycin (Gibco/Life Technologies) and 10% fetal bovine serum until they reached sub-confluence. On day 3 of culturing, primary chondrocytes from both Cre-negative and Fgfr3 cKO mice were treated with 1 μM 4OH-tamoxifen (Sigma-Aldrich) for 48 h. For IHH signaling inhibitor treatment in vitro, the SMOi GDC-0449 (Selleck Chemicals, Houston, TX, USA) was reconstituted in dimethyl sulfoxide (Sigma-Aldrich) and applied in chondrocytes at a final concentration of 1 μM for 24 h after 4OH-tamoxifen treatment, and in the presence of it.
7
Curcumin-Loaded Lipid Nanoparticles for Cancer Treatment
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Polyoxyethylene(40)stearate (Myrj52), stearic acid, chloroform, lecithin, Tween-80, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 2-(4-amidinophenyl)-6- indolecarbamidine dihydrochloride were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, People’s Republic of China). Cur (≥98%) was purchased from Aladdin Chemistry Co. Ltd. (Shanghai, People’s Republic of China). β-Actin, caspase-3, caspase-9, Bax, and Bcl-2 antibodies were purchased from Cell Signaling Technology Inc. Nucleoprotein and cytoplasmic protein extraction kit, bicinchoninic acid protein assay reagent, Annexin V-APC/7-amino-actinomycin D (7-AAD) apoptosis detection kit, cell cycle detection kit, 3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) apoptosis detection kit, and ROS detection kit were purchased from KeyGEN Biotech (Nanjin, People’s Republic of China). Dimethyl sulfoxide was purchased from Sigma Chemical Co. (St Louis, MO, USA), and MitoTracker-Red was from Invitrogen (Carlsbad, CA, USA). Water was prepared using a Millipore Milli-Q® system (Bedford, MA, USA) and decarbonated by boiling. Roswell Park Memorial Institute (RPMI)-1640, penicillin–streptomycin, fetal calf serum, and trypsinase were purchased from Gibco (BRL, Grand Island, NY, USA).
8
Chondrocyte Apoptosis Assay Protocol
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CS (molecular weight, 150 kDa; deacetylation: 98%), HA (molecular weight, 500–730 kDa), and sodium tripolyphosphate (STPP) were provided by Sigma-Aldrich (St. Louis, MO, USA). Recombinant rat IL-1β and IL-1Ra were purchased from PeproTech (Rocky Hill, NJ, USA). Trypsinase, collagenase II, Dulbecco's modified Eagle's medium (DMEM)/F12, foetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 6-diamidino-2-phenylindole dihydrochloride (DAPI), and penicillin/streptomycin were obtained from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Rabbit monoclonal antibody (IgG) for Bcl-2-associated X protein (Bax, Cat. number 14796) and rabbit polyclonal antibodies (IgG) for B-cell lymphoma 2 (Bcl-2, Cat. number 2876) and caspase-3 (Cat. number 9662) were purchased from Cell Signal Technology (Beverly, MA, USA). An in situ cell apoptosis detection kit was purchased from Roche Diagnostics (Cat. number 11684795910, Basel, Switzerland). All other chemicals used in this study were of analytical grade and obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.
9
Inflammatory Cytokine Signaling Assay
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Trypsinase, Penicillin–Streptomycin, Roswell Park Memorial Institute (RPMI) 1640 medium and fetal bovine serum (FBS) were obtained from Gibco (New York, USA). Phorbol 12-myristate-13-acetate (PMA) was purchased from Biogems (New Jersey, USA). MCC950 was purchased from APExBIO (Houston, USA). Ox-LDL was purchased from Yiyuan Biotech Inc (YB-002-1, Guangzhou, China). The primary antibodies against IL-1β and GAPDH were purchased from Abcam (Cambridge, UK). The antibodies against TLR-4, ASC, Caspase-1 and GSDMD were purchased from Santa Cruz (Dallas, USA). The primary antibodies against NLRP3 and IL18 were purchased from Invitrogen (Carlsbad, USA). High-fat diets were purchased from Jiangsu Medicience bio-pharmaceutical Co., Ltd. All reagents used in this study were of analytical grade.
10
Isolation of Primary Chondrocytes from Mice
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Primary chondrocytes were isolated from the knee articular cartilage of 5‐day‐old mice. Knee joints were first digested by 0.25% trypsinase (Gibco/Life Technologies, Carlsbad, CA, USA) at 37 °C for 15 min, and adjacent muscles, ligaments and bone tissues were removed using a stereomicroscope (Olympus BX51, Tokyo, Japan). Chondrocytes were isolated from the cartilage by additional digestion with 0.1% collagenase Π (Gibco/Life Technologies) overnight at 37 °C in a 5% CO2 incubator 21 . Cells were seeded in 35‐mm‐diameter dishes and cultured in Dulbecco’s modified Eagle’s medium/F12 (1 : 1) supplemented with 1% penicillin/streptomycin (HyClone, Logan, UT, USA) and 10% FBS (HyClone) and incubated in a humidified atmosphere of 5% CO2 at 37 °C. The culture medium was changed every 2 days.
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