Polydatin (C20H22O8; MW, 390.39; purity, > 95%); resveratrol (C14H12O3; MW, 228.24; purity, 99%) were purchased from Aladdin Co., Ltd. (Shanghai, China). Ribavirin was provided by the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). RD cells were purchased from Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). DMSO was ordered from Borunlaite Co., Ltd. (Beijing, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Scientific HyClone (UT, USA). Hybond C membrane and ECL Western blot detection system were purchased from Pierce Company (Rockford, IL, USA). Rabbit polyclonal antibodies against IKKα, p-IKKα, IKKβ, p-IKKβ, IKKγ, p-IKKγ, IKBα, p-IKBα, NF-κB p50, p-NF-κB p50, NF-κB p65, p-NF-κB p65, and horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG secondary antibodies were purchased from SAB Company (Pearland, TX, USA). Antibodies against anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from ProteinTECH Group (Chicago, IL, USA). Rabbit polyclonal antibody against EV71/VP1 was purchased from Abcam Company (Cambridge, UK).
Dulbecco s modified eagle s medium dmem
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, United Kingdom, Germany, Canada, France, Australia, Japan, India, Spain, Switzerland, Israel, Italy, Belgium, Austria, New Zealand, Brazil, Argentina, Denmark, Ireland, Singapore, Egypt
Dulbecco's modified Eagle's medium (DMEM) is a cell culture medium commonly used for the in vitro cultivation of various cell types. It provides a balanced salt solution, amino acids, vitamins, and other nutrients required for cell growth and maintenance.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2486 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (743 mentions)
Streptomycin, by Thermo Fisher Scientific (506 mentions)
Penicillin, by Thermo Fisher Scientific (496 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (333 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (234 mentions)
Trypsin edta, by Thermo Fisher Scientific (227 mentions)
Trypsin, by Thermo Fisher Scientific (185 mentions)
L glutamine, by Thermo Fisher Scientific (180 mentions)
Rpmi 1640, by Thermo Fisher Scientific (164 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (141 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (134 mentions)
Fetal bovine serum (fbs), by Merck Group (123 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (111 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (102 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (81 mentions)
Dexamethasone (dex), by Merck Group (79 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (76 mentions)
Streptomycin, by Merck Group (75 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (75 mentions)
4 408 protocols using dulbecco s modified eagle s medium dmem
1
Polydatin and Resveratrol Anti-EV71 Assay
2
Western Blot Analysis of Signaling Pathways
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Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and RPMI 1640 were purchased from Thermo Scientific HyClone (UT, USA). Hybond C membrane and ECL Western blot detection system were from Pierce (Rockford, IL, USA). Rabbit polyclonal antibodies against MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, c-Fos, p-c-Fos, c-Jun, p-c-Jun, p-c-myc, c-myc, Elk1, p-Elk1, and HRP conjugated goat anti-rabbit IgG were purchased from SAB (Pearland, TX, USA). SOS1 mouse monoclonal antibody was provided from abnova Company (Taiwan). Antibodies against anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from ProteinTECH Group (Chicago, IL, USA). Rabbit polyclonal antibody against EV71/VP1 was purchased from Abcam Company (Cambridge, UK). The MEK1/2 specific inhibitor U0126 was acquired from LC Laboratories (Woburn, MA, USA) and freshly prepared as DMSO solution.
3
Inflammatory Signaling Pathway Analysis
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Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin-streptomycin, and fetal bovine serum (FBS) were obtained from Thermo Scientific HyClone (USA). Lipopolysaccharides from Escherichia coli 0111:B4 were obtained from Sigma (USA). The antibody against β-actin was purchased from Santa Cruz Biotech (USA). Antibodies specific for iNOS, COX-2, IKKα, phospho-IKKα/β (Ser176/180), IκBα, phospho-IκBα (Ser32), NF-κB p65, phospho- NF-κB p65 (Ser536), p44/42 (Erk1/2), phospho-p44/42 (Erk1/2) (Thr202/Thr204), SAPK/JNK, phospho-SAPK/JNK (Thr183/Tyr185), p38 MAPK, phospho-p38 MAPK (Thr180/182), and α/β-tubulin were purchased from Cell Signaling Biotechnology (USA). The antibody against Lamin B1 was obtained from Abcam (UK).
4
Immunofluorescence Analysis of Cellular Proteins
5
Lipopolysaccharide-Induced Inflammatory Signaling
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Dulbecco's Modified Eagle's Medium (DMEM), penicillin-streptomycin, and fetal bovine serum (FBS) were obtained from Thermo Scientific HyClone (USA). Lipopolysaccharides from Escherichia coli 0111:B4 were obtained from Sigma (USA). The antibody against β-actin was purchased from Santa Cruz Biotech (USA). Antibodies specific for iNOS, COX-2, IKKα, phospho-IKKα/β (Ser176/180), IκBα, phospho-IκBα (Ser32), NF-κB p65, phospho-NF-κB p65 (Ser536), p44/42 (Erk1/2), phospho-p44/42 (Erk1/2) (Thr202/Thr204), SAPK/ JNK, phospho-SAPK/JNK (Thr183/Tyr185), p38 MAPK, phospho-p38 MAPK (Thr180/182), and α/β-tubulin were purchased from Cell Signaling Biotechnology (USA). The antibody against Lamin B1 was obtained from Abcam (UK).
6
Cell Culture Reagents and Antibodies
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Dulbecco’s modified Eagle’s medium (DMEM), containing glucose at 4.5 mg/ml (25 mM), Dulbecco’s modified Eagle’s medium (DMEM), containing glucose at 0.5 mg/ml (2.8 mM), l -glutamine, penicillin, streptomycin, trypsin–EDTA, FBS Gold, trypsin–EDTA were provided by Gibco (San Diego, USA), passive lysis buffer by Promega (Madison, USA), BCA Protein Assay Kit by Thermo Scientific (Rockford, USA), PE Annexin V Apoptosis Detection Kit I by BD Pharmingen™ (CA, USA), Sigma-Fast BCIP/NBT reagent, 4′,6-diamidino-2-phenylindole dihydrochloride—DAPI, donkey serum, acridine orange, ethidium bromide, camptothecin by Sigma (St Louis, MO, USA), monoclonal (mouse) anti-human ORP150 antibody by IBL (Gunma, Japan), medium coverquick by Hygeco (USA), polyclonal (rabbit) anti-human NF-κB2 p100/p52 antibody, monoclonal (rabbit) anti-human CHOP antibody, monoclonal (rabbit) anti-human β-tubulin antibody, alkaline phosphatase-labeled anti-rabbit immunoglobulin G were provided by Cell Signaling Technology (Boston, USA), monoclonal (mouse) anti-human mTOR antibody (Merck-Millipore, USA), alkaline phosphatase-labeled anti-mouse immunoglobulin G by Rockland (PA, USA), polyclonal (rabbit) anti-human LC3 by Abgen, donkey anti-rabbit IgG conjugated with Alexa Fluor 488 by Molecular Probes (USA).
7
Cell Line Characterization and Culture Protocol
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Cell lines used in this study were: MMQ (ATCC, CRL-10609), GC [51] (link), GHFT [52] (link), HEK293T (ATCC, CRL-11268) and SH-SY5Y (ATCC, CRL-2266). Antibodies used in this work included: anti-GH, anti-Prl (prolactin), anti-TSHβ, anti-Pit-1 (1769), anti-TRα/β (Santa Cruz, sc-772, fl-408), anti-FLAG (Sigma, Cat. # F3165), anti-E-cadherin (Santa Cruz, sc-7870, H-108) and anti-SmcHD1 antibody (Abcam, Cat. # ab31865). 5-Azacytidine was purchased from Invitrogen.
MMQ cells from rat pituitary were cultured in Ultraculture media (Biowhittaker, Cat. #12-725F) without L-glutamine and supplemented with 5% fetal bovine serum (FBS) and were maintained in a humidified atmosphere containing 5% CO2 at 37°C. HEK293T, SH-SY5Y and GHFT cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco) containing 4.5 g/L Glucose and L-Glutamine (Biowhittaker, Cat. # 12-604F) and supplemented with 10% fetal bovine serum (FBS). GC cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco) containing 4.5 g/L Glucose and Glutamax (Invitrogen, Cat. # 10566-016) supplemented with 12.5% horse serum and 2.5% FBS. Where indicated, cells were treated with 0.25% 5-azaC every 24 hrs for a minimum of 72 hrs.
MMQ cells from rat pituitary were cultured in Ultraculture media (Biowhittaker, Cat. #12-725F) without L-glutamine and supplemented with 5% fetal bovine serum (FBS) and were maintained in a humidified atmosphere containing 5% CO2 at 37°C. HEK293T, SH-SY5Y and GHFT cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco) containing 4.5 g/L Glucose and L-Glutamine (Biowhittaker, Cat. # 12-604F) and supplemented with 10% fetal bovine serum (FBS). GC cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco) containing 4.5 g/L Glucose and Glutamax (Invitrogen, Cat. # 10566-016) supplemented with 12.5% horse serum and 2.5% FBS. Where indicated, cells were treated with 0.25% 5-azaC every 24 hrs for a minimum of 72 hrs.
8
HEp-2 Cell Culture and Virus Propagation
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HEp-2 (human laryngeal carcinoma) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin (Gibco) and 100 µg/ml streptomycin (Gibco) at 37°C in 5% CO2. The virus strains used were HSV-1 F gifted by Professor Rapp. F from Hershey Medical Center, USA in 1990 and stored in our laboratory ever since. The virus was propagated in HEp-2 cells and stored at −80°C for further studies. R. tanguticum nanoparticles were dissolved in (DMSO) (Sigma-Aldrich, St Louis, MO) followed by dilution in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY) to prepare a stock concentration of 1 mg/ml solution.
9
Mouse Embryonic Fibroblast Culturing Protocol
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Mouse strains and cell lines used in the study are listed in Key Resource Table . Mouse Embryonic Fibroblasts were produced at embryonic day 13.5 from timed breeding between 8-12 weeks old RTEL1fl/flPole4+/− males and females. All animal experimentations were undertaken in compliance with UK Home Office legislation (project license number 70/8527) under the Animals (Scientific Procedures) Act 1986. Rtel1F/FPole4+/+and Pole4−/− primary mouse embryonic fibroblasts (MEFs) were cultured at 37°C/ 5% CO2/ 5% O2 in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 15% fetal bovine serum (Sigma) and 1% penicillin-streptomycin (Invitrogen). 293 cells were cultured in 37°C/ 5% CO2/ 5% O2 in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 15% fetal bovine serum (Sigma) and 1% penicillin-streptomycin (Invitrogen). Human 293 cells were cultured in DMEM 10% FBS (SIGMA) at 37°C/ 5% CO2.
10
Rat Mast Cell Cultivation and Assay
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PD (purity, 98%) was obtained from TW Reagent Co., Ltd (Shanghai, China). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). 3- (4,5-Dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT) and triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical reagent grade. Anti-dinitrophenyl (DNP) IgE, DNP human serum albumin (HSA), Percoll solution was purchased from Pharmacia (Uppsala, Sweden). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). RBL-2H3 cells and rat peritoneal mast cells (RPMCs) were grown at 37 °C in 5% CO2 in DMEM supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin G, 100 μg/ml streptomycin.
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