NiV (Malaysian strain; GenBank no. AF212302) was kindly provided by the Special Pathogens Branch of the Centers for Disease Control and Prevention, Atlanta, USA. Virus was propagated and titrated on VeroE6 cells (European Collection of Cell Cultures, Salisbury, UK) grown using Dulbecco’s minimal essential medium (DMEM; Gibco, Paisley, UK) supplemented with 2% fetal bovine serum (Gibco, Paisley, UK) at 37 °C. All infectious work was performed in a class III biological safety cabinet line in the containment level (CL) 4 laboratory at UKHSA, Porton Down.
Dulbecco s minimal essential medium dmem
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany
Dulbecco's minimal essential medium (DMEM) is a cell culture medium commonly used for the maintenance and growth of various cell lines. It provides a balanced salt solution and essential nutrients required for cell proliferation. DMEM is formulated to support a wide range of cell types, making it a versatile tool for cell culture applications.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (13 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
Penicillin, by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (3 mentions)
Penicillin, by Quality Biological (2 mentions)
Streptomycin, by Quality Biological (2 mentions)
Glutamine, by Quality Biological (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Geneticin g418, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (2 mentions)
Veroe6 cells, by European Collection of Cell Cultures (1 mentions)
Coomassie brilliant blue r 250, by Bio-Rad (1 mentions)
L 5 3h proline, by Cytiva (1 mentions)
30 protocols using dulbecco s minimal essential medium dmem
1
Propagation and Titration of NiV in VeroE6 Cells
2
Integrin-Mediated Cell Signaling Pathway
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Alkaline phosphatase-labeled anti-mouse IgG and anti-rabbit IgG antibodies, bacterial collagenase, 3-(4,5-di-methylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Fast BCIP/NBT reagent, l -glycyl-proline, l -proline, monoclonal (mouse) anti-IGF-IR, anti-pERK1/2 and polyclonal (rabbit) anti-β-actin antibodies, sodium pyruvate, and oxythiamine, were provided by Sigma Corp., USA., as were most other chemicals and buffers used. Dulbecco’s minimal essential medium (DMEM) and fetal bovine serum (FBS) used in cell culture were products of Gibco, USA. Glutamine, penicillin, and streptomycin were obtained from Quality Biological Inc., USA. Nitrocellulose membrane (0.2 μm), sodium dodecyl sulphate (SDS), polyacrylamide, molecular weight standards, and Coomassie Brilliant Blue R-250 were received from Bio-Rad Laboratories, USA. L-5[3H] proline (28 Ci/mmol) was purchased from Amersham, UK. Monoclonal (mouse) anti-β1 and polyclonal (rabbit) anti-α2-integrin and anti-NFκB p65 antibodies were the products of Santa Cruz Biotechnology Inc., USA. Polyclonal (rabbit) anti-ERK1/2 and monoclonal (rabbit) anti-pAkt antibodies were the products of Cell Signaling Inc., USA. Polyclonal anti-human prolidase antibody was donated by Dr. James Phang (NCI-Frederick Cancer Research and Development Center, Frederick, MD, USA). 3-mercaptopicolinic acid (3-MPA) was purchased from Applichem GmbH, Germany.
3
Culturing Human Liver Cancer Cell Lines
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Different types of the human HCC cell lines (Huh-7, well-differentiated, epithelial phenotypic and mutation in p53; LM-3, high-metastatic, epithelial phenotypic and mutation in p53; SNU-387, poor-differentiated, mesenchymal phenotypic and mutation in p53; SNU-449, poor-differentiated, mesenchymal phenotypic and mutation in p53) were purchased from Shanghai Institute for Biological Science (Shanghai, China). Huh-7 and LM-3 cells were cultured in Dulbecco’s minimal essential medium (DMEM; Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin. SNU-387 and SNU-449 cells were cultured in RPMI 1640 Medium (1640; Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin. For normoxia, cells were maintained at 37 °C in 5% CO2 and 95% air; for hypoxia, cells were maintained at atmosphere containing 0.2% O2 and 5% CO2 in a hypoxia incubator.
4
Antiplatelet and Antioxidant Assays
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Accordingly, 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), Dulbecco’s minimal essential medium (DMEM), RPMI1640 medium, phosphate buffered saline pH 7.0 (PBS), fetal bovine serum (FBS) and other materials for cell culturing were purchased from Gibco, Life Technologies (Eugene, OR. USA). Histopaque®-1077, dimethyl sulfoxide solution (DMSO), adenosine 5′-diphosphate (ADP) sodium salt, epinephrine, sodium arachidonate (AA), Thrombin receptor agonist peptide (TRAP), 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F2α (U46619), gallic acid (GA), penicillin-streptomycin, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), chlorogenic acid, and caffeine were purchased from Sigma-Aldrich Chemicals Company (Gillingham, UK). Acetonitrile, phosphoric acid, Folin-Ciocalteau reagent, potassium thiosulfate (K2SsO8) and sodium carbonate (Na2CO3) were purchased from Merck KGaA (Darmstadt, Germany). Collagen suspension was obtained from Helena Biosciences (Gateshead, UK). Competitive enzyme linked immunosorbent assay (cELISA) kit for ovine/human cyclooxygenase (COX) (No.560131) was purchased from Cayman Chemicals Company (Ann Arbor, MI, USA). UltraPure water and other chemicals and solvents that were used in this study were of the highest analytical grade.
5
SARS-CoV-2 Isolation and Infection Assay
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BetaCoV/Korea/KCDC03/2020 (GISSAID accession ID: EPI_ISL_407193) was obtained from the National Culture Collection for Pathogens of Korea Center for Disease Control and prevention (KCDC)44 (link). Vero cells were purchased from the American Type Culture Collection (ATCC CCL-81, Manassas, VA, USA) and cultured in Dulbecco’s minimal essential medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 2 (infection media) or 10% (growth media) v/v heat inactivated fetal bovine serum (FBS; Gibco) and 1% v/v penicillin/streptomycin (p/s; Gibco) in a humidified incubator of 5% CO2 atmosphere at 37 °C. OUA, CHQ and DIG were purchased from Sigma-Aldrich (St. Louis, MO, USA). REM was purchased from MedChemExpress (Monmouth Junction, NJ, USA). DIG tablets (inno.N, Seoul, Korea) were provided by Dr. Sang il Kim. OUA and DIG tablets were dissolved in H2O and CHQ, DIG and REM were dissolved in DMSO. Confluent Vero cells were infected at an MOI of 0.01 of BetaCoV/Korea/KCDC03/2020 in DMEM containing 2% FBS and 1% p/s for 1 h. Following incubation, cells were washed twice with phosphate buffered saline (PBS; Gibco) and incubated in the infection media. All experiments were performed in biosafety level 3 facilities according to KCDC guidelines.
6
Cell Viability and Luciferase Assays
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Cell culture flasks and 96-well plates were purchased from Sarstedt (Nürnbrecht, Germany). Cell culture media (Dulbecco’s Minimal Essential Medium (DMEM) and Eagle’s Minimal Essential Medium (EMEM) without phenol red) and supplements (fetal bovine serum (FBS), charcoal–dextran stripped FBS (CD-FBS), blasticidin S HCl and geneticin (G418)) were produced from Gibco and obtained from Thermo Fisher Scientific (Waltham/MA, USA). ZEN, α-ZEL, E2, (Z)-4-hydroxytamoxifen (4-OH-TAM) and sulforhodamine B (SRB) were purchased from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany). DAI, EQ, and GEN were obtained from Extrasynthese (Genay Cedex, France) whereas dimethly sulfoxide (DMSO), NaCl, KCl, Na2HPO4, Na2HPO4 * 2 H2O, and KH2PO4 were purchased from Carl Roth GmbH + Co. KG (Karlsruhe, Germany). The CellTiter-Blue® Cell Viability Assay Kit and ONE-Glo™ EX luciferase assay system were obtained from Promega Corporation (Madison/WI, USA).
7
HUVEC and U937 Cell Assays
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Human umbilical vein endothelial cells (HUVECs) and human monocytic cells (U937) were purchased from Pasteur Institute (Tehran, Iran). Dulbecco’s minimal essential medium (DMEM), RPMI 1640 cell culture medium, fetal bovine serum (FBS), trypsin-EDTA and [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylt- etrazolium bromide] (MTT) were obtained from Gibco. phorbol myristate acetate (PMA), lipopolysaccharide (LPS) from Escherichia coli 055:B5, dimethyl sulfoxide (DMSO), and dexamethasone were obtained from Sigma-Aldrich.
Fluvoxamine maleate was a gift from Abidi Pharmaceutical Co. (Tehran, Iran) and was dissolved in phosphate buffer saline (PBS) for cells and in isotonic saline for rats. Carrageenan (lambda) was purchased from Fluka Chemical (Switzerland) and was dissolved in isotonic saline.
Fluvoxamine maleate was a gift from Abidi Pharmaceutical Co. (Tehran, Iran) and was dissolved in phosphate buffer saline (PBS) for cells and in isotonic saline for rats. Carrageenan (lambda) was purchased from Fluka Chemical (Switzerland) and was dissolved in isotonic saline.
8
Zika Virus Propagation and Titration
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Vero cells (KCLB, Seoul, Korea) were grown in Minimum Essential Medium (MEM)-α supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), and Vero 76 cells (ATCC, Manassas, VA, USA) were grown in Dulbecco’s Minimal Essential Medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% FBS at 37 °C in 5% CO2 until they formed monolayers. Spodoptera frugiperda 9 (Sf9) and ExpiSf9 insect cells were cultured in Sf-900III SFM medium (Gibco, Grand Island, NY) at 27 °C in an orbital shaker. The ZIKV strains PRVABC59 (BEI Resources No. NR-50240) and MR766 (BEI Resources No. NR-50065) were obtained from BEI Resources. The ZIKV PRVABC59 strain was propagated in Vero cells and the ZIKV MR766 strain in Vero 76 cells at a multiplicity of infection of 0.01. ZIKV stocks were titrated by plaque assay using both cell lines and stored at –80 °C.
9
Optimized Lung Tissue Culture Protocol
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Dulbecco’s phosphate buffered salt solution (DPBS) was obtained from Lonza (Wuppertal, Germany). Dulbecco’s minimal essential medium (DMEM), supplemented with 100 U/mL penicillin and streptomycin, all obtained from Gibco (Life Technologies, Darmstadt, Germany) was used as medium for PCLS preparation. For infection experiments, the following medium—further referred to as ‘culture medium’ was used: Gibco Minimal Essential Medium (1× MEM, Life Technologies, Darmstadt, Germany) was supplemented with 2% penicillin/streptomycin (Sigma Aldrich, Munich, Germany), 2 mM l -glutamine and non-essential amino acids (1× NEAA; both Gibco, Life Technologies, Darmstadt, Germany). Low-gelling temperature agarose, protease inhibitor cocktail P1860, Triton X-100, and Earle’s Balanced Salt Solution (EBSS) were purchased from Sigma-Aldrich (Munich, Germany). Human rhinovirus 1b was obtained from Virapur (Lot J1323A, San Diego, CA, USA). The LDH Cytotoxicity Assay Kit was obtained from Roche (Mannheim, Germany). The Pierce™ BCA (bicinchoninic acid) total protein kit was purchased from Thermo Scientific/Life Technologies (Darmstadt, Germany).
10
Breast Cancer Cell Line Assay
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5-aminoimidazole carboxamide ribonucleotide (AICAR) was from Sigma-Aldrich, Poole, Dorset, UK. InSolution™ Akt Inhibitor VIII (Isozyme-Selective, Akt-1/2), was purchased from Calbiochem®, Watford, UK. Dulbecco's minimal essential medium (DMEM) was obtained from Gibco BRL, Paisley, UK and fetal calf serum (FCS) was from Autogenbioclean, UK Ltd. Phosphate-buffered saline and trypsin/EDTA were obtained from Sigma-Aldrich, Irvine, Ayrshire, UK. CellTiter 96® Aqueous One Solution cell proliferation assay was purchased from Promega, Madison, WI, USA. T25 and T75 tissue culture flasks were from Nunc™, Denmark and tissue culture sterile 6-, 24- and 96-well plates were from Costar®, USA. Anti-phospho-AMPK (Thr172) and anti-phospho-Akt (Ser473) antibodies were purchased from Cell Signaling Technology; Danvers, MA, USA. All blotting reagents were purchased from Bio-Rad® (CA, USA and München, Germany) except TBST and transfer buffer, which were prepared from their components. Leptin, human, recombinant expressed in E. coli (#L4146) was purchased from Sigma-Aldrich, GmbH Germany.
Breast cancer cell lines MCF-7 (p53+ and ER+), T47D (p53 mutant and ER+) and MDA-MB-231 (p53 mutant and ER−) were purchased from the European Collection of Cell Cultures (ECACC, Porton Down, UK).
Breast cancer cell lines MCF-7 (p53+ and ER+), T47D (p53 mutant and ER+) and MDA-MB-231 (p53 mutant and ER−) were purchased from the European Collection of Cell Cultures (ECACC, Porton Down, UK).
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