McCoy’s 5A and Dulbecco’s Modified Eagle Medium-F12 (DMEM-F12) were obtained from Thermo Fisher Scientific (Waltham, MA). Hygromycin B, zeocin, blasticidin hydrochloride, and doxycycline hydrochloride also were from Thermo Fisher Scientific. HyClone fetal bovine serum was obtained from GE Healthcare Life Sciences (Pittsburgh, PA). Zinc chloride was obtained from Sigma (St. Louis, MO).
Dulbecco s modified eagle medium f12 dmem f12
Manufactured by Thermo Fisher Scientific
Sourced in United States, Italy
Dulbecco's Modified Eagle Medium/F12 (DMEM/F12) is a cell culture medium used to support the growth and maintenance of a wide variety of cell types. It is a basal medium that provides essential nutrients, amino acids, vitamins, and other components required for cell proliferation and survival.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Hygromycin b, by Thermo Fisher Scientific (3 mentions)
Zeocin, by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Blasticidin, by Thermo Fisher Scientific (2 mentions)
Fluo 4, by Thermo Fisher Scientific (2 mentions)
Tryple enzyme, by Thermo Fisher Scientific (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Doxycycline hydrochloride, by Thermo Fisher Scientific (1 mentions)
Blasticidin hydrochloride, by Thermo Fisher Scientific (1 mentions)
Zinc chloride, by Merck Group (1 mentions)
Mccoy s 5a, by Thermo Fisher Scientific (1 mentions)
Hyclone fetal bovine serum, by GE Healthcare (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
15 protocols using dulbecco s modified eagle medium f12 dmem f12
1
Cell Culture Media Procurement
2
Lung Adenocarcinoma Tissue and Cell Lines
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Three fresh specimens of human early lung adenocarcinoma, seven frozen specimens of human lung adenocarcinoma in situ, and six frozen specimens of human invasive lung adenocarcinoma, together with paired samples of adjacent normal lung tissue, were obtained from patients who had undergone surgical resection at Tsukuba University Hospital (Ibaraki, Japan). Informed consent for study of their materials had been obtained from all of the patients. The A549 and PC‐9 human lung adenocarcinoma cell lines were purchased from RIKEN Cell Bank (Ibaraki, Japan). A549 was maintained in Dulbecco's modified Eagle medium/F12 (DMEM/F12) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Corning, NY, USA). PC‐9 was maintained in RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% FBS. All cells were cultured in a 5% CO2 incubator at 37°C.
3
Establishing GBM Cell Line Cultures
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Early Passage New Zealand Glioblastoma cell lines. NZB11, NZB12, NZB13, and NZB19 cell lines were provided in collaboration with the Auckland Cancer Society Research Centre. The cells were cultured at 37 °C in 5% O2, 5% CO2 as adherent monolayers on uncoated 25 cm2 culture flasks until 80–90% confluent in alpha-Minimal Essential Medium (MEM) (ThermoFisher, Auckland New Zealand) supplemented with serum (5% FBS (Moregate)) and 1× insulin-transferrin-selenium (ITS) (Sigma) (herein referred to as serum-derived cultures).
Adherent GBM Cancer Stem Cell-Like Cells (gCSC). Adherent gCSCs were expanded for experimental use and routinely cultured at 37 °C in 5% O2, 5% CO2. NZB11, NZB19, NZB12, and NZB13 cell lines were cultured in 25 cm2 culture flasks coated with 10 μg/mL laminin (ThermoFisher, Auckland, New Zealand) in Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12) (ThermoFisher, Auckland, New Zealand) supplemented with 0.5× B-27 minus vitamin A (ThermoFisher, Auckland New Zealand), 0.5× N2 supplement (ThermoFisher, Auckland New Zealand), 20 ng/mL bFGF (Peprotech), and 20 ng/mL EGF (Novus Biologicals) (herein referred to as gCSC cultures). The phenotypes of the serum-derived and gCSC cultures have been detailed extensively [14 (link)].
Adherent GBM Cancer Stem Cell-Like Cells (gCSC). Adherent gCSCs were expanded for experimental use and routinely cultured at 37 °C in 5% O2, 5% CO2. NZB11, NZB19, NZB12, and NZB13 cell lines were cultured in 25 cm2 culture flasks coated with 10 μg/mL laminin (ThermoFisher, Auckland, New Zealand) in Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12) (ThermoFisher, Auckland, New Zealand) supplemented with 0.5× B-27 minus vitamin A (ThermoFisher, Auckland New Zealand), 0.5× N2 supplement (ThermoFisher, Auckland New Zealand), 20 ng/mL bFGF (Peprotech), and 20 ng/mL EGF (Novus Biologicals) (herein referred to as gCSC cultures). The phenotypes of the serum-derived and gCSC cultures have been detailed extensively [14 (link)].
4
Bhas 42 Cell Isolation and Culture
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The reagents used for cell isolation, culture, and analysis were as follows: Bhas 42 cell from JCRB cell bank (Japan); phosphate-buffered saline (PBS) from Thermo Fisher Scientific (USA); 0.25w/v% Trypsin Solution with Phenol Red from FUJIFILM Wako Pure Chemical (Japan); Minimum essential medium (MEM) from Thermo Fisher Scientific (USA); Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12) from Thermo Fisher Scientific (USA); Fetal bovine serum (FBS) from Moregate biotech (Australia); Giemsa's azur eosin methylene blue solution from Merck (USA); Petri Dish for Cell/Tissue Culture 90Φ (deep) from Sumitomo Bakelite (Japan); Multiwell Plate for Cell/Tissue Culture 6F with lid from Sumitomo Bakelite (Japan); Dimethyl sulfoxide for molecular biology (DMSO) from Sigma-Aldrich (USA); Methanol from FUJIFILM Wako Pure Chemical (Japan); PMA for use in molecular biology applications (TPA), ≥ 99% (HPLC) from Sigma-Aldrich (USA); Lithocholic acid (LCA) from Tokyo Chemical Industry (Japan); 1- Nitropyrene (1-NP) from Tokyo Chemical Industry (Japan).
5
Quantitative Analysis of Neuronal Signaling Molecules
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All chemicals were of analytical grade. The following chemicals were from Merck Sigma-Aldrich (Milan, Italy): QA (Cat # P63204), L-glutamate (G1626), glycine (G7403), and probenecid (Cat # P8761). Ketamine was from Merial (Cat # Imalgene 1000). Esmethadone hydrochloride (CAS: 15284-15-8) was from SpecGx (Webster Groves, MO, USA; Cat # 8514). Fluo-4 (F14202) was from Invitrogen (Milan, Italy). The following cell culture chemicals were from Gibco Life Technologies (Milan, Italy): Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12; Cat # 21331), heat-inactivated fetal bovine serum (Cat # 10500), penicillin-streptomycin (Cat # 15140), L-glutamine (Cat # 25030), MEM nonessential amino acids (Cat # 11140), blasticidin (Cat # A11139), G418 (Cat # 10131), Zeocin™ (Cat # R250), and TrypLE™ enzyme (Cat # 12604). Hygromycin B (Cat # 10687) was from Invitrogen. Gentamicin sulphate concentrations were expressed in μg/mL as gentamicin sulphate was a mixture of three major components designated as C1, C1a, and C2. Calculated and corrected for purity, the molecular weight was 681.58 Da, such that 100 µg/mL corresponded to 146.7 µM gentamicin sulphate.
6
Isolation and Expansion of Umbilical Cord-Derived Cells
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UC (n = 12) was obtained from normal full-term-delivery women. Briefly, after disinfection with 75% ethanol, the fresh UC was transferred to PBS where excess blood on the surface was removed. After removal of blood vessels, 2 cm of UC was sliced into very small pieces followed by treatment with 0.1% collagenase II (Gibco, USA), containing antibiotic solution (100 U/mL penicillin and 100 μg/mL streptomycin; CSPC, China) in Dulbecco's Modified Eagle Medium/F12 (DMEM/F12) (Gibco) overnight at 37°C. The cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum (Hyclone, China) in 5% CO2 in a 37°C incubator for 3–4 days to allow cells to adhere. The adherent cells formed colonies and grew rapidly, exhibiting spindle-shaped morphology. The medium was replaced every three days. When the cells reached to 60–80% confluence, they were harvested with trypsin-EDTA solution (Neuronbc, China) and subcultured at a density of 3–6×103 cells/cm2 [38 (link)]. Amplification of pluripotent stem cell markers, such as Oct-4 and Rex-1, was used to demonstrate primitive properties of the cultured cells, as described previously [39 (link)].
7
Chrysophanic Acid Modulates Apoptosis Pathways
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Chrysophanic Acid (CHR) and CDDP were purchased from Sellect (Houston, TX, United States). Dulbecco’s modified Eagle medium F12 (DMEM/F12), Trypsin-EDTA (0.25%), and Antibiotic-Antimycotic solution were purchased from Gibco (United States). Fetal bovine serum (FBS) was supplied by CellMax (Beijing, China). Other consumable materials for cell culture were acquired from Greiner Bio-One (Essen, Germany). F4/80 (GB11027; Servicebio, Wuhan, China), c-caspase3 (GB11532; Servicebio, Wuhan, China), and Lipocalin-2/neutrophil gelatinase-associated lipocalin (NGAL; ab125075; abcam, Shanghai, China) antibody were used for immunohistochemistry (IHC). Antibody of Lipocalin-2/NGAL (NBP1-45682) was provided by Novus Biologicals (Littleton, United States). Antibodies of Bax (50599-2-Ig,) and Bcl2 (26593-1-AP) were provided by Proteintech (Wuhan, China). Antibodies of cleaved Caspase 3 (cst9664), NF-κB p65 (cst6956), Phospho-NF-κB p65 (cst 3033), IκBα (cst4814), phospho-IκBα (cst9246), IKKβ (cst2678), phospho-IKKα/β (cst2697), and GAPDH (cst2118) were provided by cell signaling technology Inc. (MSA, United States).
8
Antagonist-based Screening of Neuroprotective Compounds
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All chemicals were of analytical grade. Dextromethorphan and (±)-ketamine were from Merck Sigma-Aldrich (Milan, Italy). Memantine and (+)-MK-801 were from Bio-Techne/Tocris (Milan, Italy). REL-1017 (dextromethadone HCl) was from SpecGx (Webster Groves, MO, USA).
Cell culture consumables were from Gibco Life Technologies (Milan, Italy), including Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12; Cat # 21331), heat-inactivated fetal bovine serum (FBS; Cat # 10500), penicillin-streptomycin (Cat # 15140), L-glutamine (Cat # 25030), MEM nonessential amino acids (NEAAs; Cat # 11140), blasticidin (Cat # A11139), G418 (Cat # 10131), Zeocin™ (Cat # R250), and TrypLE™ enzyme (Cat # 12604). Hygromycin B (Cat# 10687) and Fluo-4 (F14202) were from Invitrogen (Waltham, MA, USA).
Cell culture consumables were from Gibco Life Technologies (Milan, Italy), including Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12; Cat # 21331), heat-inactivated fetal bovine serum (FBS; Cat # 10500), penicillin-streptomycin (Cat # 15140), L-glutamine (Cat # 25030), MEM nonessential amino acids (NEAAs; Cat # 11140), blasticidin (Cat # A11139), G418 (Cat # 10131), Zeocin™ (Cat # R250), and TrypLE™ enzyme (Cat # 12604). Hygromycin B (Cat# 10687) and Fluo-4 (F14202) were from Invitrogen (Waltham, MA, USA).
9
Isolation of Adipose-Derived Stem Cells
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Subcutaneous adipose tissue was harvested under sterile conditions and placed in 1x PBS (Gibco, Grand Island, NY, USA) with 10% penicillin/streptomycin (Gibco). It was then immediately taken out. Under a stereo-dissecting microscope, the subcutaneous adipose tissues were dissected away from the connective tissue and blood vessels with sterile forceps and scissors. The adipose tissue was cut into small pieces. The minced tissue blocks were digested with 0.25% collagenase I (Sigma, Kawasaki City, Japan) and 0.1% dispase II (Roche, Basel, Switzerland) for 1–2 h at 37 °C in a water bath. The digested mixture was filtered through an 80-μm cell strainer and centrifuged at 1500g for 10 min, and the supernatant was discarded. The cells were resuspended in complete growth medium (Dulbecco’s modified Eagle medium/F-12 (DMEM/F-12), Gibco) with 15% fetal bovine serum (Gibco) and 1% penicillin/streptomycin) and seeded in 60 mm Petri dishes, and the medium was changed every two days.
10
Derivation and Cultivation of Tenon's Capsule Fibroblast-Derived Induced Pluripotent Stem Cells
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The human Tenon’s capsule fibroblasts (HTFs)-derived iPSCs (TiPSCs) used in this paper have been established before [28 (link)]. Tenon’s capsule samples were obtained with written informed consent in adherence with the Declaration of Helsinki and the ARVO statement on human subjects, and with approval from the institutional review board at ZhongShan Ophthalmic Center (ID: 20140311). HTFs were reprogrammed to TiPSCs by retroviral transduction of OCT4, SOX2, C-Myc and KIF4. The resulting TiPSC colonies were maintained on mitomycin C-inactived mouse embryonic fibroblast (MEF) feeder cells in hESC medium as previously described: Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12, 1:1; Gibco; Carlsbad, CA) supplemented with 20% knockout serum replacement, 0.1 mM nonessential amino acids, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 50 units/ml penicillin, 50 μg/ml streptomycin (all from Gibco), 10 ng/ml basic fibroblast growth factor (bFGF; PeproTech, Rocky Hill, NJ), and 5 μg/ml Plasmocin (InvivoGen, San Diego, CA). The medium was changed daily. For further culture, morphologically identifiable differentiated cells were mechanically removed, and TiPSCs were passaged every 5 to 6 days. All of the cells used in this study were cultured in a 37 °C humidified incubator containing 5% CO2.
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