The 2 patient-derived SyS cell lines harboring the SSX1 translocation (Yamato-SS and Aska-SS) were generously provided by K. Itoh. The 2 patient-derived SyS cell lines harboring the SSX2 translocation (SYO-1 and CME-1) were generously provided by A. Kawai and C. Lanzi, respectively. Yamato-SS and Aska-SS were cultured in DMEM 4.5 g/l glucose (Lonza), 1 mM sodium pyruvate (PAA) medium, SYO-1 in DMEM 4.5 g/l glucose, and CME-1 in RPMI-1640 medium (Lonza). Media were supplemented with 10 % fetal bovine serum (FBS, Gibco) and 1 % penicillin/streptomycin (Lonza). All cells were maintained at 37 °C in a humidified atmosphere of 5 % CO2/95 % air.
Dmem 4.5 g l glucose
Manufactured by Lonza
Sourced in France
DMEM 4.5 g/l glucose is a cell culture medium that provides essential nutrients for the growth and maintenance of various cell types. It contains 4.5 grams per liter of glucose as the primary energy source. This product is suitable for use in a wide range of cell culture applications.
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Lab products found in correlation
L glutamine, by Lonza (3 mentions)
Penicillin streptomycin, by Lonza (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Lonza (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
Cholera toxin, by Thermo Fisher Scientific (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Adenine, by Merck Group (1 mentions)
Transferrin, by Merck Group (1 mentions)
Hepes buffer, by Lonza (1 mentions)
Hyclone fetalclone 2 serum, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
Ham s f12, by Lonza (1 mentions)
Rock inhibitor y 27632, by Merck Group (1 mentions)
Mitomycin c, by Merck Group (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Gelatin, by Merck Group (1 mentions)
Mercaptoethanol, by Merck Group (1 mentions)
9 protocols using dmem 4.5 g l glucose
1
Synovial Sarcoma Cell Line Culture
2
Culturing Human Epidermal Keratinocytes
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Normal human epidermal keratinocytes, strain NHEK-131 (GIBCO-Invitrogen, Paisley, UK), were derived from a pool of a minimum of three neonatal foreskins and obtained at 6.8 mean population doublings (MPDs). Keratinocytes were cultured at 37°C in a 10% CO2/90% air with lethally irradiated 3T3 feeder cells in flavin-Adenine enriched medium (FAD-). FAD- consists of 3 parts DMEM 4.5 g/L glucose (Lonza, Slough, UK), 1 part Ham’s F12 (Lonza), 10% (v/v) Hyclone Fetalclone II serum (Fisher Scientific, Loughborough, UK), 20 mM HEPES buffer (Lonza), 100 U/ml penicillin, 100 U/ml streptomycin (Lonza) and 2 mM L-Glutamine (Lonza), supplemented with 1.8 × 10-4 M Adenine (Sigma-Aldrich, Poole, Dorset, UK), 5 μg/ml insulin (Sigma-Aldrich), 5 μg/ml transferrin (Sigma-Aldrich), 0.4 μg/ml hydrocortisone (Sigma-Aldrich) and 8.4 ng/ml cholera toxin (Fisher Scientific, Loughborough, UK). Medium was replenished every third or fourth day with FAD+ complete medium, which consists of FAD- supplemented with 10 ng/ml of epidermal growth factor (Sigma-Aldrich).
3
Protein Induced Pluripotent Stem Cell Culture
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Protein induced pluripotent stem cells (piPS, System Biosciences, USA), were cultured on Gelatin (Sigma-Aldrich, St. Louis, MO, USA) coated dishes with feeder layer of mitomycin C (Sigma-Aldrich, USA) inactivated mouse embryonic fibroblasts (MEFs) in serum-free iPS medium DMEM/F12 supplemented with 20% KSR, 2 mM GLUTAMAX, 100 µM non-essential amino acids, 100 U/mL/100 µg/mL Penicillin/Streptomycin, 10 ng/mL bFGF (all from ThermoFisher Scientific, USA) and 100 µM -mercaptoethanol (Sigma-Aldrich). The medium was changed every day. When the culture reached about 80% confluence, iPS cells were subcultured with Accutase (Lonza, Basel, Switzerland) in proportion 1:4–1:10 and seeded onto fresh feeder layer in presence of 10 µM ROCK inhibitor Y-27632 (Sigma-Aldrich). For immunocytochemical stainings, iPS cells were cultured in feeder-free conditions on growth factor-reduced Matrigel (Corning, New York, NY, USA) coated dishes in MEF-conditioned iPS medium. MEFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM; 4.5 g/L glucose; Lonza), supplemented with 10% v/v FBS, 2 mM L-glutamine and 100 U/mL/100 µg/mL Penicillin/Streptomycin antibiotics solution (all from ThermoFisher Scientific, Waltham, MA, USA).
4
Culturing Human Colon Cancer Cells
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SW48, Caco-2 and Colo320 human colon cancer cell lines were obtained from the American Type Culture Collection (ATCC). Cells were grown in DMEM 4, 5 g/L glucose (Lonza, Levallois, France) supplemented with 10% (vol/vol) fetal bovine serum (FBS; Lonza) in an atmosphere of 95% air and 5% CO2 at 37°C.
5
Culturing Colorectal Cancer Cell Lines
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The human colorectal carcinoma HCT116, colorectal adenocarcinoma HT29, HCT8, SW480, SW620, LoVo and SW48 cell lines were obtained from the American Type Culture Collection (ATCC). Some cell lines were grown in RPMI 1640 with ultraglutamine (Lonza, Levallois, France) (HCT116, HT29, HCT8, SW480) or in DMEM 4.5g/L glucose (Lonza) (SW620, LoVo, SW48) supplemented with 10% (vol/vol) fetal bovine serum (FBS; Lonza). The human normal mucosa cell line NCM460 was obtained from INCELL corporation LLC (San Antonio, TX) and was grown in M3Base medium (INCELL corporation LLC) supplemented with 10% FBS. All cell lines were grown in an atmosphere of 95% air and 5% CO2 at 37°C.
6
Cell Culture Protocol for HEK 293, HeLa, and NHDF
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HEK 293 cells were cultured in Dulbecco’s modified Eagle medium with high glucose (DMEM 4.5 g/l glucose, Lonza). The HeLa and NHDF cells were cultured in minimum essential medium alpha (MEMα, Lonza). All media were supplemented with 10 % fetal bovine serum (Sigma), GlutaMAX supplement (Gibco), antibiotics, and antimycotics (Gibco). The HEK 293 and HeLa cell lines were purchased from the Cell Line Collection of the Polish Academy of Sciences, Institute of Immunology and Experimental Therapy in Wroclaw. The NHDF cell line was purchased from Lonza.
7
Rat L6 myoblasts differentiation
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Rat L6 myoblasts (ATCC, Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM 4.5 g/l glucose, Lonza, Basel, Switzerland) supplemented with 10% FBS (Hyclone, Pasching, Austria), 2 mM L-glutamine (Lonza) and 1% penicillin/streptomycin (Lonza) at +37°C in a humidified atmosphere of 5% CO
2. The cells were seeded in multiwell plates at 2 × 10
4 cells/cm
2 one day before starting the differentiation. Myoblasts were differentiated into myotubes by switching into α-MEM media (Gibco, Paisley, UK) supplemented with 2% horse serum (Gibco) and 1% penicillin/streptomycin. GR24 (3E,3aR,8bS)-3-[[(2 S)-4-methyl-5-oxo-2H-furan-2-yl] oxymethylidene]-4,8b-dihydro-3aH-indeno[1,2-b]furan-2-one, Chiralix, Nijmegen, Netherlands) was dissolved in DMSO. The control samples had equivalent DMSO concentration.
2. The cells were seeded in multiwell plates at 2 × 10
4 cells/cm
2 one day before starting the differentiation. Myoblasts were differentiated into myotubes by switching into α-MEM media (Gibco, Paisley, UK) supplemented with 2% horse serum (Gibco) and 1% penicillin/streptomycin. GR24 (3E,3aR,8bS)-3-[[(2 S)-4-methyl-5-oxo-2H-furan-2-yl] oxymethylidene]-4,8b-dihydro-3aH-indeno[1,2-b]furan-2-one, Chiralix, Nijmegen, Netherlands) was dissolved in DMSO. The control samples had equivalent DMSO concentration.
8
Bmpr1b Expression in NIH/3T3 Cells
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NIH/3 T3 cells were seeded into a 24-well plate in growth medium on cover glasses (Marienfeld). 24 h later Bmpr1b expression vectors were transfected into the NIH/3 T3 cells using Lipofectamine 2000 (Invitrogen, Life Technologies) following the manufacturer´s instructions. Another 24 h later cells were incubated under serum free conditions (DMEM 4.5 g/l glucose (Lonza), 2 mM L-glutamine (Lonza)) for 1 h, subsequently fixed with 4 % paraformaldehyde in PBS and blocked in PBS containing 10 % FBS superior (Biochrom) over night. Non-permeated cells were incubated with a rabbit anti-HA antibody (H6908, Sigma-Aldrich; diluted 1:100 in PBS containing 10 % FBS superior (Biochrom)) for 1 h. After washing with PBS the cells were incubated with the secondary antibody anti-rabbit-Alexa Fluor 488 (A11008, Molecular Probes Life Technologies) and with DAPI (Invitrogen, Life Technologies) for 1 h. The cover glasses containing stained cells were mounted on microscope slides (‘SuperFrost Plus’, Menzel) using Fluoromount-G (Southern Biotech). Confocal microscopy was performed using Zeiss Axio Imager.M2 equipped with a LSM700 confocal module (Carl Zeiss, 63-fold magnification).
9
Isolation and Culture of Human Oral Fibroblasts
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The normal human oral fibroblast line NHOF-1 fibroblast cells were obtained from a buccal mucosa biopsy, under ethical approval of the Dental Teaching Hospital, Peradeniya, Sri Lanka, following informed patient consent (Ethical Clearance Certificate No. FDS-ERC/2008/02/TIL) [23] . The NHOF-1 were tested for mycoplasma following isolation with the MycoplsmaAlert ® (Lonza, Switzerland) and found to be negative, and re-tested at the time of use. The cells were cultured and maintained at 37 o C in a humidified atmosphere of 10% CO2/90% air in Dulbecco's Modified Eagle's Medium (DMEM), 4.5g/l glucose (Lonza) supplemented with 10% vol/vol hyclone II fetal bovine serum (FBS -Thermoscientific), penicillin, streptomycin 50units/ml (Life Technologies) and 2mM glutamine (Life Technologies).
Cells were sub-cultured and prepared for experiments by washing once with 0.02% EDTA (ethylenediaminetetraacetic acid) in calcium-and magnesium-free phosphate buffered saline (PBS) and then incubating with 0.01% trypsin/0.01% EDTA in PBS at 37 o C, until the cells detached. Following this, the trypsin/EDTA mixture was neutralized with three times the volume of DMEM/FBS medium, and the cells counted in a haemocytometer prior to resuspension, dilution and plating for the experiments.
Cells were seeded in a 96 well plate at a density of 9.32 x 10 3 /cm 2 in each well.
Cells were sub-cultured and prepared for experiments by washing once with 0.02% EDTA (ethylenediaminetetraacetic acid) in calcium-and magnesium-free phosphate buffered saline (PBS) and then incubating with 0.01% trypsin/0.01% EDTA in PBS at 37 o C, until the cells detached. Following this, the trypsin/EDTA mixture was neutralized with three times the volume of DMEM/FBS medium, and the cells counted in a haemocytometer prior to resuspension, dilution and plating for the experiments.
Cells were seeded in a 96 well plate at a density of 9.32 x 10 3 /cm 2 in each well.
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