Equine dermal (ED) cells were grown in DMEM medium (PAN Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (FBS; PAN Biotech) and 100 U ml−1 penicillin (Roth, Karlsruhe, Germany) and 100 μg ml−1 streptomycin (Alfa Aesar, Kandel, Germany) in a 37°C incubator with 5% CO2 atmosphere. Cells were grown to confluency in a 100 mm tissue culture dish, washed with phosphate buffer saline (PBS), trypsinized with 0.25% trypsin supplemented with 2.5 μmol L−1 EDTA and counted in a Neubauer counting chamber.
Dmem medium
Manufactured by PAN Biotech
Sourced in Germany, United States
DMEM (Dulbecco's Modified Eagle's Medium) is a commonly used cell culture medium. It provides the essential nutrients, vitamins, and salts required for the growth and maintenance of a variety of cell types in in vitro cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by PAN Biotech (10 mentions)
Fetal bovine serum (fbs), by PAN Biotech (7 mentions)
Rpmi 1640 medium, by PAN Biotech (5 mentions)
Fetal calf serum (fcs), by PAN Biotech (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Fetal calf serum (fcs), by GE Healthcare (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Nocodazole, by Merck Group (2 mentions)
Dmem f12 50 50 medium, by PAN Biotech (2 mentions)
S trityl l cysteine, by Thermo Fisher Scientific (2 mentions)
Mycoalert mycoplasma detection kit, by Lonza (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by PAN Biotech (2 mentions)
Streptomycin, by PAN Biotech (2 mentions)
Collagen g, by Harvard Bioscience (2 mentions)
Dmem f12 medium, by PAN Biotech (2 mentions)
Penicillin, by Carl Roth (1 mentions)
Streptomycin, by Carl Roth (1 mentions)
31 protocols using dmem medium
1
Equine Dermal Cell Cultivation Protocol
2
Lentiviral Transduction of HEK293 Cells
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Human embryonic kidney (HEK) 293 cells (ATTC, VA, USA) were maintained in DMEM medium (PAN Biotech, Aidenbach, Germany) supplemented with 10% FBS (Anprotec, Bruckberg, Germany) and 1% penicillin-streptomycin (PAN Biotech, Aidenbach, Germany) at 37°C in a 5% CO 2 atmosphere. Stable cell lines expressing pRRL-CMV-EGFP-PRE-SIN, FUGW-mCherry-Annexin-A2
or FUGW-mCherry were developed by lentiviral infection of HEK 293 cells. Cells were incubated during 5 days with the virus and after three passages the infection rate was confirmed by microscopy (more than 90% of positive cells).
or FUGW-mCherry were developed by lentiviral infection of HEK 293 cells. Cells were incubated during 5 days with the virus and after three passages the infection rate was confirmed by microscopy (more than 90% of positive cells).
3
Cultivation and Transfection of RPE1, HEK293T, and U2OS Cells
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Human telomerase immortalized retinal pigment epithelium cells (hTERT-RPE1, ATCC) were cultured with Dulbecco’s modified Eagle’s Medium DMEM/F12 50/50 medium (Pan Biotech) supplemented with 10% fetal bovine serum (FBS, Life Technologies) and1% penicillin-streptomycin (Gibco). Human embryonic kidney (HEK293T, ATCC) and osteosarcoma epithelial (U2OS, ATCC) cells were cultured with DMEM medium (Pan Biotech) supplemented with10% FBS and 1% penicillin-streptomycin. All cell lines were authenticated by Multiplex Cell Line Authentication (MCA) and were tested for mycoplasma by MycoAlert Mycoplasma Detection Kit (Lonza). U2OS cells were transfected with the plasmids using Lipofectamine 2000 and according to the manufacturer’s instructions (Thermo Scientific). HEK293T cells were transfected with the plasmids using 1 mg/ml polyethylenimine, MW 25 kDa (PEI). For microtubule depolymerization experiments, cells were treated with 5 μg/ml nocodazole (Sigma-Aldrich) or vehicle (dimethyl sulfoxide) for 1 h at 37°C. For cell synchronization experiments, 5 μm (+)-S-trityl-L-cysteine (STLC) (Alfa-Aesar) was used for 16 h at 37°C.
4
Growth Kinetics of Cultured Cells
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For growth kinetic analyses 3 × 105 cells were seeded in triplicates in a 24-well plate in 500 μL DMEM medium (PAN-Biotech, Aidenbach, Germany) with supplements. After trypan blue staining, vital cells were counted manually every 24 h (6 time points: 0 h, 24 h, 48 h, 72 h, 96 h, and 168 h) in a Neubauer chamber.
5
Cell Line Cultivation and Lysis Protocol
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Cell line MV4-11 (ATCC: CRL-9591) was grown in RPMI 1640 medium (PAN Biotech). SW620 (from NCI60 panel), HeLa S3 (ATCC: CCL-2.2), and A549 (ATCC: CCL-185) were grown in DMEM medium (PAN Biotech). All media were supplemented with 10% FBS (PAN Biotech) and cell lines were internally tested for Mycoplasma contamination. Cells were lysed in lysis buffer (0.8% Igepal, 50 mM Tris-HCl pH 7.5, 5% glycerol, 1.5 mM MgCl2, 150 mM NaCl, 1 mM Na3VO4, 25 mM NaF, 1 mM DTT, supplemented with protease inhibitors (SigmaFast, Sigma) and phosphatase inhibitors (prepared in-house according to Phosphatase inhibitor cocktail 1–3 from Sigma-Aldrich)). The protein amount of cell lysates was determined by Bradford assay and adjusted to an Igepal concentration of 0.4% and protein concentration of 5 mg/mL.
6
Metabolomic Analysis Protocols and Reagents
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Methanol (MeOH), water, formic acid (Optima LC/MS grade) and glutamine were purchased from Thermo-Fisher Scientific (Illkirch, France). Isotope metabolite standards including 17α-Hydroxyprogesterone-d8 (2,2,4,6,6,21,21,21-d8), L-Thyroxine-13C6, Succinic acid-2,2,3,3-d4, Pyruvic acid-1-13C and DL-Alanine-15N with >98% purity were acquired from Sigma Aldrich (St. Quentin Fallavier, France) as well as oligomycin, carbonyl cyanide 4‐(trifluoromethoxy) phenylhydrazone (FCCP), antimycin A and aspartate. All antibodies (ab42364, EP1332Y, ab186695 and ab186696), the NAD/NADH and ATP Assay Kit (ab65348 and ab83355) were obtained from Abcam (Paris, France) and the Mitotracker® green from Molecular Probes (Oregon, USA). Tris-Glycine Gel was purchased from Life Technologies (Illkirch, France) and DMEM-F12 from Jacques Boy Institute of Biotechnology (Reims, France). The DMEM medium supplemented with FBS (fetal bovine serum) was acquired from PAN-biotech (Wimborne, UK) and the Seahorse XFe Base Medium from Agilent Technologies (Santa Clara, CA, USA).
7
Immortalized Retinal Epithelial Cell Culture
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Human telomerase immortalized retinal pigment epithelium cells (hTERT-RPE1, ATCC, CRL-4000) were cultured with Dulbecco's modified Eagle's Medium DMEM/F12 50/50 medium (Pan Biotech,Cat. # P04-41250) supplemented with 10% Fetal Bovine Serum (FBS, Life Technologies, Ref. # 10270-106, Lot # 42Q5283K) and1% penicillin-streptomycin (GIBCO, Cat. # 1540-122). Human embryonic kidney (HEK293T, ATCC, CRL-3216), and osteosarcoma epithelial (U2OS, ATCC, HTB-96) cells were cultured with DMEM medium (Pan Biotech, Cat. # P04-03590) supplemented with10% FBS and 1% penicillin-streptomycin. All cell lines were authenticated by Multiplex Cell Line Authentication (MCA) and were tested for mycoplasma by MycoAlert Mycoplasma Detection Kit (Lonza). U2OS cells were transfected with the plasmids using Lipofectamine 2000 and according to the manufacturer's instructions (Thermo Scientific Scientific). HEK293T cells were transfected with the plasmids using 1mg/ml polyethylenimine, MW 25 kDa (PEI). For microtubule depolymerization experiments, cells were treated with 5 µg/ml nocodazole (Sigma-Aldrich, Cat. #M1404) or vehicle (dimethyl sulfoxide) for one hour at 37 C. For cell synchronization experiments, 5 µM (+)-Strityl-L-cysteine (STLC) (Alfa-Aesar, Cat. #2799-07-7) was used for 16 h at 37 C.
8
HEK 293T Cell Transfection Protocol
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Human embryonic kidney (HEK 293T) cells were cultured and maintained in DMEM medium (PAN Biotech), supplemented with 10% FCS (PAN Biotech) and 1% Penicillin-Streptomycin (PAN Biotech) at 37°C with 5% CO2. HEK 293 transfections were performed by incubating cells with Polyethylenimine (PEI) (100 mg/mL PEI diluted 1:100 in 150mM NaCl) and the respective plasmids (10 µg for 10 cm dishes, 2 µg for 6-well plates) for 6-8 hours into fresh medium. Then, the medium was discarded and fresh medium was added.
9
Skin Biopsy Fibroblast Culture Protocol
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Skin biopsy sample was obtained from the University Hospital Regensburg (Ethic vote number 14101 0001) and given the designation Re5. The skin biopsy was put whole on a Primaria cell culture dish (#353801, Corning, Germany), containing a single drop of DMEM-Cipro medium (DMEM medium (P04-01548, PAN Biotech, Germany) containing 10% FCS (AC-SM-0161, Anprotec, Germany), 1 g/L (5.5 mM) glucose (G8644, Sigma-Aldrich/Merck, Germany), 1 mM pyruvate (#11360070, Thermo Fischer, Germany), 2 mM l-glutamine (G7513, Sigma-Aldrich/Merck, Germany) and 1% Ciprofloxacin (200 mg/100 ml infusion solution, 64,689.00.00, Fresenius Kabi, Germany)) and incubated for 30 min at 37 °C to ensure that the skin sample sticks on the dish surface. Afterward, 3 ml DMEM-Cipro 1 3
was added to the skin sample. It was cultivated at 37°, 5% CO 2 until fibroblast-outgrowth was enough for a transfer to a T25 cell culture flask where they were cultured in DMEM without Ciprofloxacin, and used for further experiments.
Before irradiation, the cells were seeded at density of 5 × 10 4 cells per well on a 6-well plate in DMEM with and without pyruvate (1 mM/0 mM) and incubated overnight.
was added to the skin sample. It was cultivated at 37°, 5% CO 2 until fibroblast-outgrowth was enough for a transfer to a T25 cell culture flask where they were cultured in DMEM without Ciprofloxacin, and used for further experiments.
Before irradiation, the cells were seeded at density of 5 × 10 4 cells per well on a 6-well plate in DMEM with and without pyruvate (1 mM/0 mM) and incubated overnight.
10
Cell Culture Protocols for Diverse Cell Lines
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The human keratinocyte cell line (HaCaT), COS7 and ovarian adenocarcinoma cell lines were cultured in DMEM medium (PAN Biotech No. P04–03550) with high glucose (4,5 g/dL), penicillin/streptomycin, glutamate and 5% fetal bovine serum. The ovarian adenocarcinoma cell lines were a kind gift from Prof. Baki Akgül (Institute of Virology, Medical Faculty, University of Cologne).34,35 (link) Cells were grown in a humidified atmosphere and 5% CO2. Subcutaneous fibroblasts from a patient suffering from EDMD5 were a gift from Prof. Wehnert (Institute of Human Genetics and Interfaculty Institute of Genetics and Functional Genomics, University Greifswald).24 (link) The fibroblasts (Passage 13–17) were cultivated in Eagle's MEM (Gibco, No. 31095–029), 20% fetal bovine serum, 7.5% Bicarbonate, 1 M HEPES, penicillin-streptomycin and glutamate.
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