Hyclone dmem

Manufactured by GE Healthcare

Sourced in Germany, United States, New Zealand

HyClone DMEM is a cell culture media formulation developed by GE Healthcare. It is designed to support the growth and maintenance of a variety of cell lines in vitro. HyClone DMEM provides the necessary nutrients, vitamins, and other components required for cell proliferation and survival.

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Lab products found in correlation

Penicillin streptomycin, by Merck Group (4 mentions) Pen strep, by Thermo Fisher Scientific (4 mentions) Non essential amino acids (neaa), by Merck Group (3 mentions) Hepes, by Merck Group (3 mentions) Pro293a medium, by Lonza (3 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Penicillin, by Thermo Fisher Scientific (3 mentions) Streptomycin, by Thermo Fisher Scientific (3 mentions) Bca kit, by Thermo Fisher Scientific (3 mentions) Arpe 19, by American Type Culture Collection (2 mentions) Fetal calf serum (fcs), by Merck Group (2 mentions) L glutamine, by Thermo Fisher Scientific (2 mentions) Padvantage vector, by Promega (1 mentions) Freestyle max, by Thermo Fisher Scientific (1 mentions) Non essential amino acids (neaa), by PAN Biotech (1 mentions) Hepes, by PAN Biotech (1 mentions) Rpmi 1640, by Merck Group (1 mentions) Ht22 cells, by Merck Group (1 mentions) Rpmi 1640 media, by Merck Group (1 mentions) Penicillin streptomycin, by American Type Culture Collection (1 mentions)

17 protocols using hyclone dmem

Cultivation and Treatment of Ocular Cell Lines

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The uveal melanoma cell line OMM-1 [42 (link)] was provided by Dr. Sarah Coupland. RPMI 1640 (Merck, Darmstadt, Germany) was used for cultivation (with 10% fetal calf serum (Linaris GmbH, Wertheim-Bettingen, Germany) and 1% penicillin/streptomycin (Merck, Darmstadt, Germany)).
The human RPE cell line ARPE-19 [43 (link)] was purchased from ATCC. The cultivation medium was HyClone DMEM (GE Healthcare, München, Germany) with 1% penicillin/streptomycin, 1.2% HEPES, 1% non-essential amino acids (all from Merck, Darmstadt, Germany), and 10% fetal calf serum, as previously described [11 (link)].
Primary porcine RPE cells were prepared as described before [30 (link),44 (link)]. RPE cells were detached from porcine eyes by trypsin incubation and cultivated in HyClone DMEM (GE Healthcare, München, Germany) supplemented with penicillin/streptomycin (1%), HEPES (2.5%), non-essential amino acids (1%) (all Merck, Darmstadt, Germany), and 10% fetal calf serum.
ARPE-19 and RPE were treated at confluence, and OMM-1 cells were treated at subconfluence.

Dörschmann P., Kopplin G., Roider J, & Klettner A. (2019). Effects of Sulfated Fucans from Laminaria hyperborea Regarding VEGF Secretion, Cell Viability, and Oxidative Stress and Correlation with Molecular Weight. Marine Drugs, 17(10), 548.

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Transient Cell Transfection Protocol

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For assays with transiently-transfected cells, cells were generated by bulk transfection. At the time of transfection, 293-F or 293F.GCaMP6s.blast cells were diluted to 1 million/mL with FreeStyle-293 medium. Transfection was performed by combining equal amounts of pAdvantage vector (Promega Corp., Madison WI) and target DNA. Total DNA was 50 µg per 40 mL transfection. The transfection reagent was Freestyle MAX (Invitrogen). Cell viability at 24 hours was above 80% for transfections to be considered successful. Cells were spun down and resuspended in HyClone™ DMEM (GE Healthcare, Little Chalfont, UK) supplemented with 10% fetal bovine serum (FBS; Omega Scientific, Tarzana, CA), then seeded into 384-well poly-D-lysine-coated plates at 10–15k cells/well at 16–24 hours after transfection, and used for assays 24–48 hours after transfection.

Wu N., Nishioka W.K., Derecki N.C, & Maher M.P. (2019). High-throughput-compatible assays using a genetically-encoded calcium indicator. Scientific Reports, 9, 12692.

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Isolation and Culture of Primary Porcine RPE Cells

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The preparation of primary RPE cells from pigs’ eyes was conducted as previously established [58 (link),59 (link)]. In brief, after removing the cornea, retina and vitreous, cells were detached with trypsin and ethylenediaminetetraacetic acid (EDTA). The cultivation medium was HyClone DMEM (GE Healthcare, Chicago, IL, USA) with addition of 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA), 2.5% HEPES (PAN-Biotech, Aidenbach, Germany), 10% fetal calf serum (Linaris GmbH, Wertheim-Bettingen, Germany) and 1% non-essential amino acids (PAN-Biotech). Cells were incubated at 37 °C and 5% CO₂. RPE cells were treated two weeks after preparation, after they reached confluence. The Norwegian LH fucoidan Fuc1 (1548.6 kDa) from past studies was used [5 (link),13 (link)], provided by Alginor ASA. The extraction and solving procedure as well as chemical characteristics such as monosaccharide composition, sulfate content, molecular weight and structure was described by Dörschmann et al. 2019 [13 (link)].

Dörschmann P., Kopplin G., Roider J, & Klettner A. (2023). Interaction of High-Molecular Weight Fucoidan from Laminaria hyperborea with Natural Functions of the Retinal Pigment Epithelium. International Journal of Molecular Sciences, 24(3), 2232.

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Cell Culture Protocols for Various Cell Lines

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The human RPE cell line ARPE-19 [54 (link)] (ATCC, RRID: CVCL_0145) was cultured in 96-well plates (15,000 cells/well) in HyClone DMEM (GE Healthcare) containing 10% fetal calf serum (Linaris GmbH, Wertheim-Bettingen, Germany), 1% penicillin/streptomycin (Merck), 2.5% HEPES (Merck) and 1% non-essential amino acids (Merck). The uveal melanoma cell line OMM-1 (RRID: CVCL_6939) [55 (link)], provided by Dr. Sarah Coupland, was cultivated in 96-well plates (15,000 cells/well) in RPMI 1640 media (Merck), supplemented with 10% fetal calf serum (Linaris GmbH) and 1% penicillin/streptomycin (Merck). Immortalized hippocampal neuroblasts (HT-22 cells, RRID:CVCL_0321, Merck Millipore, Burlington, MA, USA) and SH-SY5Y cells (Depositor: JL Biedler, RRID:CVCL_0019, American Type Culture Collection (ATCC), Manassas, VA, USA) were cultured in 96-well plates in DMEM containing 10% fetal calf serum and 1% penicillin/streptomycin (4000 cells/well and 20,000 cells/well, respectively). Cell lines were treated 24 h after plating when the density reached at least 70% confluency. All cells were cultured at 37 °C in a humidified 5% CO₂ atmosphere.

Dörschmann P., Apitz S., Hellige I., Neupane S., Alban S., Kopplin G., Ptak S., Fretté X., Roider J., Zille M, & Klettner A. (2021). Evaluation of the Effects of Fucoidans from Fucus Species and Laminaria hyperborea against Oxidative Stress and Iron-Dependent Cell Death. Marine Drugs, 19(10), 557.

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Isolation and Culture of Primary Porcine RPE Cells

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Primary porcine RPE cells were prepared and cultivated as previously described [51 (link)] with modifications [52 (link)]. In brief, eyes were obtained from a local slaughterhouse, cleaned, the anterior segment and retina were discarded, and RPE cells harvested by trypsin digestion. Cells were used in the first passage at confluence, morphology, and confluency observed in light microscopy. Cells were maintained in HyClone DMEM (GE Healthcare, Munich, Germany, supplemented with penicillin/streptomycin (1%), HEPES (2.5%), sodium pyruvate (110 mg/mL), and 10% fetal calf serum, Linaris GmbH, Wertheim-Bettingen, Germany). The immortal RPE cell line ARPE19 was obtained from ATCC and cultivated in DMEM (Merck, Darmstadt, Germany), supplemented with penicillin/streptomycin (1%), non-essential amino acids (1%), and 10% fetal calf serum.

Rohwer K., Neupane S., Bittkau K.S., Pérez M.G., Dörschmann P., Roider J., Alban S, & Klettner A. (2019). Effects of Crude Fucus distichus Subspecies evanescens Fucoidan Extract on Retinal Pigment Epithelium Cells―Implications for Use in Age-Related Macular Degeneration. Marine Drugs, 17(9), 538.

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Cultivation of Uveal Melanoma and RPE Cells

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OMM-1, a uveal melanoma cell line [36 (link)] was kindly donated by Dr. Sarah Coupland. The cultivation mediumRPMI 1640 (Merck, Darmstadt, Germany) was added with 10% fetal calf serum (Linaris GmbH, Wertheim-Bettingen, Germany) and 1% penicillin/streptomycin (Merck). The human RPE cell line ARPE-19 [37 (link)] was bought from ATCC and cultivated in HyClone DMEM (GE Healthcare, München, Germany), supplemented with 1% penicillin/streptomycin, 2.5% HEPES (Merck), 1% non-essential amino acids (Merck) and 10% fetal calf serum. ARPE-19 cells were treated at confluence and OMM-1 cells were treated at subconfluence.

Dörschmann P., Mikkelsen M.D., Thi T.N., Roider J., Meyer A.S, & Klettner A. (2020). Effects of a Newly Developed Enzyme-Assisted Extraction Method on the Biological Activities of Fucoidans in Ocular Cells. Marine Drugs, 18(6), 282.

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Differentiation and Manipulation of C2C12 Myoblasts

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C2C12 cell lines were differentiated in myotubes and RT-PCR with primers specific for murine muscle was performed for cell identity confirmation. The cells were subjected to MycoAlert Mycoplasma detection kit from Lonza (LT07-318). C2C12 myoblasts were plated on Matrigel (Corning)-coated 35 mm dishes and grown in Hyclone DMEM (GE healthcare) containing 4.5 g/l glucose, supplemented with 15% FBS (PAA) and penicillin/streptomycin. Cells were maintained at 37°C in a saturated humidity atmosphere containing 5% CO2. For the differentiation of C2C12 myoblasts, fetal bovine serum was replaced by 2% horse serum (Biowest) when myoblasts reached 70–80% confluence. Cells were differentiated for 6 days.
Sh-Creld1 and control myoblast lines were obtained by selecting puromycin-resistant clones of C2C12 myoblasts, respectively transfected with shRNA clone set against mouse Creld1 or scrambled control clone (GeneCopoeia).

D'Alessandro M., Richard M., Stigloher C., Gache V., Boulin T., Richmond J.E, & Bessereau J.L. (2018). CRELD1 is an evolutionarily-conserved maturational enhancer of ionotropic acetylcholine receptors. eLife, 7, e39649.

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Efflux Assay in HEK293 Cells

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HEK293 cells were passaged in HyClone DMEM (GE) supplemented with 5% FBS and 100 units pen/strep per ml (Gibco) at 37^°C and 5% CO₂ under humidified conditions. Efflux experiments were performed in 6-well plates. 500,000 cells were seeded per well and 2 days later the experiment was started by replacing the culture medium with 2.5 ml Pro293a medium (Lonza), supplemented with 2 mM L-glutamine and 100 units pen/strep (Gibco) per ml. Samples were taken at the indicated time points. The presence of equal numbers of cells at the time of the experiment was confirmed by quantifying relative intracellular ATP levels per well (S2 Fig).

Szeri F., Lundkvist S., Donnelly S., Engelke U.F., Rhee K., Williams C.J., Sundberg J.P., Wevers R.A., Tomlinson R.E., Jansen R.S, & van de Wetering K. (2020). The membrane protein ANKH is crucial for bone mechanical performance by mediating cellular export of citrate and ATP. PLoS Genetics, 16(7), e1008884.

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ARPE-19 Cell Culture Protocol

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ARPE-19 cells (ATCC, Wesel, Germany), an immortal human RPE cell line [17], were cultivated in Hyclone DMEM (GE Healthcare, München, Germany), supplemented with penicillin/streptomycin (1%), nonessential amino acids (1%) (all Biochrom, Berlin, Germany), and 10% fetal calf serum (LINARISblue). For all experiments, confluent cells were used. All experiments were carried out in 12-well plates except for subcellular fractioning, for which ARPE-19 cells were cultured in flasks (T75, Sarstedt, Nümbrecht, Germany). For microscopy, cells were cultivated on collagen-coated (Collagen A, Biochrom) coverslips (21 × 26 mm, Menzel GmbH), as previously described [9] .

Borchers L., Roider J, & Klettner A. (2021). Differences in Uptake and Intracellular Fate between Bevacizumab and Aflibercept after Repetitive Long-Term Treatment in the Retinal Pigment Epithelium. Ophthalmic research, 64(3).

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Generation and Characterization of Rat ABCC6 Mutant

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HEK293 cells were cultured at 37 o C and 5% CO2 in humidifying conditions in HyClone DMEM (GE) completed with 100 units pen/strep per ml (Gibco) and 5% FBS. Site-specific mutations were introduced into in pEntr223-rAbcc6 plasmid (3) by Uracil excision-based (USER) cloning (33) with the following reverse and forward primers: ACCTTTCCGUACACCAGGCCAGTGATGGC and ACGGAAAGGUCCTGGTCCTGTCCAGTGGTTCCA, respectively. The cDNA encoding the pEnter223-rAbcc6 R389G mutant were sequenced and subsequently subcloned into a Gateway compatible pQCXIP expression vector and were transfected into HEK293 cells with calcium phosphate precipitation method. The transfected cells were selected in completed DMEM medium also containing 2 µM puromycin. After puromycin selection the expression of the R389G rat ABCC6 in isolated cell clones was confirmed by immunoblot analysis and compared to that of the wild-type rat ABCC6 cell line. For the functional assays HEK293 cell lines were seeded in poly-D-lysine-coated 96-well plates in 100µl of completed DMEM, and experiments were conducted with wells using cells forming confluent monolayers.

Szeri F., Mikó Á., Navasiolava N., Kaposi A., Verschuere S., Li Q., Terry S.F., Boraldi F., Uitto J., Wetering K.v.d., Martin L., Quaglino D., Vanakker O., Tory K., & Arányi T. (2020). The pathogenic p.R391G ABCC6 displays incomplete penetrance implying the necessity of an interacting partner for the development of pseudoxanthoma elasticum.

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