The HEK 293 cells were seeded at a density 2 × 104 per well on a 96-well plate and incubated in high glucose DMEM medium (Lonza) supplemented with 10% FBS and 50 U/ml of penicillin–streptomycin at 37°C in 5% CO2. After 24 h, cells were covered with a fresh medium containing tested peptides at different concentrations (4, 8, 16, and 32 μM). After 24 or 48 h of incubation, cells viability was tested using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Merck). Briefly, 10 μl of 5 mg/ml MTT was added to each well and incubated for 4 h. Formazan produced from MTT was solubilized during overnight incubation in 100 μl of 10% SDS in 0.01 M HCl. Absorbance at 590 nm was measured using Microplate Reader Biotek (Winooski, VT, United States). The experiment was performed in triplicates.
Dmem high glucose medium
Manufactured by Lonza
Sourced in Switzerland, United States
DMEM/high glucose medium is a cell culture medium that provides a nutrient-rich environment for the growth and maintenance of various cell types. It contains a high concentration of glucose, which serves as a primary energy source for cells. The medium is designed to support the optimal growth and proliferation of cells in in vitro culture systems.
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Lab products found in correlation
Fetal bovine serum (fbs), by Lonza (4 mentions)
Penicillin, by Merck Group (4 mentions)
Streptomycin, by Merck Group (3 mentions)
L glutamine, by Merck Group (3 mentions)
Rpmi 1640 medium, by Lonza (2 mentions)
Min6 cell line, by American Type Culture Collection (2 mentions)
Insulin, by Merck Group (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Hat medium, by Merck Group (1 mentions)
Essential 8 medium, by Thermo Fisher Scientific (1 mentions)
Matrigel coated six well plates, by BD (1 mentions)
Dmem high glucose, by Lonza (1 mentions)
Ham f 10 medium, by Merck Group (1 mentions)
Antibiotic antimycotic, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Dmem glutamax medium, by Thermo Fisher Scientific (1 mentions)
Thp 1 monocytes, by American Type Culture Collection (1 mentions)
Raw264.7 macrophage, by American Type Culture Collection (1 mentions)
10 protocols using dmem high glucose medium
1
Cytotoxicity of Peptides on HEK 293 Cells
2
Establishing Hybrid Cell Lines from Fibroblasts
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Fibroblasts from the LN patient were obtained from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research (GM 20394). Human dermal fibroblasts (HDFs) from a normal donor (Thermo Fisher Scientific, Waltham, MA, USA) and the LN fibroblasts were maintained in high-glucose DMEM medium, supplemented with 10% fetal bovine serum (FBS; Lonza, Basel, Switzerland).
The A9 mouse fibroblast line, Hprt− derivative of strain L (Sigma-Aldrich),40 (link) was grown in high-glucose DMEM (Lonza) supplemented with 10% FBS (Lonza). The A9/human hybrid cell line was obtained by whole-cell fusion between A9 cells and HDF cells; clones with a normal human X chromosome were selected in 1× HAT medium (Sigma-Aldrich, Munich, Germany).
Hprt-defective CHO cells were kindly provided by Dr. E. Raimondi (University of Pavia, Pavia, Italy). The CHO cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Lonza), and their microcell-fused derivative cells were selected in 1× HAT medium (Sigma-Aldrich).
Human iPSCs were cultured in feeder-free conditions, using Essential 8 medium (Thermo Fisher Scientific) on human ESC (hESC)-qualified matrix Matrigel-coated six-well plates (BD Biosciences, San Jose, CA, USA) and their microcell-fused derivative cells were selected in HAT medium (Sigma-Aldrich).
The A9 mouse fibroblast line, Hprt− derivative of strain L (Sigma-Aldrich),40 (link) was grown in high-glucose DMEM (Lonza) supplemented with 10% FBS (Lonza). The A9/human hybrid cell line was obtained by whole-cell fusion between A9 cells and HDF cells; clones with a normal human X chromosome were selected in 1× HAT medium (Sigma-Aldrich, Munich, Germany).
Hprt-defective CHO cells were kindly provided by Dr. E. Raimondi (University of Pavia, Pavia, Italy). The CHO cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Lonza), and their microcell-fused derivative cells were selected in 1× HAT medium (Sigma-Aldrich).
Human iPSCs were cultured in feeder-free conditions, using Essential 8 medium (Thermo Fisher Scientific) on human ESC (hESC)-qualified matrix Matrigel-coated six-well plates (BD Biosciences, San Jose, CA, USA) and their microcell-fused derivative cells were selected in HAT medium (Sigma-Aldrich).
3
Modulation of MBNL-mediated Splicing in Muscle Cells
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Human HeLa and mouse C2C12 cells were grown in high-glucose DMEM medium (Lonza) supplemented with 10% fetal bovine serum (Sigma) and 1x antibiotic/antimycotic (Sigma). Human skeletal myoblast (HSkM) cells were grown in HAM F-10 medium (Sigma) supplemented with 20% FBS, 1x antibiotic/antimycotic, 0.39 µg/ml dexamethasone (Sigma) and 10 ng/ml epidermal growth (Sigma). All cells were grown at 37 °C and in a 5% atmosphere of CO2. HeLa, C2C12 or HSkM cells plated in 12-well plates were transfected at 50–60% confluence with Lipofectamine® 2000 (Invitrogen™) according to the manufacturer’s protocol. Single transfection was conducted with an siRNA mix against MBNL1 and MBNL2 (25 nM each) (FUTURE synthesis and RiboTask™, respectively67 (link),68 (link)), 50 nM AllStars negative control siRNA (Qiagen), or a specific AON at 125 nM, in which AON-Ctrl was 2′OMe-PS unspecific to a tested transcript. Co-transfection was conducted with 200 ng of the minigene and 500 ng (or as indicated in the figures) of the MBNL1, SRSF1 or GFP expressing vector. For verification of the MBNL-binding regions and inhibition of the MBNL/(CUG)exp interaction, the co-transfection was followed by a 4-hour incubation and transfection with selected AONs. Erythromycin was added directly to the medium. The cells were harvested 48 hours after transfection.
4
Culturing Human and Murine Cell Lines
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Human hepatic stellate cells (LX2 cells), provided by Prof. Scott Friedman (Mount Sinai Hospital, New York, NY, USA), were cultured in DMEM‐Glutamax medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Lonza, Verviers, Belgium) and antibiotics (50 U/ml Penicillin and 50 μg/ml streptomycin, Sigma, St. Louis, MO, USA). Murine RAW 264.7 macrophages and human THP1 monocytes, obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Lonza) supplemented with 10% FBS (Lonza), 2 mM L‐glutamine (Sigma) and antibiotics. Human HepG2 cells (ATCC) were cultured in DMEM‐high glucose medium (Lonza) supplemented with 10% FBS and antibiotics. All the cell lines used in this study were authenticated with STR profiling and were tested regularly for the absence of mycoplasma contamination by PCR.
5
Streptozotocin Impact on Pancreatic β Cells
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The MIN6 cell line (pancreatic β cells) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). MIN6 cells (passages 30–45) were cultured in DMEM High glucose medium (Lonza, Basal, Switzerland), containing 15% fetal bovine serum (FBS, Lonza), 50 μmol/L β-mercaptoethanol, 2 mmol/L l -glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin (all from Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured at 37 °C in a humidified atmosphere containing 95% air and 5% CO2. The effect of streptozotocin (STZ) and the derivatives on β cell viability was determined by the cell proliferation reagent WST-1 (Roche, Indianapolis, USA). Briefly, MIN6 cells (1 × 105 cells/well) were seeded in 96-well plates. Cells were cultured overnight and the following day incubated with or without STZ or analogue (Sigma-Aldrich) at 5 mM for 48 and 72 h followed by WST-1 assay. After 30 min incubation with WST-1 (10 μL/well) at 37 °C, the absorbance was measured at 450 nm using a Bio-Rad Benchmark Plus microplate spectrophotometer reader (Bio-Rad Laboratories B.V, Veenendaal, The Netherlands.
6
MIN6 Cell Line Cultivation Protocol
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The MIN6 cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). MIN6 cells (passages 30–45) were cultured in (Dulbecco’s Modified Eagle Medium) DMEM High-glucose medium (Lonza, Basal, Switzerland), containing 15% fetal bovine serum (FBS, Lonza), 50 µmol/L β-mercaptoethanol, 2 mmol/L l -glutamine, 50 U/mL penicillin, and 50 µg/mL streptomycin (all from Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured at 37 °C in a humidified atmosphere containing 95% air and 5% CO2.
7
Breast Cancer Cell Line Maintenance
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Breast carcinoma cell lines (MCF7, T47D, HCC1806, HCC1937, BT549, MDA-MB-453, SUM149, MDA-MB-468, SKBR3, BT20, and BT549) were kindly provided by Dr. M. Götte (Department of Gynecology and Obstetrics, Münster, Germany) and Dr. B. Greve (Department of Radiation Oncology, Münster, Germany). Cells were maintained in DMEM/high glucose medium (Lonza, Basel, Switzerland) (SKBr3, MDA-MB-453, and MDA-MB-468) or in RPMI medium (MCF7, T47D, HCC1806, HCC1937, BT20, and BT549). Both media were supplemented with 10% fetal calf serum (FCS; Gibco, Waltham, MA, USA) and 100 U/mL penicillin–streptomycin (PS; Gibco). SUM149 cells were cultured in Dulbecco’s modified Eagle’s medium-F12 (1:1) with 5% FCS, insulin (5 μg/mL) (Merk, Darmstadt, Germany), and hydrocortisone (1 μg/mL; Qiagen, Hilden, Germany). MDA-MB-231 cell line was obtained from DSMZ (Leibniz Institute DSMZ–German Collection of Microorganisms and Cell Cultures) and was grown in DMEM/high glucose medium containing 10% FCS and 100 U/mL PS. HUVECs were kindly provided by Dr. D. Vestweber (Max Planck Institute of Molecular Biomedicine, Münster, Germany) or purchased from Promocell (Heidelberg, Germany) and cultured up to passage five in ECGM-2 medium supplemented with SupplementPack (PromoCell).
8
Cell Culture Protocols for HT1080 and HEK-293T Cells
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Human fibrosarcoma HT1080 cells (ATCC, Manassas, VA, USA) and human embryonic kidney (HEK)-293T cells were cultured in DMEM/high glucose medium (Lonza, Basel, Switzerland) containing 10% (v/v) heat-inactivated fetal calf serum (FCS) (Gibco, Waltham, MA, USA), 100 U/mL penicillin and streptomycin (Gibco), at 37 °C and 5% and 7% CO2 (for HT1080 and HEK-293T cells, respectively). The HEK-293T cell medium was supplemented with 0.1 mM nonessential amino acids (PAA Laboratories, Cölbe, Germany), 6 mM l -glutamine (Gibco), and 1 mM sodium pyruvate (PAA Laboratories). The Drosophila Schneider’s cell line-2 was grown in SF-900 II SFM medium (Gibco) containing 10% (v/v) heat-inactivated FCS.
9
Culturing Cancer Cell Lines on PMMA Films
10
Characterization of RMS and HUVEC Cells
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RMS cell lines (RH30, RH41, RD, SMS-CTR) were kindly provided by Dr. PJ Houghton (Center for Childhood Cancer, Columbus, OH, USA). The cells were cultured in DMEM high-glucose medium (Lonza Group Ltd., Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS, EURx, Gdansk, Poland) and 50 μg/mL gentamicin (Lonza) at 37 °C, 5% CO2 and 95% humidity.
Human umbilical vein endothelial cells (HUVEC) have been ordered from Becton Dickinson Biosciences. They were cultured in endothelial cell growth medium (PromoCell, Heidelberg, Germany), with endothelial cell growth supplement (PromoCell).
The cell lines were routinely tested for Mycoplasma spp. contamination using by MycoAlert™ Mycoplasma Detection Kit (Lonza). RMS cell line authentication was performed by STR profiling using AmpFlSTR SGM PLUS Kit (Applied Biosystems, Foster City, CA, USA) and sequencing apparatus ABI Prism 310 Genetic Analyser (Applied Biosystems) according to the manufacturer’s protocol.
Human umbilical vein endothelial cells (HUVEC) have been ordered from Becton Dickinson Biosciences. They were cultured in endothelial cell growth medium (PromoCell, Heidelberg, Germany), with endothelial cell growth supplement (PromoCell).
The cell lines were routinely tested for Mycoplasma spp. contamination using by MycoAlert™ Mycoplasma Detection Kit (Lonza). RMS cell line authentication was performed by STR profiling using AmpFlSTR SGM PLUS Kit (Applied Biosystems, Foster City, CA, USA) and sequencing apparatus ABI Prism 310 Genetic Analyser (Applied Biosystems) according to the manufacturer’s protocol.
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