Knockout dmem

Manufactured by Merck Group

Sourced in United States

Knockout DMEM is a specialized cell culture medium developed by Merck Group. It is designed to support the growth and maintenance of pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells. The medium is formulated to maintain the cells in an undifferentiated state, allowing for their self-renewal and expansion.

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8 protocols using knockout dmem

Quantitative Cell Migration Assay

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Cancer cell migration/invasion was performed by a quantitative cell migration assay (ECM500; Chemicon, Temecula, CA, USA) according to the manufacturer’s instructions. Warm Knockout DMEM (Sigma) in the amount of 200 μl was applied to the extracellular matrix (ECM) layer to hydrate for 2 h at room temperature. AGS cells were then dislodged by trypsinization (0.25% trypsin; Sigma) and dispersed into a homogeneous single-cell suspension at the concentration of 5×10⁵ cells/ml, followed by washing and resuspension in Knockout DMEM. Then, cell suspension of 200 μl was allowed to adhere to the surface at 37°C for 60 min. The migration mediums containing cyclopamine were then put into the bottom chamber. Following 24 h of incubation at 37°C, 5% CO₂ in air, the cells in the upper chamber were stained for 20 min, and dissolved in 10% acetic acid and the optical density (OD) was read at 560 nm on a standard reader.

BAI R., ZHAO H., ZHANG X, & DU S. (2014). Characterization of sonic hedgehog inhibition in gastric carcinoma cells. Oncology Letters, 7(5), 1381-1384.

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Culturing C57BL6 MEFs, ESCs, and iPSCs

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C57BL6 MEFs were derived from embryonic day 13.5 mice embryos [35 ] and maintained in Dulbecco's modified Eagle's medium (DMEM, high glucose) containing 10% fetal bovine serum. ESCs and iPSCs were cultured on γ-irradiated MEFs in ES medium containing Knockout DMEM supplemented with 20% Knockout Serum Replacement, 0.1 mM NEAA, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM β-mercaptoethanol, 50 U and 50 mg/mL penicillin/streptomycin, and 1,000 U/mL leukemia inhibitory factor (Millipore, Billerica, MA, USA). All other reagents were purchased from Invitrogen Corp. (Carlsbad, CA, USA).

Dong F., Song Z., Yu J., Zhang B., Jiang B., Shen Y., Lu Y., Song C., Cong P, & Liu H. (2015). Dynamic Changes in Occupancy of Histone Variant H2A.Z during Induced Somatic Cell Reprogramming. Stem Cells International, 2016, 3162363.

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Murine Embryonic Stem Cell Culture

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R1 murine embryonic stem cells (mESCs, ATCC) were cultured in Knockout DMEM (Millipore) supplemented with 15% (v/v) Knockout Serum Replacement (KSR), 100 U/mL antibiotics (Invitrogen), 1000 U/ml leukemia inhibitory factor (LIF) (Life Technologies), 10 μg/ml gentamicin (Sigma), 0.1 mM ß-mercaptoethanol (Sigma), and 4 mM L-glutamine (Sigma) on a 0.1% gelatin coated T-75 culture flask; ES medium was changed daily. PA6 cells (from Dr. Jose Otero at The Ohio State University) were cultured in alpha-MEM (Millipore) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), and 100 U/mL antibiotics. Alpha-MEM was stored in the dark, and medium was changed every two days. Cells were passaged with trypsin/EDTA (Invitrogen) once 70% confluence was reached and used for encapsulation studies or passaged for future use. All cells were cultured in a T-75 flask at 37°C in a humidified 5% CO₂ incubator.

Dumbleton J., Shamul J.G., Jiang B., Agarwal P., Huang H., Jia X, & He X. (2021). Oxidation and RGD modification affect the early neural differentiation of murine embryonic stem cells cultured in core-shell alginate hydrogel microcapsules. Cells, tissues, organs, 211(3), 294-303.

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Culturing Dnmt-Deficient Embryonic Stem Cells

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All cell culture media components were purchased from Invitrogen (Paisley, UK). Dnmt1 KO and Dnmt3a/3b double KO cells with matching WT were kind gifts from Dr. Masaki Okano (RIKEN Center for Developmental Biology, Kobe, Japan). ESCs were maintained on Nunc plates (Davidson & Hardy, Belfast, UK) treated with 0.1% gelatin (Sigma-Aldrich, Dorset, UK) and cultured in Knockout DMEM plus 15% knockout serum replacement, 1% ESC-qualified Foetal Bovine Serum, 1× NEAA, 2 mM l-glutamine, 0.1 mM β-mercaptoethanol (Sigma-Aldrich, Dorset, UK) and 1000U/ml LIF (Merck Millipore, Hertfordshire, UK).

Thakur A., Mackin S.J., Irwin R.E., O’Neill K.M., Pollin G, & Walsh C. (2016). Widespread recovery of methylation at gametic imprints in hypomethylated mouse stem cells following rescue with DNMT3A2. Epigenetics & Chromatin, 9, 53.

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Sterilization and Culture of Embryonic Stem Cells

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We sterilized the nanofibrous scaffolds in 70%
ethanol for 2 hours after which they were washed
3 times with PBS for 20 minutes and subsequently
incubated with DMEM/F12 (Sigma-Aldrich, MO,
USA) for 24 hours before cell seeding. Mouse
embryonic fibroblast (MEF) cells were prepared
according to guidelines the Laboratory Animal
Ethical Commission of Tarbiat Modares University
and cultured in DMEM F12 supplemented
with 10% FBS (Sigma-Aldrich, MO, USA). After
reaching 70% confluency, the cells were chemically
inactivated with mitomycin C (10 μg/ml; Gibco-
Invitrogen, CA, USA) for 2 hours and washed
3 times with PBS to remove any remaining mitomycin
C. mESCs, obtained from the Stem Cell
Technology Research Center (17 (link)), were grown on
a gelatinized plate that contained one layer of inactive
feeder cells (MEFs) in ES culture medium
that included Knock-Out DMEM (Sigma-Aldrich,
MO, USA) which consisted of sodium pyruvate
supplemented with 20% ESC qualified fetal bovine
serum (Gibco-Invitrogen, CA, USA), 1000
IU/ml LIF (Gibco-Invitrogen, CA, USA), 1 mM
NEAA, 2 mM L-glutamine, 0.1 mM β-ME, and
100 μg/ml pen/strep antibiotics (Gibco-Invitrogen,
CA, USA). Cells were maintained in a humidified
CO₂ incubator at 37˚C. Media was changed daily
until the appropriate confluency of ESC colonies
was attained.

Abbasi N., Soudi S., Hayati-Roodbari N., Dodel M, & Soleimani M. (2014). The Effects of Plasma Treated Electrospun Nanofibrous Poly (ε-caprolactone) Scaffolds with Different Orientations on Mouse Embryonic Stem Cell Proliferation. Cell Journal (Yakhteh), 16(3), 245-254.

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NIH3T3 and E14.1 Cell Culture and Transcription Inhibition

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NIH3T3 cells were cultured in DMEM (Gibco, Life Technologies, USA) supplemented with 10% FBS (Gibco, Life Technologies, USA) and 1X pen/strep (100 units of penicillin, 100 μg of streptomycin; Gibco, Life Technologies, USA). E14.1 Mouse embryonic cells were maintained by culturing them on gelatin (0.1%) coated dishes with reconstituted media. Reconstituted media was made from ES cell Knockout DMEM supplemented with 15% Knockout Fetal Bovine Serum, 1 mM sodium pyruvate (Sigma Aldrich, USA), 0.1 mM nonessential amino acids, 2 mM L-Glutamine, 0.1 mM β-mercaptoethanol (Sigma Aldrich, USA) and 500 U/ml leukaemia inhibitory factor (Merck Millipore) and penicillin–streptomycin. All cell culture reagents, unless otherwise indicated, are from Life Technologies. All cells were maintained at 37°C in 5% CO₂ incubator. Transcription was inhibited using a transcription initiation inhibitor, α-amanitin (Sigma Aldrich, USA) at 40 μg/ml for either 3 or 12 h. Cells were treated with 100 nM of Trichostatin A (TSA, Sigma Aldrich, USA), a histone deacetylase inhibitor that leads to hyper-acetylation of chromatin during the 60 h incubation period.

Maharana S., Iyer K.V., Jain N., Nagarajan M., Wang Y, & Shivashankar G.V. (2016). Chromosome intermingling—the physical basis of chromosome organization in differentiated cells. Nucleic Acids Research, 44(11), 5148-5160.

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Murine Embryonic and Fibroblast Cell Culture

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A murine embryonic stem cell line, ES-D3 (No. CRL-1934) and a murine fibroblast cell line, BALB/3T3 clone A31 (No. RCB0005) were provided by American Type Culture Collection (ATCC) and RIKEN BioResource Center through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, respectively. Prior to use, ES-D3 cell line was grown to habituate as feeder-less culture in the medium composed of KnockOut D-MEM supplemented with 15% Knockout Serum Replacement (KSR), 100 μM non-essential amino acids, 2 mM glutamine, 1000 units/ml mLIF, 100 U/ml penicillin, 100 μg/ml streptomycin, which each reagents were purchased from Thermo Fisher Scientific (Walthan, MA, USA) and 0.1 mM 2-mercaptoethanol (Sigma–Aldrich, Saint Louis, MO, USA) on the Geltrex matrix (Thermo Fisher Scientific) in a cell culture flask of 25 cm². The BALB/3T3 cell line was maintained by D-MEM (Thermo Fisher Scientific) and 10% fetal bovine serum (BioWest, Miami, FL, USA), 4 mM glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific). In this study, ES-D3 is a surrogate of an embryonic cell and BALB/3T3 is a surrogate of a differentiated cell model, according to the EST method [23] (link).

Yoshie S., Ogasawara Y., Ikehata M., Ishii K., Suzuki Y., Wada K., Wake K., Nakasono S., Taki M, & Ohkubo C. (2016). Evaluation of biological effects of intermediate frequency magnetic field on differentiation of embryonic stem cell. Toxicology Reports, 3, 135-140.

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Cell Culture Protocol for HeLa, HCT116, and R63 ES Cells

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HeLa (ATCC) and HCT116 [37] (kind gift of R. Farber) cells were maintained in 1 g/L glucose D-MEM (Invitrogen), both supplemented with 10% FBS (Invitrogen) and 1x NEAA (Invitrogen). R63 ES cells [38] (kind gift of H. Wheadon) were maintained on Nunc tissue culture plates (Davidson & Hardy Ltd., Belfast, UK) which were gelatinised with 0.1% porcine gelatin (Sigma-Aldrich, Dorset, UK). Components of ES cell medium are from Invitrogen unless otherwise stated. ES cells were cultured in KnockOut™ D-MEM supplemented with 15% KnockOut™ Serum Replacement, 1% ES-cell qualified fetal bovine serum, 1x NEAA, 2 mM L-glutamine and 0.1 mM β-mercaptoethanol (Sigma-Aldrich). Stable CPEB1 knockdown cells were created using the Knockout™ Single Vector (Clontech, Sainte-Germaine-en-Laye, France) as per the manufacturer’s instructions, and were maintained under the same culture conditions as the parental HeLa cells with the addition of 400 µg/µl geneticin (Invitrogen). All cells were cultured at 37°C, 5% CO₂ in a humidified atmosphere.

Rutledge C.E., Lau H.T., Mangan H., Hardy L.L., Sunnotel O., Guo F., MacNicol A.M., Walsh C.P, & Lees-Murdock D.J. (2014). Efficient Translation of Dnmt1 Requires Cytoplasmic Polyadenylation and Musashi Binding Elements. PLoS ONE, 9(2), e88385.

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