knockout dulbecco s modified eagle medium ko dmem  (Thermo Fisher)


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    Name:
    KnockOut DMEM
    Description:
    KnockOut D MEM is a basal medium optimized for growth of undifferentiated embryonic and induced pluripotent stem cells 1 The osmolarity is optimized to approximate that of mouse embryonic tissue Contains no L glutamine
    Catalog Number:
    10829018
    Price:
    None
    Category:
    Cell Culture Transfection Reagents
    Applications:
    Cell Culture|Embryonic Stem Cell Culture|Induced Pluripotent Stem Cell Culture|Mammalian Cell Culture|Mesenchymal Stem Cell Culture|Stem Cell Culture|Stem Cell Research
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    Thermo Fisher knockout dulbecco s modified eagle medium ko dmem
    Hepatic differentiation of iPSCs through the definitive endoderm (DE) stage. (A) A schematic representation of the hepatic differentiation protocol (modified from Hay et al., 2008 ) and cell culture media used at each stage. See text for details of the media. (B) Phase-contrast images showing sequential morphological changes from iPSCs (day 0) to DE (day 5) and finally hepatocyte-like cells (HLCs) (day 20) during differentiation. (C,D) Immunocytochemistry of the iPSC-HLCs differentiated from three patient lines (UTA.10100.EURCAs, UTA.11104.EURCAs and UTA.11304.EURCCs) at day 20 showing the expression of (C) LDL receptor (LDL-R) and asialoglycoprotein receptor (ASGR), as well as (D) α-fetoprotein (AFP) and albumin (ALB). Nuclei are stained with DAPI. (E) The iPSC-HLCs were able to uptake LDL at day 20 of differentiation and (F) produce and secrete albumin during differentiation. Values are normalised per 1 million cells per 24 h. Bars represent means±s.d. of three biological replicates. Gene expression levels of (G) apolipoprotein B ( ApoB ) and (H) apolipoprotein AI ( ApoAI ) at different time points during the iPSC to HLC differentiation normalised to the housekeeping gene GAPDH , and expressed relative to day 0. Each sample was run in triplicate and bars represent means±s.d. of three studied cell lines. iPSC, induced pluripotent stem cell; <t>KO-DMEM,</t> KnockOut <t>Dulbecco's</t> modified Eagle medium; DMSO, dimethyl sulfoxide; HGF, hepatocyte growth factor; OSM, oncostatin M; PHH, primary human hepatocyte. Scale bars: 200 μm.
    KnockOut D MEM is a basal medium optimized for growth of undifferentiated embryonic and induced pluripotent stem cells 1 The osmolarity is optimized to approximate that of mouse embryonic tissue Contains no L glutamine
    https://www.bioz.com/result/knockout dulbecco s modified eagle medium ko dmem/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    knockout dulbecco s modified eagle medium ko dmem - by Bioz Stars, 2021-04
    86/100 stars

    Images

    1) Product Images from "Lipidomic profiling of patient-specific iPSC-derived hepatocyte-like cells"

    Article Title: Lipidomic profiling of patient-specific iPSC-derived hepatocyte-like cells

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.030841

    Hepatic differentiation of iPSCs through the definitive endoderm (DE) stage. (A) A schematic representation of the hepatic differentiation protocol (modified from Hay et al., 2008 ) and cell culture media used at each stage. See text for details of the media. (B) Phase-contrast images showing sequential morphological changes from iPSCs (day 0) to DE (day 5) and finally hepatocyte-like cells (HLCs) (day 20) during differentiation. (C,D) Immunocytochemistry of the iPSC-HLCs differentiated from three patient lines (UTA.10100.EURCAs, UTA.11104.EURCAs and UTA.11304.EURCCs) at day 20 showing the expression of (C) LDL receptor (LDL-R) and asialoglycoprotein receptor (ASGR), as well as (D) α-fetoprotein (AFP) and albumin (ALB). Nuclei are stained with DAPI. (E) The iPSC-HLCs were able to uptake LDL at day 20 of differentiation and (F) produce and secrete albumin during differentiation. Values are normalised per 1 million cells per 24 h. Bars represent means±s.d. of three biological replicates. Gene expression levels of (G) apolipoprotein B ( ApoB ) and (H) apolipoprotein AI ( ApoAI ) at different time points during the iPSC to HLC differentiation normalised to the housekeeping gene GAPDH , and expressed relative to day 0. Each sample was run in triplicate and bars represent means±s.d. of three studied cell lines. iPSC, induced pluripotent stem cell; KO-DMEM, KnockOut Dulbecco's modified Eagle medium; DMSO, dimethyl sulfoxide; HGF, hepatocyte growth factor; OSM, oncostatin M; PHH, primary human hepatocyte. Scale bars: 200 μm.
    Figure Legend Snippet: Hepatic differentiation of iPSCs through the definitive endoderm (DE) stage. (A) A schematic representation of the hepatic differentiation protocol (modified from Hay et al., 2008 ) and cell culture media used at each stage. See text for details of the media. (B) Phase-contrast images showing sequential morphological changes from iPSCs (day 0) to DE (day 5) and finally hepatocyte-like cells (HLCs) (day 20) during differentiation. (C,D) Immunocytochemistry of the iPSC-HLCs differentiated from three patient lines (UTA.10100.EURCAs, UTA.11104.EURCAs and UTA.11304.EURCCs) at day 20 showing the expression of (C) LDL receptor (LDL-R) and asialoglycoprotein receptor (ASGR), as well as (D) α-fetoprotein (AFP) and albumin (ALB). Nuclei are stained with DAPI. (E) The iPSC-HLCs were able to uptake LDL at day 20 of differentiation and (F) produce and secrete albumin during differentiation. Values are normalised per 1 million cells per 24 h. Bars represent means±s.d. of three biological replicates. Gene expression levels of (G) apolipoprotein B ( ApoB ) and (H) apolipoprotein AI ( ApoAI ) at different time points during the iPSC to HLC differentiation normalised to the housekeeping gene GAPDH , and expressed relative to day 0. Each sample was run in triplicate and bars represent means±s.d. of three studied cell lines. iPSC, induced pluripotent stem cell; KO-DMEM, KnockOut Dulbecco's modified Eagle medium; DMSO, dimethyl sulfoxide; HGF, hepatocyte growth factor; OSM, oncostatin M; PHH, primary human hepatocyte. Scale bars: 200 μm.

    Techniques Used: Modification, Cell Culture, Immunocytochemistry, Expressing, Staining, Knock-Out

    Related Articles

    Knock-Out:

    Article Title: Aldehyde dehydrogenase 1 positive glioblastoma cells show brain tumor stem cell capacity
    Article Snippet: The human GBM cell lines LN18, LN229, LNZ308, and G139 were cultured in the Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM l -glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (Invitrogen) under the standard cell culture conditions at 37°C and 5% CO2 . .. To investigate the neurosphere formation, the cells were grown in a serum-free neurobasal medium consisting of Knockout DMEM (Invitrogen), Nutrient Mixture Ham's F12 + l -glutamine (Invitrogen), and 10% Knockout-Serum Replacement (Invitrogen) with 1% N2-Supplement (Invitrogen), 1% Non-Essential Amino-Acids (Invitrogen), 0.1% natural mouse laminin (Invitrogen), 2 mM l -glutamine (Biochrom), 100 U/mL penicillin (Biochrom), 100 µg/mL streptomycin (Biochrom), 10 ng/mL basic fibroblast growth factor (bFGF; Invitrogen), and 50 ng/mL epidermal growth factor (EGF; human recombinant, Millipore). .. For nonadherent growth conditions, the culture dishes were covered with 0.1% gelatin type A (Sigma-Aldrich).

    Article Title: Vascular Smooth Muscle Cells Derived from Inbred Swine Induced Pluripotent Stem Cells for Vascular Tissue Engineering
    Article Snippet: On day 0, fibroblast medium was replaced with medium containing lentiviral particles (1 mL fresh fibroblast medium mixed with 1.4 mL viral supernatant containing hSTEMCCA and 0.6 mL supernatant containing rtTA, plus 5 μg/mL polybrene (SigmaAldrich)). .. 48 hours after the first infection, medium was switched to pluripotency-promoting medium (Knockout DMEM (ThermoFisher), 10% Knockout Serum Replacement (KOSR, ThermoFisher), 10% (v/v) FBS, 20 ng/mL human leukemia growth factor (LIF, Peprotech), 20 ng/mL human basic fibroblast growth factor (bFGF, SigmaAldrich), 2 μg/mL doxycycline (Stemgent), 1% (v/v) NEAA, 1% (v/v) pen/strep, 2 mM L-glutamine and 0.1 mM β-mercaptoethanol (SigmaAldrich)) for 48 hours. .. Then the infected SEFs were dissociated on day 4 with 0.05% trypsin EDTA (SigmaAldrich) and seeded onto irradiated mouse embryonic fibroblasts (MEFs, 40,000 cells/cm2 as feeder layer) at 4,000 cells/cm2 , and cultured in siPSC medium (Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12, ThermoFisher), 20% (v/v) KOSR, 20 ng/mL LIF, 20 ng/mL bFGF, 2 μg/mL doxycycline, 2 mM L-glutamine, 1% (v/v) NEAA, 1% (v/v) pen/strep and 0.1 mM β-mercaptoethanol).

    Article Title: Idiopathic autism: Cellular and molecular phenotypes in pluripotent stem cell derived-neurons
    Article Snippet: Irradiated CF1 mouse embryonic fibroblasts (MEFs) were from Applied StemCell and incubated in MEF medium: DMEM with 10% heat inactivated-FBS, 1% GlutaMAX (Life Technologies), and 1% antibiotics. .. Generation, maintenance, and characterizations of iPSCs were performed as described elsewhere [ ], with specific modifications described in the . iPSCs and hESC were cultured on MEF feeders in iPSC medium (Knockout DMEM (Life Technologies), containing 20% Knockout SR (Life Technologies), 1% GlutaMAX, 0.1 mM β-mercaptoethanol (Life Technologies), 1% antibiotics, 1% NEAA, and 10 ng/ml basic fibroblast growth factor (bFGF) (Peprotech). ..

    Article Title: Demarcation of Stable Subpopulations within the Pluripotent hESC Compartment
    Article Snippet: Fractionation of hESC based on REX1Venus expression reveals a previously hidden hierarchy within the pluripotent compartment, comprising undifferentiated and differentiation primed cells, which lacks the metastability observed in murine ESCs .. Human ESC culture and differentiation Human ESC line H1 (WiCell) was grown on mitotically-inactivated MEFs in hESC media (Knockout DMEM supplemented with 15% Knockout SR, 1× Non Essential Amino Acids, 1× Glutamax, 1× 2ME (all Invitrogen) and 16 ng/ml bFGF (Peprotech) and passaged with Collagenase type IV (Invitrogen). .. For antibiotic selection experiments, cells were cultured in hESC media with or without the addition of 1.5 ug/ml puromycin.

    Article Title: Generation of neural progenitor cells by chemical cocktails and hypoxia
    Article Snippet: HUCs were collected and cultured in REGM (Lonza, CC-4127) as described , . .. Generation of ciNPCs For neural progenitor cell induction from MEFs and TTFs, initial cells cultured in DMEM for 24 h were transferred into KSR medium including knockout DMEM (Life Technologies, 10829-018), 15% knockout serum replacement (Life Technologies, 10828), 1% NEAA (Life Technologies, 35050), 1% GlutaMax (Life Technologies, 35050-061), 1% sodium pyruvate (Life Technologies, 11360), 0.1 mM β-mercaptoethanol (Life Technologies, 21985-023) and 1 000 U/ml leukemia inhibitory factor (LIF) (Chemicon, ESG1107). ..

    Article Title: Competence of In Vitro Cultured Mouse Embryonic Stem Cells for Myogenic Differentiation and Fusion with Myoblasts
    Article Snippet: Next, ES-GFP cells were seeded onto MEFs and cultured under various experimental conditions. .. Standard culture was conducted in DMEM+LIF medium, consisting of Knockout DMEM (Life Technologies) supplemented with 15% ES-qualified FBS (Life Technologies), 0.1 mM nonessential amino acids (Sigma-Aldrich), 2 mM L-glutamine (Life Technologies), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), 50 U/mL penicillin (Life Technologies), 50 μg/mL streptomycin (Life Technologies), and 500 U/mL leukaemia inhibitory factor (LIF; Chemicon). .. Next, ES-GFP cells were cultured in media commonly used for myoblast culture: DMEM+FBS medium (high-glucose DMEM supplemented with 10% FBS and antibiotics) or DMEM+HS (high-glucose DMEM supplemented with 2% horse serum [HS; Life Technologies] and antibiotics) or DMEM+FBS+HS (low-glucose DMEM [Life Technologies] supplemented with 20% FBS, 10% HS, 0.5% chicken embryo extract (Sera Laboratories), and antibiotics).

    Article Title: Zinc finger proteins orchestrate active gene silencing during embryonic stem cell differentiation
    Article Snippet: Embryonic stem cells were cultured and passaged on 0.1% gelatinized (Sigma-Aldrich, St. Louis, MO, USA) dishes, as reported previously ( ). .. E14 mouse ESCs were cultured in DMEM (Hyclone, Logan, Utah) or KNOCK-OUT™ DMEM (Gibco, Grand Island, NY, USA) supplemented with 15% FBS (Gibco, Grand Island, NY, USA), 2 mM l -glutamine, 55 μM β-mercaptoethanol, 1% (v/v) non-essential amino acid, 100 U/ml penicillin and 100 μg/ml streptomycin (all from Gibco, Grand Island, New York) and 500 U/ml ESGRO LIF (Millipore, Germany). ..

    Recombinant:

    Article Title: Aldehyde dehydrogenase 1 positive glioblastoma cells show brain tumor stem cell capacity
    Article Snippet: The human GBM cell lines LN18, LN229, LNZ308, and G139 were cultured in the Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM l -glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (Invitrogen) under the standard cell culture conditions at 37°C and 5% CO2 . .. To investigate the neurosphere formation, the cells were grown in a serum-free neurobasal medium consisting of Knockout DMEM (Invitrogen), Nutrient Mixture Ham's F12 + l -glutamine (Invitrogen), and 10% Knockout-Serum Replacement (Invitrogen) with 1% N2-Supplement (Invitrogen), 1% Non-Essential Amino-Acids (Invitrogen), 0.1% natural mouse laminin (Invitrogen), 2 mM l -glutamine (Biochrom), 100 U/mL penicillin (Biochrom), 100 µg/mL streptomycin (Biochrom), 10 ng/mL basic fibroblast growth factor (bFGF; Invitrogen), and 50 ng/mL epidermal growth factor (EGF; human recombinant, Millipore). .. For nonadherent growth conditions, the culture dishes were covered with 0.1% gelatin type A (Sigma-Aldrich).

    Infection:

    Article Title: Vascular Smooth Muscle Cells Derived from Inbred Swine Induced Pluripotent Stem Cells for Vascular Tissue Engineering
    Article Snippet: On day 0, fibroblast medium was replaced with medium containing lentiviral particles (1 mL fresh fibroblast medium mixed with 1.4 mL viral supernatant containing hSTEMCCA and 0.6 mL supernatant containing rtTA, plus 5 μg/mL polybrene (SigmaAldrich)). .. 48 hours after the first infection, medium was switched to pluripotency-promoting medium (Knockout DMEM (ThermoFisher), 10% Knockout Serum Replacement (KOSR, ThermoFisher), 10% (v/v) FBS, 20 ng/mL human leukemia growth factor (LIF, Peprotech), 20 ng/mL human basic fibroblast growth factor (bFGF, SigmaAldrich), 2 μg/mL doxycycline (Stemgent), 1% (v/v) NEAA, 1% (v/v) pen/strep, 2 mM L-glutamine and 0.1 mM β-mercaptoethanol (SigmaAldrich)) for 48 hours. .. Then the infected SEFs were dissociated on day 4 with 0.05% trypsin EDTA (SigmaAldrich) and seeded onto irradiated mouse embryonic fibroblasts (MEFs, 40,000 cells/cm2 as feeder layer) at 4,000 cells/cm2 , and cultured in siPSC medium (Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12, ThermoFisher), 20% (v/v) KOSR, 20 ng/mL LIF, 20 ng/mL bFGF, 2 μg/mL doxycycline, 2 mM L-glutamine, 1% (v/v) NEAA, 1% (v/v) pen/strep and 0.1 mM β-mercaptoethanol).

    Cell Culture:

    Article Title: Idiopathic autism: Cellular and molecular phenotypes in pluripotent stem cell derived-neurons
    Article Snippet: Irradiated CF1 mouse embryonic fibroblasts (MEFs) were from Applied StemCell and incubated in MEF medium: DMEM with 10% heat inactivated-FBS, 1% GlutaMAX (Life Technologies), and 1% antibiotics. .. Generation, maintenance, and characterizations of iPSCs were performed as described elsewhere [ ], with specific modifications described in the . iPSCs and hESC were cultured on MEF feeders in iPSC medium (Knockout DMEM (Life Technologies), containing 20% Knockout SR (Life Technologies), 1% GlutaMAX, 0.1 mM β-mercaptoethanol (Life Technologies), 1% antibiotics, 1% NEAA, and 10 ng/ml basic fibroblast growth factor (bFGF) (Peprotech). ..

    Article Title: Generation of neural progenitor cells by chemical cocktails and hypoxia
    Article Snippet: HUCs were collected and cultured in REGM (Lonza, CC-4127) as described , . .. Generation of ciNPCs For neural progenitor cell induction from MEFs and TTFs, initial cells cultured in DMEM for 24 h were transferred into KSR medium including knockout DMEM (Life Technologies, 10829-018), 15% knockout serum replacement (Life Technologies, 10828), 1% NEAA (Life Technologies, 35050), 1% GlutaMax (Life Technologies, 35050-061), 1% sodium pyruvate (Life Technologies, 11360), 0.1 mM β-mercaptoethanol (Life Technologies, 21985-023) and 1 000 U/ml leukemia inhibitory factor (LIF) (Chemicon, ESG1107). ..

    Article Title: Zinc finger proteins orchestrate active gene silencing during embryonic stem cell differentiation
    Article Snippet: Embryonic stem cells were cultured and passaged on 0.1% gelatinized (Sigma-Aldrich, St. Louis, MO, USA) dishes, as reported previously ( ). .. E14 mouse ESCs were cultured in DMEM (Hyclone, Logan, Utah) or KNOCK-OUT™ DMEM (Gibco, Grand Island, NY, USA) supplemented with 15% FBS (Gibco, Grand Island, NY, USA), 2 mM l -glutamine, 55 μM β-mercaptoethanol, 1% (v/v) non-essential amino acid, 100 U/ml penicillin and 100 μg/ml streptomycin (all from Gibco, Grand Island, New York) and 500 U/ml ESGRO LIF (Millipore, Germany). ..

    Stable Transfection:

    Article Title: Generation of liver-specific TGF-α/c-Myc-overexpressing porcine induced pluripotent stem-like cells and blastocyst formation using nuclear transfer
    Article Snippet: In our preliminary study, Knockout DMEM containing 15% FBS (ES grade; Hyclone, Logan, UT, U.S.A.) with bFGF was not suitable for initial iPS cell-like colony generation. .. The initial iPS cell colonies were manually passaged onto the MEF feeder and stably maintained over 35 passages with medium containing Knockout DMEM, 15% FBS, 0.1 mM nonessential amino acid (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), 10n g/ml bFGF and 40 n g/ml SCF. .. In order to confirm the pluripotency of the transgenic porcine iPS-like cells, a characterization of the cells was carried out.

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  • 99
    Thermo Fisher knock out dmem medium
    In vitro and in vivo pluripotency of FGF-iPSCs. (A) FGF-iPSCs were detached with collagenase IV from the substrate to induce embryoid body (EB) formation. After 5 days in culture, the majority of EBs downregulate the GFP transgene. (B) EBs plated in <t>N2-medium</t> generate nestin positive neural progenitor cells as highlighted by immunofluorescence with its specific antibody. (C) EBs were plated on gelatin coated dishes containing <t>DMEM</t> medium supplemented with 20% FBS. After 20 days in such condition, islands of beating cardiomyocite became evident (see Movie S1 ). Cell differentiation was confirmed by immunoistochemistry for the mesodermal marker smooth muscle actin (Sma) (C) and for the endodermal marker Sox17 (D). (E–H) Ematoxylin and Eosin histological staining of teratomas, derived by subcutaneously injection of 1 million FGF-iPSCs, showed cartilage (E), adipose tissue (F), gut-like epithelium (G) and muscle (H). (I) Oct4-GFP positive FGF-iPSCs integrate into the inner cell mass of mouse blastocyst after morula aggregation and contribute, thereafter, to viable highly chimaeric animals (on the right) as compared to unmanipulated mice (on the left) (J). (K) Germiline contribution of FGF-iPS #5 and #9 chimera. (L) Genotyping demontrate the presence of the Oct4-GFP transgene in the offspring.
    Knock Out Dmem Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/knock out dmem medium/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    knock out dmem medium - by Bioz Stars, 2021-04
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    97
    Thermo Fisher ksr medium
    <t>hiPS</t> cells generated with an mRNA-based integration-free method display typical characteristics of hES cells. a Timeline and essential steps for the reprogramming of human fibroblasts into mRNA-induced iPS cells. Human fibroblasts were plated 1 day before the first transfection in FBSm media, on a human feeder-coated dish. Cells were transfected daily with synthetic mRNA encoding the factors OCT4, SOX2, KLF4, C-MYC, and LIN28 in Pluriton™ reprogramming medium plus B18R (all provided by Stemgent) and kept in norm-oxygen (21 %) conditions. iPS colonies appeared between days 15 and 21, when they were mechanically picked and moved onto feeders in <t>KSR</t> medium. b Morphological changes of human fibroblasts throughout the reprogramming period in norm-oxygen. Typical fibroblast morphology at the start (day 0), transitioning cells with epithelial morphology half-way (day 10), until embryonic stem cell-like colonies have formed (day 19). Magnification: 40× ( background images ), digital zoom ( smaller windows ). c hiPS cell lines express typical intracellular and extracellular pluripotency markers. Immunofluorescence staining with monoclonal antibodies of stem cell markers OCT4, TRA-1-81, and SSEA4 ( red ) and Hoechst DNA counterstain ( blue ) shown for iPS cell lines MIFF1 and MIFF3. Magnification: 40× ( background images ), digital zoom ( smaller windows ). d Established hiPS cells are able to differentiate and induce markers of three germ layers in a 7-day EB differentiation assay. Early ectoderm marker PAX6, mesoderm marker brachyury, and endoderm marker AFP are upregulated in day 7 EBs, as analyzed by RT-PCR. Nanog, a marker of undifferentiated hES cells, decreases its expression upon differentiation. Beta-actin is used as a housekeeping gene. e Representative images of H E-stained microsections of a teratoma generated after injection of hiPS cells into immunocompromised mice. Teratomas were extracted 9–12 weeks after injection and fixed in formaldehyde before embedding and sectioning. Sections show the presence of cartilage (mesoderm), intestinal glandular-like structure (endoderm), and neural tissue (ectoderm), representing derivative of the three germ layers. Magnification: 160×. AFP alpha-fetoprotein, UD undifferentiated cells, EB embryoid body, FBS fetal bovine serum, hES human embryonic stem, MIFF mRNA-induced foreskin fibroblast, PAX6 paired box 6, SSEA stage-specific embryonic antigen
    Ksr Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ksr medium/product/Thermo Fisher
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    99
    Thermo Fisher knock out dmem
    Zfp217 and Zfp516 double <t>knock-out</t> ESCs fail to differentiate properly. ( A and B ) Self-renewal assay and alkaline phosphatase (AP) staining of wild-type, Zfp516 KO, Zfp217 KO and Zfp217 and Zfp516 double knock-out (DKO) ESCs in undifferentiated and differentiated conditions. Double knock-out of Zfp217 and Zfp516 leads to incomplete exit from pluripotency during ESC differentiation by sustaining more positive AP colonies than wild-type ESCs ( n = 3). Presented as means ± SEM. ( C and D ) The protein and mRNA levels of Zfp217, Zfp516 and pluripotency-associated genes in wild-type, Zfp516 KO, Zfp217 KO and DKO ESCs upon ESC differentiation by LIF withdrawal for 6 days. Expression was detected by indicated antibodies; Actin was used as a control ( n = 3). Presented as means ± SEM (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). ( E and F ) Embryoid body (EB) formation of wild-type, Zfp516 KO, Zfp217 KO and DKO ESCs. Diameter of EBs were measured for size determination. Representative cells are shown at 40 × magnification from EB cultures ( n = 8). Presented as means ± SEM. ( G ) After forming embryonic bodies (EB) (4 and 8 days) of wild-type, Zfp516 KO, Zfp217 KO and DKO ESCs, the mRNA levels of Nanog and the three germ layer markers [mesoderm ( Sma, T, Pitx2 ) (labeled in green); endoderm ( Afp, Sox17,Gata4, Gata6 ) (labeled in red); ectoderm ( Sox1, Fgf5 ) (labeled in blue)] were analyzed by qRT-PCR ( n = 3). Presented as means ± SEM (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001).
    Knock Out Dmem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    In vitro and in vivo pluripotency of FGF-iPSCs. (A) FGF-iPSCs were detached with collagenase IV from the substrate to induce embryoid body (EB) formation. After 5 days in culture, the majority of EBs downregulate the GFP transgene. (B) EBs plated in N2-medium generate nestin positive neural progenitor cells as highlighted by immunofluorescence with its specific antibody. (C) EBs were plated on gelatin coated dishes containing DMEM medium supplemented with 20% FBS. After 20 days in such condition, islands of beating cardiomyocite became evident (see Movie S1 ). Cell differentiation was confirmed by immunoistochemistry for the mesodermal marker smooth muscle actin (Sma) (C) and for the endodermal marker Sox17 (D). (E–H) Ematoxylin and Eosin histological staining of teratomas, derived by subcutaneously injection of 1 million FGF-iPSCs, showed cartilage (E), adipose tissue (F), gut-like epithelium (G) and muscle (H). (I) Oct4-GFP positive FGF-iPSCs integrate into the inner cell mass of mouse blastocyst after morula aggregation and contribute, thereafter, to viable highly chimaeric animals (on the right) as compared to unmanipulated mice (on the left) (J). (K) Germiline contribution of FGF-iPS #5 and #9 chimera. (L) Genotyping demontrate the presence of the Oct4-GFP transgene in the offspring.

    Journal: PLoS ONE

    Article Title: An ES-Like Pluripotent State in FGF-Dependent Murine iPS cells

    doi: 10.1371/journal.pone.0016092

    Figure Lengend Snippet: In vitro and in vivo pluripotency of FGF-iPSCs. (A) FGF-iPSCs were detached with collagenase IV from the substrate to induce embryoid body (EB) formation. After 5 days in culture, the majority of EBs downregulate the GFP transgene. (B) EBs plated in N2-medium generate nestin positive neural progenitor cells as highlighted by immunofluorescence with its specific antibody. (C) EBs were plated on gelatin coated dishes containing DMEM medium supplemented with 20% FBS. After 20 days in such condition, islands of beating cardiomyocite became evident (see Movie S1 ). Cell differentiation was confirmed by immunoistochemistry for the mesodermal marker smooth muscle actin (Sma) (C) and for the endodermal marker Sox17 (D). (E–H) Ematoxylin and Eosin histological staining of teratomas, derived by subcutaneously injection of 1 million FGF-iPSCs, showed cartilage (E), adipose tissue (F), gut-like epithelium (G) and muscle (H). (I) Oct4-GFP positive FGF-iPSCs integrate into the inner cell mass of mouse blastocyst after morula aggregation and contribute, thereafter, to viable highly chimaeric animals (on the right) as compared to unmanipulated mice (on the left) (J). (K) Germiline contribution of FGF-iPS #5 and #9 chimera. (L) Genotyping demontrate the presence of the Oct4-GFP transgene in the offspring.

    Article Snippet: Feeder-free culture of FGF-iPSCs FGF-iPS cells were cultivated on fibronectin (Sigma) coated dishes (5 ug/ml) in a knock-out DMEM medium (Invitrogen) containing N2 and B27 and supplemented with 100 ng/ml bFGF (Invitrogen), 50 ng/ml ActivinA (Peprotech), 2-β-mercaptoethanol, L-glutamine and non-essential amino acids (Invitrogen).

    Techniques: In Vitro, In Vivo, Immunofluorescence, Cell Differentiation, Marker, Staining, Derivative Assay, Injection, Mouse Assay

    hiPS cells generated with an mRNA-based integration-free method display typical characteristics of hES cells. a Timeline and essential steps for the reprogramming of human fibroblasts into mRNA-induced iPS cells. Human fibroblasts were plated 1 day before the first transfection in FBSm media, on a human feeder-coated dish. Cells were transfected daily with synthetic mRNA encoding the factors OCT4, SOX2, KLF4, C-MYC, and LIN28 in Pluriton™ reprogramming medium plus B18R (all provided by Stemgent) and kept in norm-oxygen (21 %) conditions. iPS colonies appeared between days 15 and 21, when they were mechanically picked and moved onto feeders in KSR medium. b Morphological changes of human fibroblasts throughout the reprogramming period in norm-oxygen. Typical fibroblast morphology at the start (day 0), transitioning cells with epithelial morphology half-way (day 10), until embryonic stem cell-like colonies have formed (day 19). Magnification: 40× ( background images ), digital zoom ( smaller windows ). c hiPS cell lines express typical intracellular and extracellular pluripotency markers. Immunofluorescence staining with monoclonal antibodies of stem cell markers OCT4, TRA-1-81, and SSEA4 ( red ) and Hoechst DNA counterstain ( blue ) shown for iPS cell lines MIFF1 and MIFF3. Magnification: 40× ( background images ), digital zoom ( smaller windows ). d Established hiPS cells are able to differentiate and induce markers of three germ layers in a 7-day EB differentiation assay. Early ectoderm marker PAX6, mesoderm marker brachyury, and endoderm marker AFP are upregulated in day 7 EBs, as analyzed by RT-PCR. Nanog, a marker of undifferentiated hES cells, decreases its expression upon differentiation. Beta-actin is used as a housekeeping gene. e Representative images of H E-stained microsections of a teratoma generated after injection of hiPS cells into immunocompromised mice. Teratomas were extracted 9–12 weeks after injection and fixed in formaldehyde before embedding and sectioning. Sections show the presence of cartilage (mesoderm), intestinal glandular-like structure (endoderm), and neural tissue (ectoderm), representing derivative of the three germ layers. Magnification: 160×. AFP alpha-fetoprotein, UD undifferentiated cells, EB embryoid body, FBS fetal bovine serum, hES human embryonic stem, MIFF mRNA-induced foreskin fibroblast, PAX6 paired box 6, SSEA stage-specific embryonic antigen

    Journal: Stem Cell Research & Therapy

    Article Title: Apoptosis and failure of checkpoint kinase 1 activation in human induced pluripotent stem cells under replication stress

    doi: 10.1186/s13287-016-0279-2

    Figure Lengend Snippet: hiPS cells generated with an mRNA-based integration-free method display typical characteristics of hES cells. a Timeline and essential steps for the reprogramming of human fibroblasts into mRNA-induced iPS cells. Human fibroblasts were plated 1 day before the first transfection in FBSm media, on a human feeder-coated dish. Cells were transfected daily with synthetic mRNA encoding the factors OCT4, SOX2, KLF4, C-MYC, and LIN28 in Pluriton™ reprogramming medium plus B18R (all provided by Stemgent) and kept in norm-oxygen (21 %) conditions. iPS colonies appeared between days 15 and 21, when they were mechanically picked and moved onto feeders in KSR medium. b Morphological changes of human fibroblasts throughout the reprogramming period in norm-oxygen. Typical fibroblast morphology at the start (day 0), transitioning cells with epithelial morphology half-way (day 10), until embryonic stem cell-like colonies have formed (day 19). Magnification: 40× ( background images ), digital zoom ( smaller windows ). c hiPS cell lines express typical intracellular and extracellular pluripotency markers. Immunofluorescence staining with monoclonal antibodies of stem cell markers OCT4, TRA-1-81, and SSEA4 ( red ) and Hoechst DNA counterstain ( blue ) shown for iPS cell lines MIFF1 and MIFF3. Magnification: 40× ( background images ), digital zoom ( smaller windows ). d Established hiPS cells are able to differentiate and induce markers of three germ layers in a 7-day EB differentiation assay. Early ectoderm marker PAX6, mesoderm marker brachyury, and endoderm marker AFP are upregulated in day 7 EBs, as analyzed by RT-PCR. Nanog, a marker of undifferentiated hES cells, decreases its expression upon differentiation. Beta-actin is used as a housekeeping gene. e Representative images of H E-stained microsections of a teratoma generated after injection of hiPS cells into immunocompromised mice. Teratomas were extracted 9–12 weeks after injection and fixed in formaldehyde before embedding and sectioning. Sections show the presence of cartilage (mesoderm), intestinal glandular-like structure (endoderm), and neural tissue (ectoderm), representing derivative of the three germ layers. Magnification: 160×. AFP alpha-fetoprotein, UD undifferentiated cells, EB embryoid body, FBS fetal bovine serum, hES human embryonic stem, MIFF mRNA-induced foreskin fibroblast, PAX6 paired box 6, SSEA stage-specific embryonic antigen

    Article Snippet: Successfully reprogrammed colonies were selected by morphology and picked mechanically for the first three passages, and then propagated using Collagenase IV. hiPS cells were maintained on inactivated mouse embryonic fibroblast feeder cells in KSR medium (DMEM–F12 supplemented with 20 % KnockOut™ Serum Replacement (Life Technologies, Carlsbad, CA, USA ), 1 % non-essential amino acids, 1 % l -glutamine, 0.1 mM beta-mercaptoethanol, 4 ng/ml basic fibroblast growth factor).

    Techniques: Generated, Transfection, Immunofluorescence, Staining, Differentiation Assay, Marker, Reverse Transcription Polymerase Chain Reaction, Expressing, Injection, Mouse Assay

    Conversion of uMSCs into myogenic lineage. ( A ) Myogenic differentiation was induced by culturing UCB MSCs in myogenic differentiation medium M1 for 3, 6, and 10 days to detect Pax7, MyoD, Myogenin, and MyHC by ( i ) FACS and by ( ii ) immunocytochemistry. Nuclei were counterstained with DAPI. ( iii ) Myf5 mRNA expression in control and differentiated UCB MSCs after 3 days (* p = 0.03). ( iv ) Comparative immunofluorescent analysis of Pax7 and MyoD at 3 days, Myogenin at 7 days and MyHC at 10 days between UCT and UCB MSCs. UCT MSCs show increased expression of MyoD, Myogenin, and MyHC expressing cells compared to UCB MSCs at their corresponding time points (* p = 0.03). ( B ) Myogenin and MyHC mRNA expression in control UCT MSCs or UCT MSCs induced to differentiate in M1 medium with enhanced adhesion or M2 medium for 10 and 20 days (** p = 0.002). Robust expression of MyHC was observed in M1 medium with enhanced adhesion at 10 and 20 days.

    Journal: Scientific Reports

    Article Title: Umbilical cord tissue is a robust source for mesenchymal stem cells with enhanced myogenic differentiation potential compared to cord blood

    doi: 10.1038/s41598-020-75102-9

    Figure Lengend Snippet: Conversion of uMSCs into myogenic lineage. ( A ) Myogenic differentiation was induced by culturing UCB MSCs in myogenic differentiation medium M1 for 3, 6, and 10 days to detect Pax7, MyoD, Myogenin, and MyHC by ( i ) FACS and by ( ii ) immunocytochemistry. Nuclei were counterstained with DAPI. ( iii ) Myf5 mRNA expression in control and differentiated UCB MSCs after 3 days (* p = 0.03). ( iv ) Comparative immunofluorescent analysis of Pax7 and MyoD at 3 days, Myogenin at 7 days and MyHC at 10 days between UCT and UCB MSCs. UCT MSCs show increased expression of MyoD, Myogenin, and MyHC expressing cells compared to UCB MSCs at their corresponding time points (* p = 0.03). ( B ) Myogenin and MyHC mRNA expression in control UCT MSCs or UCT MSCs induced to differentiate in M1 medium with enhanced adhesion or M2 medium for 10 and 20 days (** p = 0.002). Robust expression of MyHC was observed in M1 medium with enhanced adhesion at 10 and 20 days.

    Article Snippet: After reaching a confluence of 70%, myogenic differentiation was induced by adding M1 medium (DMEM, 5% horse serum, 0.1 µM dexamethasone, and 50 µM hydrocortisone) or M2 medium (DMEM, 15% KnockOut Serum Replacement (KSR, ThermoFisher Scientific), 0.5% DMSO (Merck), CHIRON99021 (Tocris) at 1 μM and 0.1 μM LDN193189 (Tocris) for 5 days followed by DMEM, 15% KSR, 0.1% BSA supplemented with 10 ng/ml HGF, 2 ng/ml IGF-1, 20 ng/ml FGF-2 (Peprotech, Merck) and 0.1 μM LDN193189 for the rest of the incubation period.

    Techniques: FACS, Immunocytochemistry, Expressing

    Zfp217 and Zfp516 double knock-out ESCs fail to differentiate properly. ( A and B ) Self-renewal assay and alkaline phosphatase (AP) staining of wild-type, Zfp516 KO, Zfp217 KO and Zfp217 and Zfp516 double knock-out (DKO) ESCs in undifferentiated and differentiated conditions. Double knock-out of Zfp217 and Zfp516 leads to incomplete exit from pluripotency during ESC differentiation by sustaining more positive AP colonies than wild-type ESCs ( n = 3). Presented as means ± SEM. ( C and D ) The protein and mRNA levels of Zfp217, Zfp516 and pluripotency-associated genes in wild-type, Zfp516 KO, Zfp217 KO and DKO ESCs upon ESC differentiation by LIF withdrawal for 6 days. Expression was detected by indicated antibodies; Actin was used as a control ( n = 3). Presented as means ± SEM (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). ( E and F ) Embryoid body (EB) formation of wild-type, Zfp516 KO, Zfp217 KO and DKO ESCs. Diameter of EBs were measured for size determination. Representative cells are shown at 40 × magnification from EB cultures ( n = 8). Presented as means ± SEM. ( G ) After forming embryonic bodies (EB) (4 and 8 days) of wild-type, Zfp516 KO, Zfp217 KO and DKO ESCs, the mRNA levels of Nanog and the three germ layer markers [mesoderm ( Sma, T, Pitx2 ) (labeled in green); endoderm ( Afp, Sox17,Gata4, Gata6 ) (labeled in red); ectoderm ( Sox1, Fgf5 ) (labeled in blue)] were analyzed by qRT-PCR ( n = 3). Presented as means ± SEM (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001).

    Journal: Nucleic Acids Research

    Article Title: Zinc finger proteins orchestrate active gene silencing during embryonic stem cell differentiation

    doi: 10.1093/nar/gky454

    Figure Lengend Snippet: Zfp217 and Zfp516 double knock-out ESCs fail to differentiate properly. ( A and B ) Self-renewal assay and alkaline phosphatase (AP) staining of wild-type, Zfp516 KO, Zfp217 KO and Zfp217 and Zfp516 double knock-out (DKO) ESCs in undifferentiated and differentiated conditions. Double knock-out of Zfp217 and Zfp516 leads to incomplete exit from pluripotency during ESC differentiation by sustaining more positive AP colonies than wild-type ESCs ( n = 3). Presented as means ± SEM. ( C and D ) The protein and mRNA levels of Zfp217, Zfp516 and pluripotency-associated genes in wild-type, Zfp516 KO, Zfp217 KO and DKO ESCs upon ESC differentiation by LIF withdrawal for 6 days. Expression was detected by indicated antibodies; Actin was used as a control ( n = 3). Presented as means ± SEM (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). ( E and F ) Embryoid body (EB) formation of wild-type, Zfp516 KO, Zfp217 KO and DKO ESCs. Diameter of EBs were measured for size determination. Representative cells are shown at 40 × magnification from EB cultures ( n = 8). Presented as means ± SEM. ( G ) After forming embryonic bodies (EB) (4 and 8 days) of wild-type, Zfp516 KO, Zfp217 KO and DKO ESCs, the mRNA levels of Nanog and the three germ layer markers [mesoderm ( Sma, T, Pitx2 ) (labeled in green); endoderm ( Afp, Sox17,Gata4, Gata6 ) (labeled in red); ectoderm ( Sox1, Fgf5 ) (labeled in blue)] were analyzed by qRT-PCR ( n = 3). Presented as means ± SEM (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001).

    Article Snippet: E14 mouse ESCs were cultured in DMEM (Hyclone, Logan, Utah) or KNOCK-OUT™ DMEM (Gibco, Grand Island, NY, USA) supplemented with 15% FBS (Gibco, Grand Island, NY, USA), 2 mM l -glutamine, 55 μM β-mercaptoethanol, 1% (v/v) non-essential amino acid, 100 U/ml penicillin and 100 μg/ml streptomycin (all from Gibco, Grand Island, New York) and 500 U/ml ESGRO LIF (Millipore, Germany).

    Techniques: Knock-Out, Staining, Expressing, Labeling, Quantitative RT-PCR

    Loss of Zfp217 retards differentiation of embryonic stem cells. ( A and B ) The protein and mRNA levels of Zfp217 and pluripotency-associated genes in wild-type and Zfp217 KO ESCs (clone #6 and #14) upon LIF withdrawal for indicated days. Expression was detected by indicated antibodies; Actin was used as a control. ( n = 3) Presented as means ± SEM (* P ≤ 0.05, ** P ≤ 0.01). ( C ) Zfp217 expression was rescued by Flag-tagged Zfp217 overexpression in Zfp217 KO ESCs (Zfp217-rescued ESCs). Western blot analysis of Zfp217 KO and Zfp217-rescued ESCs, using Flag and Zfp217 antibodies. Expression was detected by indicated antibodies; Actin was used as a control. ( D ) Self-renewal assay and alkaline phosphatase (AP) staining of Zfp217 KO and Zfp217-rescued ESCs in undifferentiated and differentiated conditions. Knock-out of Zfp217 leads to incomplete exit from pluripotency during ESC differentiation; this phenotype was rescued upon Zfp217 introduction. ( E and F ) The protein and mRNA levels of Zfp217 , and pluripotency-associated genes in wild-type, Zfp217 KO, and Zfp217-rescued ESCs upon LIF withdrawal for indicated days. Expression was detected by indicated antibodies; Actin was used as a control ( n = 3) Presented as means ± SEM (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001)

    Journal: Nucleic Acids Research

    Article Title: Zinc finger proteins orchestrate active gene silencing during embryonic stem cell differentiation

    doi: 10.1093/nar/gky454

    Figure Lengend Snippet: Loss of Zfp217 retards differentiation of embryonic stem cells. ( A and B ) The protein and mRNA levels of Zfp217 and pluripotency-associated genes in wild-type and Zfp217 KO ESCs (clone #6 and #14) upon LIF withdrawal for indicated days. Expression was detected by indicated antibodies; Actin was used as a control. ( n = 3) Presented as means ± SEM (* P ≤ 0.05, ** P ≤ 0.01). ( C ) Zfp217 expression was rescued by Flag-tagged Zfp217 overexpression in Zfp217 KO ESCs (Zfp217-rescued ESCs). Western blot analysis of Zfp217 KO and Zfp217-rescued ESCs, using Flag and Zfp217 antibodies. Expression was detected by indicated antibodies; Actin was used as a control. ( D ) Self-renewal assay and alkaline phosphatase (AP) staining of Zfp217 KO and Zfp217-rescued ESCs in undifferentiated and differentiated conditions. Knock-out of Zfp217 leads to incomplete exit from pluripotency during ESC differentiation; this phenotype was rescued upon Zfp217 introduction. ( E and F ) The protein and mRNA levels of Zfp217 , and pluripotency-associated genes in wild-type, Zfp217 KO, and Zfp217-rescued ESCs upon LIF withdrawal for indicated days. Expression was detected by indicated antibodies; Actin was used as a control ( n = 3) Presented as means ± SEM (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001)

    Article Snippet: E14 mouse ESCs were cultured in DMEM (Hyclone, Logan, Utah) or KNOCK-OUT™ DMEM (Gibco, Grand Island, NY, USA) supplemented with 15% FBS (Gibco, Grand Island, NY, USA), 2 mM l -glutamine, 55 μM β-mercaptoethanol, 1% (v/v) non-essential amino acid, 100 U/ml penicillin and 100 μg/ml streptomycin (all from Gibco, Grand Island, New York) and 500 U/ml ESGRO LIF (Millipore, Germany).

    Techniques: Expressing, Over Expression, Western Blot, Staining, Knock-Out

    Zfp217 and Zfp516 facilitate Ctbp2-mediated repression on active ESC genes during differentiation. ( A ) ChIP-qPCR analysis of Ctbp2 on active ESC gene regions in wild-type, Zfp516 KO, Zfp217 KO and Zfp217 and Zfp516 double knock-out (DKO) ESCs ( n = 3). Presented as means ± SEM. Zfp217 KO, Zfp516 KO ESCs and DKO ESCs showed lower enrichment of Ctbp2 than wild-type ESCs ( n = 3). Presented as means ± SEM (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). ( B ) ChIP-qPCR analysis of Lsd1, Chd4, and Hdac1 on active ESC gene regions in wild-type, Zfp516 KO, Zfp217 KO and Zfp217 and Zfp516 double knock-out (DKO) ESCs ( n = 3). Presented as means ± SEM. Zfp217 KO, Zfp516 KO ESCs and DKO ESCs showed lower enrichment of Chd4 and Lsd1 than wild-type ESCs, but not Hdac1 ( n = 3). Presented as means ± SEM (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). ( C–E ) ChIP-qPCR analysis of H3K27ac, Ezh2 and H3K27me3 on active ESC gene regions in wild-type, Zfp516 KO, Zfp217 KO and DKO ESCs in undifferentiated and differentiated (LIF withdrawal for 6 days) conditions ( n = 3). Presented as means ± SEM (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001).

    Journal: Nucleic Acids Research

    Article Title: Zinc finger proteins orchestrate active gene silencing during embryonic stem cell differentiation

    doi: 10.1093/nar/gky454

    Figure Lengend Snippet: Zfp217 and Zfp516 facilitate Ctbp2-mediated repression on active ESC genes during differentiation. ( A ) ChIP-qPCR analysis of Ctbp2 on active ESC gene regions in wild-type, Zfp516 KO, Zfp217 KO and Zfp217 and Zfp516 double knock-out (DKO) ESCs ( n = 3). Presented as means ± SEM. Zfp217 KO, Zfp516 KO ESCs and DKO ESCs showed lower enrichment of Ctbp2 than wild-type ESCs ( n = 3). Presented as means ± SEM (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). ( B ) ChIP-qPCR analysis of Lsd1, Chd4, and Hdac1 on active ESC gene regions in wild-type, Zfp516 KO, Zfp217 KO and Zfp217 and Zfp516 double knock-out (DKO) ESCs ( n = 3). Presented as means ± SEM. Zfp217 KO, Zfp516 KO ESCs and DKO ESCs showed lower enrichment of Chd4 and Lsd1 than wild-type ESCs, but not Hdac1 ( n = 3). Presented as means ± SEM (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). ( C–E ) ChIP-qPCR analysis of H3K27ac, Ezh2 and H3K27me3 on active ESC gene regions in wild-type, Zfp516 KO, Zfp217 KO and DKO ESCs in undifferentiated and differentiated (LIF withdrawal for 6 days) conditions ( n = 3). Presented as means ± SEM (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001).

    Article Snippet: E14 mouse ESCs were cultured in DMEM (Hyclone, Logan, Utah) or KNOCK-OUT™ DMEM (Gibco, Grand Island, NY, USA) supplemented with 15% FBS (Gibco, Grand Island, NY, USA), 2 mM l -glutamine, 55 μM β-mercaptoethanol, 1% (v/v) non-essential amino acid, 100 U/ml penicillin and 100 μg/ml streptomycin (all from Gibco, Grand Island, New York) and 500 U/ml ESGRO LIF (Millipore, Germany).

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Knock-Out