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  • 93
    Thermo Fisher anti ssea1
    Generation of miPSCs with SeVdp Expressing ESC-Enriched miRNAs (A) Enhanced miPSC colony formation in MEFs co-infected with SeVdp(K/O/S) and SeVdp expressing ESC-enriched miRNA(s). <t>SSEA1+</t> colonies were counted on day 14 after infection. **p
    Anti Ssea1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam antibodies ssea 1
    Characteristics of cESCs in a long culture period. This culture system can stably maintain the stem-like character of cESCs in vitro for a long term. (a) Epitope characteristic of cultured cESCs in passages 3, 5, and 20. The pluripotency surface marker <t>SSEA-1</t> (green) was stained. Meanwhile, DAPI (blue) was used to stain the nucleus. (b) SSEA-1 expression was maintained at a relatively stable level. The ImageXpress Micro XLS Widefield High-Content Analysis System was used. The bars indicated the positive SSEA-1 mean cell average intensity of cultured cESCs in passages 3, 5, and 20. (c) Lin28a , Nanog , and PouV gene mRNA relative expression level of cultured cESCs in passages 3, 6, 9, and 20. ∗ Compared to the expression level of cultured cESCs in passage 3, P
    Antibodies Ssea 1, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore ssea1
    Expression of pluripotency-associated proteins Oct-4 and <t>SSEA1</t> in e-Pc with immunofluorescent staining. The scale bar represents 50 μm. Oct-4 and SSEA1 proteins were found in P9 (week 4), not in P18 (week 8) cells.
    Ssea1, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 370 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology ssea1
    De novo FA synthesis mediated by Acc1 affects mitochondrial fission via Fis1 acetylation or ER stress/UPR qRT‐PCR analysis showed the mRNA expression of Acc1 and Fis1 in Acc1 overexpressing MEF cells. Western blotting showing the expression of FIS1 in 10 μM TSA‐ and 5 mM NAM‐treated 293T cells in the presence or absence of MG132. EV‐ or ACC1‐overexpressing MEFs were infected with viruses expressing four factors in the presence or absence of acetate treatment for 12 days. AP‐ and <t>SSEA1‐positive</t> colonies were counted on day 14 after virus infection. AP staining of iPSC colonies is shown on the top. Cellular AcCoA levels were measured in MEF cells treated with oleic acid by ELISA Kit. The values were normalized by cellular protein. Equal amount of proteins from Flag‐Fis1‐overexpressing MEF cells treated with or without OA were used for immunoprecipitation with anti‐Flag antibody, followed by blotting with anti‐acetylated‐lysine or anti‐Fis1. Western blot analysis of ER stress or UPR related protein Atf6, Chop, Atf4, and Bip in MEF cells treated with OA. Mitochondrial fission protein Drp1, Fis1, Mff, Mid51, and Mid49 were analyzed by Western blot in MEF cells treated with OA in the presence or absence of 0.5 mM 4‐PBA for 24 h. OA or/and 4‐PBA was added into the medium of MEF cells starting from 2 days after infection with viruses expressing four factors. AP staining (upper panel) showed the formed iPSC colonies. AP‐ and SSEA1‐positive iPSC colonies were counted (lower panel). Data information: Data are presented as mean (± SD). * P
    Ssea1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Developmental Studies Hybridoma Bank anti ssea1
    PGCs are reduced in Tbx4 -null embryos. Tbx4 +/− and Tbx4 −/− embryos at the 30- to 33-somite stage were isolated and stained with <t>anti-SSEA1</t> antibody to identify PGCs. Right lateral views of the trunk region with anterior to the
    Anti Ssea1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 90/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Becton Dickinson anti ssea1
    Cellular Pluripotency Marker Expression Levels in EpiSCs Derived by the IWP-2 Method (A–J) Immunofluorescence images for <t>SSEA1</t> (A and B), PECAM1 (red) and OCT4 (green, C and D), NANOG (E and F), and histone H3K27 trimethylation (G and H) in mESCs (A, C, E, and G) and EpiSCs (B, D, F, and H). Nuclear staining is shown using TO-PRO3 (blue). Cytograms of EpiSCs and mESCs are shown using anti-SSEA1 (I) and anti-PECAM1 (J) antibodies. (K) Relative expression levels of marker genes in EpiSCs detected by qRT-PCR compared with J1 mESCs. For each gene, technical triplicate assays and two independent experiments were performed. Error bars represent the standard SEM. (L) Growth curves of EpiSCs. At the indicated time points, 2.0 × 10 4 cells were plated and counted. Averaged data from three independent experiments were plotted. Scale bars, 20 μm (A, B, C, D, G, and H) and 50 μm (E and F).
    Anti Ssea1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Stemgent anti ssea1
    In vitro characterization of the pluripotent state of EiPS cells. ( a ). Nanog, <t>SSEA1,</t> SSEA4, TRA-1-60 and TRA-1-81 detected by fluorescent immunohistochemistry. Yellow scale bar 10 μm, white scalebar 20 μm. ( b ). Detection of gene expression of key pluripotency markers using RT-PCR primers specific for equine endogenous genes
    Anti Ssea1, supplied by Stemgent, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti ssea1
    Characterization of B6 MOG/mESCs and induction of the differentiation of the cells into TEPs in vitro . (A) MOG/mESCs and control mESCs were analyzed for GFP and MOG expression by immunofluorescence with an anti-MOG antibody. The expression of GFP (green) and MOG (red) was observed under a confocal microscope. Scale bars: 50 μm. (B, C) MOG/mESCs and control mESCs were examined for the expression of (B) MOG protein by Western blot, and pluripotent markers (C) AP by AP staining and (D) Sox2 and <t>SSEA1</t> by immunofluorescence. (E) Parent B6 mESC, MOG/mESCs and control mESCs (5X10 5 cells/well) were induced to differentiate into definitive endoderm and then TEPs in the presence or absence of BFFE, rFOXN1 and rHOXA3 proteins. The mESC-derived cells were analyzed for the expression of EpCAM1, K5 and K8 proteins on day 16 by flow cytometry. The number of EpCAM + K5 + K8 + cells was shown. (F) B6 mice were injected i.t. with EpCAM1 + MOG/mESC-TEPs (5×10 4 ), or control mESC-TEPs (5×10 4 ). MOG protein expression in the thymus was examined by Western blot on day 90 after the injection. The data are presented from 3 independent experiments.
    Anti Ssea1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend anti ssea1
    Characterization of B6 MOG/mESCs and induction of the differentiation of the cells into TEPs in vitro . (A) MOG/mESCs and control mESCs were analyzed for GFP and MOG expression by immunofluorescence with an anti-MOG antibody. The expression of GFP (green) and MOG (red) was observed under a confocal microscope. Scale bars: 50 μm. (B, C) MOG/mESCs and control mESCs were examined for the expression of (B) MOG protein by Western blot, and pluripotent markers (C) AP by AP staining and (D) Sox2 and <t>SSEA1</t> by immunofluorescence. (E) Parent B6 mESC, MOG/mESCs and control mESCs (5X10 5 cells/well) were induced to differentiate into definitive endoderm and then TEPs in the presence or absence of BFFE, rFOXN1 and rHOXA3 proteins. The mESC-derived cells were analyzed for the expression of EpCAM1, K5 and K8 proteins on day 16 by flow cytometry. The number of EpCAM + K5 + K8 + cells was shown. (F) B6 mice were injected i.t. with EpCAM1 + MOG/mESC-TEPs (5×10 4 ), or control mESC-TEPs (5×10 4 ). MOG protein expression in the thymus was examined by Western blot on day 90 after the injection. The data are presented from 3 independent experiments.
    Anti Ssea1, supplied by BioLegend, used in various techniques. Bioz Stars score: 90/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Clinisciences anti ssea1 pe
    Characterization of B6 MOG/mESCs and induction of the differentiation of the cells into TEPs in vitro . (A) MOG/mESCs and control mESCs were analyzed for GFP and MOG expression by immunofluorescence with an anti-MOG antibody. The expression of GFP (green) and MOG (red) was observed under a confocal microscope. Scale bars: 50 μm. (B, C) MOG/mESCs and control mESCs were examined for the expression of (B) MOG protein by Western blot, and pluripotent markers (C) AP by AP staining and (D) Sox2 and <t>SSEA1</t> by immunofluorescence. (E) Parent B6 mESC, MOG/mESCs and control mESCs (5X10 5 cells/well) were induced to differentiate into definitive endoderm and then TEPs in the presence or absence of BFFE, rFOXN1 and rHOXA3 proteins. The mESC-derived cells were analyzed for the expression of EpCAM1, K5 and K8 proteins on day 16 by flow cytometry. The number of EpCAM + K5 + K8 + cells was shown. (F) B6 mice were injected i.t. with EpCAM1 + MOG/mESC-TEPs (5×10 4 ), or control mESC-TEPs (5×10 4 ). MOG protein expression in the thymus was examined by Western blot on day 90 after the injection. The data are presented from 3 independent experiments.
    Anti Ssea1 Pe, supplied by Clinisciences, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Kyowa Hakko Kirin anti ssea1 antibody
    Effect of FCS and LIF on the differentiation into trophoblast. The effect of FCS: the cells were cultured in BMP4-supplemented ESF5 medium with 10% FCS (indicating as “BMP4 with FCS”) or without FCS (indicating as “BMP4 only”) for 4 d. ( A ) Quantitative real-time RT–PCR analysis of the expression of trophoblast-specific transcription factors. The gene expressions were normalized by the amount of Gapdh . The values are the mean ± SEM ( n = 4). ( B ) Immunocytochemistry with Cdh3 antibodies. Immunopositive reaction of Cdh3 antibody was visualized with AlexaFluor-488-conjugated secondary antibodies ( green ). Nuclei were stained with DAPI ( blue ). Scale bars are 50 µm. The effect of LIF: the cells were cultured in BMP4-supplemented ESF5 medium with 10 ng/ml of LIF (indicating as “BMP4 with LIF”) or without LIF (indicating as “BMP4 only”) for 4 d. ( C ) Quantitative real-time RT–PCR analysis of the expression of trophoblast-specific transcription factors. ( D ) Immunocytochemistry with Cdh3 antibodies. ( E ) Immunocytochemistry with anti-Nanog or <t>anti-SSEA1</t> antibodies. Immunopositive reaction of anti-Nanog or anti-SSEA1 antibody was visualized with AlexaFluor-488-conjugated secondary antibodies ( green ).
    Anti Ssea1 Antibody, supplied by Kyowa Hakko Kirin, used in various techniques. Bioz Stars score: 88/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Generation of miPSCs with SeVdp Expressing ESC-Enriched miRNAs (A) Enhanced miPSC colony formation in MEFs co-infected with SeVdp(K/O/S) and SeVdp expressing ESC-enriched miRNA(s). SSEA1+ colonies were counted on day 14 after infection. **p

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: A Sendai Virus-Based Cytoplasmic RNA Vector as a Novel Platform for Long-Term Expression of MicroRNAs

    doi: 10.1016/j.omtm.2019.10.012

    Figure Lengend Snippet: Generation of miPSCs with SeVdp Expressing ESC-Enriched miRNAs (A) Enhanced miPSC colony formation in MEFs co-infected with SeVdp(K/O/S) and SeVdp expressing ESC-enriched miRNA(s). SSEA1+ colonies were counted on day 14 after infection. **p

    Article Snippet: The following primary antibodies were used, anti-SSEA1 (1:200; eBioScience, San Diego, CA, USA), anti-NANOG (1:200; Abcam, Cambridge, UK), anti-OCT4 (1:400; Abcam), anti-SOX2 (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti-KLF4 (1:200; Santa Cruz Biotechnology), and anti-SeV-NP (1:1,000).

    Techniques: Expressing, Infection

    Characteristics of cESCs in a long culture period. This culture system can stably maintain the stem-like character of cESCs in vitro for a long term. (a) Epitope characteristic of cultured cESCs in passages 3, 5, and 20. The pluripotency surface marker SSEA-1 (green) was stained. Meanwhile, DAPI (blue) was used to stain the nucleus. (b) SSEA-1 expression was maintained at a relatively stable level. The ImageXpress Micro XLS Widefield High-Content Analysis System was used. The bars indicated the positive SSEA-1 mean cell average intensity of cultured cESCs in passages 3, 5, and 20. (c) Lin28a , Nanog , and PouV gene mRNA relative expression level of cultured cESCs in passages 3, 6, 9, and 20. ∗ Compared to the expression level of cultured cESCs in passage 3, P

    Journal: Stem Cells International

    Article Title: An Alternative Method for Long-Term Culture of Chicken Embryonic Stem Cell In Vitro

    doi: 10.1155/2018/2157451

    Figure Lengend Snippet: Characteristics of cESCs in a long culture period. This culture system can stably maintain the stem-like character of cESCs in vitro for a long term. (a) Epitope characteristic of cultured cESCs in passages 3, 5, and 20. The pluripotency surface marker SSEA-1 (green) was stained. Meanwhile, DAPI (blue) was used to stain the nucleus. (b) SSEA-1 expression was maintained at a relatively stable level. The ImageXpress Micro XLS Widefield High-Content Analysis System was used. The bars indicated the positive SSEA-1 mean cell average intensity of cultured cESCs in passages 3, 5, and 20. (c) Lin28a , Nanog , and PouV gene mRNA relative expression level of cultured cESCs in passages 3, 6, 9, and 20. ∗ Compared to the expression level of cultured cESCs in passage 3, P

    Article Snippet: The primary antibodies SSEA-1 (Abcam, 1 : 100) and Nanog (Abcam, 1 : 150), both of which were diluted in the blocking buffer, were incubated along with the fixed cells overnight at 4°C.

    Techniques: Stable Transfection, In Vitro, Cell Culture, Marker, Staining, Expressing, High Content Screening

    The growth of chicken blastodermal cells (cBCs) in E8 medium. a The cells have large nuclei and pronounced nucleoli ( arrows ), and grow in a monolayer with clearly distinguishable individual cells. b The cells form tight and compact colonies of multilayer cells arranged in clusters after FGF9 and TGF-β are withdrawn. c Immunofluorescence staining of SSEA-1 ( green ), nuclei were counterstained with DAPI ( blue )

    Journal: BMC Biotechnology

    Article Title: Retinoic acid promotes expression of germline-specific genes in chicken blastoderm cells by stimulating Smad1/5 phosphorylation in a feeder-free culture system

    doi: 10.1186/s12896-017-0332-y

    Figure Lengend Snippet: The growth of chicken blastodermal cells (cBCs) in E8 medium. a The cells have large nuclei and pronounced nucleoli ( arrows ), and grow in a monolayer with clearly distinguishable individual cells. b The cells form tight and compact colonies of multilayer cells arranged in clusters after FGF9 and TGF-β are withdrawn. c Immunofluorescence staining of SSEA-1 ( green ), nuclei were counterstained with DAPI ( blue )

    Article Snippet: Immunofluorescence detection Adherent cells were washed with PBS and fixed in 4% paraformaldehyde at room temperature for 20 min, permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO) in PBS for 10 min. After incubation with blocking buffer containing 5% newborn bovine serum (BSA, Solarbio, Beijing, China) for 20 min, the cells were then stained with anti-SSEA-1 antibodies (1:100, Abcam, Cambridge, UK) at 4 °C overnight and at rewarmed at 37 °C for 45 min. Alexa Fluor 488 goat anti-mouse IgG (1:1000, Abcam) was added and incubated at room temperature for 1 h. The nuclei were stained with 10 μM DAPI (Sigma-Aldrich) for 30 min.

    Techniques: Immunofluorescence, Staining

    Characterization of iPSCs in vitro . ( A ) Flow cytometry was performed to detect Oct4, Sox2, Nanog, and SSEA1 (n = 3). ( B ) Immunofluorescent staining was performed for the detection of Oct4, Sox2, Nanog, and SSEA1 by confocal microscopy (n = 3). Scale bar: 50 μm. ( C ) Western blotting for the detection of Oct4, Sox2, and Nanog (n = 3). ( D ) Q-PCR analysis (n = 3). Q-PCR analysis was used to show that the iPSCs express endogenous pluripotency genes, including Oct4, Sox2, Klf4, c-Myc, Nanog , Spalt-Like Transcription Factor 4 ( Sall4 ), dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 ( DAXR1 ), developmental pluripotency-associated 5 ( Dppa5 ), zinc finger protein ( ZFP ), undifferentiated embryonic cell transcription factor ( UTF ), cysteine-rich PDZ-binding protein ( CRIPTO ), reduced expression ( REX ). The parental MEF group was used as a control. ( E ) me3H3K27 immunostaining of MEFs and iPSCs. Red color indicates the staining of me3H3k27. Blue color indicates the DAPI staining. Scale bar: 10 μm.

    Journal: Scientific Reports

    Article Title: EpEX/EpCAM and Oct4 or Klf4 alone are sufficient to generate induced pluripotent stem cells through STAT3 and HIF2α

    doi: 10.1038/srep41852

    Figure Lengend Snippet: Characterization of iPSCs in vitro . ( A ) Flow cytometry was performed to detect Oct4, Sox2, Nanog, and SSEA1 (n = 3). ( B ) Immunofluorescent staining was performed for the detection of Oct4, Sox2, Nanog, and SSEA1 by confocal microscopy (n = 3). Scale bar: 50 μm. ( C ) Western blotting for the detection of Oct4, Sox2, and Nanog (n = 3). ( D ) Q-PCR analysis (n = 3). Q-PCR analysis was used to show that the iPSCs express endogenous pluripotency genes, including Oct4, Sox2, Klf4, c-Myc, Nanog , Spalt-Like Transcription Factor 4 ( Sall4 ), dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 ( DAXR1 ), developmental pluripotency-associated 5 ( Dppa5 ), zinc finger protein ( ZFP ), undifferentiated embryonic cell transcription factor ( UTF ), cysteine-rich PDZ-binding protein ( CRIPTO ), reduced expression ( REX ). The parental MEF group was used as a control. ( E ) me3H3K27 immunostaining of MEFs and iPSCs. Red color indicates the staining of me3H3k27. Blue color indicates the DAPI staining. Scale bar: 10 μm.

    Article Snippet: Cells were stained with antibodies against Oct4, Sox2, Nanog, SSEA1 (1:100) (ab107156, Abcam), HIF2α (1:200) (NB100-122, Novus), me3H3k27 (1:200) (ab6147, Abcam) respectively for 60 min at RT, and then washed with PBS.

    Techniques: In Vitro, Flow Cytometry, Cytometry, Staining, Confocal Microscopy, Western Blot, Polymerase Chain Reaction, Binding Assay, Expressing, Immunostaining

    Testing the identity of the iPS cell by molecular markers. The iPS cells that derived from frozen tail (FT)/fibroblasts (FF) had strong alkaline phosphatase activity ( a ) and had strong expression of mouse ES cell surface marker SSEA1 ( b ). They showed similar expression pattern as the C57BL/6J wild type (WT)ES cells. Magnification ×20. Scale bar in panel a and b, 50 μm

    Journal: Transgenic Research

    Article Title: Rederivation of transgenic mice from iPS cells derived from frozen tissue

    doi: 10.1007/s11248-010-9390-9

    Figure Lengend Snippet: Testing the identity of the iPS cell by molecular markers. The iPS cells that derived from frozen tail (FT)/fibroblasts (FF) had strong alkaline phosphatase activity ( a ) and had strong expression of mouse ES cell surface marker SSEA1 ( b ). They showed similar expression pattern as the C57BL/6J wild type (WT)ES cells. Magnification ×20. Scale bar in panel a and b, 50 μm

    Article Snippet: The cells were incubated with the blocking solution (10% goat serum) for 30 min and then with the SSEA1 antibody (Abcam) in 1:300 dilution for 3 h. Staining was performed using the Vectastain ABC kit (Vector Laboratory) according to the manufacturer’s recommendations.

    Techniques: Derivative Assay, Activity Assay, Expressing, Marker

    Expression of pluripotency-associated proteins Oct-4 and SSEA1 in e-Pc with immunofluorescent staining. The scale bar represents 50 μm. Oct-4 and SSEA1 proteins were found in P9 (week 4), not in P18 (week 8) cells.

    Journal: Molecular Vision

    Article Title: Modulation of rabbit corneal epithelial cells fate using embryonic stem cell extract

    doi:

    Figure Lengend Snippet: Expression of pluripotency-associated proteins Oct-4 and SSEA1 in e-Pc with immunofluorescent staining. The scale bar represents 50 μm. Oct-4 and SSEA1 proteins were found in P9 (week 4), not in P18 (week 8) cells.

    Article Snippet: The primary antibodies were mouse anti-goat Oct-4 recognizing multiclonal Ab (mAb; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), SSEA1 (Chemicon), K3/K12 mAb (Chemicon), p63 mAb (Chemicon), ABCG2 mAb (Santa Cruz Biotechnology, Inc.), and Vimentin mAb (Abcam, Cambridge, UK).

    Techniques: Expressing, Staining

    Determination of specific gene markers in pAF-MSCs. (A) The expression of cell surface antigens was examined by flow cytometry. The antibodies of CD34, CD117 and CD166 were labeled with PE, and CD45, CD44 and CD54 were labeled with FITC. (B) The mRNA expressions of CD90, CD117 and HLA-abc, but not CD45 were detectable in pAF-MSCs by semi-quantitative RT-PCR assay. The RNA samples were prepared from pAF-MSCs at 4 th , 8 th , 12 th and 16 th passages. The β-actin was used as internal control. (C) The pluripotent markers of ES cell were determined in pAF-MSCs (at 6 th passage) by immunofluorescence assay. The antibodies against Oct4, Nanog, SSEA1, SSEA4, Tra-1-60 and Tra-1-81 were conducted and the positive staining showed green fluorescence (FITC). The nuclei were stained by Hoechst 33342 (blue fluorescence).

    Journal: PLoS ONE

    Article Title: Isolation and Characterization of Porcine Amniotic Fluid-Derived Multipotent Stem Cells

    doi: 10.1371/journal.pone.0019964

    Figure Lengend Snippet: Determination of specific gene markers in pAF-MSCs. (A) The expression of cell surface antigens was examined by flow cytometry. The antibodies of CD34, CD117 and CD166 were labeled with PE, and CD45, CD44 and CD54 were labeled with FITC. (B) The mRNA expressions of CD90, CD117 and HLA-abc, but not CD45 were detectable in pAF-MSCs by semi-quantitative RT-PCR assay. The RNA samples were prepared from pAF-MSCs at 4 th , 8 th , 12 th and 16 th passages. The β-actin was used as internal control. (C) The pluripotent markers of ES cell were determined in pAF-MSCs (at 6 th passage) by immunofluorescence assay. The antibodies against Oct4, Nanog, SSEA1, SSEA4, Tra-1-60 and Tra-1-81 were conducted and the positive staining showed green fluorescence (FITC). The nuclei were stained by Hoechst 33342 (blue fluorescence).

    Article Snippet: The primary antibodies used in this experiment were as below: anti-Oct4 (ab13840, Abcam, UK), anti-Nanog (ab21603, Abcam, UK), anti-SSEA1 (90230, Millipore, USA), anti-SSEA4 (90231, Millipore, USA), anti-Tra-1-60 (90232, Millipore, USA), anti-Tra-1-81 (90233, Millipore, USA), anti-NF-200 (ab82259, Abcam, UK), anti-Nestin (MAB5326, Chemicon), anti-TERT (sc-7212, Santa Cruz, UAS), anti-α-actin (A2172, Sigma, USA), anti-β-tublin (bs-0210R, Beijing Biosynthesis Biotechnology Co, LTD, China ), anti-Nkx2.5 (ab97355, Abcam, UK).

    Techniques: Expressing, Flow Cytometry, Cytometry, Labeling, Quantitative RT-PCR, Immunofluorescence, Staining, Fluorescence

    De novo FA synthesis mediated by Acc1 affects mitochondrial fission via Fis1 acetylation or ER stress/UPR qRT‐PCR analysis showed the mRNA expression of Acc1 and Fis1 in Acc1 overexpressing MEF cells. Western blotting showing the expression of FIS1 in 10 μM TSA‐ and 5 mM NAM‐treated 293T cells in the presence or absence of MG132. EV‐ or ACC1‐overexpressing MEFs were infected with viruses expressing four factors in the presence or absence of acetate treatment for 12 days. AP‐ and SSEA1‐positive colonies were counted on day 14 after virus infection. AP staining of iPSC colonies is shown on the top. Cellular AcCoA levels were measured in MEF cells treated with oleic acid by ELISA Kit. The values were normalized by cellular protein. Equal amount of proteins from Flag‐Fis1‐overexpressing MEF cells treated with or without OA were used for immunoprecipitation with anti‐Flag antibody, followed by blotting with anti‐acetylated‐lysine or anti‐Fis1. Western blot analysis of ER stress or UPR related protein Atf6, Chop, Atf4, and Bip in MEF cells treated with OA. Mitochondrial fission protein Drp1, Fis1, Mff, Mid51, and Mid49 were analyzed by Western blot in MEF cells treated with OA in the presence or absence of 0.5 mM 4‐PBA for 24 h. OA or/and 4‐PBA was added into the medium of MEF cells starting from 2 days after infection with viruses expressing four factors. AP staining (upper panel) showed the formed iPSC colonies. AP‐ and SSEA1‐positive iPSC colonies were counted (lower panel). Data information: Data are presented as mean (± SD). * P

    Journal: The EMBO Journal

    Article Title: Fatty acid synthesis is critical for stem cell pluripotency via promoting mitochondrial fission

    doi: 10.15252/embj.201695417

    Figure Lengend Snippet: De novo FA synthesis mediated by Acc1 affects mitochondrial fission via Fis1 acetylation or ER stress/UPR qRT‐PCR analysis showed the mRNA expression of Acc1 and Fis1 in Acc1 overexpressing MEF cells. Western blotting showing the expression of FIS1 in 10 μM TSA‐ and 5 mM NAM‐treated 293T cells in the presence or absence of MG132. EV‐ or ACC1‐overexpressing MEFs were infected with viruses expressing four factors in the presence or absence of acetate treatment for 12 days. AP‐ and SSEA1‐positive colonies were counted on day 14 after virus infection. AP staining of iPSC colonies is shown on the top. Cellular AcCoA levels were measured in MEF cells treated with oleic acid by ELISA Kit. The values were normalized by cellular protein. Equal amount of proteins from Flag‐Fis1‐overexpressing MEF cells treated with or without OA were used for immunoprecipitation with anti‐Flag antibody, followed by blotting with anti‐acetylated‐lysine or anti‐Fis1. Western blot analysis of ER stress or UPR related protein Atf6, Chop, Atf4, and Bip in MEF cells treated with OA. Mitochondrial fission protein Drp1, Fis1, Mff, Mid51, and Mid49 were analyzed by Western blot in MEF cells treated with OA in the presence or absence of 0.5 mM 4‐PBA for 24 h. OA or/and 4‐PBA was added into the medium of MEF cells starting from 2 days after infection with viruses expressing four factors. AP staining (upper panel) showed the formed iPSC colonies. AP‐ and SSEA1‐positive iPSC colonies were counted (lower panel). Data information: Data are presented as mean (± SD). * P

    Article Snippet: Primary antibodies against the following proteins were used: cMyc (Epitomics), Oct4 (Stemgene), Sox2 (Millipore), DRP1 (Novus), SSEA1 (Santa cruz), HRP‐HA (Abcam); Acc1, Acly, Fasn, Klf4, Fis1, Mff, Mid49, Mid51, Actin, Opa1, Mfn1, Mfn2, Gpi1, Tpi1, Gapdh, Pgk1, Pgam1, Eno1, and Ldha (Proteintech).

    Techniques: Quantitative RT-PCR, Expressing, Western Blot, Infection, Staining, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

    Mitochondrial fission is involved in cellular reprogramming Western blot analysis of Fis1, Drp1, Mid51, Mid49, and Mff protein levels in MEF cells infected with viruses expressing four factors (Klf4/Sox2/Oct4/c‐Myc) on days 0, 2, 4, and 6 during reprogramming. Western blot analysis of mitochondrial fission‐related protein Fis1, Drp1, Mid51, Mid49, Mff, and mitochondrial fusion‐related protein Opa1, Mfn1, and Mfn2 expression in E14 cells cultured in RA differentiation medium for 0, 24, and 48 h. MEF cells with Fis1, Drp1, or Mff knocked down by shRNAs were infected with viruses expressing four factors to induce iPSCs. AP staining (upper panel) showed the formed iPSC colonies. AP‐ and SSEA1‐positive iPSC numbers were counted (lower panel). Fis1, Drp1, or Mff overexpressing MEF cells were infected with viruses expressing four factors to induce iPSCs. AP staining (upper panel) showed the formed iPSC colonies. AP‐ and SSEA1‐positive iPSC numbers were counted (lower panel). Data information: Data are presented as mean (± SD). * P

    Journal: The EMBO Journal

    Article Title: Fatty acid synthesis is critical for stem cell pluripotency via promoting mitochondrial fission

    doi: 10.15252/embj.201695417

    Figure Lengend Snippet: Mitochondrial fission is involved in cellular reprogramming Western blot analysis of Fis1, Drp1, Mid51, Mid49, and Mff protein levels in MEF cells infected with viruses expressing four factors (Klf4/Sox2/Oct4/c‐Myc) on days 0, 2, 4, and 6 during reprogramming. Western blot analysis of mitochondrial fission‐related protein Fis1, Drp1, Mid51, Mid49, Mff, and mitochondrial fusion‐related protein Opa1, Mfn1, and Mfn2 expression in E14 cells cultured in RA differentiation medium for 0, 24, and 48 h. MEF cells with Fis1, Drp1, or Mff knocked down by shRNAs were infected with viruses expressing four factors to induce iPSCs. AP staining (upper panel) showed the formed iPSC colonies. AP‐ and SSEA1‐positive iPSC numbers were counted (lower panel). Fis1, Drp1, or Mff overexpressing MEF cells were infected with viruses expressing four factors to induce iPSCs. AP staining (upper panel) showed the formed iPSC colonies. AP‐ and SSEA1‐positive iPSC numbers were counted (lower panel). Data information: Data are presented as mean (± SD). * P

    Article Snippet: Primary antibodies against the following proteins were used: cMyc (Epitomics), Oct4 (Stemgene), Sox2 (Millipore), DRP1 (Novus), SSEA1 (Santa cruz), HRP‐HA (Abcam); Acc1, Acly, Fasn, Klf4, Fis1, Mff, Mid49, Mid51, Actin, Opa1, Mfn1, Mfn2, Gpi1, Tpi1, Gapdh, Pgk1, Pgam1, Eno1, and Ldha (Proteintech).

    Techniques: Western Blot, Infection, Expressing, Cell Culture, Staining

    Expression of pluripotent markers and CDX2 in CDX2-KD and control bESCs. OCT4 ( A , A′ – C , C′ ), SOX2 ( D , D′ – F , F′ ), NANOG ( G , G′ – I , I′ ), E-CAD ( J , J′ – L , L′ ), SSEA1 ( M , M′ – O , O′ ), SSEA4 ( P , P′ – R , R′ ) and CDX2 ( S , S′ – U , U′ ). GFP expression in CDX2-KD bESCs was shown in inserted picture. Nuclear was stained by DAPI. Bar = 100 μm.

    Journal: Scientific Reports

    Article Title: Establishment of bovine embryonic stem cells after knockdown of CDX2

    doi: 10.1038/srep28343

    Figure Lengend Snippet: Expression of pluripotent markers and CDX2 in CDX2-KD and control bESCs. OCT4 ( A , A′ – C , C′ ), SOX2 ( D , D′ – F , F′ ), NANOG ( G , G′ – I , I′ ), E-CAD ( J , J′ – L , L′ ), SSEA1 ( M , M′ – O , O′ ), SSEA4 ( P , P′ – R , R′ ) and CDX2 ( S , S′ – U , U′ ). GFP expression in CDX2-KD bESCs was shown in inserted picture. Nuclear was stained by DAPI. Bar = 100 μm.

    Article Snippet: Cells were incubated in primary antibody overnight at 4 °C and secondary antibody at 37 °C for 1 h. Primary antibodies were used that were anti-OCT-3/4 (Santa Cruz), anti-NANOG (Abcam), anti-SOX2 (Cell signaling), anti-SSEA1 (Santa Cruz), anti-SSEA4 (Santa Cruz), anti-E-CADHERIN (BD Bioscience), anti-KLF4 (Stemgent), anti-CDX2 (Biogenex).

    Techniques: Expressing, Staining

    Loss of Pten promotes reprogramming of MEFs into iPSCs. ( a ) Representative image of an iPSC colony derived from Pten −/− MEFs by transduction of mouse OKSM. Scale bar = 100 μm. ( b ) Immunocytochemical staining of SSEA1 on iPSCs induced

    Journal: Molecular Therapy

    Article Title: Inhibition of PTEN Tumor Suppressor Promotes the Generation of Induced Pluripotent Stem Cells

    doi: 10.1038/mt.2013.60

    Figure Lengend Snippet: Loss of Pten promotes reprogramming of MEFs into iPSCs. ( a ) Representative image of an iPSC colony derived from Pten −/− MEFs by transduction of mouse OKSM. Scale bar = 100 μm. ( b ) Immunocytochemical staining of SSEA1 on iPSCs induced

    Article Snippet: Staining was carried out by incubation with an anti-SSEA1 antibody (Santa Cruz Biotechnology) overnight at 4 °C.

    Techniques: Derivative Assay, Transduction, Staining

    PGCs are reduced in Tbx4 -null embryos. Tbx4 +/− and Tbx4 −/− embryos at the 30- to 33-somite stage were isolated and stained with anti-SSEA1 antibody to identify PGCs. Right lateral views of the trunk region with anterior to the

    Journal: Biology of Reproduction

    Article Title: Investigating the Role of Tbx4 in the Female Germline in Mice 1

    doi: 10.1095/biolreprod.113.107649

    Figure Lengend Snippet: PGCs are reduced in Tbx4 -null embryos. Tbx4 +/− and Tbx4 −/− embryos at the 30- to 33-somite stage were isolated and stained with anti-SSEA1 antibody to identify PGCs. Right lateral views of the trunk region with anterior to the

    Article Snippet: Primary antibodies used included: anti-Stella (ab19878; 1:200; Abcam), anti-SSEA1 (1:200; Developmental Studies Hybridoma Bank), anti-phospho-Histone H3 (anti-phH3; H9908; 1:100; Sigma-Aldrich), and anti-cleaved caspase-3 (#9661; 1:200; Cell Signaling).

    Techniques: Isolation, Staining

    Lentiviral strategy for the purification of iPS cell-derived cardiomyocytes. ( a ) The lentiviral construct contains a puromycin resistance gene ( PurR ) and a GFP reporter gene separated by a 2A self-cleaving peptide sequence (2A) under the control of the cardiac α-MHC promoter, as well as a neomycin resistance gene ( NeoR ) under the control of the Rex1 promoter; ( b ) strategy of lentivirus gene transfer into iPS cells and purification of cardiomyocytes for electrophysiological analysis; ( c , d ) after lentiviral gene transfer and selection, wild-type and Scn5a∆/+ iPS cell lines maintained the characteristic embryonic stem cell-like morphology ( c ) and expressed the embryonic stem cell-specific markers, Oct3/4 ( d , green) and SSEA1 ( d , red). Nuclei are shown in blue. Scale bars: 50 μm.

    Journal: Journal of Clinical Medicine

    Article Title: Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy

    doi: 10.3390/jcm4010102

    Figure Lengend Snippet: Lentiviral strategy for the purification of iPS cell-derived cardiomyocytes. ( a ) The lentiviral construct contains a puromycin resistance gene ( PurR ) and a GFP reporter gene separated by a 2A self-cleaving peptide sequence (2A) under the control of the cardiac α-MHC promoter, as well as a neomycin resistance gene ( NeoR ) under the control of the Rex1 promoter; ( b ) strategy of lentivirus gene transfer into iPS cells and purification of cardiomyocytes for electrophysiological analysis; ( c , d ) after lentiviral gene transfer and selection, wild-type and Scn5a∆/+ iPS cell lines maintained the characteristic embryonic stem cell-like morphology ( c ) and expressed the embryonic stem cell-specific markers, Oct3/4 ( d , green) and SSEA1 ( d , red). Nuclei are shown in blue. Scale bars: 50 μm.

    Article Snippet: The primary antibodies were diluted in 0.5% donkey or goat serum, and cells were incubated for 2 h. Colonies of iPS cells were stained against Oct3/4 (rabbit, 1:100; Santa Cruz Biotechnology, Heidelberg, Germany) and SSEA1 (mouse, 1:80; Developmental Studies Hybridoma Bank, Iowa, USA).

    Techniques: Purification, Derivative Assay, Construct, Sequencing, Selection

    Cellular Pluripotency Marker Expression Levels in EpiSCs Derived by the IWP-2 Method (A–J) Immunofluorescence images for SSEA1 (A and B), PECAM1 (red) and OCT4 (green, C and D), NANOG (E and F), and histone H3K27 trimethylation (G and H) in mESCs (A, C, E, and G) and EpiSCs (B, D, F, and H). Nuclear staining is shown using TO-PRO3 (blue). Cytograms of EpiSCs and mESCs are shown using anti-SSEA1 (I) and anti-PECAM1 (J) antibodies. (K) Relative expression levels of marker genes in EpiSCs detected by qRT-PCR compared with J1 mESCs. For each gene, technical triplicate assays and two independent experiments were performed. Error bars represent the standard SEM. (L) Growth curves of EpiSCs. At the indicated time points, 2.0 × 10 4 cells were plated and counted. Averaged data from three independent experiments were plotted. Scale bars, 20 μm (A, B, C, D, G, and H) and 50 μm (E and F).

    Journal: Stem Cell Reports

    Article Title: A Simple and Robust Method for Establishing Homogeneous Mouse Epiblast Stem Cell Lines by Wnt Inhibition

    doi: 10.1016/j.stemcr.2015.02.014

    Figure Lengend Snippet: Cellular Pluripotency Marker Expression Levels in EpiSCs Derived by the IWP-2 Method (A–J) Immunofluorescence images for SSEA1 (A and B), PECAM1 (red) and OCT4 (green, C and D), NANOG (E and F), and histone H3K27 trimethylation (G and H) in mESCs (A, C, E, and G) and EpiSCs (B, D, F, and H). Nuclear staining is shown using TO-PRO3 (blue). Cytograms of EpiSCs and mESCs are shown using anti-SSEA1 (I) and anti-PECAM1 (J) antibodies. (K) Relative expression levels of marker genes in EpiSCs detected by qRT-PCR compared with J1 mESCs. For each gene, technical triplicate assays and two independent experiments were performed. Error bars represent the standard SEM. (L) Growth curves of EpiSCs. At the indicated time points, 2.0 × 10 4 cells were plated and counted. Averaged data from three independent experiments were plotted. Scale bars, 20 μm (A, B, C, D, G, and H) and 50 μm (E and F).

    Article Snippet: Primary antibodies were diluted with FBS/PBS as follows: anti-SSEA1 (0.5 μg/ml, 560079; BD Biosciences) and anti-PECAM1 (1:25, 550274; BD Biosciences).

    Techniques: Marker, Expressing, Derivative Assay, Immunofluorescence, Staining, Quantitative RT-PCR

    Immunophenotype of the miPSC-MSC. Representative flow cytometry analyses of primary mouse bone marrow MSC (mMSC), noninduced miPSC (miPSC), miPSC induced to differentiate into MSC presorting (miPSC-MSC presort) and after sorting for CD105+, and SSEA1 cells (miPSC-MSC after sort). MSC-related markers CD73, CD105, and Sca-1, pluripotency marker SSEA1, and haematopoietic markers CD34 and CD45 were assessed (solid histogram) relative to their respective isotype controls (open histogram).

    Journal: Stem Cells International

    Article Title: Potential of iPSC-Derived Mesenchymal Stromal Cells for Treating Periodontal Disease

    doi: 10.1155/2018/2601945

    Figure Lengend Snippet: Immunophenotype of the miPSC-MSC. Representative flow cytometry analyses of primary mouse bone marrow MSC (mMSC), noninduced miPSC (miPSC), miPSC induced to differentiate into MSC presorting (miPSC-MSC presort) and after sorting for CD105+, and SSEA1 cells (miPSC-MSC after sort). MSC-related markers CD73, CD105, and Sca-1, pluripotency marker SSEA1, and haematopoietic markers CD34 and CD45 were assessed (solid histogram) relative to their respective isotype controls (open histogram).

    Article Snippet: Approximately, 1 × 105 cells were incubated with specific cell surface marker antibodies reactive with mouse CD34, CD45, CD73, CD105, Sca-1, SSEA1 (BD Biosciences), or isotype control antibodies (10 mg/mL) on ice for 1 hour.

    Techniques: Flow Cytometry, Cytometry, Marker

    In vitro characterization of the pluripotent state of EiPS cells. ( a ). Nanog, SSEA1, SSEA4, TRA-1-60 and TRA-1-81 detected by fluorescent immunohistochemistry. Yellow scale bar 10 μm, white scalebar 20 μm. ( b ). Detection of gene expression of key pluripotency markers using RT-PCR primers specific for equine endogenous genes

    Journal: Stem Cell Reviews

    Article Title: Induced Pluripotent Stem Cell Lines Derived from Equine Fibroblasts

    doi: 10.1007/s12015-011-9239-5

    Figure Lengend Snippet: In vitro characterization of the pluripotent state of EiPS cells. ( a ). Nanog, SSEA1, SSEA4, TRA-1-60 and TRA-1-81 detected by fluorescent immunohistochemistry. Yellow scale bar 10 μm, white scalebar 20 μm. ( b ). Detection of gene expression of key pluripotency markers using RT-PCR primers specific for equine endogenous genes

    Article Snippet: The cells were then incubated overnight at 4°C with the following primary anti-mouse or anti-human antibodies; (i ) anti-Nanog (Reprocell #RCAB0002P-F), (ii ) anti-Oct4 (Santa Cruz #sc-5279), (iii ) anti-SSEA1 (Stemgent #09-0005), (vi) anti-SSEA4 (Stemgent #09-0006), (v ) anti-TRA-1-60 (Stemgent #09-0009), (vi ) anti-TRA-1-81 (Stemgent #09-0011).

    Techniques: In Vitro, Immunohistochemistry, Expressing, Reverse Transcription Polymerase Chain Reaction

    Characterization of B6 MOG/mESCs and induction of the differentiation of the cells into TEPs in vitro . (A) MOG/mESCs and control mESCs were analyzed for GFP and MOG expression by immunofluorescence with an anti-MOG antibody. The expression of GFP (green) and MOG (red) was observed under a confocal microscope. Scale bars: 50 μm. (B, C) MOG/mESCs and control mESCs were examined for the expression of (B) MOG protein by Western blot, and pluripotent markers (C) AP by AP staining and (D) Sox2 and SSEA1 by immunofluorescence. (E) Parent B6 mESC, MOG/mESCs and control mESCs (5X10 5 cells/well) were induced to differentiate into definitive endoderm and then TEPs in the presence or absence of BFFE, rFOXN1 and rHOXA3 proteins. The mESC-derived cells were analyzed for the expression of EpCAM1, K5 and K8 proteins on day 16 by flow cytometry. The number of EpCAM + K5 + K8 + cells was shown. (F) B6 mice were injected i.t. with EpCAM1 + MOG/mESC-TEPs (5×10 4 ), or control mESC-TEPs (5×10 4 ). MOG protein expression in the thymus was examined by Western blot on day 90 after the injection. The data are presented from 3 independent experiments.

    Journal: Cellular immunology

    Article Title: ESC-derived thymic epithelial cells expressing MOG prevents EAE by central and peripheral tolerance mechanisms

    doi: 10.1016/j.cellimm.2017.10.007

    Figure Lengend Snippet: Characterization of B6 MOG/mESCs and induction of the differentiation of the cells into TEPs in vitro . (A) MOG/mESCs and control mESCs were analyzed for GFP and MOG expression by immunofluorescence with an anti-MOG antibody. The expression of GFP (green) and MOG (red) was observed under a confocal microscope. Scale bars: 50 μm. (B, C) MOG/mESCs and control mESCs were examined for the expression of (B) MOG protein by Western blot, and pluripotent markers (C) AP by AP staining and (D) Sox2 and SSEA1 by immunofluorescence. (E) Parent B6 mESC, MOG/mESCs and control mESCs (5X10 5 cells/well) were induced to differentiate into definitive endoderm and then TEPs in the presence or absence of BFFE, rFOXN1 and rHOXA3 proteins. The mESC-derived cells were analyzed for the expression of EpCAM1, K5 and K8 proteins on day 16 by flow cytometry. The number of EpCAM + K5 + K8 + cells was shown. (F) B6 mice were injected i.t. with EpCAM1 + MOG/mESC-TEPs (5×10 4 ), or control mESC-TEPs (5×10 4 ). MOG protein expression in the thymus was examined by Western blot on day 90 after the injection. The data are presented from 3 independent experiments.

    Article Snippet: The following primary antibodies were used: anti-SSEA1, Sox2 (Cell Signaling Technology, Inc., Danvers, MA), and MOG (Abcam, Cambridge, MA).

    Techniques: In Vitro, Expressing, Immunofluorescence, Microscopy, Western Blot, Staining, Derivative Assay, Flow Cytometry, Cytometry, Mouse Assay, Injection

    Effect of FCS and LIF on the differentiation into trophoblast. The effect of FCS: the cells were cultured in BMP4-supplemented ESF5 medium with 10% FCS (indicating as “BMP4 with FCS”) or without FCS (indicating as “BMP4 only”) for 4 d. ( A ) Quantitative real-time RT–PCR analysis of the expression of trophoblast-specific transcription factors. The gene expressions were normalized by the amount of Gapdh . The values are the mean ± SEM ( n = 4). ( B ) Immunocytochemistry with Cdh3 antibodies. Immunopositive reaction of Cdh3 antibody was visualized with AlexaFluor-488-conjugated secondary antibodies ( green ). Nuclei were stained with DAPI ( blue ). Scale bars are 50 µm. The effect of LIF: the cells were cultured in BMP4-supplemented ESF5 medium with 10 ng/ml of LIF (indicating as “BMP4 with LIF”) or without LIF (indicating as “BMP4 only”) for 4 d. ( C ) Quantitative real-time RT–PCR analysis of the expression of trophoblast-specific transcription factors. ( D ) Immunocytochemistry with Cdh3 antibodies. ( E ) Immunocytochemistry with anti-Nanog or anti-SSEA1 antibodies. Immunopositive reaction of anti-Nanog or anti-SSEA1 antibody was visualized with AlexaFluor-488-conjugated secondary antibodies ( green ).

    Journal: In Vitro Cellular & Developmental Biology. Animal

    Article Title: BMP4 induction of trophoblast from mouse embryonic stem cells in defined culture conditions on laminin

    doi: 10.1007/s11626-009-9266-6

    Figure Lengend Snippet: Effect of FCS and LIF on the differentiation into trophoblast. The effect of FCS: the cells were cultured in BMP4-supplemented ESF5 medium with 10% FCS (indicating as “BMP4 with FCS”) or without FCS (indicating as “BMP4 only”) for 4 d. ( A ) Quantitative real-time RT–PCR analysis of the expression of trophoblast-specific transcription factors. The gene expressions were normalized by the amount of Gapdh . The values are the mean ± SEM ( n = 4). ( B ) Immunocytochemistry with Cdh3 antibodies. Immunopositive reaction of Cdh3 antibody was visualized with AlexaFluor-488-conjugated secondary antibodies ( green ). Nuclei were stained with DAPI ( blue ). Scale bars are 50 µm. The effect of LIF: the cells were cultured in BMP4-supplemented ESF5 medium with 10 ng/ml of LIF (indicating as “BMP4 with LIF”) or without LIF (indicating as “BMP4 only”) for 4 d. ( C ) Quantitative real-time RT–PCR analysis of the expression of trophoblast-specific transcription factors. ( D ) Immunocytochemistry with Cdh3 antibodies. ( E ) Immunocytochemistry with anti-Nanog or anti-SSEA1 antibodies. Immunopositive reaction of anti-Nanog or anti-SSEA1 antibody was visualized with AlexaFluor-488-conjugated secondary antibodies ( green ).

    Article Snippet: The primary antibodies used are as follows: anti-Cdx2 antibody (Biogenex, San Ramon, CA; 1:100), anti-Cdh3 antibody (R & D systems; 1:200), anti-CK7 antibody (Chemicon; 1:100), anti-Cx31 antibody (Chemicon; 1:100), anti-Nanog antibody (ReproCell, Tokyo, Japan; 1:200), and anti-SSEA1 antibody (Kyowa, Tokyo, Japan; 1:100).

    Techniques: Cell Culture, Quantitative RT-PCR, Expressing, Immunocytochemistry, Staining