Structured Review

R&D Systems nanog
Characterisation of <t>hESCs</t> maintained in CMp11 and media conditioned by passage 18 HFFs (CMp18). Representative immunofluorescent images of hESCs maintained for 5 days in CMp11 (n = 5) and CMp18 media (n = 5) (bar = 100 µm, TRA-1.81 bar = 200 µm). Cells cultured in CMp11 presented well defined colonies with morphology characteristic of undifferentiated cells. In contrast, CMp18 cultured cell were morphologically distinct with colonies lacking smooth and defined edges and a clearing effect from the centre. Consistent with an undifferentiated phenotype, CMp11 cultured cells were negative for the early differentiation marker SSEA-1 (green) and positive for the undifferentiation markers SSEA-4, TRA-1.60, TRA-1.81, OCT4 and <t>NANOG.</t> Cells cultured in CMp18 were positive for SSEA-1 and negative for undifferentiation markers. All cells were co-labelled with the nuclear stain DAPI (blue).
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Images

1) Product Images from "Characterisation of Human Embryonic Stem Cells Conditioning Media by 1H-Nuclear Magnetic Resonance Spectroscopy"

Article Title: Characterisation of Human Embryonic Stem Cells Conditioning Media by 1H-Nuclear Magnetic Resonance Spectroscopy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0016732

Characterisation of hESCs maintained in CMp11 and media conditioned by passage 18 HFFs (CMp18). Representative immunofluorescent images of hESCs maintained for 5 days in CMp11 (n = 5) and CMp18 media (n = 5) (bar = 100 µm, TRA-1.81 bar = 200 µm). Cells cultured in CMp11 presented well defined colonies with morphology characteristic of undifferentiated cells. In contrast, CMp18 cultured cell were morphologically distinct with colonies lacking smooth and defined edges and a clearing effect from the centre. Consistent with an undifferentiated phenotype, CMp11 cultured cells were negative for the early differentiation marker SSEA-1 (green) and positive for the undifferentiation markers SSEA-4, TRA-1.60, TRA-1.81, OCT4 and NANOG. Cells cultured in CMp18 were positive for SSEA-1 and negative for undifferentiation markers. All cells were co-labelled with the nuclear stain DAPI (blue).
Figure Legend Snippet: Characterisation of hESCs maintained in CMp11 and media conditioned by passage 18 HFFs (CMp18). Representative immunofluorescent images of hESCs maintained for 5 days in CMp11 (n = 5) and CMp18 media (n = 5) (bar = 100 µm, TRA-1.81 bar = 200 µm). Cells cultured in CMp11 presented well defined colonies with morphology characteristic of undifferentiated cells. In contrast, CMp18 cultured cell were morphologically distinct with colonies lacking smooth and defined edges and a clearing effect from the centre. Consistent with an undifferentiated phenotype, CMp11 cultured cells were negative for the early differentiation marker SSEA-1 (green) and positive for the undifferentiation markers SSEA-4, TRA-1.60, TRA-1.81, OCT4 and NANOG. Cells cultured in CMp18 were positive for SSEA-1 and negative for undifferentiation markers. All cells were co-labelled with the nuclear stain DAPI (blue).

Techniques Used: Cell Culture, Marker, Staining

2) Product Images from "Effects of antioxidants on the quality and genomic stability of induced pluripotent stem cells"

Article Title: Effects of antioxidants on the quality and genomic stability of induced pluripotent stem cells

Journal: Scientific Reports

doi: 10.1038/srep03779

Stemness of iPS cells after 2 months of culture. The expression of stem cell markers Oct3/4, Nanog, SSEA-4, and ALP were detected by staining, and representative images of expressions in 201B7 (A) and 253G1 (B) iPS cell lines were shown. Western blot analysis on the expressions of Nanog and Oct3/4 in 201B7 (C) and 253G1 (D) iPS cell lines was also done, and representative images that cropped from full-length blots ( Supplementary Figure 1 ) was shown. Abbreviations: AOS, proprietary antioxidant supplement from Sigma-Aldrich; AOH, Homemade antioxidant cocktail.
Figure Legend Snippet: Stemness of iPS cells after 2 months of culture. The expression of stem cell markers Oct3/4, Nanog, SSEA-4, and ALP were detected by staining, and representative images of expressions in 201B7 (A) and 253G1 (B) iPS cell lines were shown. Western blot analysis on the expressions of Nanog and Oct3/4 in 201B7 (C) and 253G1 (D) iPS cell lines was also done, and representative images that cropped from full-length blots ( Supplementary Figure 1 ) was shown. Abbreviations: AOS, proprietary antioxidant supplement from Sigma-Aldrich; AOH, Homemade antioxidant cocktail.

Techniques Used: Expressing, ALP Assay, Staining, Western Blot

3) Product Images from "Stemness of the CT-2A Immunocompetent Mouse Brain Tumor Model: Characterization In Vitro"

Article Title: Stemness of the CT-2A Immunocompetent Mouse Brain Tumor Model: Characterization In Vitro

Journal: Journal of Cancer

doi: 10.7150/jca.4149

CT-2A cells and neurospheres also express the SC markers: Oct4, Nanog and Nestin . A) Immunofluorescence analysis of SC marker expression (Oct4, Nanog and Nestin) in monolayer (left panel), 1week neurospheres (middle panel) and 2week neurospheres (right panel). Oct 4 was present in monolayer cells, and both 1week and 2week neurospheres. Nanog absent in the monolayer, but present in both 1 and 2week monospheres. Nestin staining was present in monolayer, 1week and 2week neurospheres. A and B: magnification, 10x; scale bar, 100 μm. B) RT-PCR corroborated the above findings in the robust expression of all 3 SC markers at 2 weeks.
Figure Legend Snippet: CT-2A cells and neurospheres also express the SC markers: Oct4, Nanog and Nestin . A) Immunofluorescence analysis of SC marker expression (Oct4, Nanog and Nestin) in monolayer (left panel), 1week neurospheres (middle panel) and 2week neurospheres (right panel). Oct 4 was present in monolayer cells, and both 1week and 2week neurospheres. Nanog absent in the monolayer, but present in both 1 and 2week monospheres. Nestin staining was present in monolayer, 1week and 2week neurospheres. A and B: magnification, 10x; scale bar, 100 μm. B) RT-PCR corroborated the above findings in the robust expression of all 3 SC markers at 2 weeks.

Techniques Used: Immunofluorescence, Marker, Expressing, Staining, Reverse Transcription Polymerase Chain Reaction

4) Product Images from "Inducible CRISPR genome editing platform in naive human embryonic stem cells reveals JARID2 function in self-renewal"

Article Title: Inducible CRISPR genome editing platform in naive human embryonic stem cells reveals JARID2 function in self-renewal

Journal: Cell Cycle

doi: 10.1080/15384101.2018.1442621

JARID2 KO phenotype in naïve hESC. (A) Western blot analysis of EZH2, OCT4, NANOG, and H3K27Me3 marks in JARID2 Knockout clones of 2iL-I-F naïve hESC. (B) Schematic representation of mutation in JARID2 KO Clone2. (C-D) Confocal imaging of wild type Elf1-iCas9 cells and Elf1-iCas9 cells transfected with JARID2 sgRNA6.1 (2iL-I-F(C); 5iLA(D)). Blue: DAPI; Green: Oct4, Red: Nanog and Magenta: JARID2 (higher concentration of Ab used for 5iLA to detect lower levels of JARID2: JARID2 Ab 1:200 in 2iL-I-F; 1:100 in 5iLA). The arrows indicate JARID2 KO cells, Scale bar 50 µm (C) or 10 µm (D).
Figure Legend Snippet: JARID2 KO phenotype in naïve hESC. (A) Western blot analysis of EZH2, OCT4, NANOG, and H3K27Me3 marks in JARID2 Knockout clones of 2iL-I-F naïve hESC. (B) Schematic representation of mutation in JARID2 KO Clone2. (C-D) Confocal imaging of wild type Elf1-iCas9 cells and Elf1-iCas9 cells transfected with JARID2 sgRNA6.1 (2iL-I-F(C); 5iLA(D)). Blue: DAPI; Green: Oct4, Red: Nanog and Magenta: JARID2 (higher concentration of Ab used for 5iLA to detect lower levels of JARID2: JARID2 Ab 1:200 in 2iL-I-F; 1:100 in 5iLA). The arrows indicate JARID2 KO cells, Scale bar 50 µm (C) or 10 µm (D).

Techniques Used: Western Blot, Knock-Out, Clone Assay, Mutagenesis, Imaging, Transfection, Concentration Assay

Generation Elf1-iCas9 line. (A) Principle Component Analysis of single-cell RNA-seq data from in vivo samples of cynomolgus monkeys, with human ex vivo blastocyst and in vitro , , ]). Blue and orange lines represent the homology of the constructs to PPP1R12C intron 1 on chromosome 19. The black arrows show the cleaving sites of the TALENS; SA, splice acceptor; 2A, self-cleaving 2A peptide; Puro, Puromycin resistance gene; Neo, Neomycin resistance gene; TRE, Tetracyclin responsive element. (C) Workflow of the generation of Elf1-iCas9 lines. (D) Southern blot analysis of lines #2, #11 and #12 using 3′ external and 5′ internal probes. Blue arrow indicates additional random integrations. (E) Elf1-iCas9 cells express Cas9 protein only after doxycycline treatment (2µg/ml for 2 days, Western Blot). (F) Elf1-iCas9 Clone #2 displays a normal G-banded karyotype. (G) Confocal analysis of pluripotent stem cell markers Oct4 and Nanog in the Elf1-iCas9 Clone #2 grown in EPS, 5iLA, and 2iL-I-F conditions. Scale bars represents 50 µm. (H) Flow cytometry analysis of stem cell markers Oct4 and Nanog in Elf1 2iL-I-F and Elf1-iCas9 2i-I-F. The pink peak indicates unstained cells and orange and blue peak represent stained cells for Elf1 and Elf1-iCas9 respectively.
Figure Legend Snippet: Generation Elf1-iCas9 line. (A) Principle Component Analysis of single-cell RNA-seq data from in vivo samples of cynomolgus monkeys, with human ex vivo blastocyst and in vitro , , ]). Blue and orange lines represent the homology of the constructs to PPP1R12C intron 1 on chromosome 19. The black arrows show the cleaving sites of the TALENS; SA, splice acceptor; 2A, self-cleaving 2A peptide; Puro, Puromycin resistance gene; Neo, Neomycin resistance gene; TRE, Tetracyclin responsive element. (C) Workflow of the generation of Elf1-iCas9 lines. (D) Southern blot analysis of lines #2, #11 and #12 using 3′ external and 5′ internal probes. Blue arrow indicates additional random integrations. (E) Elf1-iCas9 cells express Cas9 protein only after doxycycline treatment (2µg/ml for 2 days, Western Blot). (F) Elf1-iCas9 Clone #2 displays a normal G-banded karyotype. (G) Confocal analysis of pluripotent stem cell markers Oct4 and Nanog in the Elf1-iCas9 Clone #2 grown in EPS, 5iLA, and 2iL-I-F conditions. Scale bars represents 50 µm. (H) Flow cytometry analysis of stem cell markers Oct4 and Nanog in Elf1 2iL-I-F and Elf1-iCas9 2i-I-F. The pink peak indicates unstained cells and orange and blue peak represent stained cells for Elf1 and Elf1-iCas9 respectively.

Techniques Used: RNA Sequencing Assay, In Vivo, Ex Vivo, In Vitro, Construct, TALENs, Southern Blot, Western Blot, Flow Cytometry, Cytometry, Staining

5) Product Images from "Characterization of iPSCs derived from low grade gliomas revealed early regional chromosomal amplifications during gliomagenesis"

Article Title: Characterization of iPSCs derived from low grade gliomas revealed early regional chromosomal amplifications during gliomagenesis

Journal: Journal of neuro-oncology

doi: 10.1007/s11060-018-03047-1

Generation of iPSCs from primary LGG cells. ( A, C ) Bright field images of primary BT01 and BT03 cells. ( B, D ) Images of representative iPSC colonies derived from BT01 and BT03 cells. ( E ) LGG-iPSCs express alkaline phosphatase. ( F-H ) Immunostaining demonstrated that LGG-iPSCs express pluripotency-associated transcription factors (F) OCT4, (G) NANOG, and (H) SOX2. ( I ) Flow cytometry analysis confirmed LGG-iPSCs expressing the pluripotency-specific surface marker Tra-1–60. ( J-L ) Teratoma formation assay demonstrated LGG-iPSCs can differentiated into (J) ectoderm (neuroepithelium), (K) mesoderm (cartilage), and (L) endoderm (glandular structure). Scale bars, 100 μm. ( M ) Representative sequencing results of the BT01- and BT03-iPSC clones. More than 15 independent clones of each LGG samples were sequenced. None carried the IDH1 mutations.
Figure Legend Snippet: Generation of iPSCs from primary LGG cells. ( A, C ) Bright field images of primary BT01 and BT03 cells. ( B, D ) Images of representative iPSC colonies derived from BT01 and BT03 cells. ( E ) LGG-iPSCs express alkaline phosphatase. ( F-H ) Immunostaining demonstrated that LGG-iPSCs express pluripotency-associated transcription factors (F) OCT4, (G) NANOG, and (H) SOX2. ( I ) Flow cytometry analysis confirmed LGG-iPSCs expressing the pluripotency-specific surface marker Tra-1–60. ( J-L ) Teratoma formation assay demonstrated LGG-iPSCs can differentiated into (J) ectoderm (neuroepithelium), (K) mesoderm (cartilage), and (L) endoderm (glandular structure). Scale bars, 100 μm. ( M ) Representative sequencing results of the BT01- and BT03-iPSC clones. More than 15 independent clones of each LGG samples were sequenced. None carried the IDH1 mutations.

Techniques Used: Derivative Assay, Immunostaining, Flow Cytometry, Cytometry, Expressing, Marker, Teratoma Formation Assay, Sequencing, Clone Assay

6) Product Images from "Dental follicle stem cells in bone regeneration on titanium implants"

Article Title: Dental follicle stem cells in bone regeneration on titanium implants

Journal: BMC Biotechnology

doi: 10.1186/s12896-015-0229-6

Immunocytochemical staining for characteristic stem cells markers, nuclei were counterstained with DAPI. ( a ) strong expression for CD 44 FITC and weak positivity for CD 105 PE; ( b ) negative staining for CD 34 FITC and CD 117 PE; ( c ) strong positivity for CD 90 FITC, weak expression of CD 49 PE; ( d ) strong positivity for early embryonic antigen SSEA-4 FITC; ( e ) the self-renewal embryonic proteins Oct3/4 FITC strong expression and ( f ) Nanog FITC positive in some cells (magnification ×400)
Figure Legend Snippet: Immunocytochemical staining for characteristic stem cells markers, nuclei were counterstained with DAPI. ( a ) strong expression for CD 44 FITC and weak positivity for CD 105 PE; ( b ) negative staining for CD 34 FITC and CD 117 PE; ( c ) strong positivity for CD 90 FITC, weak expression of CD 49 PE; ( d ) strong positivity for early embryonic antigen SSEA-4 FITC; ( e ) the self-renewal embryonic proteins Oct3/4 FITC strong expression and ( f ) Nanog FITC positive in some cells (magnification ×400)

Techniques Used: Staining, Expressing, Negative Staining

7) Product Images from "Development of histocompatible primate induced pluripotent stem cells for neural transplantation"

Article Title: Development of histocompatible primate induced pluripotent stem cells for neural transplantation

Journal: Stem Cells (Dayton, Ohio)

doi: 10.1002/stem.662

Generation of Cynomolgus Monkey iPSC (A–B) Phase contrast images showing ESC-like morphology of CM-iPSCs on MEF. (C) High-magnification image of CM-iPSCs showing the high nucleus/cytoplasm ratio and prominent nucleoli. (D) Immunofluorescence staining for the pluripotency markers Nanog, Oct4, SSEA-4, TRA-1–60, and TRA-1–81. (E) CM-iPSC colonies stained positive for alkaline phosphatase (AP). (F) Karyotype analysis of CM-iPSCs (line 27-04, passage 10). CM-iPSCs maintained a normal 42, XY karyotype after expansion. (G) Quantitative RT-PCR showing induction of endogenous transcripts of Sox2, Oct4, Klf4, and c-Myc. Primers specific for endogenously (black) or viral exogenous (red) encoded transcripts of the four reprogramming factors. Monkey dermal fibroblasts, four days after transduction with the four retroviruses (RV-FIB), were used as a positive control for expression of the viral transgenes. Values were normalized by the averaged value of β-actin. Values are expressed as averages + SEM of 3 independent experiments. The value of hESCs (H9) was set to 1 in each experiment. Scale bar, 200 µm (A–B, D–E); 20 µm (C).
Figure Legend Snippet: Generation of Cynomolgus Monkey iPSC (A–B) Phase contrast images showing ESC-like morphology of CM-iPSCs on MEF. (C) High-magnification image of CM-iPSCs showing the high nucleus/cytoplasm ratio and prominent nucleoli. (D) Immunofluorescence staining for the pluripotency markers Nanog, Oct4, SSEA-4, TRA-1–60, and TRA-1–81. (E) CM-iPSC colonies stained positive for alkaline phosphatase (AP). (F) Karyotype analysis of CM-iPSCs (line 27-04, passage 10). CM-iPSCs maintained a normal 42, XY karyotype after expansion. (G) Quantitative RT-PCR showing induction of endogenous transcripts of Sox2, Oct4, Klf4, and c-Myc. Primers specific for endogenously (black) or viral exogenous (red) encoded transcripts of the four reprogramming factors. Monkey dermal fibroblasts, four days after transduction with the four retroviruses (RV-FIB), were used as a positive control for expression of the viral transgenes. Values were normalized by the averaged value of β-actin. Values are expressed as averages + SEM of 3 independent experiments. The value of hESCs (H9) was set to 1 in each experiment. Scale bar, 200 µm (A–B, D–E); 20 µm (C).

Techniques Used: Immunofluorescence, Staining, Quantitative RT-PCR, Transduction, Positive Control, Expressing

8) Product Images from "Multipotent mesenchymal stem cells in lung fibrosis"

Article Title: Multipotent mesenchymal stem cells in lung fibrosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0181946

Characterization of mesenchymal stem cells. Primary human mesenchymal stem cells immunostained for Oct3/4 (A), and Nanog (B). Cells were fixed and permeabilized. Primary antibodies were detected by addition of fluorescein-labelled (FITC) (green) and Alexa Fluor 594-labelled (red) secondary antibodies. E, G-I: Double immunofluorescence staining for CD90 (E), CD105 (G, H, I) and C-X-C-chemokine receptor type 4 (CXCR4) (panels E, G, H, I) in lung tissue sections from patients with histologically confirmed IPF/UIP and chronic fibrotic hypersensitivity pneumonitis. Primary antibodies were detected by addition of secondary antibodies labelled with fluorescein FITC (green) for CD105 and CXCR4 detection or Cy3-labelled (red) for CD90 and CXCR4 detection. Magnification x40. Images were acquired using LSM 510 confocal microscope. Panel H and I is a 3D reconstruction to show more clear co stainings on the same cell. F: Co-staining with CD44 (pink) and CXCR4 (dark red) (Scale bar 2μm, Magnification x100 (oil)). (C, D) Oct3/4 (C) and Nanog (D) mRNA expression in mesenchymal stem cells (black bars) and in fibroblasts (grey bars). RNA expression was assessed by quantitative real time RT-PCR. Data are presented as mean ± SEM of independent experiments performed in three different cell lines.
Figure Legend Snippet: Characterization of mesenchymal stem cells. Primary human mesenchymal stem cells immunostained for Oct3/4 (A), and Nanog (B). Cells were fixed and permeabilized. Primary antibodies were detected by addition of fluorescein-labelled (FITC) (green) and Alexa Fluor 594-labelled (red) secondary antibodies. E, G-I: Double immunofluorescence staining for CD90 (E), CD105 (G, H, I) and C-X-C-chemokine receptor type 4 (CXCR4) (panels E, G, H, I) in lung tissue sections from patients with histologically confirmed IPF/UIP and chronic fibrotic hypersensitivity pneumonitis. Primary antibodies were detected by addition of secondary antibodies labelled with fluorescein FITC (green) for CD105 and CXCR4 detection or Cy3-labelled (red) for CD90 and CXCR4 detection. Magnification x40. Images were acquired using LSM 510 confocal microscope. Panel H and I is a 3D reconstruction to show more clear co stainings on the same cell. F: Co-staining with CD44 (pink) and CXCR4 (dark red) (Scale bar 2μm, Magnification x100 (oil)). (C, D) Oct3/4 (C) and Nanog (D) mRNA expression in mesenchymal stem cells (black bars) and in fibroblasts (grey bars). RNA expression was assessed by quantitative real time RT-PCR. Data are presented as mean ± SEM of independent experiments performed in three different cell lines.

Techniques Used: Double Immunofluorescence Staining, Microscopy, Staining, Expressing, RNA Expression, Quantitative RT-PCR

9) Product Images from "The Role of Controlled Surface Topography and Chemistry on Mouse Embryonic Stem Cell Attachment, Growth and Self-Renewal"

Article Title: The Role of Controlled Surface Topography and Chemistry on Mouse Embryonic Stem Cell Attachment, Growth and Self-Renewal

Journal: Materials

doi: 10.3390/ma10091081

mES cells were grown on glass coverslips treated with 16 nm nanoparticles precoated with fibronectin for 2 h, 24 h, and 72 h. Cells were fixed, then stained with Oct4 and Nanog and counterstained with DAPI. Presented as representative images from each sample set Scale bar = 50 mm.
Figure Legend Snippet: mES cells were grown on glass coverslips treated with 16 nm nanoparticles precoated with fibronectin for 2 h, 24 h, and 72 h. Cells were fixed, then stained with Oct4 and Nanog and counterstained with DAPI. Presented as representative images from each sample set Scale bar = 50 mm.

Techniques Used: Staining

mES cells seeded on glass coverslips surface modified with allylamine, acrylic acid and octadiene, precoated with fibronectin. Cells were cultured for 72 h, fixed then stained with Oct4 and Nanog and counterstained with DAPI. Presented as representative images. Scale bar = 50 mm.
Figure Legend Snippet: mES cells seeded on glass coverslips surface modified with allylamine, acrylic acid and octadiene, precoated with fibronectin. Cells were cultured for 72 h, fixed then stained with Oct4 and Nanog and counterstained with DAPI. Presented as representative images. Scale bar = 50 mm.

Techniques Used: Modification, Cell Culture, Staining

mES cells seeded on glass coverslips with and without treatment with 16, 38, and 68 nm particles and subsequent surface chemical modification with allylamine, acrylic acid and octadiene, precoated with fibronectin. Cells were cultured for 2, 24, and 72 h, fixed and stained with Oct4 and Nanog and counterstained with DAPI. The % of cells stained with Oct4/nanog to DAPI was counted in three random fields of view on each sample. Presented as mean ± SD n = 3 (* = p
Figure Legend Snippet: mES cells seeded on glass coverslips with and without treatment with 16, 38, and 68 nm particles and subsequent surface chemical modification with allylamine, acrylic acid and octadiene, precoated with fibronectin. Cells were cultured for 2, 24, and 72 h, fixed and stained with Oct4 and Nanog and counterstained with DAPI. The % of cells stained with Oct4/nanog to DAPI was counted in three random fields of view on each sample. Presented as mean ± SD n = 3 (* = p

Techniques Used: Modification, Cell Culture, Staining

10) Product Images from "A Euploid Line of Human Embryonic Stem Cells Derived from a 43,XX,dup(9q),+12,-14,-15,-18,-21 Embryo"

Article Title: A Euploid Line of Human Embryonic Stem Cells Derived from a 43,XX,dup(9q),+12,-14,-15,-18,-21 Embryo

Journal: PLoS ONE

doi: 10.1371/journal.pone.0140999

Characterization of pluripotency of BR-6. (A) Immunofluorescence using antibodies against (a) OCT4 in red, (b) NANOG in green and (c) SSEA-4 in red. Nuclei were counterstained with (d) DAPI.; (B) Flow cytometry showing cells positive for NANOG, OCT3/4 and SOX2 (red curves). Black curves correspond to cells with control isotypes. Percentage of positive cells are indicated; (C) ScoreCard results: change in expression levels of genes characteristic of self-renewal and of each germ layer in embryoid bodies in relation to hESCs are indicated by color code (from red–upregulation, to blue–downregulation. See “Fold change legend”).
Figure Legend Snippet: Characterization of pluripotency of BR-6. (A) Immunofluorescence using antibodies against (a) OCT4 in red, (b) NANOG in green and (c) SSEA-4 in red. Nuclei were counterstained with (d) DAPI.; (B) Flow cytometry showing cells positive for NANOG, OCT3/4 and SOX2 (red curves). Black curves correspond to cells with control isotypes. Percentage of positive cells are indicated; (C) ScoreCard results: change in expression levels of genes characteristic of self-renewal and of each germ layer in embryoid bodies in relation to hESCs are indicated by color code (from red–upregulation, to blue–downregulation. See “Fold change legend”).

Techniques Used: Immunofluorescence, Flow Cytometry, Cytometry, Expressing

11) Product Images from "Human Embryonic and Fetal Mesenchymal Stem Cells Differentiate toward Three Different Cardiac Lineages in Contrast to Their Adult Counterparts"

Article Title: Human Embryonic and Fetal Mesenchymal Stem Cells Differentiate toward Three Different Cardiac Lineages in Contrast to Their Adult Counterparts

Journal: PLoS ONE

doi: 10.1371/journal.pone.0024164

Characterization of hESC-MSCs. (A1) Bright field image of a hESC colony in which the cells at the periphery are differentiating toward spindle-shaped fibroblast-like cells and (A2–A3) pure cultures of hESC-MSC. (B1–B2) Confirmation of the human origin of the hMSCs derived from hESC colonies with the aid of a human-specific lamin A/C antibody. Incubation of murine MSCs (mMSCs; negative control cells) with this antibody (B1) did not produce signal corroborating its species specificity. (C1–E2) Immunostaining of hESC colonies and hMSCs derived from these colonies for the embryonic stem cell marker SSEA-4 and the pluripotency markers Oct-4 and Nanog. Nuclei were detected with Hoechst.
Figure Legend Snippet: Characterization of hESC-MSCs. (A1) Bright field image of a hESC colony in which the cells at the periphery are differentiating toward spindle-shaped fibroblast-like cells and (A2–A3) pure cultures of hESC-MSC. (B1–B2) Confirmation of the human origin of the hMSCs derived from hESC colonies with the aid of a human-specific lamin A/C antibody. Incubation of murine MSCs (mMSCs; negative control cells) with this antibody (B1) did not produce signal corroborating its species specificity. (C1–E2) Immunostaining of hESC colonies and hMSCs derived from these colonies for the embryonic stem cell marker SSEA-4 and the pluripotency markers Oct-4 and Nanog. Nuclei were detected with Hoechst.

Techniques Used: Derivative Assay, Incubation, Negative Control, Immunostaining, Marker

12) Product Images from "Novel sequential ChIP and simplified basic ChIP protocols for promoter co-occupancy and target gene identification in human embryonic stem cells"

Article Title: Novel sequential ChIP and simplified basic ChIP protocols for promoter co-occupancy and target gene identification in human embryonic stem cells

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-9-59

Identification of SOX2/NANOG/OCT3/4 target genes using the simplified, basic ChIP protocol . (A) NANOG expression by immunocytochemistry (red), DAPI (green) and merged images of undifferentiated BG01V human embryonic stem cells (magnification = 20×). (B) rtPCR data from SOX2/NANOG/OCT3/4 ChIP using primers to previously known target genes. (C) PCR results from the indicated ChIP assays with undifferentiated BG01V cells. (D) Densitometric analysis of ChIP data shown in C, obtained with ImageJ software. Three independent experiments were performed. Representative results are shown. Fold enrichment = ChIP/Input DNA. Error bars represent standard deviation.
Figure Legend Snippet: Identification of SOX2/NANOG/OCT3/4 target genes using the simplified, basic ChIP protocol . (A) NANOG expression by immunocytochemistry (red), DAPI (green) and merged images of undifferentiated BG01V human embryonic stem cells (magnification = 20×). (B) rtPCR data from SOX2/NANOG/OCT3/4 ChIP using primers to previously known target genes. (C) PCR results from the indicated ChIP assays with undifferentiated BG01V cells. (D) Densitometric analysis of ChIP data shown in C, obtained with ImageJ software. Three independent experiments were performed. Representative results are shown. Fold enrichment = ChIP/Input DNA. Error bars represent standard deviation.

Techniques Used: Chromatin Immunoprecipitation, Expressing, Immunocytochemistry, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Software, Standard Deviation

Identification of known target genes for a variety of stem cell transcription factors using the simplified, basic ChIP protocol . (A) Mouse Lama1 detection from SOX17 ChIP using endoderm differentiated D3 mouse embryonic stem cells. (B) VEGF and BAX detection from RUNX2 ChIP. (C) OCT3/4 , NANOG , and B2R detection from KLF4 ChIP. (D) HOXC13 detection from BMI-1 ChIP. (E) c-MYC and GLI1 detection from SMAD2/3 ChIP. (F) P21 detection from OLIG2 ChIP. (A) - (F): Fold enrichment = ChIP/Input DNA. Error bars represent standard deviation. (B-F): using undifferentiated BG01V human embryonic stem cells.
Figure Legend Snippet: Identification of known target genes for a variety of stem cell transcription factors using the simplified, basic ChIP protocol . (A) Mouse Lama1 detection from SOX17 ChIP using endoderm differentiated D3 mouse embryonic stem cells. (B) VEGF and BAX detection from RUNX2 ChIP. (C) OCT3/4 , NANOG , and B2R detection from KLF4 ChIP. (D) HOXC13 detection from BMI-1 ChIP. (E) c-MYC and GLI1 detection from SMAD2/3 ChIP. (F) P21 detection from OLIG2 ChIP. (A) - (F): Fold enrichment = ChIP/Input DNA. Error bars represent standard deviation. (B-F): using undifferentiated BG01V human embryonic stem cells.

Techniques Used: Chromatin Immunoprecipitation, Standard Deviation

Identification of SOX2/OCT3/4/NANOG/c-MYC and RUNX2/BMI-1/SMAD2/3 as complexes in BG01V cells using the novel Sequential ChIP protocol . (A) Detection of promoter co-occupancy by SOX2/OCT3/4/NANOG, with the indicated combinations of antibodies. (B) Detection of promoter co-occupancy by SOX2/OCT3/4/NANOG/c-MYC, with the indicated combinations of antibodies. (C) SOX2 immunodepletion of BG01V lysates by three subsequent rounds of SOX2 immumoprecipitation, using ERK1/2 as a negative control. (D) c-MYC ChIP assays results using SOX2-depleted or non-depleted BG01V human embryonic stem cell lysates. (E) Detection of promoter co-occupancy by BMI-1/RUNX2/SMAD2/3, with the indicated combinations of antibodies.
Figure Legend Snippet: Identification of SOX2/OCT3/4/NANOG/c-MYC and RUNX2/BMI-1/SMAD2/3 as complexes in BG01V cells using the novel Sequential ChIP protocol . (A) Detection of promoter co-occupancy by SOX2/OCT3/4/NANOG, with the indicated combinations of antibodies. (B) Detection of promoter co-occupancy by SOX2/OCT3/4/NANOG/c-MYC, with the indicated combinations of antibodies. (C) SOX2 immunodepletion of BG01V lysates by three subsequent rounds of SOX2 immumoprecipitation, using ERK1/2 as a negative control. (D) c-MYC ChIP assays results using SOX2-depleted or non-depleted BG01V human embryonic stem cell lysates. (E) Detection of promoter co-occupancy by BMI-1/RUNX2/SMAD2/3, with the indicated combinations of antibodies.

Techniques Used: Chromatin Immunoprecipitation, Negative Control

13) Product Images from "A Defined and Xeno-Free Culture Method Enabling the Establishment of Clinical-Grade Human Embryonic, Induced Pluripotent and Adipose Stem Cells"

Article Title: A Defined and Xeno-Free Culture Method Enabling the Establishment of Clinical-Grade Human Embryonic, Induced Pluripotent and Adipose Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0010246

Morphology and immunocytochemical characterization of hESC lines. A ) Bright-field (scale bar, 500 µm) microscopic images showing undifferentiated colony morphology of hESC lines Regea 07/046 at passage 44, Regea 08/013 at passage 12, and control cell line Regea 06/040 at passage 45. B ) Fluorescent images (scale bar, 100 µm) showing qualitative immunocytochemistry of cells positive for Nanog, Oct4, Sox2, SSEA-4 and TRA-1-60. Insets represent DAPI staining. Regea 07/046 at passage 63, Regea 08/013 at passage 51, and control cell line Regea 06/040 at passage 42.
Figure Legend Snippet: Morphology and immunocytochemical characterization of hESC lines. A ) Bright-field (scale bar, 500 µm) microscopic images showing undifferentiated colony morphology of hESC lines Regea 07/046 at passage 44, Regea 08/013 at passage 12, and control cell line Regea 06/040 at passage 45. B ) Fluorescent images (scale bar, 100 µm) showing qualitative immunocytochemistry of cells positive for Nanog, Oct4, Sox2, SSEA-4 and TRA-1-60. Insets represent DAPI staining. Regea 07/046 at passage 63, Regea 08/013 at passage 51, and control cell line Regea 06/040 at passage 42.

Techniques Used: Immunocytochemistry, Staining

Analysis of pluripotency and differentiation characteristics of the hESC lines derived in xeno-free medium RegES. A ) RT-PCR analysis of in vitro -derived EBs showing transcripts for AFP and SOX17 (endodermal markers), α-cardiac actin and T (Brachyury; mesodermal markers), SOX1 and PAX6 (ectodermal markers), and β-actin . Lane 1, 50-bp DNA ladder. Regea 07/046 at passage 42, Regea 08/013 at passage 35, and Regea 06/040 at passage 101. B ) Differentiated cardiomyocytes from hESC line Regea 08/013 were stained with a) cardiac troponin T (red) and b) ventricular myosin heavy chain (green). c) Merged picture of a) and b) with DAPI staining. Scale bar is 100 µm. C ) RT-PCR analysis of neurospheres derived from hESC line Regea 08/013 cultured in RegES medium showed expression of neural precursor markers Musashi , Nestin and PAX6 ; neuronal markers MAP-2 , NF68 and OTX2 ; and astrocytic marker GFAP . No expression of pluripotent markers Oct4 and Nanog , nor endo- AFP or mesodermal markers T/Brachyury were detected. D ) Most of the cells migrating out from the plated neurospheres stained positive for neuronal marker MAP-2 (green) and few cells were positive for astrocytic marker GFAP (red). DAPI staining (blue). Scale bar is 100 µm.
Figure Legend Snippet: Analysis of pluripotency and differentiation characteristics of the hESC lines derived in xeno-free medium RegES. A ) RT-PCR analysis of in vitro -derived EBs showing transcripts for AFP and SOX17 (endodermal markers), α-cardiac actin and T (Brachyury; mesodermal markers), SOX1 and PAX6 (ectodermal markers), and β-actin . Lane 1, 50-bp DNA ladder. Regea 07/046 at passage 42, Regea 08/013 at passage 35, and Regea 06/040 at passage 101. B ) Differentiated cardiomyocytes from hESC line Regea 08/013 were stained with a) cardiac troponin T (red) and b) ventricular myosin heavy chain (green). c) Merged picture of a) and b) with DAPI staining. Scale bar is 100 µm. C ) RT-PCR analysis of neurospheres derived from hESC line Regea 08/013 cultured in RegES medium showed expression of neural precursor markers Musashi , Nestin and PAX6 ; neuronal markers MAP-2 , NF68 and OTX2 ; and astrocytic marker GFAP . No expression of pluripotent markers Oct4 and Nanog , nor endo- AFP or mesodermal markers T/Brachyury were detected. D ) Most of the cells migrating out from the plated neurospheres stained positive for neuronal marker MAP-2 (green) and few cells were positive for astrocytic marker GFAP (red). DAPI staining (blue). Scale bar is 100 µm.

Techniques Used: Derivative Assay, Reverse Transcription Polymerase Chain Reaction, In Vitro, Staining, Cell Culture, Expressing, Marker

Characterization of iPSC lines. A ) Bright-field microscopic images (scale bar, 500 µm) showing undifferentiated colony morphology of iPSCs cultured in hES and RegES media. Cell line FiPS 5-7 in hES medium is at passage 15, in RegES medium at passage 5 and FiPS 6-14 in hES medium at passage 18, in RegES medium at passage 9. B ) Fluorescent microscopic images (scale bar, 100 µm) of iPSCs cultured in RegES medium showing cells positive for Nanog, Oct4, Sox2, SSEA-4 and TRA-1-60. Insets represent DAPI staining. Cell line FiPS 5-7 is at passage 23 and FiPS 6-14 at passage 26. C ) Quantitative flow cytometry analyses indicating expression of SSEA-4 and TRA-1-81 of iPSC lines cultured in hES and RegES media. Cell samples cultured in hES medium are from 6 day old colonies, cell samples from cell line FiPS 5-7 cultured in RegES from 7 day old colonies and samples from cell line FiPS 6-14 from 8 day old colonies. Cell line FiPS 5-7 in hES medium at passage 15, in RegES medium at passage 14 and FiPS 6-14 in hES medium at passage 16, in RegES medium at passage 7. D ) Quantitative RT-PCR analysis of Nanog , Oct4 , GABRB3 , GDF3 , DNMT3B and TDGF1 expression of day 6 colonies in iPSC lines FiPS 5-7 in hES medium at passage 10, in RegES medium at passage 7 and FiPS 6-14 in hES medium at passage 11, in RegES medium at passage 8. No significant differences (over 2-fold) were detected between iPSC lines. E ) RT-PCR analysis of in vitro -derived EBs showing transcripts for AFP and SOX17 (endodermal markers), α-cardiac actin and T (Brachyury; mesodermal markers), SOX1 and PAX6 (ectodermal markers), and β-actin . Lane 1, 50-bp DNA ladder. Both cell lines at passage 10.
Figure Legend Snippet: Characterization of iPSC lines. A ) Bright-field microscopic images (scale bar, 500 µm) showing undifferentiated colony morphology of iPSCs cultured in hES and RegES media. Cell line FiPS 5-7 in hES medium is at passage 15, in RegES medium at passage 5 and FiPS 6-14 in hES medium at passage 18, in RegES medium at passage 9. B ) Fluorescent microscopic images (scale bar, 100 µm) of iPSCs cultured in RegES medium showing cells positive for Nanog, Oct4, Sox2, SSEA-4 and TRA-1-60. Insets represent DAPI staining. Cell line FiPS 5-7 is at passage 23 and FiPS 6-14 at passage 26. C ) Quantitative flow cytometry analyses indicating expression of SSEA-4 and TRA-1-81 of iPSC lines cultured in hES and RegES media. Cell samples cultured in hES medium are from 6 day old colonies, cell samples from cell line FiPS 5-7 cultured in RegES from 7 day old colonies and samples from cell line FiPS 6-14 from 8 day old colonies. Cell line FiPS 5-7 in hES medium at passage 15, in RegES medium at passage 14 and FiPS 6-14 in hES medium at passage 16, in RegES medium at passage 7. D ) Quantitative RT-PCR analysis of Nanog , Oct4 , GABRB3 , GDF3 , DNMT3B and TDGF1 expression of day 6 colonies in iPSC lines FiPS 5-7 in hES medium at passage 10, in RegES medium at passage 7 and FiPS 6-14 in hES medium at passage 11, in RegES medium at passage 8. No significant differences (over 2-fold) were detected between iPSC lines. E ) RT-PCR analysis of in vitro -derived EBs showing transcripts for AFP and SOX17 (endodermal markers), α-cardiac actin and T (Brachyury; mesodermal markers), SOX1 and PAX6 (ectodermal markers), and β-actin . Lane 1, 50-bp DNA ladder. Both cell lines at passage 10.

Techniques Used: Cell Culture, Staining, Flow Cytometry, Cytometry, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, In Vitro, Derivative Assay

Stem cell marker expression and proliferation of hESC lines. A ) Quantitative flow cytometry analyses showing comparable expression levels of SSEA-4 and TRA-1-81 in all hESC lines at day 7. Regea 07/046 at passage 45, Regea 08/013 at passage 41, and Regea 06/040 at passage 26. B ) Cell proliferation analysis demonstrating comparable growth rates of hESC lines Regea 06/040 at passage 29, Regea 07/046 at passage 53 and Regea 08/013 at passage 41. C ) Quantitative RT-PCR analysis of Nanog , Oct4 , GABRB3 , GDF3 , DNMT3B and TDGF1 expression in hESC lines Regea 07/046 at passage 52, Regea 08/013 at passage 45, and Regea 06/040 at passage 33. No significant differences (over 2-fold) were detected between hESC lines.
Figure Legend Snippet: Stem cell marker expression and proliferation of hESC lines. A ) Quantitative flow cytometry analyses showing comparable expression levels of SSEA-4 and TRA-1-81 in all hESC lines at day 7. Regea 07/046 at passage 45, Regea 08/013 at passage 41, and Regea 06/040 at passage 26. B ) Cell proliferation analysis demonstrating comparable growth rates of hESC lines Regea 06/040 at passage 29, Regea 07/046 at passage 53 and Regea 08/013 at passage 41. C ) Quantitative RT-PCR analysis of Nanog , Oct4 , GABRB3 , GDF3 , DNMT3B and TDGF1 expression in hESC lines Regea 07/046 at passage 52, Regea 08/013 at passage 45, and Regea 06/040 at passage 33. No significant differences (over 2-fold) were detected between hESC lines.

Techniques Used: Marker, Expressing, Flow Cytometry, Cytometry, Quantitative RT-PCR

14) Product Images from "A thermoresponsive and chemically defined hydrogel for long-term culture of human embryonic stem cells"

Article Title: A thermoresponsive and chemically defined hydrogel for long-term culture of human embryonic stem cells

Journal: Nature Communications

doi: 10.1038/ncomms2341

Screening procedure for selection of polymers that support hESC culture. ( a ) Outline of the screening procedure used to select polymers that support hESC culture and passaging by thermodetachment. Cell attachment (nuclear count following DAPI stain), Oct3/4 and Nanog expression levels (immunocytochemistry) and thermodetachment ability (30 min at 15 °C) were assessed after culture of cells on polymer arrays. Successful polymers were selected for longer-term culture studies. ( b ) Cell detachment following lowering ambient temperature to 15 °C of hESC cultured on the top three polymers or Matrigel as control. Mean±s.d., n =3. ( c ) Chemical structure of polymers HG19, 20 and 21 with 1:3, 1:1 and 3:1 ratios of AEtMA-Cl/DEAEA, respectively. ( d ) Following thermodetachment of RH1 hESC from HG21 the majority of the remaining cells, visualized by DAPI-stained nuclei (blue) and phase-contrast light microscopy, are negative for Oct3/4 (green) and Nanog (red) (immunocytochemistry). Scale bar, 100 μm. ( e ) Cell growth curves of RH1 hESC cultured on HG21 or MG, plotted as mean fold increase (±s.e.m., n =3 biological replicates) relative to when cells were plated (day 0). *** P
Figure Legend Snippet: Screening procedure for selection of polymers that support hESC culture. ( a ) Outline of the screening procedure used to select polymers that support hESC culture and passaging by thermodetachment. Cell attachment (nuclear count following DAPI stain), Oct3/4 and Nanog expression levels (immunocytochemistry) and thermodetachment ability (30 min at 15 °C) were assessed after culture of cells on polymer arrays. Successful polymers were selected for longer-term culture studies. ( b ) Cell detachment following lowering ambient temperature to 15 °C of hESC cultured on the top three polymers or Matrigel as control. Mean±s.d., n =3. ( c ) Chemical structure of polymers HG19, 20 and 21 with 1:3, 1:1 and 3:1 ratios of AEtMA-Cl/DEAEA, respectively. ( d ) Following thermodetachment of RH1 hESC from HG21 the majority of the remaining cells, visualized by DAPI-stained nuclei (blue) and phase-contrast light microscopy, are negative for Oct3/4 (green) and Nanog (red) (immunocytochemistry). Scale bar, 100 μm. ( e ) Cell growth curves of RH1 hESC cultured on HG21 or MG, plotted as mean fold increase (±s.e.m., n =3 biological replicates) relative to when cells were plated (day 0). *** P

Techniques Used: Selection, Passaging, Cell Attachment Assay, Staining, Expressing, Immunocytochemistry, Cell Culture, Light Microscopy

Characterization of RH1 hESC cultured on thermomodulatable HG21. ( a ) Representative micrographs of hESC colonies cultured on HG21 (left) and Matrigel (MG, right). Scale bar, 100 μm. ( b ) Immunocytochemistry (ICC) for Nanog (red) and Oct3/4 (green) expression in RH1 hESC cultured on HG21 for 10 and 27 passages. Scale bar, 100 μm. ( c ) qPCR analysis of RNA expression of the pluripotency markers Oct3/4, Nanog and Sox2 for RH1 hESC cultured on HG21 for 10 and 20 passages. Expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase, and data represented as fold-change compared with expression in MG-cultured RH1 hESC. Data are representative examples of minimally two independent qPCR experiments per gene. ( d ) SSEA-1, SSEA-4 and TRA-1-60R cell surface markers are expressed at similar levels on RH1 hESC cultured on HG21 (dashed line) and MG (solid line, grey area), as determined by flow cytometry. M1 and M2 gates discriminate background to signal fluorescence thresholds. Apposing table depicts cell percentages with fluorescence signal intensity above background. ( e ) After 20 passages on HG21, RH1 hESC can differentiate to all three germ layers, as shown by ICC for β-tubulin III (ectoderm; green), α-fetoprotein (endoderm; green) and α-smooth muscle actin (mesoderm; green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. ( f ) Kidney capsule teratomas, induced by injection of RH1 hESC cultured for 20 passages on HG21 into NOD/SCID mice, contain tissue derived from all three primary germ layers. Clockwise: islands of undifferentiated EC/ES cells, neural rosettes (ectoderm), gut epithelium (endoderm) and cartilage (mesoderm). (Masson’s trichrome stain). Scale bar, 100 μm. ( g ) Schematic summary of gene variations detected by CGH analysis of RH1 hESC using a NimblegenTM 135 K probe array following culture on HG21 for 10 and 21 passages (p10, p21) and age-matched MG (MG) (21 passages). Depicted are microdeletions and insertions ( > no.
Figure Legend Snippet: Characterization of RH1 hESC cultured on thermomodulatable HG21. ( a ) Representative micrographs of hESC colonies cultured on HG21 (left) and Matrigel (MG, right). Scale bar, 100 μm. ( b ) Immunocytochemistry (ICC) for Nanog (red) and Oct3/4 (green) expression in RH1 hESC cultured on HG21 for 10 and 27 passages. Scale bar, 100 μm. ( c ) qPCR analysis of RNA expression of the pluripotency markers Oct3/4, Nanog and Sox2 for RH1 hESC cultured on HG21 for 10 and 20 passages. Expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase, and data represented as fold-change compared with expression in MG-cultured RH1 hESC. Data are representative examples of minimally two independent qPCR experiments per gene. ( d ) SSEA-1, SSEA-4 and TRA-1-60R cell surface markers are expressed at similar levels on RH1 hESC cultured on HG21 (dashed line) and MG (solid line, grey area), as determined by flow cytometry. M1 and M2 gates discriminate background to signal fluorescence thresholds. Apposing table depicts cell percentages with fluorescence signal intensity above background. ( e ) After 20 passages on HG21, RH1 hESC can differentiate to all three germ layers, as shown by ICC for β-tubulin III (ectoderm; green), α-fetoprotein (endoderm; green) and α-smooth muscle actin (mesoderm; green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. ( f ) Kidney capsule teratomas, induced by injection of RH1 hESC cultured for 20 passages on HG21 into NOD/SCID mice, contain tissue derived from all three primary germ layers. Clockwise: islands of undifferentiated EC/ES cells, neural rosettes (ectoderm), gut epithelium (endoderm) and cartilage (mesoderm). (Masson’s trichrome stain). Scale bar, 100 μm. ( g ) Schematic summary of gene variations detected by CGH analysis of RH1 hESC using a NimblegenTM 135 K probe array following culture on HG21 for 10 and 21 passages (p10, p21) and age-matched MG (MG) (21 passages). Depicted are microdeletions and insertions ( > no.

Techniques Used: Cell Culture, Immunocytochemistry, Expressing, Real-time Polymerase Chain Reaction, RNA Expression, Flow Cytometry, Cytometry, Fluorescence, Staining, Injection, Mouse Assay, Derivative Assay

15) Product Images from "DMH1, a Highly Selective Small Molecule BMP Inhibitor Promotes Neurogenesis of hiPSCs: Comparison of PAX6 and SOX1 Expression during Neural Induction"

Article Title: DMH1, a Highly Selective Small Molecule BMP Inhibitor Promotes Neurogenesis of hiPSCs: Comparison of PAX6 and SOX1 Expression during Neural Induction

Journal: ACS Chemical Neuroscience

doi: 10.1021/cn300029t

Noggin and DMH1-induced regulation of OCT4, Nanog, andPAX6 proteinexpression. Expression of OCT4, Nanog, and PAX6 proteins was assessedon days 1, 3, 5, and 7 of neuralization induced with SB431542 andeither Noggin or DMH1. (A) Cells (SM3) were stained
Figure Legend Snippet: Noggin and DMH1-induced regulation of OCT4, Nanog, andPAX6 proteinexpression. Expression of OCT4, Nanog, and PAX6 proteins was assessedon days 1, 3, 5, and 7 of neuralization induced with SB431542 andeither Noggin or DMH1. (A) Cells (SM3) were stained

Techniques Used: Expressing, Staining

Noggin andDMH1-induced regulation of pluripotency and neural precursormarker mRNAs. (A) Expression of two hiPSC markers ( OCT4 , Nanog ) and four neural precursor markers ( PAX6 , SOX1 , FOXG1 , and OTX2 ) was assessed during neuralization using dual-SMADinhibition
Figure Legend Snippet: Noggin andDMH1-induced regulation of pluripotency and neural precursormarker mRNAs. (A) Expression of two hiPSC markers ( OCT4 , Nanog ) and four neural precursor markers ( PAX6 , SOX1 , FOXG1 , and OTX2 ) was assessed during neuralization using dual-SMADinhibition

Techniques Used: Expressing

16) Product Images from "Loss of Tenomodulin Results in Reduced Self-Renewal and Augmented Senescence of Tendon Stem/Progenitor Cells"

Article Title: Loss of Tenomodulin Results in Reduced Self-Renewal and Augmented Senescence of Tendon Stem/Progenitor Cells

Journal: Stem Cells and Development

doi: 10.1089/scd.2014.0314

Expression profile of mTSPC. (A) Immunocytochemical detection of stem cell markers CD146, CD105, CD90.2, CD73, CD44, Nestin, Nanog, and Sca-1 and tenogenic markers Scx and Tnmd. Insets show nuclear DAP1 stainings. (B) Quantitative PCR for Scx. ** P
Figure Legend Snippet: Expression profile of mTSPC. (A) Immunocytochemical detection of stem cell markers CD146, CD105, CD90.2, CD73, CD44, Nestin, Nanog, and Sca-1 and tenogenic markers Scx and Tnmd. Insets show nuclear DAP1 stainings. (B) Quantitative PCR for Scx. ** P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

17) Product Images from "Nuclear localized Akt enhances breast cancer stem-like cells through counter-regulation of p21Waf1/Cip1 and p27kip1"

Article Title: Nuclear localized Akt enhances breast cancer stem-like cells through counter-regulation of p21Waf1/Cip1 and p27kip1

Journal: Cell Cycle

doi: 10.1080/15384101.2015.1041692

Expression of stem cell reprogramming markers. ( A ) Western blot detection of Oct4, cMyc, Nanog and Sox2 were increased in Akt-WT and Akt-NLS compared to control. In case of KLF4, a protein level was approximately the same as in Akt-WT and Akt-NLS cells compared to control. ( B ) Gene expression of Oct4, cMyc, Nanog and Sox2 were increased in Akt WT compared to Akt-NLS and control. In contrast, KLF4 expression is increased in Akt-NLS as compared to Akt WT and control but not significant in SKBR3 mammospheres. ( C ) Nuclear Akt enhanced the colony forming ability (mammosphere formation) of SKBR3 and MDA-MB468 as compared to mock cells. The mammosphere formation assay was conducted in soft agar. ( D ) Quantitative representation of data “C” a significantly increased number of colonies (mammosphere formation) of SKBR3 and MDA-MB468 as compared to, * P
Figure Legend Snippet: Expression of stem cell reprogramming markers. ( A ) Western blot detection of Oct4, cMyc, Nanog and Sox2 were increased in Akt-WT and Akt-NLS compared to control. In case of KLF4, a protein level was approximately the same as in Akt-WT and Akt-NLS cells compared to control. ( B ) Gene expression of Oct4, cMyc, Nanog and Sox2 were increased in Akt WT compared to Akt-NLS and control. In contrast, KLF4 expression is increased in Akt-NLS as compared to Akt WT and control but not significant in SKBR3 mammospheres. ( C ) Nuclear Akt enhanced the colony forming ability (mammosphere formation) of SKBR3 and MDA-MB468 as compared to mock cells. The mammosphere formation assay was conducted in soft agar. ( D ) Quantitative representation of data “C” a significantly increased number of colonies (mammosphere formation) of SKBR3 and MDA-MB468 as compared to, * P

Techniques Used: Expressing, Western Blot, Multiple Displacement Amplification, Tube Formation Assay

18) Product Images from "Characterization of the interface between adsorbed fibronectin and human embryonic stem cells"

Article Title: Characterization of the interface between adsorbed fibronectin and human embryonic stem cells

Journal: Journal of the Royal Society Interface

doi: 10.1098/rsif.2013.0139

Immunostaining of HUES7 cells for pluripotency markers Oct3/4 (in red) and Nanog (in green) on TCPS dishes coated with fibronectin and its fragments, 70 kDa (160 µg ml −1 ) and 120 kDa (10 µg ml −1 ), after 4 days of culture.
Figure Legend Snippet: Immunostaining of HUES7 cells for pluripotency markers Oct3/4 (in red) and Nanog (in green) on TCPS dishes coated with fibronectin and its fragments, 70 kDa (160 µg ml −1 ) and 120 kDa (10 µg ml −1 ), after 4 days of culture.

Techniques Used: Immunostaining

Immunostaining of HUES7 cells for pluripotency markers Oct3/4 (in red) and Nanog (in green) after five passages on dishes coated with fibronectin and its fragments: 70 kDa (160 mg ml −1 ) and 120 kDa (10 mg ml −1 ). Nuclei are stained in blue
Figure Legend Snippet: Immunostaining of HUES7 cells for pluripotency markers Oct3/4 (in red) and Nanog (in green) after five passages on dishes coated with fibronectin and its fragments: 70 kDa (160 mg ml −1 ) and 120 kDa (10 mg ml −1 ). Nuclei are stained in blue

Techniques Used: Immunostaining, Staining

19) Product Images from "Klotho Deficiency Accelerates Stem Cells Aging by Impairing Telomerase Activity"

Article Title: Klotho Deficiency Accelerates Stem Cells Aging by Impairing Telomerase Activity

Journal: The Journals of Gerontology Series A: Biological Sciences and Medical Sciences

doi: 10.1093/gerona/gly261

Pluripotent-markers expression of adipose-derived stem cells (ADSC) in KL -/- and wild-type animals. ( A ) ADSC were analyzed for the expression of pluripotent-markers of Sox2, Oct4, stage-specific embryonic antigen (SSEA1), and Nanog. Representative histograms revealed lower in % expression of pluripotent-markers in KL -/- ADSC compared to wild type. ( B ) Histogram peaks showed lesser in % expression, of Sox2, Oct4, SSEA1 and Nanog in KL -/- -derived ADSC. ( C ) Immunofluorescence labeled anti-mouse primary antibodies were used to determine the protein expression of Sox2, Oct4, SSEA1, and Nanog markers to know the level/intensity of expression in KL -/- group and wild-type ADSC. Nuclei were counter stained with DAPI (blue). ( D ) Senescence β-Galactosidase staining was used to differentiate between senescent and healthy cells in KL -/- and wild-type-derived ADSC. Mean ± SEM, statistically significant values are: * p = .05; ** p = .01; *** p = .001, scale bar: 100 μm.
Figure Legend Snippet: Pluripotent-markers expression of adipose-derived stem cells (ADSC) in KL -/- and wild-type animals. ( A ) ADSC were analyzed for the expression of pluripotent-markers of Sox2, Oct4, stage-specific embryonic antigen (SSEA1), and Nanog. Representative histograms revealed lower in % expression of pluripotent-markers in KL -/- ADSC compared to wild type. ( B ) Histogram peaks showed lesser in % expression, of Sox2, Oct4, SSEA1 and Nanog in KL -/- -derived ADSC. ( C ) Immunofluorescence labeled anti-mouse primary antibodies were used to determine the protein expression of Sox2, Oct4, SSEA1, and Nanog markers to know the level/intensity of expression in KL -/- group and wild-type ADSC. Nuclei were counter stained with DAPI (blue). ( D ) Senescence β-Galactosidase staining was used to differentiate between senescent and healthy cells in KL -/- and wild-type-derived ADSC. Mean ± SEM, statistically significant values are: * p = .05; ** p = .01; *** p = .001, scale bar: 100 μm.

Techniques Used: Expressing, Derivative Assay, Immunofluorescence, Labeling, Staining

20) Product Images from "Valproic Acid Modulates the Multipotency in Periodontal Ligament Stem Cells via p53-Mediated Cell Cycle"

Article Title: Valproic Acid Modulates the Multipotency in Periodontal Ligament Stem Cells via p53-Mediated Cell Cycle

Journal: Tissue Engineering and Regenerative Medicine

doi: 10.1007/s13770-017-0027-4

PDLSCs expressed OCT4 and NANOG after cyclic VPA treatment as measured by immunofluorescence and flow cytometry (n=3). A , B OCT4 and NANOG expression was higher when treated with VPA for 24 hours and 48 hours rest. Bar 20 μm (×400). C , D Flow cytometry for OCT3/4- and NANOG-expressing PDLSCs. C OCT4-positive PDLSC showed the highest percentage after 0.5 mM VPA treatment. D NANOG-positive PDLSCs showed the highest percentage after 0.5 mM VPA treatment
Figure Legend Snippet: PDLSCs expressed OCT4 and NANOG after cyclic VPA treatment as measured by immunofluorescence and flow cytometry (n=3). A , B OCT4 and NANOG expression was higher when treated with VPA for 24 hours and 48 hours rest. Bar 20 μm (×400). C , D Flow cytometry for OCT3/4- and NANOG-expressing PDLSCs. C OCT4-positive PDLSC showed the highest percentage after 0.5 mM VPA treatment. D NANOG-positive PDLSCs showed the highest percentage after 0.5 mM VPA treatment

Techniques Used: Immunofluorescence, Flow Cytometry, Cytometry, Expressing

21) Product Images from "Dynamic regulation of the cancer stem cell compartment by Cripto-1 in colorectal cancer"

Article Title: Dynamic regulation of the cancer stem cell compartment by Cripto-1 in colorectal cancer

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2015.19

Cripto-1 is expressed in normal and neoplastic colon stem cells. ( a ) Immunofluorescence staining for CD133 and cytokeratin-20 (CK20) on colon cancer spheroids (stem) and sphere-derived adherent cells (SDAC). Line Pt1, 60 × magnification, 1,4 × zoom ( b ) Immunofluorescence detection of stem cell antigens on permeabilized spheroids (Stem) and SDAC, line Pt2. 60 × magnification, 2x zoom ( c ) Immunofluorescence detection of CR1 on non-permeabilized spheroids (Stem) and sphere-derived adherent cells (SDAC), line Pt2. 60 × magnification ( d ) Flow cytometry analysis of CR1 expression on spheroid lines derived from different patients (detailed in Supplementary Table 1 ) ( e ) Flow cytometry staining of CR1 and either Nanog, Ephrin B2 receptor (EphB2) or Lgr5 on spheroid line Pt1. Numbers refer to the percentage of double-positive cells ( f ) Whole-mount in situ hybridization of CR1 on mouse neonatal colon. Magnification 20 ×, inset 40 × ( g ) Anti-CR1 immunohistochemistry on adult mouse normal colon (left panel) and high-grade adenoma (right panel). Arrows indicate cells with membrane CR1 staining ( h ) Immunofluorescence staining of CR1/Lgr5 (left) and CR1/EphB2 (right) expression on FFPE sections of normal colon and colon cancer. Images of colon cancer were taken both from central areas (Colon cancer) and from the invasive edge of the tumor (Invasion front). Magnification 40 ×, detail (yellow square) 3 × zoom
Figure Legend Snippet: Cripto-1 is expressed in normal and neoplastic colon stem cells. ( a ) Immunofluorescence staining for CD133 and cytokeratin-20 (CK20) on colon cancer spheroids (stem) and sphere-derived adherent cells (SDAC). Line Pt1, 60 × magnification, 1,4 × zoom ( b ) Immunofluorescence detection of stem cell antigens on permeabilized spheroids (Stem) and SDAC, line Pt2. 60 × magnification, 2x zoom ( c ) Immunofluorescence detection of CR1 on non-permeabilized spheroids (Stem) and sphere-derived adherent cells (SDAC), line Pt2. 60 × magnification ( d ) Flow cytometry analysis of CR1 expression on spheroid lines derived from different patients (detailed in Supplementary Table 1 ) ( e ) Flow cytometry staining of CR1 and either Nanog, Ephrin B2 receptor (EphB2) or Lgr5 on spheroid line Pt1. Numbers refer to the percentage of double-positive cells ( f ) Whole-mount in situ hybridization of CR1 on mouse neonatal colon. Magnification 20 ×, inset 40 × ( g ) Anti-CR1 immunohistochemistry on adult mouse normal colon (left panel) and high-grade adenoma (right panel). Arrows indicate cells with membrane CR1 staining ( h ) Immunofluorescence staining of CR1/Lgr5 (left) and CR1/EphB2 (right) expression on FFPE sections of normal colon and colon cancer. Images of colon cancer were taken both from central areas (Colon cancer) and from the invasive edge of the tumor (Invasion front). Magnification 40 ×, detail (yellow square) 3 × zoom

Techniques Used: Immunofluorescence, Staining, Derivative Assay, Flow Cytometry, Cytometry, Expressing, In Situ Hybridization, Immunohistochemistry, Formalin-fixed Paraffin-Embedded

22) Product Images from "The Effect of Human and Mouse Fibroblast Feeder Cells on Cardiac Differentiation of Human Pluripotent Stem Cells"

Article Title: The Effect of Human and Mouse Fibroblast Feeder Cells on Cardiac Differentiation of Human Pluripotent Stem Cells

Journal: Stem Cells International

doi: 10.1155/2012/875059

Undifferentiated H7 hESC colonies (on passage 15) 3 days after passaging on hFF feeder cells (a) and after 55 passages on MEF feeder cells (b). H7 cells passaged 11 times on hFF feeder cells expressed Nanog (c and d), Oct4 (e and f), SSEA4 (g and h), and TRA-1-60 (i and j). H7 cells on passage 45 on MEF feeder cells expressed Nanog (k and l), Oct4 (m and n), and SSEA4 (o and p). Scale bars 200 μ m.
Figure Legend Snippet: Undifferentiated H7 hESC colonies (on passage 15) 3 days after passaging on hFF feeder cells (a) and after 55 passages on MEF feeder cells (b). H7 cells passaged 11 times on hFF feeder cells expressed Nanog (c and d), Oct4 (e and f), SSEA4 (g and h), and TRA-1-60 (i and j). H7 cells on passage 45 on MEF feeder cells expressed Nanog (k and l), Oct4 (m and n), and SSEA4 (o and p). Scale bars 200 μ m.

Techniques Used: Passaging

23) Product Images from "Cryopreservation of Human iPS Cell Aggregates in a DMSO-Free Solution—An Optimization and Comparative Study"

Article Title: Cryopreservation of Human iPS Cell Aggregates in a DMSO-Free Solution—An Optimization and Comparative Study

Journal: Frontiers in Bioengineering and Biotechnology

doi: 10.3389/fbioe.2020.00001

Immunocytochemistry of human induced pluripotent stem cells (hiPSCs) cryopreserved using the optimized dimethyl sulfoxide (DMSO)-free solution. Monochromatic images with pseudo-coloring matching the real color of respective fluorescent dye. (A) Quantitative fluorescent microscopy (counterstained with nuclear dye Hoechst 33342, blue) and forward vs. side scatter-gated flow cytometry of cryopreserved hiPSCs showing high expression of transcription factors NANOG (red), OCT4 (green), and pluripotency surface marker TRA-1-60. Scale bar: 100 μm. (B) Immunocytochemistry images showing trilineage differentiation of cryopreserved hiPSCs into three germ layers and expression of endodermal markers, FOXA2 and SOX17, mesodermal markers, T and HAND1, and ectodermal markers, PAX6 and NESTIN. Scale bar: 100 μm. (C) A representative image of normal male karyotype without numerical or structural chromosomal abnormality from the 16 metaphase cells available for analysis.
Figure Legend Snippet: Immunocytochemistry of human induced pluripotent stem cells (hiPSCs) cryopreserved using the optimized dimethyl sulfoxide (DMSO)-free solution. Monochromatic images with pseudo-coloring matching the real color of respective fluorescent dye. (A) Quantitative fluorescent microscopy (counterstained with nuclear dye Hoechst 33342, blue) and forward vs. side scatter-gated flow cytometry of cryopreserved hiPSCs showing high expression of transcription factors NANOG (red), OCT4 (green), and pluripotency surface marker TRA-1-60. Scale bar: 100 μm. (B) Immunocytochemistry images showing trilineage differentiation of cryopreserved hiPSCs into three germ layers and expression of endodermal markers, FOXA2 and SOX17, mesodermal markers, T and HAND1, and ectodermal markers, PAX6 and NESTIN. Scale bar: 100 μm. (C) A representative image of normal male karyotype without numerical or structural chromosomal abnormality from the 16 metaphase cells available for analysis.

Techniques Used: Immunocytochemistry, Microscopy, Flow Cytometry, Expressing, Marker

24) Product Images from "Generation of Neuronal Progenitor Cells and Neurons from Mouse Sleeping Beauty Transposon-Generated Induced Pluripotent Stem Cells"

Article Title: Generation of Neuronal Progenitor Cells and Neurons from Mouse Sleeping Beauty Transposon-Generated Induced Pluripotent Stem Cells

Journal: Cellular Reprogramming

doi: 10.1089/cell.2012.0010

Immunocytochemistry analysis of iPS cell line. Mouse SB-mediated iPS cells were maintained in an undifferentiated stage before neuronal differentiation. The cells were stained with the pluripotent markers Oct4, Sox2, and Nanog. Scale bars, 100 μm.
Figure Legend Snippet: Immunocytochemistry analysis of iPS cell line. Mouse SB-mediated iPS cells were maintained in an undifferentiated stage before neuronal differentiation. The cells were stained with the pluripotent markers Oct4, Sox2, and Nanog. Scale bars, 100 μm.

Techniques Used: Immunocytochemistry, Staining

25) Product Images from "Induced pluripotent stem cells offer new approach to therapy in thalassemia and sickle cell anemia and option in prenatal diagnosis in genetic diseases"

Article Title: Induced pluripotent stem cells offer new approach to therapy in thalassemia and sickle cell anemia and option in prenatal diagnosis in genetic diseases

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0904689106

iPS cell colonies reprogrammed with 3 factors ( 3sy ) or 4 factors ( 4sy ) from the skin fibroblasts of a patient with homozygous β–thalassemia (β -thal ) compared with hESC colonies ( hES ). They were stained for alkaline phosphatase ( AP , Inset ) and ES cell markers Nanog, SSEA3, SSEA4, Tra-1–60, and Tra-1–81.
Figure Legend Snippet: iPS cell colonies reprogrammed with 3 factors ( 3sy ) or 4 factors ( 4sy ) from the skin fibroblasts of a patient with homozygous β–thalassemia (β -thal ) compared with hESC colonies ( hES ). They were stained for alkaline phosphatase ( AP , Inset ) and ES cell markers Nanog, SSEA3, SSEA4, Tra-1–60, and Tra-1–81.

Techniques Used: Staining

26) Product Images from "Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems"

Article Title: Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems

Journal: PLoS ONE

doi: 10.1371/journal.pone.0151264

hiPS cell maintenance in E8 medium and vitronectin. (A) Colonies cultured with E8 medium in Geltrex (left panel), and in vitronectin (right panel). Scale bars—100μm. (B) Cumulative fold increase in total cell numbers over four passages with E8 medium in Geltrex (green), in vitronectin (purple), in Synthemax ® (blue) or in CELLStart™ (red). (C) Average fold increase per passage. The fold increase was 5.2 for cells growing in Geltrex, 4.3 for vitronectin, 4.5 for Synthemax ® and 1.9 for CELLStart™. Error bars represent SEM (standard error of the mean). Four passages and two independent experiments were performed. (D) Immunostaining of colonies cultured with E8 medium in vitronectin. Colonies were stained for pluripotency markers OCT4, SOX2, and NANOG, and nuclei counterstained with DAPI. Scale bars—100μm. (E) Immunostaining of colonies cultured with E8 medium in vitronectin. Colonies were stained for pluripotency surface markers TRA-1-60, TRA-1-81, and SSEA4. Scale bars—100μm. (F) and (G) Flow cytometry analysis of pluripotency surface markers (TRA-1-60 and SSEA4) (F) and transcription factors (OCT4, NANOG, SOX2) (G) in cells expanded with E8 medium and vitronectin.
Figure Legend Snippet: hiPS cell maintenance in E8 medium and vitronectin. (A) Colonies cultured with E8 medium in Geltrex (left panel), and in vitronectin (right panel). Scale bars—100μm. (B) Cumulative fold increase in total cell numbers over four passages with E8 medium in Geltrex (green), in vitronectin (purple), in Synthemax ® (blue) or in CELLStart™ (red). (C) Average fold increase per passage. The fold increase was 5.2 for cells growing in Geltrex, 4.3 for vitronectin, 4.5 for Synthemax ® and 1.9 for CELLStart™. Error bars represent SEM (standard error of the mean). Four passages and two independent experiments were performed. (D) Immunostaining of colonies cultured with E8 medium in vitronectin. Colonies were stained for pluripotency markers OCT4, SOX2, and NANOG, and nuclei counterstained with DAPI. Scale bars—100μm. (E) Immunostaining of colonies cultured with E8 medium in vitronectin. Colonies were stained for pluripotency surface markers TRA-1-60, TRA-1-81, and SSEA4. Scale bars—100μm. (F) and (G) Flow cytometry analysis of pluripotency surface markers (TRA-1-60 and SSEA4) (F) and transcription factors (OCT4, NANOG, SOX2) (G) in cells expanded with E8 medium and vitronectin.

Techniques Used: Cell Culture, Immunostaining, Staining, Flow Cytometry, Cytometry

hiPS cell expansion on microcarriers in E8 medium, under static conditions. (A) Three different inoculation strategies: a. accutase, b. EDTA and c. EDTA+ROCKi were compared. (B) Seeding yield after 5 days of culture of hiPS cells on Geltrex coated polystyrene microcarriers (GM) and plate (GP), vitronectin coated polystyrene microcarriers (VtnM) and plate (VtnP), using the three different inoculation strategies: accutase (green), EDTA (purple) and EDTA+ROCKi (blue). Cells were inoculated at a 5x10 4 cells/cm 2 density. Error bars represent SEM (standard error of the mean). Three replicate wells were performed for each condition. (C) Immunostaining of hiPS cells cultured on microcarriers with E8 medium at day 5. Cells were stained for pluripotency markers NANOG (left panel) and OCT4 (right panel), and nuclei counterstained with DAPI. Scale bars– 100 μm.
Figure Legend Snippet: hiPS cell expansion on microcarriers in E8 medium, under static conditions. (A) Three different inoculation strategies: a. accutase, b. EDTA and c. EDTA+ROCKi were compared. (B) Seeding yield after 5 days of culture of hiPS cells on Geltrex coated polystyrene microcarriers (GM) and plate (GP), vitronectin coated polystyrene microcarriers (VtnM) and plate (VtnP), using the three different inoculation strategies: accutase (green), EDTA (purple) and EDTA+ROCKi (blue). Cells were inoculated at a 5x10 4 cells/cm 2 density. Error bars represent SEM (standard error of the mean). Three replicate wells were performed for each condition. (C) Immunostaining of hiPS cells cultured on microcarriers with E8 medium at day 5. Cells were stained for pluripotency markers NANOG (left panel) and OCT4 (right panel), and nuclei counterstained with DAPI. Scale bars– 100 μm.

Techniques Used: Immunostaining, Cell Culture, Staining

hiPS cell expansion in E8 medium, in a 50 mL spinner flask. EDTA clump inoculation was performed using 55,000 cells/cm 2 and a continuous agitation at 44 rpm (optimal values obtained solving the quadratic model). (A) Total cell numbers during the 10 days expansion. Maximum cell yield obtained solving the quadratic model is represented by the black line. Error bars represent SEM (standard error of the mean) of duplicate samples. (B) Immunostaining of hiPS cells cultured on microcarriers in the spinner flask with E8 medium at day 10. Cells were stained for pluripotency markers NANOG (left panel) and OCT4 (right panel), and nuclei counterstained with DAPI. Scale bars– 100 μm. (C) Immunostaining of hiPS cells harvested from microcarriers after 10 days expansion in spinner flask and re-plated on GP. Cells were stained after 3 days for intracellular pluripotency markers NANOG, OCT4 and SOX2, and nuclei counterstained with DAPI; and for surface markers SSEA4 and TRA-1-60. Scale bars– 100 μm. (D) Flow cytometry analysis of hiPS cells harvested from microcarriers after 10 days expansion in spinner flask. Cells were stained for OCT4 and NANOG. (E) mRNA was isolated from hiPSC at day 0 and at the end of the spinner flask culture (day 12) on microcarriers, and the undifferentiated hiPSC marker transcripts ( OCT4 and NANOG ) were analysed by RT-PCR. (F) Quantitative RT-PCR analysis of spontaneous differentiated EB of hiPSC cultured in spinner flask. The relative expression of each gene was measured against the same gene prior to differentiation. (G) Immunostaining showing the formation of cells expressing SOX17 (endoderm), TUJ1 (ectoderm) and α-SMA (mesoderm) after EB formation and spontaneous differentiation assay with hiPSC cultured in spinner flask. Nuclei were counterstained with DAPI. Scale bar: 50 μm.
Figure Legend Snippet: hiPS cell expansion in E8 medium, in a 50 mL spinner flask. EDTA clump inoculation was performed using 55,000 cells/cm 2 and a continuous agitation at 44 rpm (optimal values obtained solving the quadratic model). (A) Total cell numbers during the 10 days expansion. Maximum cell yield obtained solving the quadratic model is represented by the black line. Error bars represent SEM (standard error of the mean) of duplicate samples. (B) Immunostaining of hiPS cells cultured on microcarriers in the spinner flask with E8 medium at day 10. Cells were stained for pluripotency markers NANOG (left panel) and OCT4 (right panel), and nuclei counterstained with DAPI. Scale bars– 100 μm. (C) Immunostaining of hiPS cells harvested from microcarriers after 10 days expansion in spinner flask and re-plated on GP. Cells were stained after 3 days for intracellular pluripotency markers NANOG, OCT4 and SOX2, and nuclei counterstained with DAPI; and for surface markers SSEA4 and TRA-1-60. Scale bars– 100 μm. (D) Flow cytometry analysis of hiPS cells harvested from microcarriers after 10 days expansion in spinner flask. Cells were stained for OCT4 and NANOG. (E) mRNA was isolated from hiPSC at day 0 and at the end of the spinner flask culture (day 12) on microcarriers, and the undifferentiated hiPSC marker transcripts ( OCT4 and NANOG ) were analysed by RT-PCR. (F) Quantitative RT-PCR analysis of spontaneous differentiated EB of hiPSC cultured in spinner flask. The relative expression of each gene was measured against the same gene prior to differentiation. (G) Immunostaining showing the formation of cells expressing SOX17 (endoderm), TUJ1 (ectoderm) and α-SMA (mesoderm) after EB formation and spontaneous differentiation assay with hiPSC cultured in spinner flask. Nuclei were counterstained with DAPI. Scale bar: 50 μm.

Techniques Used: Immunostaining, Cell Culture, Staining, Flow Cytometry, Cytometry, Isolation, Marker, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Differentiation Assay

27) Product Images from "Characterization of iPSCs derived from low grade gliomas revealed early regional chromosomal amplifications during gliomagenesis"

Article Title: Characterization of iPSCs derived from low grade gliomas revealed early regional chromosomal amplifications during gliomagenesis

Journal: Journal of neuro-oncology

doi: 10.1007/s11060-018-03047-1

Generation of iPSCs from primary LGG cells. ( A, C ) Bright field images of primary BT01 and BT03 cells. ( B, D ) Images of representative iPSC colonies derived from BT01 and BT03 cells. ( E ) LGG-iPSCs express alkaline phosphatase. ( F-H ) Immunostaining demonstrated that LGG-iPSCs express pluripotency-associated transcription factors (F) OCT4, (G) NANOG, and (H) SOX2. ( I ) Flow cytometry analysis confirmed LGG-iPSCs expressing the pluripotency-specific surface marker Tra-1–60. ( J-L ) Teratoma formation assay demonstrated LGG-iPSCs can differentiated into (J) ectoderm (neuroepithelium), (K) mesoderm (cartilage), and (L) endoderm (glandular structure). Scale bars, 100 μm. ( M ) Representative sequencing results of the BT01- and BT03-iPSC clones. More than 15 independent clones of each LGG samples were sequenced. None carried the IDH1 mutations.
Figure Legend Snippet: Generation of iPSCs from primary LGG cells. ( A, C ) Bright field images of primary BT01 and BT03 cells. ( B, D ) Images of representative iPSC colonies derived from BT01 and BT03 cells. ( E ) LGG-iPSCs express alkaline phosphatase. ( F-H ) Immunostaining demonstrated that LGG-iPSCs express pluripotency-associated transcription factors (F) OCT4, (G) NANOG, and (H) SOX2. ( I ) Flow cytometry analysis confirmed LGG-iPSCs expressing the pluripotency-specific surface marker Tra-1–60. ( J-L ) Teratoma formation assay demonstrated LGG-iPSCs can differentiated into (J) ectoderm (neuroepithelium), (K) mesoderm (cartilage), and (L) endoderm (glandular structure). Scale bars, 100 μm. ( M ) Representative sequencing results of the BT01- and BT03-iPSC clones. More than 15 independent clones of each LGG samples were sequenced. None carried the IDH1 mutations.

Techniques Used: Derivative Assay, Immunostaining, Flow Cytometry, Cytometry, Expressing, Marker, Teratoma Formation Assay, Sequencing, Clone Assay

28) Product Images from "Mesenchymal stromal cells derived from whole human umbilical cord exhibit similar properties to those derived from Wharton's jelly and bone marrow"

Article Title: Mesenchymal stromal cells derived from whole human umbilical cord exhibit similar properties to those derived from Wharton's jelly and bone marrow

Journal: FEBS Open Bio

doi: 10.1002/2211-5463.12104

Immunocytochemistry and flow cytometry showing the presence of pluripotency markers and stage‐specific embryonic antigens on BM‐MSC, MC‐MSC and WJ‐MSC. (A) Immunocytochemical staining of BM‐MSC, MC‐MSC, WJ‐MSC and H9 ESC cell line with the pluripotency markers, OCT3/4, nanog and REX‐1. Scale bars represent 100 μm. (B) The presence of stage‐specific embryonic antigens (SSEA)‐1 and 4, tumour repressor antigens (TRA)‐1‐60, TRA‐1‐81 and the pluripotency marker alkaline phosphatase on MC‐MSC, WJ‐MSC and BM‐MSC, assessed by flow cytometry. n = 4 for each cell type. Bars represent average percentage of positively stained cells ± SEM. The ESC H9 cell line was also used as a positive control for the markers shown in B (data not shown). Levels of significance indicated are * P
Figure Legend Snippet: Immunocytochemistry and flow cytometry showing the presence of pluripotency markers and stage‐specific embryonic antigens on BM‐MSC, MC‐MSC and WJ‐MSC. (A) Immunocytochemical staining of BM‐MSC, MC‐MSC, WJ‐MSC and H9 ESC cell line with the pluripotency markers, OCT3/4, nanog and REX‐1. Scale bars represent 100 μm. (B) The presence of stage‐specific embryonic antigens (SSEA)‐1 and 4, tumour repressor antigens (TRA)‐1‐60, TRA‐1‐81 and the pluripotency marker alkaline phosphatase on MC‐MSC, WJ‐MSC and BM‐MSC, assessed by flow cytometry. n = 4 for each cell type. Bars represent average percentage of positively stained cells ± SEM. The ESC H9 cell line was also used as a positive control for the markers shown in B (data not shown). Levels of significance indicated are * P

Techniques Used: Immunocytochemistry, Flow Cytometry, Cytometry, Staining, Marker, Positive Control

29) Product Images from "HIGH-THROUGHPUT SCREENING ASSAY FOR THE IDENTIFICATION OF COMPOUNDS REGULATING SELF-RENEWAL AND DIFFERENTIATION IN HUMAN EMBRYONIC STEM CELLS"

Article Title: HIGH-THROUGHPUT SCREENING ASSAY FOR THE IDENTIFICATION OF COMPOUNDS REGULATING SELF-RENEWAL AND DIFFERENTIATION IN HUMAN EMBRYONIC STEM CELLS

Journal: Cell stem cell

doi: 10.1016/j.stem.2008.05.010

HTS assay development. ( A ) HESCs (H9 line) maintained on matrigel prior to 384 well plating exhibit typical undifferentiated cell morphology (phase contrast), express Oct4 and incorporate the S-phase marker BrdU. ( B ) Cell titration on matrigel coated 384 well plates followed Alamar Blue assay to determine the number of metabolically active cells (RFU: Relative Fluorescence Units). Data are presented as mean ± S.E.M. Cell density refers to seeding density at the time of plating; AlamarBlue is added at day 7, RFU is measured at day 8. ( C ) Upper panel: Quantification of Oct4 signal after treatment of hESCs in 384 well plates with BMP4 (low control) and FGF2 (high control). Lower panel: Corresponding immunocytochemical analysis for Oct4 and Nanog in BMP4 and FGF2-treated cells. A.U. corresponds to “arbitrary units”. ( D ) Schematic representation of HTS assay: Undifferentiated hESCs were grown on matrigel, dissociated into single cells using Accutase and plated onto matrigel coated 384-well plates at a density of 6000 cells/well. After 48hrs, compounds ( yellow ) and high and low controls ( red: FGF2; green: BMP4 ) were added for an additional 5 days. At day 7, automated immunocytochemistry for Oct4 was performed and signal intensity was quantified using automated laser scanning confocal microscopy (GE InCell Analyzer 3000). The specific image shown corresponds to a culture well at day 7 after plating maintained in the presence of FGF2. E ) Control experiments in HTS conditions were carried out using IGF2 for similar effect as FGF2 and BMP2 and PD98059 for loss of Oct4 expression. Scale bar in A corresponds to 500μm in A (left panel), 50μm in A (right panel), and 100 μm in C (lower panels).
Figure Legend Snippet: HTS assay development. ( A ) HESCs (H9 line) maintained on matrigel prior to 384 well plating exhibit typical undifferentiated cell morphology (phase contrast), express Oct4 and incorporate the S-phase marker BrdU. ( B ) Cell titration on matrigel coated 384 well plates followed Alamar Blue assay to determine the number of metabolically active cells (RFU: Relative Fluorescence Units). Data are presented as mean ± S.E.M. Cell density refers to seeding density at the time of plating; AlamarBlue is added at day 7, RFU is measured at day 8. ( C ) Upper panel: Quantification of Oct4 signal after treatment of hESCs in 384 well plates with BMP4 (low control) and FGF2 (high control). Lower panel: Corresponding immunocytochemical analysis for Oct4 and Nanog in BMP4 and FGF2-treated cells. A.U. corresponds to “arbitrary units”. ( D ) Schematic representation of HTS assay: Undifferentiated hESCs were grown on matrigel, dissociated into single cells using Accutase and plated onto matrigel coated 384-well plates at a density of 6000 cells/well. After 48hrs, compounds ( yellow ) and high and low controls ( red: FGF2; green: BMP4 ) were added for an additional 5 days. At day 7, automated immunocytochemistry for Oct4 was performed and signal intensity was quantified using automated laser scanning confocal microscopy (GE InCell Analyzer 3000). The specific image shown corresponds to a culture well at day 7 after plating maintained in the presence of FGF2. E ) Control experiments in HTS conditions were carried out using IGF2 for similar effect as FGF2 and BMP2 and PD98059 for loss of Oct4 expression. Scale bar in A corresponds to 500μm in A (left panel), 50μm in A (right panel), and 100 μm in C (lower panels).

Techniques Used: HTS Assay, Marker, Titration, Alamar Blue Assay, Metabolic Labelling, Fluorescence, Immunocytochemistry, Confocal Microscopy, Expressing

Small molecules promoting hESC self-renewal. ( A ) Expression of Oct4 and Nanog upon treatment with the four activator compounds was confirmed in H9 and in H1 hESCs. ( B ) Repeated passaging of hESCs (H9) showed an increase in the percentage of undifferentiated colonies in presence of the self-renewal drugs, compared to HES medium alone (∅). The graph represents the relative increase of undifferentiated colonies comparing HES medium alone (defined as 0%) versus MEF-conditioned medium supplemented with FGF2 (defined as 100%). ( C ) Mouse ES cell self-renewal was tested after three passages using an Oct4::eGFP BAC transgenic mouse ES cell line. The percentage of eGFP+ cells was quantified by flow cytometry and the levels of Oct4 cells achieved in mouse ES cell medium alone (defined as ∅ =0%) were compared to those treated with LIF (defined as 100%). ( D ). Quantification of the ratio of Oct4/DAPI staining under different culture conditions. Statistical analysis with ANOVA and post hoc Dunnett’s test showed that the BMP4 group was significantly different from HES control (p
Figure Legend Snippet: Small molecules promoting hESC self-renewal. ( A ) Expression of Oct4 and Nanog upon treatment with the four activator compounds was confirmed in H9 and in H1 hESCs. ( B ) Repeated passaging of hESCs (H9) showed an increase in the percentage of undifferentiated colonies in presence of the self-renewal drugs, compared to HES medium alone (∅). The graph represents the relative increase of undifferentiated colonies comparing HES medium alone (defined as 0%) versus MEF-conditioned medium supplemented with FGF2 (defined as 100%). ( C ) Mouse ES cell self-renewal was tested after three passages using an Oct4::eGFP BAC transgenic mouse ES cell line. The percentage of eGFP+ cells was quantified by flow cytometry and the levels of Oct4 cells achieved in mouse ES cell medium alone (defined as ∅ =0%) were compared to those treated with LIF (defined as 100%). ( D ). Quantification of the ratio of Oct4/DAPI staining under different culture conditions. Statistical analysis with ANOVA and post hoc Dunnett’s test showed that the BMP4 group was significantly different from HES control (p

Techniques Used: Expressing, Passaging, BAC Assay, Transgenic Assay, Flow Cytometry, Cytometry, Staining

30) Product Images from "Expression of Oct3/4 and Nanog in the head and neck squamous carcinoma cells and its clinical implications for delayed neck metastasis in stage I/II oral tongue squamous cell carcinoma"

Article Title: Expression of Oct3/4 and Nanog in the head and neck squamous carcinoma cells and its clinical implications for delayed neck metastasis in stage I/II oral tongue squamous cell carcinoma

Journal: BMC Cancer

doi: 10.1186/s12885-015-1732-9

Expression of stem cell markers in SP and MP cells. a : The mRNA expressions of Oct3/4 , Nanog , Sox2 , Notch1 , Bmi-1 , ABCG2 , and GAPDH in both SP and MP cells were separately analyzed using RT-PCR. b : The PCR products visualized under UV-light were semi-quantified using ImageJ. The relative expression levels of the genes were compared after normalization using those of GAPDH . Gene expression levels in the MP cells were defined as 1.0 in each comparison for convenience. c and d : The protein expressions of Oct3/4 and Nanog in both SP and MP cells were separately evaluated using immunofluorescent staining in the SCC4 ( c ) and SAS ( d ) cells. Nuclei were stained with DAPI. Scale bar: 100 μm
Figure Legend Snippet: Expression of stem cell markers in SP and MP cells. a : The mRNA expressions of Oct3/4 , Nanog , Sox2 , Notch1 , Bmi-1 , ABCG2 , and GAPDH in both SP and MP cells were separately analyzed using RT-PCR. b : The PCR products visualized under UV-light were semi-quantified using ImageJ. The relative expression levels of the genes were compared after normalization using those of GAPDH . Gene expression levels in the MP cells were defined as 1.0 in each comparison for convenience. c and d : The protein expressions of Oct3/4 and Nanog in both SP and MP cells were separately evaluated using immunofluorescent staining in the SCC4 ( c ) and SAS ( d ) cells. Nuclei were stained with DAPI. Scale bar: 100 μm

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Staining

Immunohistochemistry of Oct3/4 and Nanog in TSCC specimens. In the representative cases shown, positive staining of Oct3/4 ( a ) or Nanog ( b ) was observed in the nuclei of the tumor cells (arrows). Original magnification: ×200. Scale bar: 100 μm
Figure Legend Snippet: Immunohistochemistry of Oct3/4 and Nanog in TSCC specimens. In the representative cases shown, positive staining of Oct3/4 ( a ) or Nanog ( b ) was observed in the nuclei of the tumor cells (arrows). Original magnification: ×200. Scale bar: 100 μm

Techniques Used: Immunohistochemistry, Staining

31) Product Images from "Human neural crest cells contribute to coat pigmentation in interspecies chimeras after in utero injection into mouse embryos"

Article Title: Human neural crest cells contribute to coat pigmentation in interspecies chimeras after in utero injection into mouse embryos

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1525518113

Derivation of iPSCs from African-American (AA) donor fibroblasts. ( A ) Fibroblasts from an AA donor were reprogrammed to hiPSCs (AA-hiPSCs). Immunocytochemistry showed expression of the key pluripotency markers NANOG, OCT3/4, and SOX2 in the reprogrammed
Figure Legend Snippet: Derivation of iPSCs from African-American (AA) donor fibroblasts. ( A ) Fibroblasts from an AA donor were reprogrammed to hiPSCs (AA-hiPSCs). Immunocytochemistry showed expression of the key pluripotency markers NANOG, OCT3/4, and SOX2 in the reprogrammed

Techniques Used: Immunocytochemistry, Expressing

32) Product Images from "Spheroid-Derived Cells From Renal Adenocarcinoma Have Low Telomerase Activity and High Stem-Like and Invasive Characteristics"

Article Title: Spheroid-Derived Cells From Renal Adenocarcinoma Have Low Telomerase Activity and High Stem-Like and Invasive Characteristics

Journal: Frontiers in Oncology

doi: 10.3389/fonc.2019.01302

Expression levels of stemness and EMT genes using qRT-PCR, H E, and immunohistochemical staining in xenograft tumors derived from parental cells (PCs) and spheroid-derived cells (SDCs) and expression levels of stemness and EMT genes in PCs of ACHN cell line and their SDCs by qRT-PCR. (A) Expression of all stemness genes and some EMT-related genes were higher in SDCs from xenograft tumors compared to PCs. (B) Results of H E staining showed a clear cell RCC morphology. Immunohistochemical staining indicated increased expression of OCT4 and Nanog in tumors from SDCs compared to PCs. (C) SDCs showed significantly higher mRNA levels of all stemness genes compared to PCs. (D) In EMT-related genes, significantly higher levels of Snail1, Twist2, Zeb1, and Vimentin mRNA and significantly lower levels of Snail2, E-cadherin, and Twist1 mRNA were observed in SDCs compared to PCs. There were no significant differences in mRNA expressions of N-cadherin and Zeb2 in the two cell populations. Data are represented as mean ± SD ( n = 3 each). * P
Figure Legend Snippet: Expression levels of stemness and EMT genes using qRT-PCR, H E, and immunohistochemical staining in xenograft tumors derived from parental cells (PCs) and spheroid-derived cells (SDCs) and expression levels of stemness and EMT genes in PCs of ACHN cell line and their SDCs by qRT-PCR. (A) Expression of all stemness genes and some EMT-related genes were higher in SDCs from xenograft tumors compared to PCs. (B) Results of H E staining showed a clear cell RCC morphology. Immunohistochemical staining indicated increased expression of OCT4 and Nanog in tumors from SDCs compared to PCs. (C) SDCs showed significantly higher mRNA levels of all stemness genes compared to PCs. (D) In EMT-related genes, significantly higher levels of Snail1, Twist2, Zeb1, and Vimentin mRNA and significantly lower levels of Snail2, E-cadherin, and Twist1 mRNA were observed in SDCs compared to PCs. There were no significant differences in mRNA expressions of N-cadherin and Zeb2 in the two cell populations. Data are represented as mean ± SD ( n = 3 each). * P

Techniques Used: Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining, Derivative Assay

33) Product Images from "SHBG Is an Important Factor in Stemness Induction of Cells by DHT In Vitro and Associated with Poor Clinical Features of Prostate Carcinomas"

Article Title: SHBG Is an Important Factor in Stemness Induction of Cells by DHT In Vitro and Associated with Poor Clinical Features of Prostate Carcinomas

Journal: PLoS ONE

doi: 10.1371/journal.pone.0070558

DHT upregulates the expression of stemess factors through SHBG. (A) Stronger immunocytochemical staining of SHBG is shown in LNCaP and PC-3 cells treated with DHT (left panel, bar scale: 50 µm). Immunoblotting demonstrates higher levels of SHBG, Oct3/4 and Nanog expressions in the cells treated with DHT (right panel). (B) SHBG specific siRNA results. SHBG knockdown in both LNCaP and PC-3 cells was verified by immunoblotting (left panel); 10 nM DHT induces expressions of Oct3/4 and Nanog in the cells transfected with the siRNA control, but such an induction disappears in the cells transfected with the specific SHBG siRNA (right panel). (C) LNCaP and PC-3 cells treated with specific SHBG siRNA grow relatively slower compared to the cells cultivated with control siRNA. (* means P
Figure Legend Snippet: DHT upregulates the expression of stemess factors through SHBG. (A) Stronger immunocytochemical staining of SHBG is shown in LNCaP and PC-3 cells treated with DHT (left panel, bar scale: 50 µm). Immunoblotting demonstrates higher levels of SHBG, Oct3/4 and Nanog expressions in the cells treated with DHT (right panel). (B) SHBG specific siRNA results. SHBG knockdown in both LNCaP and PC-3 cells was verified by immunoblotting (left panel); 10 nM DHT induces expressions of Oct3/4 and Nanog in the cells transfected with the siRNA control, but such an induction disappears in the cells transfected with the specific SHBG siRNA (right panel). (C) LNCaP and PC-3 cells treated with specific SHBG siRNA grow relatively slower compared to the cells cultivated with control siRNA. (* means P

Techniques Used: Expressing, Staining, Transfection

DHT increases Oct3/4 and Nanog expressions in prostate cancer cell lines. (A) The higher expressions of Oct3/4 and Nanog by immunoblotting assay are shown in LNCaP and PC-3 cells treated with 1 nM and 10 nM DHT treatments, respectively, for variable periods of times. (B) The immunocytochemical staining shows higher levels of these two factors in both cell lines treated with different concentrations of DHT. Human seminoma tissue sections were used as positive controls for these two antibodies (bar scale: 50 µm).
Figure Legend Snippet: DHT increases Oct3/4 and Nanog expressions in prostate cancer cell lines. (A) The higher expressions of Oct3/4 and Nanog by immunoblotting assay are shown in LNCaP and PC-3 cells treated with 1 nM and 10 nM DHT treatments, respectively, for variable periods of times. (B) The immunocytochemical staining shows higher levels of these two factors in both cell lines treated with different concentrations of DHT. Human seminoma tissue sections were used as positive controls for these two antibodies (bar scale: 50 µm).

Techniques Used: Staining

34) Product Images from "Fatty acid amide hydrolase deficiency limits early pregnancy events"

Article Title: Fatty acid amide hydrolase deficiency limits early pregnancy events

Journal:

doi: 10.1172/JCI28621

Expression of Cdx2, Nanog, and Oct3/4 is aberrant in Faah -deficient morulae ( A ) and early ( B ) and late blastocysts ( C ).
Figure Legend Snippet: Expression of Cdx2, Nanog, and Oct3/4 is aberrant in Faah -deficient morulae ( A ) and early ( B ) and late blastocysts ( C ).

Techniques Used: Expressing

35) Product Images from "Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long-QT syndrome"

Article Title: Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long-QT syndrome

Journal: The EMBO Journal

doi: 10.1038/emboj.2013.240

Generation of hiPSCs from a patient with type-2 long-QT syndrome. ( A ) Genetic screening in the patient revealed the heterozygous single-nucleotide mutation A→T in exon 13 of the KCNH2 gene, in position 2987 of the coding sequence (CDS) (c.A2987T, NM_000238.3), resulting in the substitution of an asparagine with an isoleucine at position 996 of the protein (N996I, NP_000229.1). ( B ) The N996I mutation (red dot) is located in the C-terminal of the HERG protein, which is made of six trans-membrane domains (S1–S6), an amino (NH2) domain, a carboxyl (COOH) domain, and a pore (P) region. ( C ) Example of a hiPSC colony harbouring the c.A2987T (N996I) KCNH2 mutation (LQT2-hiPSCs N996I ). Scale bar: 400 μm. ( D ) Immunofluorescence analysis of pluripotency markers SSEA4 (green) and NANOG (red) in a representative LQT2-hiPSC N996I clone, with nuclear staining (DNA, blue). The image on the right is a magnification of the area framed in the left image. Scale bars: 100 μm (left image); 50 μm (right image).
Figure Legend Snippet: Generation of hiPSCs from a patient with type-2 long-QT syndrome. ( A ) Genetic screening in the patient revealed the heterozygous single-nucleotide mutation A→T in exon 13 of the KCNH2 gene, in position 2987 of the coding sequence (CDS) (c.A2987T, NM_000238.3), resulting in the substitution of an asparagine with an isoleucine at position 996 of the protein (N996I, NP_000229.1). ( B ) The N996I mutation (red dot) is located in the C-terminal of the HERG protein, which is made of six trans-membrane domains (S1–S6), an amino (NH2) domain, a carboxyl (COOH) domain, and a pore (P) region. ( C ) Example of a hiPSC colony harbouring the c.A2987T (N996I) KCNH2 mutation (LQT2-hiPSCs N996I ). Scale bar: 400 μm. ( D ) Immunofluorescence analysis of pluripotency markers SSEA4 (green) and NANOG (red) in a representative LQT2-hiPSC N996I clone, with nuclear staining (DNA, blue). The image on the right is a magnification of the area framed in the left image. Scale bars: 100 μm (left image); 50 μm (right image).

Techniques Used: Mutagenesis, Sequencing, Immunofluorescence, Staining

36) Product Images from "Laser-Based Propagation of Human iPS and ES Cells Generates Reproducible Cultures with Enhanced Differentiation Potential"

Article Title: Laser-Based Propagation of Human iPS and ES Cells Generates Reproducible Cultures with Enhanced Differentiation Potential

Journal: Stem Cells International

doi: 10.1155/2012/926463

Quality of iPSC cultures after laser-mediated passage. (a) Immunocytochemical analysis of Oct4, Sox2, Nanog, SSEA4, TRA1-60, and TRA1-81 expression immediately following laser-mediated sectioning of iPSC cultures (BIMR L) into 200 μ m sections. Hoechst was used as a nuclear counterstain. Note that all markers are expressed homogeneously across iPSC clumps. Scale bar, 1 mm. (b) Immunocytochemical analysis of apoptosis markers, caspase-3, and cleaved PARP, following laser-mediated sectioning of iPSC cultures. Hoechst was used as a nuclear counterstain. Scale bar, 1 mm. (c) Analysis of iPSC (BIMR 6, left) and ESC (H9, right) growth following propagation using laser-mediated passage or collagenase passage. P indicates passage number. Data are shown as mean + s.d. ( n = 3).
Figure Legend Snippet: Quality of iPSC cultures after laser-mediated passage. (a) Immunocytochemical analysis of Oct4, Sox2, Nanog, SSEA4, TRA1-60, and TRA1-81 expression immediately following laser-mediated sectioning of iPSC cultures (BIMR L) into 200 μ m sections. Hoechst was used as a nuclear counterstain. Note that all markers are expressed homogeneously across iPSC clumps. Scale bar, 1 mm. (b) Immunocytochemical analysis of apoptosis markers, caspase-3, and cleaved PARP, following laser-mediated sectioning of iPSC cultures. Hoechst was used as a nuclear counterstain. Scale bar, 1 mm. (c) Analysis of iPSC (BIMR 6, left) and ESC (H9, right) growth following propagation using laser-mediated passage or collagenase passage. P indicates passage number. Data are shown as mean + s.d. ( n = 3).

Techniques Used: Expressing

Pluripotency and stability of stem cells after laser-mediated passage. (a) Colony morphology (brightfield, BF) and immunocytochemical analysis of Oct4, Sox2, Nanog, alkaline phosphatase (AP), SSEA4, TRA1-60, and TRA1-81 expression in human ESCs (H9) after 24 consecutive laser-mediated passages. Hoechst was used as a nuclear counterstain. Scale bar, 250 μ m. (b) Whole well brightfield images of human iPSC (BIMR 6) cultures after 10 consecutive laser-mediated passages or collagenase passages. Scale bar, 5 mm. (c) QRT-PCR analysis of stem-cell-associated gene expression in iPSCs (BIMR 6) after 10 consecutive laser-mediated passages or collagenase passages. The asterisks (*) indicate values that are statistically significant compared with the starting population of iPSCs. The data are presented as mean ± s.d. ( n = 3). Statistical analysis was performed using t -test, with a P value ≤ 0.05 considered to be significant. (d) Normal karyotype of H9 human ESCs after 24 laser-mediated passages (6 months). (e) Schematic depicting genomic abnormalities of H9 ESCs at P35 (starting population) and at P59, after 24 laser-mediated passages (6 months), as determined by aCGH. No new subkaryotypic abnormalities were detected after 24 passages. Red bars indicate a deletion. Green bars indicate an amplification. See Table 1 for complete list of aberrations found in these cells.
Figure Legend Snippet: Pluripotency and stability of stem cells after laser-mediated passage. (a) Colony morphology (brightfield, BF) and immunocytochemical analysis of Oct4, Sox2, Nanog, alkaline phosphatase (AP), SSEA4, TRA1-60, and TRA1-81 expression in human ESCs (H9) after 24 consecutive laser-mediated passages. Hoechst was used as a nuclear counterstain. Scale bar, 250 μ m. (b) Whole well brightfield images of human iPSC (BIMR 6) cultures after 10 consecutive laser-mediated passages or collagenase passages. Scale bar, 5 mm. (c) QRT-PCR analysis of stem-cell-associated gene expression in iPSCs (BIMR 6) after 10 consecutive laser-mediated passages or collagenase passages. The asterisks (*) indicate values that are statistically significant compared with the starting population of iPSCs. The data are presented as mean ± s.d. ( n = 3). Statistical analysis was performed using t -test, with a P value ≤ 0.05 considered to be significant. (d) Normal karyotype of H9 human ESCs after 24 laser-mediated passages (6 months). (e) Schematic depicting genomic abnormalities of H9 ESCs at P35 (starting population) and at P59, after 24 laser-mediated passages (6 months), as determined by aCGH. No new subkaryotypic abnormalities were detected after 24 passages. Red bars indicate a deletion. Green bars indicate an amplification. See Table 1 for complete list of aberrations found in these cells.

Techniques Used: Expressing, Quantitative RT-PCR, Amplification

37) Product Images from "Klotho Deficiency Accelerates Stem Cells Aging by Impairing Telomerase Activity"

Article Title: Klotho Deficiency Accelerates Stem Cells Aging by Impairing Telomerase Activity

Journal: The Journals of Gerontology Series A: Biological Sciences and Medical Sciences

doi: 10.1093/gerona/gly261

Pluripotent-markers expression of adipose-derived stem cells (ADSC) in KL -/- and wild-type animals. ( A ) ADSC were analyzed for the expression of pluripotent-markers of Sox2, Oct4, stage-specific embryonic antigen (SSEA1), and Nanog. Representative histograms revealed lower in % expression of pluripotent-markers in KL -/- ADSC compared to wild type. ( B ) Histogram peaks showed lesser in % expression, of Sox2, Oct4, SSEA1 and Nanog in KL -/- -derived ADSC. ( C ) Immunofluorescence labeled anti-mouse primary antibodies were used to determine the protein expression of Sox2, Oct4, SSEA1, and Nanog markers to know the level/intensity of expression in KL -/- group and wild-type ADSC. Nuclei were counter stained with DAPI (blue). ( D ) Senescence β-Galactosidase staining was used to differentiate between senescent and healthy cells in KL -/- and wild-type-derived ADSC. Mean ± SEM, statistically significant values are: * p = .05; ** p = .01; *** p = .001, scale bar: 100 μm.
Figure Legend Snippet: Pluripotent-markers expression of adipose-derived stem cells (ADSC) in KL -/- and wild-type animals. ( A ) ADSC were analyzed for the expression of pluripotent-markers of Sox2, Oct4, stage-specific embryonic antigen (SSEA1), and Nanog. Representative histograms revealed lower in % expression of pluripotent-markers in KL -/- ADSC compared to wild type. ( B ) Histogram peaks showed lesser in % expression, of Sox2, Oct4, SSEA1 and Nanog in KL -/- -derived ADSC. ( C ) Immunofluorescence labeled anti-mouse primary antibodies were used to determine the protein expression of Sox2, Oct4, SSEA1, and Nanog markers to know the level/intensity of expression in KL -/- group and wild-type ADSC. Nuclei were counter stained with DAPI (blue). ( D ) Senescence β-Galactosidase staining was used to differentiate between senescent and healthy cells in KL -/- and wild-type-derived ADSC. Mean ± SEM, statistically significant values are: * p = .05; ** p = .01; *** p = .001, scale bar: 100 μm.

Techniques Used: Expressing, Derivative Assay, Immunofluorescence, Labeling, Staining

38) Product Images from "Patient-specific induced pluripotent stem cell derived models of LEOPARD syndrome"

Article Title: Patient-specific induced pluripotent stem cell derived models of LEOPARD syndrome

Journal: Nature

doi: 10.1038/nature09005

Gene expression profile in LS-iPSC is similar to HESC a, Quantitative real-time PCR assay for the expression of endogenous h OCT4 , h NANOG and h SOX2 in iPSC and parental fibroblasts (Fib). PCR reactions were normalized against β-ACTIN and plotted relative to expression levels in HES2. Error bars indicate ± s.d. of triplicates. b, Bisulfite sequencing analyses of the OCT4 and NANOG promoters. The cell line and the percentage of methylation is indicated to the left of each cluster. c, Heat map showing hierarchical clustering of 3657 genes with at least two-fold expression change between the average of the three fibroblast cell lines versus all the iPSC lines/HES samples. Expression levels are represented by color; red indicates lower and yellow higher expression.
Figure Legend Snippet: Gene expression profile in LS-iPSC is similar to HESC a, Quantitative real-time PCR assay for the expression of endogenous h OCT4 , h NANOG and h SOX2 in iPSC and parental fibroblasts (Fib). PCR reactions were normalized against β-ACTIN and plotted relative to expression levels in HES2. Error bars indicate ± s.d. of triplicates. b, Bisulfite sequencing analyses of the OCT4 and NANOG promoters. The cell line and the percentage of methylation is indicated to the left of each cluster. c, Heat map showing hierarchical clustering of 3657 genes with at least two-fold expression change between the average of the three fibroblast cell lines versus all the iPSC lines/HES samples. Expression levels are represented by color; red indicates lower and yellow higher expression.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Methylation Sequencing, Methylation

39) Product Images from "Role of IGF1R+ MSCs in modulating neuroplasticity via CXCR4 cross-interaction"

Article Title: Role of IGF1R+ MSCs in modulating neuroplasticity via CXCR4 cross-interaction

Journal: Scientific Reports

doi: 10.1038/srep32595

Isolation and clonal expansion of IGF1R-expressed hDSCs. ( a ) In the representative figure of cytokines array (square P = positive control, square N = negative control), five different cytokines such as EGF (square #1), Angiogenin (#2), MIP-1δ (#3), RANTES (#4), and PDGF-BB (arrow) were significantly up-regulated in hUCS compared to FCS. The level of IGF1 (arrow head) was almost the same between hUCS and FCS. ( b ) In ELISA, the concentration of serum PDGF-BB showed significant increase in the hUCS compared to that of FCS, but not found in serum IGF1. ( c ) Fibroblast-like cell morphology (arrow) was found in hDSCs from dental pulp explant (arrow head, left panel). Surface marker of IGF1R and other MSCs’ markers was not only found in hDSCs, but in human fibroblast (positive control), ADMSCs, UMSCs and BMSCs. In flowcytometric analysis, hDSCs carried the surface markers of MSCs and pluripotent markers of Oct-4, Sox-2, Nanog and SSEA-4. ( d ) hDSCs were cultured in 2% hUCS or 10% FCS. In representative graph of the expansion potential profile, U-IGF1R + hDSCs revealed more fast growth rate than that of U-IGF1R ‒ hDSCs and U-IGF1R + hDSCs+PPP. hUCS-cultured hDSCs revealed rapider division than FCS-cultured hDSCs. In addition, higher cellular proliferation rate was abolished in both hUCS-cultured hDSCs (U-hDSCs) and FCS-cultured hDSCs (F-hDSCs) infected with LV-IGF1R-shRNA. ( e ) Western blot exhibited significant upregulation of Bmi1 and cdk4, as well as downregulation of p21 in U-IGF1R + hDSCs compared to that of U-IGF1R ‒ hDSCs, but no difference in cyclinD1, JunB and PTEN. ( f ) In BrdU ELISA, U-IGF1R + hDSCs showed significant increaser BrdU uptake compared to that of U-IGF1R ‒ hDSCs and U-IGF1R + hDSCs+PPP. More, hUCS-cultured hDSCs showed significantly increased BrdU incorporation compared to that of FCS-cultured hDSCs (SDF-1α as positive control), but not in LV-IGF1R-sh-hUCS-hDSCs and LV-IGF1R-sh-FCS-hDSCs. ( g ) Co-expression of IGF1R with self-renewal markers Oct-4, Sox-2, Nanog, and SSEA4 was found on hDSCs by flowcytometry. Furthermore, qRT-PCR analysis of relative expression of Oct-4, Sox-2, Nanog and SSEA4 significantly increased in p5 IGF1R + hDSCs compared to that in IGF1R – hDSCs. Data are expressed as mean ± SEM. * P
Figure Legend Snippet: Isolation and clonal expansion of IGF1R-expressed hDSCs. ( a ) In the representative figure of cytokines array (square P = positive control, square N = negative control), five different cytokines such as EGF (square #1), Angiogenin (#2), MIP-1δ (#3), RANTES (#4), and PDGF-BB (arrow) were significantly up-regulated in hUCS compared to FCS. The level of IGF1 (arrow head) was almost the same between hUCS and FCS. ( b ) In ELISA, the concentration of serum PDGF-BB showed significant increase in the hUCS compared to that of FCS, but not found in serum IGF1. ( c ) Fibroblast-like cell morphology (arrow) was found in hDSCs from dental pulp explant (arrow head, left panel). Surface marker of IGF1R and other MSCs’ markers was not only found in hDSCs, but in human fibroblast (positive control), ADMSCs, UMSCs and BMSCs. In flowcytometric analysis, hDSCs carried the surface markers of MSCs and pluripotent markers of Oct-4, Sox-2, Nanog and SSEA-4. ( d ) hDSCs were cultured in 2% hUCS or 10% FCS. In representative graph of the expansion potential profile, U-IGF1R + hDSCs revealed more fast growth rate than that of U-IGF1R ‒ hDSCs and U-IGF1R + hDSCs+PPP. hUCS-cultured hDSCs revealed rapider division than FCS-cultured hDSCs. In addition, higher cellular proliferation rate was abolished in both hUCS-cultured hDSCs (U-hDSCs) and FCS-cultured hDSCs (F-hDSCs) infected with LV-IGF1R-shRNA. ( e ) Western blot exhibited significant upregulation of Bmi1 and cdk4, as well as downregulation of p21 in U-IGF1R + hDSCs compared to that of U-IGF1R ‒ hDSCs, but no difference in cyclinD1, JunB and PTEN. ( f ) In BrdU ELISA, U-IGF1R + hDSCs showed significant increaser BrdU uptake compared to that of U-IGF1R ‒ hDSCs and U-IGF1R + hDSCs+PPP. More, hUCS-cultured hDSCs showed significantly increased BrdU incorporation compared to that of FCS-cultured hDSCs (SDF-1α as positive control), but not in LV-IGF1R-sh-hUCS-hDSCs and LV-IGF1R-sh-FCS-hDSCs. ( g ) Co-expression of IGF1R with self-renewal markers Oct-4, Sox-2, Nanog, and SSEA4 was found on hDSCs by flowcytometry. Furthermore, qRT-PCR analysis of relative expression of Oct-4, Sox-2, Nanog and SSEA4 significantly increased in p5 IGF1R + hDSCs compared to that in IGF1R – hDSCs. Data are expressed as mean ± SEM. * P

Techniques Used: Isolation, Positive Control, Negative Control, Enzyme-linked Immunosorbent Assay, Concentration Assay, Marker, Cell Culture, Infection, shRNA, Western Blot, BrdU Incorporation Assay, Expressing, Quantitative RT-PCR

40) Product Images from "Ionizing radiation induces ATM dependent checkpoint signaling and G2 but not G1 cell cycle arrest in pluripotent human embryonic stem cells"

Article Title: Ionizing radiation induces ATM dependent checkpoint signaling and G2 but not G1 cell cycle arrest in pluripotent human embryonic stem cells

Journal: Stem Cells (Dayton, Ohio)

doi: 10.1002/stem.123

Pluripotency and radiosensitivity of human ES cells (A): Human ES cells were irradiated, or left untreated, and allowed to recover for the indicated time periods prior to collection. Total RNA was isolated and the expression of pluripotency markers CD9, Nanog and POU5F1 analyzed by TaqMan® human stem cell pluripotency array. The mRNA fold changes were calculated using the −ΔΔCt method and normalized using β-actin as endogenous control. The results in non-irradiated cells were normalized to one, and the value in irradiated cells was calculated in respect to this. (B): Western blot analysis of Nanog and POU5F1 protein levels following the irradiation of human ES cells. α-tubulin served as the loading control. Confocal microscopy for (C): POU5F1, (D): Nanog and (E): SSEA-4 in human ES cells at indicated time points after irradiation. (F): Western blot analysis for cleaved caspase-3 following irradiation. α-tubulin served as the loading control. Bar = 100 μm (C), 50 μm (D,E)
Figure Legend Snippet: Pluripotency and radiosensitivity of human ES cells (A): Human ES cells were irradiated, or left untreated, and allowed to recover for the indicated time periods prior to collection. Total RNA was isolated and the expression of pluripotency markers CD9, Nanog and POU5F1 analyzed by TaqMan® human stem cell pluripotency array. The mRNA fold changes were calculated using the −ΔΔCt method and normalized using β-actin as endogenous control. The results in non-irradiated cells were normalized to one, and the value in irradiated cells was calculated in respect to this. (B): Western blot analysis of Nanog and POU5F1 protein levels following the irradiation of human ES cells. α-tubulin served as the loading control. Confocal microscopy for (C): POU5F1, (D): Nanog and (E): SSEA-4 in human ES cells at indicated time points after irradiation. (F): Western blot analysis for cleaved caspase-3 following irradiation. α-tubulin served as the loading control. Bar = 100 μm (C), 50 μm (D,E)

Techniques Used: Irradiation, Isolation, Expressing, Western Blot, Confocal Microscopy

Related Articles

Clone Assay:

Article Title: Efficient Generation of Human iPS Cells by a Synthetic Self-Replicative RNA
Article Snippet: .. Antibodies used in this research are as follows; anti-OCT4 (sc-9081), anti-KLF4 (sc-20691), anti-GLIS1 (sc-67584), anti-c-MYC (sc-42), TRA-1-60 (sc-21705), SSEA1 (sc-21702), SSEA4 (sc-21704), anti-mouse (sc-2005), anti-rabbit (sc-2004) and anti-goat (sc-2020) from Santa Cruz; anti-SOX2 (AF2018) and anti-NANOG (AF1997) from R & D Systems; TRA-1-81 (09-0011) from Stemgent; AE1/AE3 (RB-9010P0), Desmin (MS-376-S0), AFP (RB-365) and GFAP (RB-087) from Labvision; NF-1 (NB-300-155) from Novus Biological; anti-alpha-Actinin (A7811) from Sigma; anti-Cardiac Troponin T (MS-295-P0) from Thermo Scientific; Alexa Flour 488 anti-mouse (A11001) for cardiomyocytes analysis, Alexa Fluor 488 anti-mouse , Alexa Fluor 488 anti-rabbit ( ) and Alexa Fluor 488 anti-goat ( ) for immunostaining of iPS clones from Life Technologies. .. Total RNAs were isolated with RNeasy mini kit (Qiagen), and cDNA library of each cells were synthesized and analyzed as described before ( ).

Immunocytochemistry:

Article Title: The Disease-Specific Phenotype in Cardiomyocytes Derived from Induced Pluripotent Stem Cells of Two Long QT Syndrome Type 3 Patients
Article Snippet: .. Immunocytochemistry Undifferentiated ES and iPS cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.1% Triton X-100 and blocked with 5% FBS and then stained overnight at 4°C with primary antibodies specific for OCT4 (Santa Cruz Biotechnology, Heidelberg, Germany, Cat. No. sc-5279, 1:100), SOX2 (Stemgent, Cambridge, MA, USA, Cat. No. 09-0020, 1:100), Nanog (R & D Systems, Wiesbaden, Germany, Cat. No. AF1997, 1:100), TRA-1-60 (BD Biosciences, Heidelberg, Germany, Cat. No. 560121, 1:100), TRA-1-80 (Santa Cruz, Cat. No. Sc-21706, 1:200) and SSEA4 (Santa Cruz, Cat. No. Sc-21704, 1:800). .. Samples were visualized after staining with AlexaFluor 488- or AlexaFluor 555-conjugated secondary antibodies (Life Technologies) for 1 hour at room temperature.

Immunostaining:

Article Title: Efficient Generation of Human iPS Cells by a Synthetic Self-Replicative RNA
Article Snippet: .. Antibodies used in this research are as follows; anti-OCT4 (sc-9081), anti-KLF4 (sc-20691), anti-GLIS1 (sc-67584), anti-c-MYC (sc-42), TRA-1-60 (sc-21705), SSEA1 (sc-21702), SSEA4 (sc-21704), anti-mouse (sc-2005), anti-rabbit (sc-2004) and anti-goat (sc-2020) from Santa Cruz; anti-SOX2 (AF2018) and anti-NANOG (AF1997) from R & D Systems; TRA-1-81 (09-0011) from Stemgent; AE1/AE3 (RB-9010P0), Desmin (MS-376-S0), AFP (RB-365) and GFAP (RB-087) from Labvision; NF-1 (NB-300-155) from Novus Biological; anti-alpha-Actinin (A7811) from Sigma; anti-Cardiac Troponin T (MS-295-P0) from Thermo Scientific; Alexa Flour 488 anti-mouse (A11001) for cardiomyocytes analysis, Alexa Fluor 488 anti-mouse , Alexa Fluor 488 anti-rabbit ( ) and Alexa Fluor 488 anti-goat ( ) for immunostaining of iPS clones from Life Technologies. .. Total RNAs were isolated with RNeasy mini kit (Qiagen), and cDNA library of each cells were synthesized and analyzed as described before ( ).

Concentration Assay:

Article Title: The role of Nanog expression in tamoxifen-resistant breast cancer cells
Article Snippet: .. Cells were labeled with human Nanog phycoerythrin polyclonal antibody goat IgG, human Oct4 MAb mouse IgG and human Sox2 monoclonal antibody mouse IgG (R & D System), diluted in permeablization solution at a final concentration according to the manufacturer’s recommendation and incubated for 30 minutes at 2°C–8°C. .. Cells were washed three times by centrifugation at 400× g for 5 minutes and resuspended in ice cold PBS.

Incubation:

Article Title: The role of Nanog expression in tamoxifen-resistant breast cancer cells
Article Snippet: .. Cells were labeled with human Nanog phycoerythrin polyclonal antibody goat IgG, human Oct4 MAb mouse IgG and human Sox2 monoclonal antibody mouse IgG (R & D System), diluted in permeablization solution at a final concentration according to the manufacturer’s recommendation and incubated for 30 minutes at 2°C–8°C. .. Cells were washed three times by centrifugation at 400× g for 5 minutes and resuspended in ice cold PBS.

Labeling:

Article Title: The role of Nanog expression in tamoxifen-resistant breast cancer cells
Article Snippet: .. Cells were labeled with human Nanog phycoerythrin polyclonal antibody goat IgG, human Oct4 MAb mouse IgG and human Sox2 monoclonal antibody mouse IgG (R & D System), diluted in permeablization solution at a final concentration according to the manufacturer’s recommendation and incubated for 30 minutes at 2°C–8°C. .. Cells were washed three times by centrifugation at 400× g for 5 minutes and resuspended in ice cold PBS.

Mass Spectrometry:

Article Title: Efficient Generation of Human iPS Cells by a Synthetic Self-Replicative RNA
Article Snippet: .. Antibodies used in this research are as follows; anti-OCT4 (sc-9081), anti-KLF4 (sc-20691), anti-GLIS1 (sc-67584), anti-c-MYC (sc-42), TRA-1-60 (sc-21705), SSEA1 (sc-21702), SSEA4 (sc-21704), anti-mouse (sc-2005), anti-rabbit (sc-2004) and anti-goat (sc-2020) from Santa Cruz; anti-SOX2 (AF2018) and anti-NANOG (AF1997) from R & D Systems; TRA-1-81 (09-0011) from Stemgent; AE1/AE3 (RB-9010P0), Desmin (MS-376-S0), AFP (RB-365) and GFAP (RB-087) from Labvision; NF-1 (NB-300-155) from Novus Biological; anti-alpha-Actinin (A7811) from Sigma; anti-Cardiac Troponin T (MS-295-P0) from Thermo Scientific; Alexa Flour 488 anti-mouse (A11001) for cardiomyocytes analysis, Alexa Fluor 488 anti-mouse , Alexa Fluor 488 anti-rabbit ( ) and Alexa Fluor 488 anti-goat ( ) for immunostaining of iPS clones from Life Technologies. .. Total RNAs were isolated with RNeasy mini kit (Qiagen), and cDNA library of each cells were synthesized and analyzed as described before ( ).

Staining:

Article Title: The Disease-Specific Phenotype in Cardiomyocytes Derived from Induced Pluripotent Stem Cells of Two Long QT Syndrome Type 3 Patients
Article Snippet: .. Immunocytochemistry Undifferentiated ES and iPS cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.1% Triton X-100 and blocked with 5% FBS and then stained overnight at 4°C with primary antibodies specific for OCT4 (Santa Cruz Biotechnology, Heidelberg, Germany, Cat. No. sc-5279, 1:100), SOX2 (Stemgent, Cambridge, MA, USA, Cat. No. 09-0020, 1:100), Nanog (R & D Systems, Wiesbaden, Germany, Cat. No. AF1997, 1:100), TRA-1-60 (BD Biosciences, Heidelberg, Germany, Cat. No. 560121, 1:100), TRA-1-80 (Santa Cruz, Cat. No. Sc-21706, 1:200) and SSEA4 (Santa Cruz, Cat. No. Sc-21704, 1:800). .. Samples were visualized after staining with AlexaFluor 488- or AlexaFluor 555-conjugated secondary antibodies (Life Technologies) for 1 hour at room temperature.

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  • 90
    R&D Systems antibodies against nanog
    5-Lox inhibition-induced apoptosis in prostate cancer stem cells involves inhibition of PKCε, but not Akt or ERK PCS cells (3 × 10 5 per plate) were plated in complete growth medium supplemented with 10% serum in 60 mm diameter plates and allowed to grow for 48 hours. On the day of experiment, the spent culture medium was replaced with 2 ml fresh RPMI medium and the cells were treated either with MK591 (10–30 μM) or ibuprofen (30 μM) for 24 hours. Control cells were treated with vehicle only (0.2% DMSO). At the end of incubation, cells were harvested and proteins were separated in 12% SDS-PAGE. In ( A ), protein levels of PKC-epsilon and phosphorylations of Akt-pSer 473 , ERK-pThr 202/Y204 , <t>Nanog-pThr</t> 200 and Stat3-pSer 727 , and in ( B ) protein levels of <t>survivin,</t> cyclin D1, CDK4 and Bcl-xl were detected by Western blot. Data show a representative of three independent experiments with similar results.
    Antibodies Against Nanog, supplied by R&D Systems, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies against nanog/product/R&D Systems
    Average 90 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    antibodies against nanog - by Bioz Stars, 2020-07
    90/100 stars
      Buy from Supplier

    90
    R&D Systems goat anti nanog
    DNA replication fork directions in the <t>Nanog</t> segment are different for H14 hESCs compared to those for MECs and R-NSCs. The DNA segment containing the NANOG gene is much smaller (110 kb) than the one containing POU5F1 (350 kb). In general, shorter molecules
    Goat Anti Nanog, supplied by R&D Systems, used in various techniques. Bioz Stars score: 90/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti nanog/product/R&D Systems
    Average 90 stars, based on 26 article reviews
    Price from $9.99 to $1999.99
    goat anti nanog - by Bioz Stars, 2020-07
    90/100 stars
      Buy from Supplier

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    5-Lox inhibition-induced apoptosis in prostate cancer stem cells involves inhibition of PKCε, but not Akt or ERK PCS cells (3 × 10 5 per plate) were plated in complete growth medium supplemented with 10% serum in 60 mm diameter plates and allowed to grow for 48 hours. On the day of experiment, the spent culture medium was replaced with 2 ml fresh RPMI medium and the cells were treated either with MK591 (10–30 μM) or ibuprofen (30 μM) for 24 hours. Control cells were treated with vehicle only (0.2% DMSO). At the end of incubation, cells were harvested and proteins were separated in 12% SDS-PAGE. In ( A ), protein levels of PKC-epsilon and phosphorylations of Akt-pSer 473 , ERK-pThr 202/Y204 , Nanog-pThr 200 and Stat3-pSer 727 , and in ( B ) protein levels of survivin, cyclin D1, CDK4 and Bcl-xl were detected by Western blot. Data show a representative of three independent experiments with similar results.

    Journal: Oncotarget

    Article Title: Inhibition of 5-lipoxygenase downregulates stemness and kills prostate cancer stem cells by triggering apoptosis via activation of c-Jun N-terminal kinase

    doi: 10.18632/oncotarget.13422

    Figure Lengend Snippet: 5-Lox inhibition-induced apoptosis in prostate cancer stem cells involves inhibition of PKCε, but not Akt or ERK PCS cells (3 × 10 5 per plate) were plated in complete growth medium supplemented with 10% serum in 60 mm diameter plates and allowed to grow for 48 hours. On the day of experiment, the spent culture medium was replaced with 2 ml fresh RPMI medium and the cells were treated either with MK591 (10–30 μM) or ibuprofen (30 μM) for 24 hours. Control cells were treated with vehicle only (0.2% DMSO). At the end of incubation, cells were harvested and proteins were separated in 12% SDS-PAGE. In ( A ), protein levels of PKC-epsilon and phosphorylations of Akt-pSer 473 , ERK-pThr 202/Y204 , Nanog-pThr 200 and Stat3-pSer 727 , and in ( B ) protein levels of survivin, cyclin D1, CDK4 and Bcl-xl were detected by Western blot. Data show a representative of three independent experiments with similar results.

    Article Snippet: Antibodies against Nanog, c-Myc and survivin were purchased from R and D Systems (Minneapolis, MN, USA), and antibodies against, Sox2, CD44, CD133, ABCG-2, cyclin D1, CDK4, and Bcl-xl were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Inhibition, Incubation, SDS Page, Western Blot

    Culture of hESCs on feeder cell-derived ECM. A , phase-contrast images of ECMs isolated from CD1 MEFs at P4 and P9 and from ihPSFs by alkali/detergent extraction. Scale bars , 100 μm. B , phase-contrast images showing morphology of HUES1 cells cultured for five consecutive passages on ECM derived from CD1 P4 and P9, ihPSF, and hPSF. Scale bars , 100 μm. C , HUES1 cells cultured on CD1 P4 and P9 ECM stained positively for pluripotency-associated markers Nanog and Oct4 and surface marker TRA-1-81 after multiple (up to four) passages. Cell nuclei were stained with DAPI ( blue ; insets ). Scale bars , 100 μm. D , HUES1 cells cultured on ihPSF-derived ECM stained positively for pluripotency-associated markers Nanog, Oct4, and Sox2 after multiple (up to four) passages, whereas HUES1 cells cultured on hPSF-derived ECM lost the expression of pluripotency-associated markers Nanog, Oct4, and Sox2. Cell nuclei were stained with DAPI ( blue ; insets ). Scale bars , 50 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance *

    doi: 10.1074/jbc.M113.463372

    Figure Lengend Snippet: Culture of hESCs on feeder cell-derived ECM. A , phase-contrast images of ECMs isolated from CD1 MEFs at P4 and P9 and from ihPSFs by alkali/detergent extraction. Scale bars , 100 μm. B , phase-contrast images showing morphology of HUES1 cells cultured for five consecutive passages on ECM derived from CD1 P4 and P9, ihPSF, and hPSF. Scale bars , 100 μm. C , HUES1 cells cultured on CD1 P4 and P9 ECM stained positively for pluripotency-associated markers Nanog and Oct4 and surface marker TRA-1-81 after multiple (up to four) passages. Cell nuclei were stained with DAPI ( blue ; insets ). Scale bars , 100 μm. D , HUES1 cells cultured on ihPSF-derived ECM stained positively for pluripotency-associated markers Nanog, Oct4, and Sox2 after multiple (up to four) passages, whereas HUES1 cells cultured on hPSF-derived ECM lost the expression of pluripotency-associated markers Nanog, Oct4, and Sox2. Cell nuclei were stained with DAPI ( blue ; insets ). Scale bars , 50 μm.

    Article Snippet: Primary antibodies and concentrations used were anti-Nanog (2 μg/ml; R & D Systems); anti-Oct4 (2.5 μg/ml; BD Biosciences); anti-TRA-1-81 (1 μg/ml; Abcam); anti-Sox2, anti-GATA4, anti-α-smooth muscle actin, anti-βIII-tubulin, anti-Sox17, anti-brachyury, anti-vimentin, and anti-α-fetoprotein (all 5 μg/ml; all R & D Systems); anti-fibronectin (1.25 μg/ml; Sigma-Aldrich); anti-tenascin C (10 μg/ml; Millipore); anti-collagen VI (10 μg/ml; Abcam); anti-human collagen XII and anti-mouse collagen XII (1:3000 and 1:1000, respectively; kind gift of M. Koch, University of Cologne); pan-specific anti-laminin (1:100; kind gift of D. R. Garrod, University of Manchester); anti-fibrillin-1 N-19 (N-terminal region) and PRO (proline-rich region) (1:50 and 1:200, respectively; kind gift of C. M. Kielty); and anti-fibulin-2 (2.5 μg/ml; kind gift of T. Sasaki).

    Techniques: Derivative Assay, Isolation, Cell Culture, Staining, Marker, Expressing

    Culture of hESCs on single ECM substrates or on substrates in combination with 5 μg/ml fibronectin. A , HUES1 cells were successfully cultured over three passages on fibrillin-1 coated at two different concentrations (10 and 20 μg/ml), similar to feeder-free culture on fibronectin (50 μg/ml). B , fibulin-2 and perlecan supported HUES1 cell culture only in combination with 5 μg/ml fibronectin; on these substrates, hESCs were maintained over three passages. C, HUES1 and HUES7 cells were successfully cultured over five passages on fibrillin-1 (10 μg/ml) and were positive for pluripotency-associated markers Nanog and Oct4 and surface marker TRA-1-81. Cell nuclei were stained with DAPI ( blue ; insets ). Scale bars , 100 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance *

    doi: 10.1074/jbc.M113.463372

    Figure Lengend Snippet: Culture of hESCs on single ECM substrates or on substrates in combination with 5 μg/ml fibronectin. A , HUES1 cells were successfully cultured over three passages on fibrillin-1 coated at two different concentrations (10 and 20 μg/ml), similar to feeder-free culture on fibronectin (50 μg/ml). B , fibulin-2 and perlecan supported HUES1 cell culture only in combination with 5 μg/ml fibronectin; on these substrates, hESCs were maintained over three passages. C, HUES1 and HUES7 cells were successfully cultured over five passages on fibrillin-1 (10 μg/ml) and were positive for pluripotency-associated markers Nanog and Oct4 and surface marker TRA-1-81. Cell nuclei were stained with DAPI ( blue ; insets ). Scale bars , 100 μm.

    Article Snippet: Primary antibodies and concentrations used were anti-Nanog (2 μg/ml; R & D Systems); anti-Oct4 (2.5 μg/ml; BD Biosciences); anti-TRA-1-81 (1 μg/ml; Abcam); anti-Sox2, anti-GATA4, anti-α-smooth muscle actin, anti-βIII-tubulin, anti-Sox17, anti-brachyury, anti-vimentin, and anti-α-fetoprotein (all 5 μg/ml; all R & D Systems); anti-fibronectin (1.25 μg/ml; Sigma-Aldrich); anti-tenascin C (10 μg/ml; Millipore); anti-collagen VI (10 μg/ml; Abcam); anti-human collagen XII and anti-mouse collagen XII (1:3000 and 1:1000, respectively; kind gift of M. Koch, University of Cologne); pan-specific anti-laminin (1:100; kind gift of D. R. Garrod, University of Manchester); anti-fibrillin-1 N-19 (N-terminal region) and PRO (proline-rich region) (1:50 and 1:200, respectively; kind gift of C. M. Kielty); and anti-fibulin-2 (2.5 μg/ml; kind gift of T. Sasaki).

    Techniques: Cell Culture, Marker, Staining

    DNA replication fork directions in the Nanog segment are different for H14 hESCs compared to those for MECs and R-NSCs. The DNA segment containing the NANOG gene is much smaller (110 kb) than the one containing POU5F1 (350 kb). In general, shorter molecules

    Journal: Molecular and Cellular Biology

    Article Title: Single-Molecule Analysis Reveals Changes in the DNA Replication Program for the POU5F1 Locus upon Human Embryonic Stem Cell Differentiation ▿ Locus upon Human Embryonic Stem Cell Differentiation ▿ #

    doi: 10.1128/MCB.00380-10

    Figure Lengend Snippet: DNA replication fork directions in the Nanog segment are different for H14 hESCs compared to those for MECs and R-NSCs. The DNA segment containing the NANOG gene is much smaller (110 kb) than the one containing POU5F1 (350 kb). In general, shorter molecules

    Article Snippet: Cells were incubated overnight at 4°C with primary antibodies diluted in PBS-1% BSA (mouse anti-Oct3/4, 1:200 [Santa Cruz Biotechnology, Santa Cruz, CA]; goat anti-Nanog, 1:50 [R & D Systems, Minneapolis, MN]).

    Techniques:

    Immunocytochemistry of SOX2, OCT4, NANOG, EMD and SSEA1 in hESCs with and without BANF1 knockdown. CHB-4 hESCs were transduced with either Scrambled or human Banf1 shRNA lentiviral vectors, selected with puromycin to eliminate uninfected cells, and seeded

    Journal: Journal of Cell Science

    Article Title: Banf1 is required to maintain the self-renewal of both mouse and human embryonic stem cells

    doi: 10.1242/jcs.083238

    Figure Lengend Snippet: Immunocytochemistry of SOX2, OCT4, NANOG, EMD and SSEA1 in hESCs with and without BANF1 knockdown. CHB-4 hESCs were transduced with either Scrambled or human Banf1 shRNA lentiviral vectors, selected with puromycin to eliminate uninfected cells, and seeded

    Article Snippet: Other primary antibodies used are against: OCT4 (1:500, sc-8628, Santa Cruz Biotechnology), SOX2 (1:1000, ab-75179, Abcam, Cambridge, MA), NANOG (1:500, AF2729, R & D Systems, Minneapolis, MN), HDAC1 (1:5000, ab-7028, Abcam), HDAC2 (1:5000, ab-7029, Abcam).

    Techniques: Immunocytochemistry, Transduction, shRNA

    Knockdown of Banf1 in mESCs causes changes in cell morphology, elevation of gene markers of differentiation and reduction in the protein levels of Oct4, Sox2 and Nanog. ( A ) Banf1 levels were examined by western blot analysis using nuclear proteins isolated

    Journal: Journal of Cell Science

    Article Title: Banf1 is required to maintain the self-renewal of both mouse and human embryonic stem cells

    doi: 10.1242/jcs.083238

    Figure Lengend Snippet: Knockdown of Banf1 in mESCs causes changes in cell morphology, elevation of gene markers of differentiation and reduction in the protein levels of Oct4, Sox2 and Nanog. ( A ) Banf1 levels were examined by western blot analysis using nuclear proteins isolated

    Article Snippet: Other primary antibodies used are against: OCT4 (1:500, sc-8628, Santa Cruz Biotechnology), SOX2 (1:1000, ab-75179, Abcam, Cambridge, MA), NANOG (1:500, AF2729, R & D Systems, Minneapolis, MN), HDAC1 (1:5000, ab-7028, Abcam), HDAC2 (1:5000, ab-7029, Abcam).

    Techniques: Western Blot, Isolation

    Immunocytochemistry of Sox2, Oct4, Nanog, SSEA1 and Emd in mESCs with and without Banf1 knockdown. D3 mESCs were infected, selected with puromycin and seeded into a 12-well plate for immunocytochemistry as described in the Materials and Methods. Photomicrographs

    Journal: Journal of Cell Science

    Article Title: Banf1 is required to maintain the self-renewal of both mouse and human embryonic stem cells

    doi: 10.1242/jcs.083238

    Figure Lengend Snippet: Immunocytochemistry of Sox2, Oct4, Nanog, SSEA1 and Emd in mESCs with and without Banf1 knockdown. D3 mESCs were infected, selected with puromycin and seeded into a 12-well plate for immunocytochemistry as described in the Materials and Methods. Photomicrographs

    Article Snippet: Other primary antibodies used are against: OCT4 (1:500, sc-8628, Santa Cruz Biotechnology), SOX2 (1:1000, ab-75179, Abcam, Cambridge, MA), NANOG (1:500, AF2729, R & D Systems, Minneapolis, MN), HDAC1 (1:5000, ab-7028, Abcam), HDAC2 (1:5000, ab-7029, Abcam).

    Techniques: Immunocytochemistry, Infection