cd31  (Thermo Fisher)


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    Structured Review

    Thermo Fisher cd31
    Comparison of kappa values among pathologists for lymphovascular invasion (LVI) detection in colorectal cancers. While the average of LVI detection rate for each pathologist was 43% with hematoxylin and eosin (H E) only, 10% with <t>CD31,</t> 29% with D2-40, and 16% with ERG, the consensus reached 80% of LVI detection after a joint discussion about ERG patterns with LVI. a Interpreted by ERG
    Cd31, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 39 article reviews
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    cd31 - by Bioz Stars, 2022-10
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    Images

    1) Product Images from "ERG Immunohistochemistry as an Endothelial Marker for Assessing Lymphovascular Invasion"

    Article Title: ERG Immunohistochemistry as an Endothelial Marker for Assessing Lymphovascular Invasion

    Journal: Korean Journal of Pathology

    doi: 10.4132/KoreanJPathol.2013.47.4.355

    Comparison of kappa values among pathologists for lymphovascular invasion (LVI) detection in colorectal cancers. While the average of LVI detection rate for each pathologist was 43% with hematoxylin and eosin (H E) only, 10% with CD31, 29% with D2-40, and 16% with ERG, the consensus reached 80% of LVI detection after a joint discussion about ERG patterns with LVI. a Interpreted by ERG
    Figure Legend Snippet: Comparison of kappa values among pathologists for lymphovascular invasion (LVI) detection in colorectal cancers. While the average of LVI detection rate for each pathologist was 43% with hematoxylin and eosin (H E) only, 10% with CD31, 29% with D2-40, and 16% with ERG, the consensus reached 80% of LVI detection after a joint discussion about ERG patterns with LVI. a Interpreted by ERG

    Techniques Used:

    Comparison of ERG, CD31, and D2-40 endothelial markers. (A) ERG, panendothelial marker showing nuclear immunoreactivity in artery, vein, and lymphatics. (B) ERG immunostaining specific for endothelial cells without cross-reactivity. (C) D31 immunostaining showing cross-reactivity in inflammatory cells. (D) D2-40 immunostaining showing cross-reactivity in fibroblasts.
    Figure Legend Snippet: Comparison of ERG, CD31, and D2-40 endothelial markers. (A) ERG, panendothelial marker showing nuclear immunoreactivity in artery, vein, and lymphatics. (B) ERG immunostaining specific for endothelial cells without cross-reactivity. (C) D31 immunostaining showing cross-reactivity in inflammatory cells. (D) D2-40 immunostaining showing cross-reactivity in fibroblasts.

    Techniques Used: Marker, Immunostaining

    2) Product Images from "Exosomes from microRNA‐126 overexpressing mesenchymal stem cells promote angiogenesis by targeting the PIK3R2‐mediated PI3K/Akt signalling pathway, et al. Exosomes from microRNA‐126 overexpressing mesenchymal stem cells promote angiogenesis by targeting the PIK3R2‐mediated PI3K/Akt signalling pathway"

    Article Title: Exosomes from microRNA‐126 overexpressing mesenchymal stem cells promote angiogenesis by targeting the PIK3R2‐mediated PI3K/Akt signalling pathway, et al. Exosomes from microRNA‐126 overexpressing mesenchymal stem cells promote angiogenesis by targeting the PIK3R2‐mediated PI3K/Akt signalling pathway

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.16192

    Exo‐miR‐126 enhanced angiogenesis in the wound sites of mice. (A) CD31 immunofluorescence staining of wound sections from mice receiving different treatments at day 14 post‐wounding (scale bar: 50 μm). (B) Representative images of CD34 staining of wound sections from mice receiving different treatments at day 14 post‐wounding (scale bar: 50 μm). (C) Quantitative analysis of the CD31‐positive area in (A) (n = 8). (D) Quantitative analysis of the CD34‐positive area in (B) (n = 8). * P
    Figure Legend Snippet: Exo‐miR‐126 enhanced angiogenesis in the wound sites of mice. (A) CD31 immunofluorescence staining of wound sections from mice receiving different treatments at day 14 post‐wounding (scale bar: 50 μm). (B) Representative images of CD34 staining of wound sections from mice receiving different treatments at day 14 post‐wounding (scale bar: 50 μm). (C) Quantitative analysis of the CD31‐positive area in (A) (n = 8). (D) Quantitative analysis of the CD34‐positive area in (B) (n = 8). * P

    Techniques Used: Mouse Assay, Immunofluorescence, Staining

    3) Product Images from "Distribution of Bone-Marrow-Derived Endothelial and Immune Cells in a Murine Colitis-Associated Colorectal Cancer Model"

    Article Title: Distribution of Bone-Marrow-Derived Endothelial and Immune Cells in a Murine Colitis-Associated Colorectal Cancer Model

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073666

    Bone marrow-derived endothelial cells contributed to colitis-associated colon cancer angiogenesis. (A) Blood vessels were stained with 647 WGA (blue), which was injected retrorbitally. Confocal microscopy analysis from living tissue specimens showed that the BM-derived cells (green) infiltrated the stroma, and a portion of cells were hemmed by a WGA-647-positive (blue) margin at the tumor endothelium. Double-positive cells were observed lining the vessels. (B) The frozen tumors sections were stained with an anti-CD31 antibody to detect BM-derived endothelial cells (green). GFP-positive cells (green) with CD31-positive margins were seen in the tumor endothelium. C (CMT93) and D (CT26) xenograft tumor tissues isolated from BM transplantation mice were stained with anti-CD31 (red) antibodies. GFP-positive cells were observed around the tumor vessels, and some lined the endothelium.
    Figure Legend Snippet: Bone marrow-derived endothelial cells contributed to colitis-associated colon cancer angiogenesis. (A) Blood vessels were stained with 647 WGA (blue), which was injected retrorbitally. Confocal microscopy analysis from living tissue specimens showed that the BM-derived cells (green) infiltrated the stroma, and a portion of cells were hemmed by a WGA-647-positive (blue) margin at the tumor endothelium. Double-positive cells were observed lining the vessels. (B) The frozen tumors sections were stained with an anti-CD31 antibody to detect BM-derived endothelial cells (green). GFP-positive cells (green) with CD31-positive margins were seen in the tumor endothelium. C (CMT93) and D (CT26) xenograft tumor tissues isolated from BM transplantation mice were stained with anti-CD31 (red) antibodies. GFP-positive cells were observed around the tumor vessels, and some lined the endothelium.

    Techniques Used: Derivative Assay, Staining, Whole Genome Amplification, Injection, Confocal Microscopy, Isolation, Transplantation Assay, Mouse Assay

    4) Product Images from "Establishment of a Human iPSC- and Nanofiber-Based Microphysiological Blood–Brain Barrier System"

    Article Title: Establishment of a Human iPSC- and Nanofiber-Based Microphysiological Blood–Brain Barrier System

    Journal: ACS applied materials & interfaces

    doi: 10.1021/acsami.8b03962

    Cell viability and phenotypic marker expression of hiPSC-ECs and hiPSC-Astro on nanofibrous PLGA meshes: (A) Live/Dead assay images (scale bar: 100 µm) and (B,C) typical IF images, showing the expression of vWF (red), CD31 (green) for hiPSC-EC and S100B (red), GFAP (green) for hiPSC-Astro (scale bar: 100 µm).
    Figure Legend Snippet: Cell viability and phenotypic marker expression of hiPSC-ECs and hiPSC-Astro on nanofibrous PLGA meshes: (A) Live/Dead assay images (scale bar: 100 µm) and (B,C) typical IF images, showing the expression of vWF (red), CD31 (green) for hiPSC-EC and S100B (red), GFAP (green) for hiPSC-Astro (scale bar: 100 µm).

    Techniques Used: Marker, Expressing, Live Dead Assay

    Generation and characterization of the in vitro BBB model: (A) schematic of a coculture of hiPSC-ECs and hiPSC-Astro on the PLGA mesh that was electrospun onto the 3D-printed holder and fit into the transwell frame. Human iPSC-ECs were cultured on the top side of the PLGA mesh and conditioned in EGM in the upper chamber, whereas hiPSC-Astro were cultured in the bottom side in astrocyte medium in the lower chamber; (B) IF staining of cocultured hiPSC-ECs and hiPSC-Astro after 7 day culture. In the upper panel, the BBB model was costained with CD31 for hiPSC-ECs and GFAP for hiPSC-Astro, whereas in the lower panel, S100B and vWF were stained for hiPSC-Astro and hiPSC-ECs, respectively. Volume-rendered side views of the bilayer cells were also presented (scale bar: 100 µm); (C) expression of the tight junction protein ZO-1 in the cocultured hiPSC-EC (scale bar: 100 µm for general view, and 20 µm for close view); (D) hiPSC-EC morphology on the electrospun PLGA mesh after 7 day coculture (scale bar: 50 µm); (E) TEER value of the empty mesh, without cells, in the transwell insert (white), BBB model with hiPSC-Astro alone (gray), and with coculture of hiPSC-ECs and hiPSC-Astro at different time points (black) ( n = 3–5, ** p
    Figure Legend Snippet: Generation and characterization of the in vitro BBB model: (A) schematic of a coculture of hiPSC-ECs and hiPSC-Astro on the PLGA mesh that was electrospun onto the 3D-printed holder and fit into the transwell frame. Human iPSC-ECs were cultured on the top side of the PLGA mesh and conditioned in EGM in the upper chamber, whereas hiPSC-Astro were cultured in the bottom side in astrocyte medium in the lower chamber; (B) IF staining of cocultured hiPSC-ECs and hiPSC-Astro after 7 day culture. In the upper panel, the BBB model was costained with CD31 for hiPSC-ECs and GFAP for hiPSC-Astro, whereas in the lower panel, S100B and vWF were stained for hiPSC-Astro and hiPSC-ECs, respectively. Volume-rendered side views of the bilayer cells were also presented (scale bar: 100 µm); (C) expression of the tight junction protein ZO-1 in the cocultured hiPSC-EC (scale bar: 100 µm for general view, and 20 µm for close view); (D) hiPSC-EC morphology on the electrospun PLGA mesh after 7 day coculture (scale bar: 50 µm); (E) TEER value of the empty mesh, without cells, in the transwell insert (white), BBB model with hiPSC-Astro alone (gray), and with coculture of hiPSC-ECs and hiPSC-Astro at different time points (black) ( n = 3–5, ** p

    Techniques Used: In Vitro, Cell Culture, Staining, Expressing

    5) Product Images from "Enhancement of Bone-Marrow-Derived Mesenchymal Stem Cell Angiogenic Capacity by NPWT for a Combinatorial Therapy to Promote Wound Healing with Large Defect"

    Article Title: Enhancement of Bone-Marrow-Derived Mesenchymal Stem Cell Angiogenic Capacity by NPWT for a Combinatorial Therapy to Promote Wound Healing with Large Defect

    Journal: BioMed Research International

    doi: 10.1155/2017/7920265

    BMSC + NPWT accelerate the formation of vascularized granulation tissue. (a) Immunohistochemical staining for CD31 and collagen IV, representing newly formed blood vessels. (b) Quantification of newly formed blood vessels. (c) Quantification of collagen IV expression. Data is given as the mean ± SD, ∗ p
    Figure Legend Snippet: BMSC + NPWT accelerate the formation of vascularized granulation tissue. (a) Immunohistochemical staining for CD31 and collagen IV, representing newly formed blood vessels. (b) Quantification of newly formed blood vessels. (c) Quantification of collagen IV expression. Data is given as the mean ± SD, ∗ p

    Techniques Used: Immunohistochemistry, Staining, Expressing

    Assessment of angiogenesis related factors and cytokines. (a) Western blotting of NG2, VEGF, CD31, and α -SMA protein expression within wounds among different groups on day 6. (b) Quantification of western blot results on day 6. (c) Western blotting of NG2, VEGF, CD31, and α -SMA protein expression within wounds among different groups on day 9. (d) Quantification of western blot results on day 9. (e) Immunofluorescence staining of NG2 within wounds on day 9. Nuclei were stained with DAPI (blue). Data is given as the mean ± SD, ∗∗ p
    Figure Legend Snippet: Assessment of angiogenesis related factors and cytokines. (a) Western blotting of NG2, VEGF, CD31, and α -SMA protein expression within wounds among different groups on day 6. (b) Quantification of western blot results on day 6. (c) Western blotting of NG2, VEGF, CD31, and α -SMA protein expression within wounds among different groups on day 9. (d) Quantification of western blot results on day 9. (e) Immunofluorescence staining of NG2 within wounds on day 9. Nuclei were stained with DAPI (blue). Data is given as the mean ± SD, ∗∗ p

    Techniques Used: Western Blot, Expressing, Immunofluorescence, Staining

    Assessment of angiogenesis related factors and cytokines. (a) Immunofluorescence staining of CD31, VEGF, and α -SMA within wounds among different groups on day 9. (b) RT-PCR results revealed more expression of CD31, VEGF, and α -SMA in wounds treated with BMSC + NPWT group compared to other three groups. Data is given as the mean ± SD, ∗∗∗ p
    Figure Legend Snippet: Assessment of angiogenesis related factors and cytokines. (a) Immunofluorescence staining of CD31, VEGF, and α -SMA within wounds among different groups on day 9. (b) RT-PCR results revealed more expression of CD31, VEGF, and α -SMA in wounds treated with BMSC + NPWT group compared to other three groups. Data is given as the mean ± SD, ∗∗∗ p

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

    Effect of NPWT on BMSC differentiation. (a) qRT-PCR analysis of NG2, VEGF, CD31, and α -SMA gene expression under NPWT versus 2D culture with standard medium. The data of 2D culture were considered as 1. (b) Western blotting of NG2, VEGF, CD31, and α -SMA protein expression under NPWT versus 2D culture. (c) Quantification of western blotting. (d) Immunofluorescent staining demonstrated that BMSCs cultured under NPWT exhibited differentiated states compared with 2D standard. Data is given as the mean ± SD, ∗∗∗ p
    Figure Legend Snippet: Effect of NPWT on BMSC differentiation. (a) qRT-PCR analysis of NG2, VEGF, CD31, and α -SMA gene expression under NPWT versus 2D culture with standard medium. The data of 2D culture were considered as 1. (b) Western blotting of NG2, VEGF, CD31, and α -SMA protein expression under NPWT versus 2D culture. (c) Quantification of western blotting. (d) Immunofluorescent staining demonstrated that BMSCs cultured under NPWT exhibited differentiated states compared with 2D standard. Data is given as the mean ± SD, ∗∗∗ p

    Techniques Used: Quantitative RT-PCR, Expressing, Western Blot, Staining, Cell Culture

    BMSC + NPWT accelerate the formation of mature vessel. (a) Immunofluorescence staining for CD31 and a-SMA. Red and green costaining represented mature blood vessels. Nuclei were stained with DAPI (blue). (b) Quantification of mature blood vessels. (c) Granulation tissue score and wound maturity score. (d) Concentration of TGF- β 1 within wounds among different groups. Data is given as the mean ± SD, ∗∗ p
    Figure Legend Snippet: BMSC + NPWT accelerate the formation of mature vessel. (a) Immunofluorescence staining for CD31 and a-SMA. Red and green costaining represented mature blood vessels. Nuclei were stained with DAPI (blue). (b) Quantification of mature blood vessels. (c) Granulation tissue score and wound maturity score. (d) Concentration of TGF- β 1 within wounds among different groups. Data is given as the mean ± SD, ∗∗ p

    Techniques Used: Immunofluorescence, Staining, Concentration Assay

    6) Product Images from "Calcineurin inhibitors cyclosporine A and tacrolimus induce vascular inflammation and endothelial activation through TLR4 signaling"

    Article Title: Calcineurin inhibitors cyclosporine A and tacrolimus induce vascular inflammation and endothelial activation through TLR4 signaling

    Journal: Scientific Reports

    doi: 10.1038/srep27915

    CNI induce inflammation in wild-type aortas but not in aortas from TLR4 −/− mice. Aorta tissue segments extracted from wild-type or TLR4 −/− C57BL/6 mice were stimulated for 6 h with 10 μg/ml CsA or 20 μg/ml Tac. CLI-095 was added 6 h before stimulation with the CNIs. ( A,B) Confocal microphotographs showing NF-κB/p65 content and location in control or CsA or CsA plus CLI-095 treated aortic sections from wild-type mice. Activation of NF-κB/p65 was detected by intensification of the specific red fluorescence in cytoplasm and nucleus of either endothelial cells recognized by CD31 staining (green fluorescence surrounding the cell borders) ( A ) or VSMC cells expressing αSMA (cytoplasmic green fluorescence) ( B ). Endothelial cells were found lining the intima layer and facing the lumen and VSMC located in the media layer. Yellow asterisks point cells with nuclear translocation of p65 and white asterisks indicate cells with increased cytoplasmic p65 expression. White arrows point endothelial and VSMC without increased expression of p65 in control or CLI095 treated aortas. White arrowheads in A show elastin fibers (green autofluorescence) which otherwise are not apparently visualized in B because the much higher αSMA specific fluorescence. Nuclei were counterstained with DAPI. Original magnification x630. ( C,D) Gene expression of CCL2, CCL5, IL-6, TNF-α (left panel) and ICAM-1, ET-1(right panel) in cultured aorta sections from wild-type mice exposed to CsA or Tac alone or in the presence of CLI-095. Data are expressed as mean ± SEM of 4 samples. *p
    Figure Legend Snippet: CNI induce inflammation in wild-type aortas but not in aortas from TLR4 −/− mice. Aorta tissue segments extracted from wild-type or TLR4 −/− C57BL/6 mice were stimulated for 6 h with 10 μg/ml CsA or 20 μg/ml Tac. CLI-095 was added 6 h before stimulation with the CNIs. ( A,B) Confocal microphotographs showing NF-κB/p65 content and location in control or CsA or CsA plus CLI-095 treated aortic sections from wild-type mice. Activation of NF-κB/p65 was detected by intensification of the specific red fluorescence in cytoplasm and nucleus of either endothelial cells recognized by CD31 staining (green fluorescence surrounding the cell borders) ( A ) or VSMC cells expressing αSMA (cytoplasmic green fluorescence) ( B ). Endothelial cells were found lining the intima layer and facing the lumen and VSMC located in the media layer. Yellow asterisks point cells with nuclear translocation of p65 and white asterisks indicate cells with increased cytoplasmic p65 expression. White arrows point endothelial and VSMC without increased expression of p65 in control or CLI095 treated aortas. White arrowheads in A show elastin fibers (green autofluorescence) which otherwise are not apparently visualized in B because the much higher αSMA specific fluorescence. Nuclei were counterstained with DAPI. Original magnification x630. ( C,D) Gene expression of CCL2, CCL5, IL-6, TNF-α (left panel) and ICAM-1, ET-1(right panel) in cultured aorta sections from wild-type mice exposed to CsA or Tac alone or in the presence of CLI-095. Data are expressed as mean ± SEM of 4 samples. *p

    Techniques Used: Mouse Assay, Activation Assay, Fluorescence, Staining, Expressing, Translocation Assay, Cell Culture

    7) Product Images from "Quantification of Epithelial Cell Differentiation in Mammary Glands and Carcinomas from DMBA- and MNU-Exposed Rats"

    Article Title: Quantification of Epithelial Cell Differentiation in Mammary Glands and Carcinomas from DMBA- and MNU-Exposed Rats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026145

    Characterization of rat mammary epithelial cells (RMECs) based on cell surface and intracellular markers. ( A ) Representative flow cytometric histograms and dot plots showing gating for propidium iodide (PI)-negative (live) cells (left panel); exclusion of endothelial cells and leukocytes based on CD31 and CD45 expression, respectively (middle left panel); CD61 expression in CD45–CD31– RMECs (middle right panel); CD24 and CD29 expression in CD45–CD31– RMECs identifies two major populations indicated with a red or blue circle (right panel). ( B ) Dot plots of intracellular cytokeratin (CK) 14 and CK19 expression in CD45–CD31– RMECs (upper left panel); intracellular smooth muscle actin (SMA) staining with phalloidin and CD29 expression in CD45–CD31– RMECs (upper right panel); overlay of dot plots showing CD24 and CD29 expression in CK14+CK19- cells and CK19+CK14- cells (lower left panel); overlay of dot plots of phalloidin bright cells on CD24 and CD29 expression in CD45–CD31– RMECs (lower right panel). Based on CK14, CK19, and SMA expression, the luminal (red) and basal (blue) populations in CD45–CD31– RMECs are identified. ( C ) Contour plot showing binding of Peanut Lectin (PNL) or anti-Thy-1 in CD45–CD31– RMECs (left panel), overlaid histograms showing CD29 expression on PNL+Thy1-, PNL-Thy-1+ cells (middle left panel), contour plots showing anti-Thy-1 (middle right panel) or PNL binding in CD29med or CD29hi cells (right panel). For all panels, rats of 12 weeks of age were used.
    Figure Legend Snippet: Characterization of rat mammary epithelial cells (RMECs) based on cell surface and intracellular markers. ( A ) Representative flow cytometric histograms and dot plots showing gating for propidium iodide (PI)-negative (live) cells (left panel); exclusion of endothelial cells and leukocytes based on CD31 and CD45 expression, respectively (middle left panel); CD61 expression in CD45–CD31– RMECs (middle right panel); CD24 and CD29 expression in CD45–CD31– RMECs identifies two major populations indicated with a red or blue circle (right panel). ( B ) Dot plots of intracellular cytokeratin (CK) 14 and CK19 expression in CD45–CD31– RMECs (upper left panel); intracellular smooth muscle actin (SMA) staining with phalloidin and CD29 expression in CD45–CD31– RMECs (upper right panel); overlay of dot plots showing CD24 and CD29 expression in CK14+CK19- cells and CK19+CK14- cells (lower left panel); overlay of dot plots of phalloidin bright cells on CD24 and CD29 expression in CD45–CD31– RMECs (lower right panel). Based on CK14, CK19, and SMA expression, the luminal (red) and basal (blue) populations in CD45–CD31– RMECs are identified. ( C ) Contour plot showing binding of Peanut Lectin (PNL) or anti-Thy-1 in CD45–CD31– RMECs (left panel), overlaid histograms showing CD29 expression on PNL+Thy1-, PNL-Thy-1+ cells (middle left panel), contour plots showing anti-Thy-1 (middle right panel) or PNL binding in CD29med or CD29hi cells (right panel). For all panels, rats of 12 weeks of age were used.

    Techniques Used: Flow Cytometry, Expressing, Staining, Binding Assay

    Differences between the RMECs from mammary glands of untreated control rats and mammary carcinomas from rats exposed to 7,12-dimethylbenz(a)anthracene (DMBA) or N -methyl- N -nitrosourea (MNU). ( A ) Representative pseudo-color dot plots showing CD24 and CD29 expression in the RMECs from the mammary gland of an age-matched (22 weeks of age) untreated control rat (upper left panel) and a DMBA- (upper middle panel) or MNU-induced (upper right panel) carcinoma; bar graphs (lower panel) quantifying mean ± sem percentage cells in the CD24hiD29hi gate within the total (CD45–CD31–) RMECs. A significantly different percentage comparing carcinomas to mammary glands is indicated with an asterisk (p
    Figure Legend Snippet: Differences between the RMECs from mammary glands of untreated control rats and mammary carcinomas from rats exposed to 7,12-dimethylbenz(a)anthracene (DMBA) or N -methyl- N -nitrosourea (MNU). ( A ) Representative pseudo-color dot plots showing CD24 and CD29 expression in the RMECs from the mammary gland of an age-matched (22 weeks of age) untreated control rat (upper left panel) and a DMBA- (upper middle panel) or MNU-induced (upper right panel) carcinoma; bar graphs (lower panel) quantifying mean ± sem percentage cells in the CD24hiD29hi gate within the total (CD45–CD31–) RMECs. A significantly different percentage comparing carcinomas to mammary glands is indicated with an asterisk (p

    Techniques Used: Expressing

    Features of the actively dividing cells in CD45–CD31– RMECs. ( A ) Flow cytometric histogram showing gating for actively dividing cells (cells in S/G2+M phase of the cell cycle) by having > 2n cellular DNA content (left panel); representative dot plot showing the actively dividing cells overlaid on CD24 and CD29 expression in the RMECs (right panel). ( B ) Representative dot plot showing gating for RMECs expressing high levels of both CD24 and CD29 (CD24hiCD29hi gate; left panel); bar graph (right panel) showing mean ± sem percentage of cells in S/G2+M phase of the cell cycle in total RMECs or CD24hiCD29hi-gated cells (n = 24 each). A significant enrichment of actively dividing cells was detected in the CD24hiCD29hi-gated cells (p
    Figure Legend Snippet: Features of the actively dividing cells in CD45–CD31– RMECs. ( A ) Flow cytometric histogram showing gating for actively dividing cells (cells in S/G2+M phase of the cell cycle) by having > 2n cellular DNA content (left panel); representative dot plot showing the actively dividing cells overlaid on CD24 and CD29 expression in the RMECs (right panel). ( B ) Representative dot plot showing gating for RMECs expressing high levels of both CD24 and CD29 (CD24hiCD29hi gate; left panel); bar graph (right panel) showing mean ± sem percentage of cells in S/G2+M phase of the cell cycle in total RMECs or CD24hiCD29hi-gated cells (n = 24 each). A significant enrichment of actively dividing cells was detected in the CD24hiCD29hi-gated cells (p

    Techniques Used: Flow Cytometry, Expressing

    8) Product Images from "HMGA1B/2 transcriptionally activated-POU1F1 facilitates gastric carcinoma metastasis via CXCL12/CXCR4 axis-mediated macrophage polarization"

    Article Title: HMGA1B/2 transcriptionally activated-POU1F1 facilitates gastric carcinoma metastasis via CXCL12/CXCR4 axis-mediated macrophage polarization

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-021-03703-x

    POU1F1 promotes GC progression via regulating macrophage proliferation, migration, polarization, and angiogenesis. A The immunoreactivity of CD163 was assessed by IHC analysis. B Kaplan–Meier curves for overall survival of GC patients. C The correlation between CD163 and POU1F1 in GC was determined by Pearson correlation analysis. D Cell surface CD14, CD11b, F4/80, and CD11c expression were analyzed by flow cytometry. E Cell viability of macrophages was monitored by CCK-8 assay. F Cell migration of macrophages was determined by transwell migration assay. The mRNA ( G ) and protein ( H ) levels of CD163 and CD206 were detected by qRT-PCR and western blot, respectively. CD11b served as an internal control. ( I ) The immunoreactivity of CD31 was assessed by IHC analysis. J The correlations between CD31 and CD163 in GC were determined by Pearson correlation analysis. K Cell viability of HUVECs was monitored by CCK-8 assay. L In vitro angiogenesis was monitored by tube formation assay. M The protein level of VEGF was determined by western blot. GAPDH served as a loading control. Data were representative images or were expressed as the mean ± SD of n = 3 experiments. * P
    Figure Legend Snippet: POU1F1 promotes GC progression via regulating macrophage proliferation, migration, polarization, and angiogenesis. A The immunoreactivity of CD163 was assessed by IHC analysis. B Kaplan–Meier curves for overall survival of GC patients. C The correlation between CD163 and POU1F1 in GC was determined by Pearson correlation analysis. D Cell surface CD14, CD11b, F4/80, and CD11c expression were analyzed by flow cytometry. E Cell viability of macrophages was monitored by CCK-8 assay. F Cell migration of macrophages was determined by transwell migration assay. The mRNA ( G ) and protein ( H ) levels of CD163 and CD206 were detected by qRT-PCR and western blot, respectively. CD11b served as an internal control. ( I ) The immunoreactivity of CD31 was assessed by IHC analysis. J The correlations between CD31 and CD163 in GC were determined by Pearson correlation analysis. K Cell viability of HUVECs was monitored by CCK-8 assay. L In vitro angiogenesis was monitored by tube formation assay. M The protein level of VEGF was determined by western blot. GAPDH served as a loading control. Data were representative images or were expressed as the mean ± SD of n = 3 experiments. * P

    Techniques Used: Migration, Immunohistochemistry, Expressing, Flow Cytometry, CCK-8 Assay, Transwell Migration Assay, Quantitative RT-PCR, Western Blot, In Vitro, Tube Formation Assay

    POU1F1 promotes GC metastasis in lung by modulating macrophage polarization through CXCL12/CXCR4 axis. A The photos of xenograft tumors. B Quantitative analysis of tumor size. C Quantitative analysis of tumor weight. D The photos of lung tissues. Quantitative analysis of metastatic nodule numbers. E Histopathological analysis of metastatic nodules in lung. The histopathological changes were determined by H E staining. The immunoreactivities of CD31, CD163, and POU1F1 were assessed by IHC analysis.
    Figure Legend Snippet: POU1F1 promotes GC metastasis in lung by modulating macrophage polarization through CXCL12/CXCR4 axis. A The photos of xenograft tumors. B Quantitative analysis of tumor size. C Quantitative analysis of tumor weight. D The photos of lung tissues. Quantitative analysis of metastatic nodule numbers. E Histopathological analysis of metastatic nodules in lung. The histopathological changes were determined by H E staining. The immunoreactivities of CD31, CD163, and POU1F1 were assessed by IHC analysis.

    Techniques Used: Staining, Immunohistochemistry

    9) Product Images from "IL-7–dependent maintenance of ILC3s is required for normal entry of lymphocytes into lymph nodes"

    Article Title: IL-7–dependent maintenance of ILC3s is required for normal entry of lymphocytes into lymph nodes

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20170518

    Lymphocyte entry into lymph nodes is defective in the absence of ILC3s. (A–D) Mice specifically lacking ILC3s were generated by reconstitution of irradiated WT hosts with bone marrow from Rorc −/− ( n = 9) or WT ( n = 10) donor bone marrow as control. Both groups were also treated with anti-Thy1 for 2 wk after BMT. 12 wk after reconstitution, hosts were injected with 10 7 labeled WT lymphocytes from either the spleen or a mixture of the lymph nodes and spleen, and 1 or 3 h later cell numbers in the lymph nodes and spleen were determined by FACS. (A) Density plots are of IL-7R vs. Lin for total lymphocytes and Gata3 vs. RORgt by IL-7R + Lin − -gated cells. Bar charts show total numbers of ILC subpopulations in the indicated chimeras. (B) Bar charts are of total lymph node size in chimeras of WT hosts reconstituted with either Rorc −/− or WT bone marrow. (C) Bar charts show the recovery of donor T and B cells from the indicated Rorc −/− chimeras from either lymph nodes (LN) or spleen (SPN). (D) Frozen sections from lymph nodes from the indicated chimeras were analyzed for expression of PNAd (green), CD31 (red) and counterstained with nuclear DAPI staining. (E–H) CD45.1 hosts were irradiated and reconstituted with either WT or Rorc −/− bone marrow. WT chimeras were treated with either anti-IL-7R or isotype control for 1 wk after irradiation, whereas Rorc −/− chimeras all received anti-IL-7R mAb. (E) 2 wk after BMT, groups of anti-IL-7R (αIL-7R) and isotype-treated WT chimeras ( n = 3 per group) were injected with labeled lymphocytes. Bar charts show numbers of T and B cells in lymph nodes 1 h after transfer. (F) 12 wk after BMT, lymph nodes were analyzed by FACs. Bar chart shows total numbers of ILC populations in lymph nodes from isotype-treated WT chimeras (white bars), anti-IL-7R (αIL-7R)-treated WT chimeras (gray bars), and anti-IL-7R (αIL-7R)-treated Rorc −/− chimeras (black bars). (G) Density plots show CD45.1 host vs. CD45.2 donor cells among total lymphocytes, gated ILC2, CD4 + ILC3, and CD4 – ILC3 in the indicated chimeras. (H) 12 wk after BMT, chimeras ( n = 7 per group) were injected with labeled WT cells from mixture of the lymph nodes and spleen, and 1 h later the host lymph nodes and spleen were analyzed by FACS. Bar charts show the numbers of donor T and B cells recovered from the lymph nodes and spleen of the indicated chimeras. Data are pools of four (B and C) or two (A, D, and E–H) independent experiments. E, F, and H are also representative of one further experiment performed by using WT CD45.2 hosts. *, P
    Figure Legend Snippet: Lymphocyte entry into lymph nodes is defective in the absence of ILC3s. (A–D) Mice specifically lacking ILC3s were generated by reconstitution of irradiated WT hosts with bone marrow from Rorc −/− ( n = 9) or WT ( n = 10) donor bone marrow as control. Both groups were also treated with anti-Thy1 for 2 wk after BMT. 12 wk after reconstitution, hosts were injected with 10 7 labeled WT lymphocytes from either the spleen or a mixture of the lymph nodes and spleen, and 1 or 3 h later cell numbers in the lymph nodes and spleen were determined by FACS. (A) Density plots are of IL-7R vs. Lin for total lymphocytes and Gata3 vs. RORgt by IL-7R + Lin − -gated cells. Bar charts show total numbers of ILC subpopulations in the indicated chimeras. (B) Bar charts are of total lymph node size in chimeras of WT hosts reconstituted with either Rorc −/− or WT bone marrow. (C) Bar charts show the recovery of donor T and B cells from the indicated Rorc −/− chimeras from either lymph nodes (LN) or spleen (SPN). (D) Frozen sections from lymph nodes from the indicated chimeras were analyzed for expression of PNAd (green), CD31 (red) and counterstained with nuclear DAPI staining. (E–H) CD45.1 hosts were irradiated and reconstituted with either WT or Rorc −/− bone marrow. WT chimeras were treated with either anti-IL-7R or isotype control for 1 wk after irradiation, whereas Rorc −/− chimeras all received anti-IL-7R mAb. (E) 2 wk after BMT, groups of anti-IL-7R (αIL-7R) and isotype-treated WT chimeras ( n = 3 per group) were injected with labeled lymphocytes. Bar charts show numbers of T and B cells in lymph nodes 1 h after transfer. (F) 12 wk after BMT, lymph nodes were analyzed by FACs. Bar chart shows total numbers of ILC populations in lymph nodes from isotype-treated WT chimeras (white bars), anti-IL-7R (αIL-7R)-treated WT chimeras (gray bars), and anti-IL-7R (αIL-7R)-treated Rorc −/− chimeras (black bars). (G) Density plots show CD45.1 host vs. CD45.2 donor cells among total lymphocytes, gated ILC2, CD4 + ILC3, and CD4 – ILC3 in the indicated chimeras. (H) 12 wk after BMT, chimeras ( n = 7 per group) were injected with labeled WT cells from mixture of the lymph nodes and spleen, and 1 h later the host lymph nodes and spleen were analyzed by FACS. Bar charts show the numbers of donor T and B cells recovered from the lymph nodes and spleen of the indicated chimeras. Data are pools of four (B and C) or two (A, D, and E–H) independent experiments. E, F, and H are also representative of one further experiment performed by using WT CD45.2 hosts. *, P

    Techniques Used: Mouse Assay, Generated, Irradiation, Injection, Labeling, FACS, Expressing, Staining

    Normal numbers of stromal and dendritic subsets but reduced ILC populations in lymph nodes after IL-7 ablation. Il7 fx/KO R26 CreERT2 mice ( n = 4) and CreERT –ve littermate controls ( n = 4) were treated with tamoxifen for 5 d. 3 wk later, lymph nodes were recovered and cell composition analyzed by FACS. (A) Density plots are of CD45 vs. SSc on total live cells and show CD45 – gate used to display gp38 vs CD31 (PECAM-1) density plots used to identify FRCs, LECs, and BECs. Bar chart shows total numbers of these subsets recovered from R26 CreERT2 +ve (closed bars) and R26 CreERT2 –ve Il7 fx/KO mice (open bars). (B) Density plots are of Meca79 vs. anti-ICAM-1 staining by CD31 + gp38 – BECs from representative mice described in A. Meca79 was either stained ex vivo in cell suspensions (Total Meca79) or by injection of mice with Meca79 mAb before ex vivo staining and analysis (luminal Meca79). Histograms are of ICAM-1 and Meca79 staining (total vs. luminal) by cells from Cre+ (solid line) or Cre– litter mates (gray shading), gated on Meca79 + ICAM-1 + cells defined by the gate shown on density plots. (C) Frozen sections from lymph nodes from the indicated mice were analyzed for expression of PNAd (green), CD31 (red), and counterstained with nuclear DAPI staining. (D) Il7 fx/KO R26 CreERT2 mice ( n = 3) and CreERT –ve littermate controls ( n = 3) were treated with tamoxifen for 5 d. 3 wk later, total mRNA was isolated from total lymph nodes of individual mice and gene expression determined by RNAseq. Bar charts show mRNA expression level in transcripts per kilobase million (TPM) of the indicated genes. (E) Density plots are of CD11c vs. Class II MHC (MHC II) and PDCA-1 vs. CD11c, used to identify MHC II + CD11c Hi resident DC (Res), MHC II Hi CD11c + migratory DC (Mig), and PDCA-1 + CD11c + plasmacytoid DCs (pDC). Bar charts show total numbers of these DC subsets recovered from the lymph nodes and spleen of R26 CreERT2 +ve and R26 CreERT2 –ve Il7 fx/KO mice. (F) Density plots are of IL-7R vs. Lin; histograms are of Thy1 expression by IL-7R + lin – -gated cells. Density plots are of Gata3 vs. RORgt expression by Thy1 + IL-7R + lin – ILCs. Histograms are of CD4 expression by RORgt+ ILCs. Bar charts show total numbers of ILC subsets isolated from the lymph nodes and spleen of R26 CreERT2 +ve and R26 CreERT2 –ve Il7 fx/KO mice. Data are representative of three independent experiments (A–D), a pool of two experiments (E), or four independent experiments (F). *, P
    Figure Legend Snippet: Normal numbers of stromal and dendritic subsets but reduced ILC populations in lymph nodes after IL-7 ablation. Il7 fx/KO R26 CreERT2 mice ( n = 4) and CreERT –ve littermate controls ( n = 4) were treated with tamoxifen for 5 d. 3 wk later, lymph nodes were recovered and cell composition analyzed by FACS. (A) Density plots are of CD45 vs. SSc on total live cells and show CD45 – gate used to display gp38 vs CD31 (PECAM-1) density plots used to identify FRCs, LECs, and BECs. Bar chart shows total numbers of these subsets recovered from R26 CreERT2 +ve (closed bars) and R26 CreERT2 –ve Il7 fx/KO mice (open bars). (B) Density plots are of Meca79 vs. anti-ICAM-1 staining by CD31 + gp38 – BECs from representative mice described in A. Meca79 was either stained ex vivo in cell suspensions (Total Meca79) or by injection of mice with Meca79 mAb before ex vivo staining and analysis (luminal Meca79). Histograms are of ICAM-1 and Meca79 staining (total vs. luminal) by cells from Cre+ (solid line) or Cre– litter mates (gray shading), gated on Meca79 + ICAM-1 + cells defined by the gate shown on density plots. (C) Frozen sections from lymph nodes from the indicated mice were analyzed for expression of PNAd (green), CD31 (red), and counterstained with nuclear DAPI staining. (D) Il7 fx/KO R26 CreERT2 mice ( n = 3) and CreERT –ve littermate controls ( n = 3) were treated with tamoxifen for 5 d. 3 wk later, total mRNA was isolated from total lymph nodes of individual mice and gene expression determined by RNAseq. Bar charts show mRNA expression level in transcripts per kilobase million (TPM) of the indicated genes. (E) Density plots are of CD11c vs. Class II MHC (MHC II) and PDCA-1 vs. CD11c, used to identify MHC II + CD11c Hi resident DC (Res), MHC II Hi CD11c + migratory DC (Mig), and PDCA-1 + CD11c + plasmacytoid DCs (pDC). Bar charts show total numbers of these DC subsets recovered from the lymph nodes and spleen of R26 CreERT2 +ve and R26 CreERT2 –ve Il7 fx/KO mice. (F) Density plots are of IL-7R vs. Lin; histograms are of Thy1 expression by IL-7R + lin – -gated cells. Density plots are of Gata3 vs. RORgt expression by Thy1 + IL-7R + lin – ILCs. Histograms are of CD4 expression by RORgt+ ILCs. Bar charts show total numbers of ILC subsets isolated from the lymph nodes and spleen of R26 CreERT2 +ve and R26 CreERT2 –ve Il7 fx/KO mice. Data are representative of three independent experiments (A–D), a pool of two experiments (E), or four independent experiments (F). *, P

    Techniques Used: Mouse Assay, FACS, Staining, Ex Vivo, Injection, Expressing, Isolation

    10) Product Images from "RhoB controls coordination of adult angiogenesis and lymphangiogenesis following injury by regulating VEZF1-mediated transcription"

    Article Title: RhoB controls coordination of adult angiogenesis and lymphangiogenesis following injury by regulating VEZF1-mediated transcription

    Journal: Nature Communications

    doi: 10.1038/ncomms3824

    RhoB loss normalizes angiogenesis in OIR and decreases BV density in granulation tissue. ( a ) Whole-mount staining of blood vessels (BS-I lectin) in the retina of wt and RhoB −/− pups subjected to hyperoxia from P7 to P12, and then returned back to normoxia from P12 to P17 (OD, optic disc). Histological (H E) analysis of wt and RhoB −/− retinas at P17. Quantification of nuclei number interior to ILM ( n =10 mice per genotype, mean±s.e.m., unpaired two-tailed Student’s t -test). ( b ) Confocal analysis following whole-mount staining of CD31 expression in the granulation tissue present 7 days after ear wounding in adult wt and RhoB −/− mice (6–8 weeks old). Quantification of CD31-positive staining in the neovasculature of the granulation tissue (delineated with white lines) ( n =6 mice per genotype, mean±s.e.m., unpaired two-tailed Student’s t -test). ( c ) Confocal analysis following whole-mount staining of CD31 and podoplanin expression in the granulation tissue present 7 days after wounding. Arrowheads highlight lymphatic vessels and denote areas of colocalization of CD31 and podoplanin in the merged image, thus confirming the identity of the lymphatic vessels. Scale bars: whole-mount staining=100 μm, H E sections=50 μm ( a ), 200 μm ( b , c ).
    Figure Legend Snippet: RhoB loss normalizes angiogenesis in OIR and decreases BV density in granulation tissue. ( a ) Whole-mount staining of blood vessels (BS-I lectin) in the retina of wt and RhoB −/− pups subjected to hyperoxia from P7 to P12, and then returned back to normoxia from P12 to P17 (OD, optic disc). Histological (H E) analysis of wt and RhoB −/− retinas at P17. Quantification of nuclei number interior to ILM ( n =10 mice per genotype, mean±s.e.m., unpaired two-tailed Student’s t -test). ( b ) Confocal analysis following whole-mount staining of CD31 expression in the granulation tissue present 7 days after ear wounding in adult wt and RhoB −/− mice (6–8 weeks old). Quantification of CD31-positive staining in the neovasculature of the granulation tissue (delineated with white lines) ( n =6 mice per genotype, mean±s.e.m., unpaired two-tailed Student’s t -test). ( c ) Confocal analysis following whole-mount staining of CD31 and podoplanin expression in the granulation tissue present 7 days after wounding. Arrowheads highlight lymphatic vessels and denote areas of colocalization of CD31 and podoplanin in the merged image, thus confirming the identity of the lymphatic vessels. Scale bars: whole-mount staining=100 μm, H E sections=50 μm ( a ), 200 μm ( b , c ).

    Techniques Used: Staining, Mouse Assay, Two Tailed Test, Expressing

    11) Product Images from "Generation and Characterization of a Transgenic Pig Carrying a DsRed-Monomer Reporter Gene"

    Article Title: Generation and Characterization of a Transgenic Pig Carrying a DsRed-Monomer Reporter Gene

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0106864

    (a) Cytometric analyses of CD4a, CD31, CD44, and CD90 conjugated with FITC on DsRed pig amniotic fluid progenitor/stem cells (pAFPCs). pAFPCs were negative for the surface antigens CD4a and CD31, and positive for CD44 and CD90. (b) Mesoderm trilineage differentiation potential of DsRed pAFPCs. DsRed pAFPCs exhibited spindle-shaped morphology (A1) and expressed red fluorescence under fluorescent microscopy (A2). Lipid droplets were observed in the DsRed pAFPCs adipogenic differentiation culture by using Oil-Red O staining (B1). Calcium accumulation was observed in the osteogenic differentiation culture by using Alizarin red staining (C1). Collagen was detected in the chondrogenic differentiation culture by using toluidine blue staining (D1). Under fluorescent microscopy, red fluorescence was still evident after osteogenetic (B2), adipogenetic (C2), and chondrogenetic (D2) differentiation.
    Figure Legend Snippet: (a) Cytometric analyses of CD4a, CD31, CD44, and CD90 conjugated with FITC on DsRed pig amniotic fluid progenitor/stem cells (pAFPCs). pAFPCs were negative for the surface antigens CD4a and CD31, and positive for CD44 and CD90. (b) Mesoderm trilineage differentiation potential of DsRed pAFPCs. DsRed pAFPCs exhibited spindle-shaped morphology (A1) and expressed red fluorescence under fluorescent microscopy (A2). Lipid droplets were observed in the DsRed pAFPCs adipogenic differentiation culture by using Oil-Red O staining (B1). Calcium accumulation was observed in the osteogenic differentiation culture by using Alizarin red staining (C1). Collagen was detected in the chondrogenic differentiation culture by using toluidine blue staining (D1). Under fluorescent microscopy, red fluorescence was still evident after osteogenetic (B2), adipogenetic (C2), and chondrogenetic (D2) differentiation.

    Techniques Used: Fluorescence, Microscopy, Staining

    12) Product Images from "Homozygous MESP1 knock-in reporter hESCs facilitated cardiovascular cell differentiation and myocardial infarction repair"

    Article Title: Homozygous MESP1 knock-in reporter hESCs facilitated cardiovascular cell differentiation and myocardial infarction repair

    Journal: Theranostics

    doi: 10.7150/thno.42347

    Efficient tri-lineage differentiation based on optimizing MESP1-mTomato expression. ( A ) Schematic view of the tri-lineage differentiation system. ( B ) Immunostaining of α-actinin (red) and cTnT (red) in CMs, DNA in blue, scale bars, 20 µm. ( C ) Flow cytometry analysis of cTnT + cells on day 12. ( D ) Di-4-ANEPPS fluorescence intensity measurement to show the action potential change in MESP1-mTomato + cells derived CMs. Most beating cells showed ventricular-like electrophysiology characteristics (left), and some displayed nodal-like potential change (right). ( E ) Time-lapse images of Ca 2+ transients in CMs. The time was indicated on the top-right corner; the white line circled the contracting area. ( F ) Drug response of CMs differentiated from MESP1-mTomato + cells. Beating cells stained with Fluo-4 AM were first filmed. Then they were treated with 5 µmol/L Isoproterenol and filmed again. Images were recorded at 10 frames per second. Fluorescence intensity of the circled area in (E) was quantified before and after Isoproterenol addition. ( G ) Immunostaining of CD31 (red), αSMA (green) and SM22a (red). Scale bars, 100 µm. ( H ) Flow cytometry analysis of CD31-CD144 for ECs and SM22a for SMCs. ( I ) Tube formation assay of ECs. Scale bar, 10 µm. ( J ) DiI-ac-LDL (red) uptake assay, scale bar, 100 µm. ( K ) Heatmap comparison of marker gene expression among HUVEC, MESP1 cells derived EC, SMC, and HCASMC.
    Figure Legend Snippet: Efficient tri-lineage differentiation based on optimizing MESP1-mTomato expression. ( A ) Schematic view of the tri-lineage differentiation system. ( B ) Immunostaining of α-actinin (red) and cTnT (red) in CMs, DNA in blue, scale bars, 20 µm. ( C ) Flow cytometry analysis of cTnT + cells on day 12. ( D ) Di-4-ANEPPS fluorescence intensity measurement to show the action potential change in MESP1-mTomato + cells derived CMs. Most beating cells showed ventricular-like electrophysiology characteristics (left), and some displayed nodal-like potential change (right). ( E ) Time-lapse images of Ca 2+ transients in CMs. The time was indicated on the top-right corner; the white line circled the contracting area. ( F ) Drug response of CMs differentiated from MESP1-mTomato + cells. Beating cells stained with Fluo-4 AM were first filmed. Then they were treated with 5 µmol/L Isoproterenol and filmed again. Images were recorded at 10 frames per second. Fluorescence intensity of the circled area in (E) was quantified before and after Isoproterenol addition. ( G ) Immunostaining of CD31 (red), αSMA (green) and SM22a (red). Scale bars, 100 µm. ( H ) Flow cytometry analysis of CD31-CD144 for ECs and SM22a for SMCs. ( I ) Tube formation assay of ECs. Scale bar, 10 µm. ( J ) DiI-ac-LDL (red) uptake assay, scale bar, 100 µm. ( K ) Heatmap comparison of marker gene expression among HUVEC, MESP1 cells derived EC, SMC, and HCASMC.

    Techniques Used: Expressing, Immunostaining, Flow Cytometry, Fluorescence, Derivative Assay, Staining, Tube Formation Assay, Marker

    13) Product Images from "Skeletal myogenic differentiation of human periodontal ligament stromal cells isolated from orthodontically extracted premolars"

    Article Title: Skeletal myogenic differentiation of human periodontal ligament stromal cells isolated from orthodontically extracted premolars

    Journal: Korean Journal of Orthodontics

    doi: 10.4041/kjod.2012.42.5.249

    Multipotency of periodontal ligament (PDL) stromal cells. A , PDL stromal cells displayed high expression levels of the mesenchymal stem cell surface markers CD90 and CD105, but very low expression levels of the hematopoietic stem cell surface markers CD31 and CD34. Gray solid fill, isotype; black line, marker of interest. B , Calcified nodules, lipid cluster formation, and chondrogenic pellet formation were noted after osteogenic, adipogenic, and chondrogenic induction, respectively.
    Figure Legend Snippet: Multipotency of periodontal ligament (PDL) stromal cells. A , PDL stromal cells displayed high expression levels of the mesenchymal stem cell surface markers CD90 and CD105, but very low expression levels of the hematopoietic stem cell surface markers CD31 and CD34. Gray solid fill, isotype; black line, marker of interest. B , Calcified nodules, lipid cluster formation, and chondrogenic pellet formation were noted after osteogenic, adipogenic, and chondrogenic induction, respectively.

    Techniques Used: Expressing, Marker

    14) Product Images from "CD3ε Expression Defines Functionally Distinct Subsets of Vδ1 T Cells in Patients With Human Immunodeficiency Virus Infection"

    Article Title: CD3ε Expression Defines Functionally Distinct Subsets of Vδ1 T Cells in Patients With Human Immunodeficiency Virus Infection

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00940

    CD3ε lo Vδ1 T cells more frequently express programmed death-1 (PD-1), but not lymphocyte-activation gene 3 (LAG-3) or CD31, than CD3ε hi Vδ1 T cells. Peripheral blood mononuclear cell prepared from eight healthy donors and five untreated patients with human immunodeficiency virus (HIV) were enriched for γδ T cells using magnetic bead separation, stained with monoclonal antibodies specific for CD3ε, Vδ1, PD-1, LAG-3, and CD31 and analyzed by flow cytometry. (A) Flow cytometry dot plots showing PD-1, LAG-3, and CD31 expression by gated CD3ε lo and CD3ε hi Vδ1 T cells from a patient with HIV. (B) Scatter plots showing the frequencies of CD3ε lo and CD3ε hi Vδ1 T cells from the patients with HIV and control subjects that expressed PD-1 and CD31. Groups were compared using the Mann–Whitney U test.
    Figure Legend Snippet: CD3ε lo Vδ1 T cells more frequently express programmed death-1 (PD-1), but not lymphocyte-activation gene 3 (LAG-3) or CD31, than CD3ε hi Vδ1 T cells. Peripheral blood mononuclear cell prepared from eight healthy donors and five untreated patients with human immunodeficiency virus (HIV) were enriched for γδ T cells using magnetic bead separation, stained with monoclonal antibodies specific for CD3ε, Vδ1, PD-1, LAG-3, and CD31 and analyzed by flow cytometry. (A) Flow cytometry dot plots showing PD-1, LAG-3, and CD31 expression by gated CD3ε lo and CD3ε hi Vδ1 T cells from a patient with HIV. (B) Scatter plots showing the frequencies of CD3ε lo and CD3ε hi Vδ1 T cells from the patients with HIV and control subjects that expressed PD-1 and CD31. Groups were compared using the Mann–Whitney U test.

    Techniques Used: Activation Assay, Staining, Flow Cytometry, Cytometry, Expressing, MANN-WHITNEY

    15) Product Images from "Defining the role of pulmonary endothelial cell heterogeneity in the response to acute lung injury"

    Article Title: Defining the role of pulmonary endothelial cell heterogeneity in the response to acute lung injury

    Journal: eLife

    doi: 10.7554/eLife.53072

    Isolation and qRT-PCR analysis of CD34-high ECs confirms their transcriptional distinction from other ECs. ( A ) Gating strategy for isolating Car4 -high (CD34-high) ECs, other CD34-low ECs, and non-ECs (CD45 - /CD31 - cells) by FACS. After isolation of each cell population, RNA extraction and subsequent qRT-PCR analysis show that in comparison to CD34-low ECs, CD34-high ECs express higher relative levels of ( B ) Cd34 , ( C ) Car4 , ( D ) Ednrb , and ( E ) Kdr , whereas non-ECs do not express high levels of any of these genes. In contrast, CD34-low and CD34-high ECs express comparable levels of other EC genes such as ( F ) Pecam1 , ( G ) Plvap , ( H ) Gpihbp1 , and ( I ) Vwf , while non-ECs express significantly lower levels of these genes. Each point represents the average of three technical replicates for N = 4 animals. *, p
    Figure Legend Snippet: Isolation and qRT-PCR analysis of CD34-high ECs confirms their transcriptional distinction from other ECs. ( A ) Gating strategy for isolating Car4 -high (CD34-high) ECs, other CD34-low ECs, and non-ECs (CD45 - /CD31 - cells) by FACS. After isolation of each cell population, RNA extraction and subsequent qRT-PCR analysis show that in comparison to CD34-low ECs, CD34-high ECs express higher relative levels of ( B ) Cd34 , ( C ) Car4 , ( D ) Ednrb , and ( E ) Kdr , whereas non-ECs do not express high levels of any of these genes. In contrast, CD34-low and CD34-high ECs express comparable levels of other EC genes such as ( F ) Pecam1 , ( G ) Plvap , ( H ) Gpihbp1 , and ( I ) Vwf , while non-ECs express significantly lower levels of these genes. Each point represents the average of three technical replicates for N = 4 animals. *, p

    Techniques Used: Isolation, Quantitative RT-PCR, FACS, RNA Extraction

    Proliferation in CD34-low ECs increases in the first seven days following H1N1 injury. ( A ) 7 days after intranasal H1N1 administration, a single-cell suspension isolated from the lungs of a representative mouse given EdU from days 0–7 following injury had 21.3% CD45-negative non-immune cells, indicative of a robust immune response in the lung at this time point. ( B ) Of the non-immune cells, 32.9% were CD31-positive ECs. ( C ) Of these ECs, 7.49% had incorporated EdU over days 0–7 after influenza injury, indicating that endothelial proliferation is already increasing during this early time window. ( D ) Separation of CD45-negative, CD31-positive ECs into CD34-high and CD34-low populations shows that in the same representative mouse, 8.45% of non-immune cells are CD34-high ECs, a percentage intermediate between control animals and animals at 14 dpi. Within these EC subpopulations, ( E ) 0.78% of CD34-high ECs and ( F ) 7.10% of CD34-low ECs had incorporated EdU over days 0–7 following H1N1 administration, indicating that EC proliferation during this early time window following influenza injury is driven largely by CD34-low ECs. Quantification of these parameters across H1N1 cohort one is shown in Figure 5 .
    Figure Legend Snippet: Proliferation in CD34-low ECs increases in the first seven days following H1N1 injury. ( A ) 7 days after intranasal H1N1 administration, a single-cell suspension isolated from the lungs of a representative mouse given EdU from days 0–7 following injury had 21.3% CD45-negative non-immune cells, indicative of a robust immune response in the lung at this time point. ( B ) Of the non-immune cells, 32.9% were CD31-positive ECs. ( C ) Of these ECs, 7.49% had incorporated EdU over days 0–7 after influenza injury, indicating that endothelial proliferation is already increasing during this early time window. ( D ) Separation of CD45-negative, CD31-positive ECs into CD34-high and CD34-low populations shows that in the same representative mouse, 8.45% of non-immune cells are CD34-high ECs, a percentage intermediate between control animals and animals at 14 dpi. Within these EC subpopulations, ( E ) 0.78% of CD34-high ECs and ( F ) 7.10% of CD34-low ECs had incorporated EdU over days 0–7 following H1N1 administration, indicating that EC proliferation during this early time window following influenza injury is driven largely by CD34-low ECs. Quantification of these parameters across H1N1 cohort one is shown in Figure 5 .

    Techniques Used: Isolation

    ECs proliferate very little in uninjured mice. ( A ) 14 days after intranasal PBS, a single-cell suspension isolated from the lungs of a representative mouse given EdU from days 7–14 had 73.9% CD45-negative non-immune cells, ( B ) of which 40.8% were CD31-positive ECs. ( C ) Of these ECs, 1.21% had incorporated EdU over days 7–14 after PBS, indicating that sham injury elicits very little proliferative response in control mice. ( D ) Separation of CD45-negative, CD31-positive ECs into CD34-high and CD34-low populations demonstrates that in the same control mouse, only 5.27% of non-immune cells were CD34-high ECs, a much lower percentage than that seen 14 days post-H1N1 injury. Within these EC subpopulations, ( E ) 3.41% of CD34-high ECs and ( F ) 0.93% of CD34-low ECs had incorporated EdU over days 7–14 following PBS administration, again indicating very little EC proliferation in the uninjured lung. Quantification of these parameters across the entire control cohort is shown in Figure 5 . ( G, H ) In a separate control experiment to assess the reliability of EdU water administration and measurement of EdU incorporation by flow cytometry, single-cell lung suspensions isolated from two mice given H1N1 on day 0 and EdU water from days 7–14 demonstrated EdU incorporation in 15.2% and 15.4% of CD31-positive ECs. ( I ) In contrast, no EdU-positive cells were observed in the single-cell lung suspension of a mouse given H1N1 and EdU water but without fluorescent labeling of EdU. ( J, K, L ) Single-cell lung suspensions isolated from three individual mice given H1N1 but no EdU water and treated with the EdU fluorescent labeling assay demonstrated low levels of background fluorescence in the EdU channel. This indicates that although background levels of fluorescence are possible in the EdU labeling assay, the size of the changes we observe between control and flu-injured animals can be reliably detected using the EdU water administration and fluorescent labeling strategy we employed.
    Figure Legend Snippet: ECs proliferate very little in uninjured mice. ( A ) 14 days after intranasal PBS, a single-cell suspension isolated from the lungs of a representative mouse given EdU from days 7–14 had 73.9% CD45-negative non-immune cells, ( B ) of which 40.8% were CD31-positive ECs. ( C ) Of these ECs, 1.21% had incorporated EdU over days 7–14 after PBS, indicating that sham injury elicits very little proliferative response in control mice. ( D ) Separation of CD45-negative, CD31-positive ECs into CD34-high and CD34-low populations demonstrates that in the same control mouse, only 5.27% of non-immune cells were CD34-high ECs, a much lower percentage than that seen 14 days post-H1N1 injury. Within these EC subpopulations, ( E ) 3.41% of CD34-high ECs and ( F ) 0.93% of CD34-low ECs had incorporated EdU over days 7–14 following PBS administration, again indicating very little EC proliferation in the uninjured lung. Quantification of these parameters across the entire control cohort is shown in Figure 5 . ( G, H ) In a separate control experiment to assess the reliability of EdU water administration and measurement of EdU incorporation by flow cytometry, single-cell lung suspensions isolated from two mice given H1N1 on day 0 and EdU water from days 7–14 demonstrated EdU incorporation in 15.2% and 15.4% of CD31-positive ECs. ( I ) In contrast, no EdU-positive cells were observed in the single-cell lung suspension of a mouse given H1N1 and EdU water but without fluorescent labeling of EdU. ( J, K, L ) Single-cell lung suspensions isolated from three individual mice given H1N1 but no EdU water and treated with the EdU fluorescent labeling assay demonstrated low levels of background fluorescence in the EdU channel. This indicates that although background levels of fluorescence are possible in the EdU labeling assay, the size of the changes we observe between control and flu-injured animals can be reliably detected using the EdU water administration and fluorescent labeling strategy we employed.

    Techniques Used: Mouse Assay, Isolation, Flow Cytometry, Labeling, Fluorescence

    Endothelial proliferation continues to increase from 14 to 21 days following H1N1 injury. ( A ) At 21 days post-H1N1 injury, single cells isolated from the lungs of a representative mouse given EdU from days 14–21 were 32.8% CD45-negative non-immune cells, indicating that immune response in the lung is still high at this later time point. ( B ) Of the non-immune cells, 46.1% were CD31-positive ECs. ( C ) Of these ECs, 37.6% had incorporated EdU over days 14–21 of the regenerative response to H1N1 injury, indicating that EC proliferation is still ongoing and even increasing later in regeneration. ( D ) Separation of CD45-negative, CD31-positive ECs into CD34-high and CD34-low populations reveals that in the same representative mouse, 15% of non-immune cells are CD34-high ECs, with ( E ) 32.0% of CD34-high ECs and ( F ) 38.7% of CD34-low ECs incorporating EdU over days 7–14 of the regenerative response. This indicates that both EC populations continue to proliferate over the third week of regeneration following H1N1 injury in the lung. Quantification of these parameters across H1N1 cohort three is shown in Figure 5 .
    Figure Legend Snippet: Endothelial proliferation continues to increase from 14 to 21 days following H1N1 injury. ( A ) At 21 days post-H1N1 injury, single cells isolated from the lungs of a representative mouse given EdU from days 14–21 were 32.8% CD45-negative non-immune cells, indicating that immune response in the lung is still high at this later time point. ( B ) Of the non-immune cells, 46.1% were CD31-positive ECs. ( C ) Of these ECs, 37.6% had incorporated EdU over days 14–21 of the regenerative response to H1N1 injury, indicating that EC proliferation is still ongoing and even increasing later in regeneration. ( D ) Separation of CD45-negative, CD31-positive ECs into CD34-high and CD34-low populations reveals that in the same representative mouse, 15% of non-immune cells are CD34-high ECs, with ( E ) 32.0% of CD34-high ECs and ( F ) 38.7% of CD34-low ECs incorporating EdU over days 7–14 of the regenerative response. This indicates that both EC populations continue to proliferate over the third week of regeneration following H1N1 injury in the lung. Quantification of these parameters across H1N1 cohort three is shown in Figure 5 .

    Techniques Used: Isolation

    Isolation of CD45 - /CD31 + cells results in capture of some mesenchymal and epithelial cells. Identification of the top three differentially expressed genes in ( A ) cluster five and ( B ) cluster six by average log 2 fold change in gene expression indicates that these clusters are likely contaminating cell types in the analysis of CD31 + /CD45 - cells. Cluster 5 (363/15,894 cells) is defined by high expression of cytoskeleton and extracellular matrix genes such as Gsn and Mfap4 and is likely composed of mesenchymal cells. Cells in cluster 6 (80/15,894 cells) express surfactant protein genes such as Sftpa , Sftpb , and Sftpd and likely represent epithelial cells.
    Figure Legend Snippet: Isolation of CD45 - /CD31 + cells results in capture of some mesenchymal and epithelial cells. Identification of the top three differentially expressed genes in ( A ) cluster five and ( B ) cluster six by average log 2 fold change in gene expression indicates that these clusters are likely contaminating cell types in the analysis of CD31 + /CD45 - cells. Cluster 5 (363/15,894 cells) is defined by high expression of cytoskeleton and extracellular matrix genes such as Gsn and Mfap4 and is likely composed of mesenchymal cells. Cells in cluster 6 (80/15,894 cells) express surfactant protein genes such as Sftpa , Sftpb , and Sftpd and likely represent epithelial cells.

    Techniques Used: Isolation, Expressing

    Proliferating ECs express general EC marker genes, but do not express genes that are highly expressed in Car4 -high ECs. ( A ) UMAP dimension reduction of H1N1 injury scRNA-seq dataset, with red box enclosing EC clusters and representing areas of increased digital zoom displayed in ( B )-( I ). Black circle demarcates proliferating EC cluster. Proliferating ECs express genes highly expressed in all ECs or in miECs, such as ( B ) Cd31 , ( C ) Gpihbp1 , and ( D ) Plvap . However, they express moderate levels of ( E ) Cd34 and do not express other genes that are highly expressed in Car4 -high ECs, such as ( F ) Kdr or ( G ) Ednrb . Proliferating ECs do not express genes enriched in maECs, such as ( H ) Vcam1 or ( I ) Vwf.
    Figure Legend Snippet: Proliferating ECs express general EC marker genes, but do not express genes that are highly expressed in Car4 -high ECs. ( A ) UMAP dimension reduction of H1N1 injury scRNA-seq dataset, with red box enclosing EC clusters and representing areas of increased digital zoom displayed in ( B )-( I ). Black circle demarcates proliferating EC cluster. Proliferating ECs express genes highly expressed in all ECs or in miECs, such as ( B ) Cd31 , ( C ) Gpihbp1 , and ( D ) Plvap . However, they express moderate levels of ( E ) Cd34 and do not express other genes that are highly expressed in Car4 -high ECs, such as ( F ) Kdr or ( G ) Ednrb . Proliferating ECs do not express genes enriched in maECs, such as ( H ) Vcam1 or ( I ) Vwf.

    Techniques Used: Marker

    16) Product Images from "The human lung during the embryonic period: vasculogenesis and primitive erythroblasts circulation"

    Article Title: The human lung during the embryonic period: vasculogenesis and primitive erythroblasts circulation

    Journal: Journal of Anatomy

    doi: 10.1111/joa.12042

    Confocal images of double immunofluorescence for CD34 and CD31 in human embryonic lungs at 35 days pf. (A) CD34 + cells (green) in the vascular plexus around the terminal bud. (B) Immunoreactivity for CD31 (red) in some endothelial cells. (C) The merged
    Figure Legend Snippet: Confocal images of double immunofluorescence for CD34 and CD31 in human embryonic lungs at 35 days pf. (A) CD34 + cells (green) in the vascular plexus around the terminal bud. (B) Immunoreactivity for CD31 (red) in some endothelial cells. (C) The merged

    Techniques Used: Immunofluorescence

    17) Product Images from "Expansion of functional personalized cells with specific transgene combinations"

    Article Title: Expansion of functional personalized cells with specific transgene combinations

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03408-4

    Cell-type-specific and reproducible cell expansion. a Cumulative population doubling levels of HUVEC lines e-hUVEC-2, e-hUVEC-7, and e-hUVEC-9. For e-hUVEC-2 and e-hUVEC-7, primary cells were derived from different donors, but generated using the same gene set (ID1, ID2, and MYC). e-hUVEC-9 cells were transduced with ID2, FOS, and MYC. Graphs for e-hUVEC-2 were taken from Fig. 1b . b Four independent endothelial cell lines generated from the same donor upon transduction with MYC, ID1, and ID2 (technical replicates to e-hUVEC-2), and one cell line generated using the same gene set but derived from a different donor (biological replicate to e-hUVEC-2), were analyzed after 80 cumulative population doublings for acLDL uptake, CD31 expression, and eNOS activity. c Four independent endothelial cell lines generated from the same donor upon transduction with ID2, FOS, and MYC (technical replicates to e-hUVEC-9) were analyzed after 80 cumulative population doublings for acLDL uptake, CD31 expression, and eNOS activity. Unstained or isotype control is shown in gray, antibody stained samples are depicted in black
    Figure Legend Snippet: Cell-type-specific and reproducible cell expansion. a Cumulative population doubling levels of HUVEC lines e-hUVEC-2, e-hUVEC-7, and e-hUVEC-9. For e-hUVEC-2 and e-hUVEC-7, primary cells were derived from different donors, but generated using the same gene set (ID1, ID2, and MYC). e-hUVEC-9 cells were transduced with ID2, FOS, and MYC. Graphs for e-hUVEC-2 were taken from Fig. 1b . b Four independent endothelial cell lines generated from the same donor upon transduction with MYC, ID1, and ID2 (technical replicates to e-hUVEC-2), and one cell line generated using the same gene set but derived from a different donor (biological replicate to e-hUVEC-2), were analyzed after 80 cumulative population doublings for acLDL uptake, CD31 expression, and eNOS activity. c Four independent endothelial cell lines generated from the same donor upon transduction with ID2, FOS, and MYC (technical replicates to e-hUVEC-9) were analyzed after 80 cumulative population doublings for acLDL uptake, CD31 expression, and eNOS activity. Unstained or isotype control is shown in gray, antibody stained samples are depicted in black

    Techniques Used: Derivative Assay, Generated, Transduction, Expressing, Activity Assay, Staining

    Comparable phenotype of primary and expanded endothelial cells. a Phase contrast microscopy shows the expandable HUVEC cell line e-hUVEC-2 (MYC, ID1, and ID2). Scale bar 100 µm. b CD31 expression of human endothelial cell populations immortalized with three different gene sets as indicated. c Global gene expression analysis was performed on three different HUVEC lines (e-hUVEC-2, 8 and 9; in duplicate) (3, 4, 5, respectively) and two independent primary HUVEC populations (2) as well as four primary gingiva fibroblast populations (1). Expression data was processed with GeneSpring 11.5.1 software and a Standard Pearson Correlation was determined for each gene versus all other genes. The correlation heatmap depicts the pair-wise correlation coefficient between the given samples and displays the relationship between the different samples. The samples are clustered based on the pair-wise correlation coefficients between all entities. d Phenotypic stability of e-hUVEC-2 cells after 45 and 90 cumulative population doublings was evaluated by CD31 (also known as PECAM1) expression, acetylated LDL uptake, and eNOS activity. Gray fill: antibody isotype control; black outline: stained sample. MFI: median fluorescence intensity. e Immunofluorescence-based detection of CD31 and CD146 (also known as MCAM) in expandable HUVECs counterstained for nuclei with DAPI. Scale bars, 100 µm. f The phenotype and the functionality of cell line e-hUVEC-2 (cumulative population doubling 80) was compared to primary HUVECs based on flow cytometric analysis of CD31, TIE1, TIE2, and CD309 (also known as VEGFR2) expression. Gray fill: antibody isotype control; black outline: stained sample. MFI: median fluorescence intensity. g The angiogenic potential of primary HUVECs and e-hUVEC-2 was determined in vitro by a matrigel tube formation assay. Scale bars, 200 µm. h Spheroids in matrigel of primary HUVEC and e-hUVEC-2 were subcutaneously injected into Rag2 -/- Il2r g −/− mice. After two weeks the implants were dissected and stained for human CD31 (brown color). e-hUVEC-2 organized into human CD31 positive microvessels similar to primary HUVEC. Scale bars, 100 µm
    Figure Legend Snippet: Comparable phenotype of primary and expanded endothelial cells. a Phase contrast microscopy shows the expandable HUVEC cell line e-hUVEC-2 (MYC, ID1, and ID2). Scale bar 100 µm. b CD31 expression of human endothelial cell populations immortalized with three different gene sets as indicated. c Global gene expression analysis was performed on three different HUVEC lines (e-hUVEC-2, 8 and 9; in duplicate) (3, 4, 5, respectively) and two independent primary HUVEC populations (2) as well as four primary gingiva fibroblast populations (1). Expression data was processed with GeneSpring 11.5.1 software and a Standard Pearson Correlation was determined for each gene versus all other genes. The correlation heatmap depicts the pair-wise correlation coefficient between the given samples and displays the relationship between the different samples. The samples are clustered based on the pair-wise correlation coefficients between all entities. d Phenotypic stability of e-hUVEC-2 cells after 45 and 90 cumulative population doublings was evaluated by CD31 (also known as PECAM1) expression, acetylated LDL uptake, and eNOS activity. Gray fill: antibody isotype control; black outline: stained sample. MFI: median fluorescence intensity. e Immunofluorescence-based detection of CD31 and CD146 (also known as MCAM) in expandable HUVECs counterstained for nuclei with DAPI. Scale bars, 100 µm. f The phenotype and the functionality of cell line e-hUVEC-2 (cumulative population doubling 80) was compared to primary HUVECs based on flow cytometric analysis of CD31, TIE1, TIE2, and CD309 (also known as VEGFR2) expression. Gray fill: antibody isotype control; black outline: stained sample. MFI: median fluorescence intensity. g The angiogenic potential of primary HUVECs and e-hUVEC-2 was determined in vitro by a matrigel tube formation assay. Scale bars, 200 µm. h Spheroids in matrigel of primary HUVEC and e-hUVEC-2 were subcutaneously injected into Rag2 -/- Il2r g −/− mice. After two weeks the implants were dissected and stained for human CD31 (brown color). e-hUVEC-2 organized into human CD31 positive microvessels similar to primary HUVEC. Scale bars, 100 µm

    Techniques Used: Microscopy, Expressing, Software, Activity Assay, Staining, Fluorescence, Immunofluorescence, Flow Cytometry, In Vitro, Tube Formation Assay, Injection, Mouse Assay

    18) Product Images from "FOXF1 transcription factor promotes lung regeneration after partial pneumonectomy"

    Article Title: FOXF1 transcription factor promotes lung regeneration after partial pneumonectomy

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-11175-3

    Decreased MMP14 activity in PDGFb-iCre/Foxf1 fl /+ lungs after PNX. ( A ) FACS-sorted endothelial cells (CD45 − CD31 + CD326 − ) showed decreased Foxf1 but increased Timp3 mRNAs in PDGFb-iCre/Foxf1 fl /+ lungs compared to controls. ( B ) TIMP3 staining was increased in PDGFb-iCre/Foxf1 fl /+ mice after PNX. ( C ) Representative zymography gel showing MMP14 gelatinase activity in lung samples collected after sham or PNX surgery in control and PDGFb-iCre/Foxf1 fl /+ mice. A cropped gel is presented here with full gel available in Supplemental Fig. 9 . ( D ) Quantification of zymography gels shows decreased MMP14 activity in PDGFb-iCre/Foxf1 fl /+ lungs compared to controls. ( E ) ChIPseq analysis showing multiple FOXF1 binding sites within Timp3 gene locus. ( F ) Diagram showing multiple pathways whereby FOXF1 induces lung regeneration after PNX. *p
    Figure Legend Snippet: Decreased MMP14 activity in PDGFb-iCre/Foxf1 fl /+ lungs after PNX. ( A ) FACS-sorted endothelial cells (CD45 − CD31 + CD326 − ) showed decreased Foxf1 but increased Timp3 mRNAs in PDGFb-iCre/Foxf1 fl /+ lungs compared to controls. ( B ) TIMP3 staining was increased in PDGFb-iCre/Foxf1 fl /+ mice after PNX. ( C ) Representative zymography gel showing MMP14 gelatinase activity in lung samples collected after sham or PNX surgery in control and PDGFb-iCre/Foxf1 fl /+ mice. A cropped gel is presented here with full gel available in Supplemental Fig. 9 . ( D ) Quantification of zymography gels shows decreased MMP14 activity in PDGFb-iCre/Foxf1 fl /+ lungs compared to controls. ( E ) ChIPseq analysis showing multiple FOXF1 binding sites within Timp3 gene locus. ( F ) Diagram showing multiple pathways whereby FOXF1 induces lung regeneration after PNX. *p

    Techniques Used: Activity Assay, FACS, Staining, Mouse Assay, Zymography, Binding Assay

    Identification of FOXF1 target genes in endothelial cells from regenerating lungs. ( A ) FACS-sorting strategy to isolate populations of endothelial (CD45 − CD31 + CD326 − ) and epithelial (CD45 − CD31 − CD326 + ) cells, showing GFP expression in PDGFb-iCre expressing mice. ( B ) Foxf1 mRNA was highly expressed in endothelial cells but not in epithelial cells. Endothelial cells from PDGFb-iCre/Foxf1 fl /+ mice had significantly less Foxf1 mRNA than endothelial cells from control mice. ( C ) Heat map showing significant changes in expression of 1047 genes in endothelial cells from Foxf1 fl /+ and PDGFb-iCre/Foxf1 fl /+ lungs after PNX. ( D ) Western blot analysis showed increased protein levels for CDKN1A (P21 Cip1 ) and CDKN2B (P15 Ink4b ) in PDGFb-iCre/Foxf1 fl /+ lungs in sham mice and 3 days after PNX. Cropped gels are presented here with full gel available in Supplemental Fig. 8 . ( E ) qRT-PCR analysis showed significant changes in mRNA expression of several FOXF1 target genes in endothelial cells from PDGFb-iCre/Foxf1 fl /+ lungs. Cdkn2b was not detectable (N.D.) in control samples. *p
    Figure Legend Snippet: Identification of FOXF1 target genes in endothelial cells from regenerating lungs. ( A ) FACS-sorting strategy to isolate populations of endothelial (CD45 − CD31 + CD326 − ) and epithelial (CD45 − CD31 − CD326 + ) cells, showing GFP expression in PDGFb-iCre expressing mice. ( B ) Foxf1 mRNA was highly expressed in endothelial cells but not in epithelial cells. Endothelial cells from PDGFb-iCre/Foxf1 fl /+ mice had significantly less Foxf1 mRNA than endothelial cells from control mice. ( C ) Heat map showing significant changes in expression of 1047 genes in endothelial cells from Foxf1 fl /+ and PDGFb-iCre/Foxf1 fl /+ lungs after PNX. ( D ) Western blot analysis showed increased protein levels for CDKN1A (P21 Cip1 ) and CDKN2B (P15 Ink4b ) in PDGFb-iCre/Foxf1 fl /+ lungs in sham mice and 3 days after PNX. Cropped gels are presented here with full gel available in Supplemental Fig. 8 . ( E ) qRT-PCR analysis showed significant changes in mRNA expression of several FOXF1 target genes in endothelial cells from PDGFb-iCre/Foxf1 fl /+ lungs. Cdkn2b was not detectable (N.D.) in control samples. *p

    Techniques Used: FACS, Expressing, Mouse Assay, Western Blot, Quantitative RT-PCR

    19) Product Images from "A 3D Bioprinted in vitro Model of Neuroblastoma Recapitulates Dynamic Tumor‐Endothelial Cell Interactions Contributing to Solid Tumor Aggressive Behavior, A 3D Bioprinted in vitro Model of Neuroblastoma Recapitulates Dynamic Tumor‐Endothelial Cell Interactions Contributing to Solid Tumor Aggressive Behavior"

    Article Title: A 3D Bioprinted in vitro Model of Neuroblastoma Recapitulates Dynamic Tumor‐Endothelial Cell Interactions Contributing to Solid Tumor Aggressive Behavior, A 3D Bioprinted in vitro Model of Neuroblastoma Recapitulates Dynamic Tumor‐Endothelial Cell Interactions Contributing to Solid Tumor Aggressive Behavior

    Journal: Advanced Science

    doi: 10.1002/advs.202200244

    Evaluating HUVECs viability and growth within the 3D bioprinted gelMA channels. A) Live/dead results from various regions within the 3D channel structure on day 14 of culture demonstrate channel endothelialization ( n = 3). B) CD31 (red), connexin‐43 (CX43, green) and DAPI (blue) staining of HUVECs from longitudinal (left) and tangential/perpendicular (right) views. C) Quantification of Live/Dead assay at days 1, 4, 7, and 14 of culture ( n = 3). D) AlamarBlue assay to measure HUVECs metabolic activity, as a measure of cell viability and growth ( n = 4). E) Quantification of the fluorescence intensity of CD31 performed on the confocal images in B ( n = 3). Scale bars represent 200 µm. *: p
    Figure Legend Snippet: Evaluating HUVECs viability and growth within the 3D bioprinted gelMA channels. A) Live/dead results from various regions within the 3D channel structure on day 14 of culture demonstrate channel endothelialization ( n = 3). B) CD31 (red), connexin‐43 (CX43, green) and DAPI (blue) staining of HUVECs from longitudinal (left) and tangential/perpendicular (right) views. C) Quantification of Live/Dead assay at days 1, 4, 7, and 14 of culture ( n = 3). D) AlamarBlue assay to measure HUVECs metabolic activity, as a measure of cell viability and growth ( n = 4). E) Quantification of the fluorescence intensity of CD31 performed on the confocal images in B ( n = 3). Scale bars represent 200 µm. *: p

    Techniques Used: Staining, Live Dead Assay, Alamar Blue Assay, Activity Assay, Fluorescence

    NB‐HUVEC coculture in 3D bioprinted gelMA constructs. A) Schematic illustration of the coculture. B) Bright‐field images of NB spheroids cultured for 1 and 14 days in the gelMA constructs, in the absence (NB‐only) and presence of HUVECs (NB‐HUVEC). C) Quantification of NB spheroids shape, i.e., the spheroid diameter, perimeter, and circularity of spheroids cultured in suspension (control) versus those grown in bioprinted models with or without HUVECs ( n = 6 per group). D,E) Immunohistochemical imaging of the cocultured constructs at days 7 (D) and 14 (E). Confocal images show the staining results for EC‐specific CD31 (red), synaptophysin (green) and DAPI (blue). White arrows point to the NB invasion into the gelMA, and yellow arrows highlight EC infiltration into the spheroid. The gelMA boundary is depicted by dotted white line. F–H) Quantification of synaptophysin (F), NB cell invasion distance into the gelMA (G), and EC infiltration distance into the cancer spheroid (H), based on the confocal images ( n = 3). I) Quantification of NB cell invasion extent into the gelMA matrix in monoculture (NB‐only) versus coculture (NB‐HUVEC) at day 14 of culture, reported as %area of gelMA tissue outside the spheroid ( n = 3). Scale bars in (B) represent 500 µm and in (D) and (E) represent 200 µm. *: p
    Figure Legend Snippet: NB‐HUVEC coculture in 3D bioprinted gelMA constructs. A) Schematic illustration of the coculture. B) Bright‐field images of NB spheroids cultured for 1 and 14 days in the gelMA constructs, in the absence (NB‐only) and presence of HUVECs (NB‐HUVEC). C) Quantification of NB spheroids shape, i.e., the spheroid diameter, perimeter, and circularity of spheroids cultured in suspension (control) versus those grown in bioprinted models with or without HUVECs ( n = 6 per group). D,E) Immunohistochemical imaging of the cocultured constructs at days 7 (D) and 14 (E). Confocal images show the staining results for EC‐specific CD31 (red), synaptophysin (green) and DAPI (blue). White arrows point to the NB invasion into the gelMA, and yellow arrows highlight EC infiltration into the spheroid. The gelMA boundary is depicted by dotted white line. F–H) Quantification of synaptophysin (F), NB cell invasion distance into the gelMA (G), and EC infiltration distance into the cancer spheroid (H), based on the confocal images ( n = 3). I) Quantification of NB cell invasion extent into the gelMA matrix in monoculture (NB‐only) versus coculture (NB‐HUVEC) at day 14 of culture, reported as %area of gelMA tissue outside the spheroid ( n = 3). Scale bars in (B) represent 500 µm and in (D) and (E) represent 200 µm. *: p

    Techniques Used: Construct, Cell Culture, Immunohistochemistry, Imaging, Staining

    20) Product Images from "CD8+ T Cell Responses Against Hemoglobin-? Prevent Solid Tumor Growth"

    Article Title: CD8+ T Cell Responses Against Hemoglobin-? Prevent Solid Tumor Growth

    Journal:

    doi: 10.1158/0008-5472.CAN-08-0387

    Regressing CMS4 tumors in HBB peptide vaccinated mice exhibit elevated CD8+ T cell infiltration and reduced expression of CD31 VEC, SMA+ pericytes and HBB protein in situ
    Figure Legend Snippet: Regressing CMS4 tumors in HBB peptide vaccinated mice exhibit elevated CD8+ T cell infiltration and reduced expression of CD31 VEC, SMA+ pericytes and HBB protein in situ

    Techniques Used: Mouse Assay, Expressing, In Situ

    21) Product Images from "Clinicopathologic features of Stewart-Treves syndrome"

    Article Title: Clinicopathologic features of Stewart-Treves syndrome

    Journal: International Journal of Clinical and Experimental Pathology

    doi:

    Positive expression of CD31 (IHC ×200).
    Figure Legend Snippet: Positive expression of CD31 (IHC ×200).

    Techniques Used: Expressing, Immunohistochemistry

    22) Product Images from "ERG Immunohistochemistry as an Endothelial Marker for Assessing Lymphovascular Invasion"

    Article Title: ERG Immunohistochemistry as an Endothelial Marker for Assessing Lymphovascular Invasion

    Journal: Korean Journal of Pathology

    doi: 10.4132/KoreanJPathol.2013.47.4.355

    Comparison of kappa values among pathologists for lymphovascular invasion (LVI) detection in colorectal cancers. While the average of LVI detection rate for each pathologist was 43% with hematoxylin and eosin (H E) only, 10% with CD31, 29% with D2-40, and 16% with ERG, the consensus reached 80% of LVI detection after a joint discussion about ERG patterns with LVI. a Interpreted by ERG
    Figure Legend Snippet: Comparison of kappa values among pathologists for lymphovascular invasion (LVI) detection in colorectal cancers. While the average of LVI detection rate for each pathologist was 43% with hematoxylin and eosin (H E) only, 10% with CD31, 29% with D2-40, and 16% with ERG, the consensus reached 80% of LVI detection after a joint discussion about ERG patterns with LVI. a Interpreted by ERG

    Techniques Used:

    Comparison of ERG, CD31, and D2-40 endothelial markers. (A) ERG, panendothelial marker showing nuclear immunoreactivity in artery, vein, and lymphatics. (B) ERG immunostaining specific for endothelial cells without cross-reactivity. (C) D31 immunostaining showing cross-reactivity in inflammatory cells. (D) D2-40 immunostaining showing cross-reactivity in fibroblasts.
    Figure Legend Snippet: Comparison of ERG, CD31, and D2-40 endothelial markers. (A) ERG, panendothelial marker showing nuclear immunoreactivity in artery, vein, and lymphatics. (B) ERG immunostaining specific for endothelial cells without cross-reactivity. (C) D31 immunostaining showing cross-reactivity in inflammatory cells. (D) D2-40 immunostaining showing cross-reactivity in fibroblasts.

    Techniques Used: Marker, Immunostaining

    23) Product Images from "CD44 Regulation of Endothelial Cell Proliferation and Apoptosis via Modulation of CD31 and VE-cadherin Expression *"

    Article Title: CD44 Regulation of Endothelial Cell Proliferation and Apoptosis via Modulation of CD31 and VE-cadherin Expression *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.529313

    CD44 and CD31 play a key role in endothelial cell proliferation. A , growth curves of WT ( red squares ), CD44KO-BEC ( blue triangles ), WT-BEC-VO ( pink triangles ), CD44KO-BEC-VO ( green diamonds ), CD44KO-BEC-mCD44 ( orange circles ), CD44KO-BEC-mCD31 ( black
    Figure Legend Snippet: CD44 and CD31 play a key role in endothelial cell proliferation. A , growth curves of WT ( red squares ), CD44KO-BEC ( blue triangles ), WT-BEC-VO ( pink triangles ), CD44KO-BEC-VO ( green diamonds ), CD44KO-BEC-mCD44 ( orange circles ), CD44KO-BEC-mCD31 ( black

    Techniques Used:

    Immunofluorescence evidence of CD44 and CD31 playing key roles in endothelial cell proliferation by modulating Hippo pathway activation. Shown are phase ( top row ) and merged DAPI and CD31, DAPI and VE-cadherin, DAPI and F-actin, DAPI and survivin, and
    Figure Legend Snippet: Immunofluorescence evidence of CD44 and CD31 playing key roles in endothelial cell proliferation by modulating Hippo pathway activation. Shown are phase ( top row ) and merged DAPI and CD31, DAPI and VE-cadherin, DAPI and F-actin, DAPI and survivin, and

    Techniques Used: Immunofluorescence, Activation Assay

    Western blotting evidence of CD44 and CD31 playing key roles in endothelial cell proliferation by modulating Hippo pathway activation. Shown are Western blotting and densitometric analyses of lysates of WT-BEC-VO, CD44KO-BEC-VO, CD44KO-BEC-mCD44, CD44KO-BEC-mCD31
    Figure Legend Snippet: Western blotting evidence of CD44 and CD31 playing key roles in endothelial cell proliferation by modulating Hippo pathway activation. Shown are Western blotting and densitometric analyses of lysates of WT-BEC-VO, CD44KO-BEC-VO, CD44KO-BEC-mCD44, CD44KO-BEC-mCD31

    Techniques Used: Western Blot, Activation Assay

    Working model for the involvement of CD44 and CD31 in the modulation of proliferation and active caspase cascades in endothelial cells. A , in WT-BEC, in the presence of CD44, there is appropriate adherens junction formation that facilitates Merlin interactions
    Figure Legend Snippet: Working model for the involvement of CD44 and CD31 in the modulation of proliferation and active caspase cascades in endothelial cells. A , in WT-BEC, in the presence of CD44, there is appropriate adherens junction formation that facilitates Merlin interactions

    Techniques Used:

    CD31 extracellular domain plays an important role in endothelial cell morphology and proliferation as well as caspase-3 and -8 activation. A , WT brain endothelial cells were incubated without ( left column ) and with 5 μg/ml prebleed ( PB ) rabbit
    Figure Legend Snippet: CD31 extracellular domain plays an important role in endothelial cell morphology and proliferation as well as caspase-3 and -8 activation. A , WT brain endothelial cells were incubated without ( left column ) and with 5 μg/ml prebleed ( PB ) rabbit

    Techniques Used: Activation Assay, Incubation

    24) Product Images from "Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors"

    Article Title: Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors

    Journal: bioRxiv

    doi: 10.1101/660977

    OT1-iT cell therapy suppresses the solid tumor growth in mice transplanted with E.G7-OVA cells a Schematic diagram of OT1 engineered iT cells for anti-tumor therapy. Mouse MEF cells were isolated from CD45.2 + C57BL/6 mouse and reprogrammed into iPSC with Oct4, Klf4, and Sox2 retro-viruses. Then a rtTA-TRE-Runx1-Hoxa9-HygroR DNA cassette was inserted into the Rosa26 locus . Next, a CAG-OT1-IRES-GFP-PuroR expression element was inserted into the Hipp11 locus of iR9 -iPSC. OT1- iR9 -iPSC results in the production of CD8 + T cells carrying TCRVα2 and TCRVβ5 (MHC class I-restricted, ovalbumin-specific TCR). OT1- iR9 -iPSC-derived iHEC were induced into iHPC (OT1-iHPC) as described in material and method sections. The iHPC were injected into irradiated (4.5 Gy) Rag1 −/− recipient mice (3 million/mouse, 8-10-week-old C57BL/6 background). E.G7-OVA tumor cell line (C57BL/6 background) were transplanted into the groin of the Rag1 −/− (n = 8) or OT1-iT- Rag1 −/− (n = 8) by subcutaneous injection (0.2 million/mouse) six weeks after OT1-iHPC transplantation. b TCRVα2 and TCRVβ5 expression in OT1- iR9 -iPSC measured by intracellular staining. The iR9 -iPSC was used as negative control. c Sorting gates of the OT1 - iR9 -iPSC-derived iHEC population at day 11. The cells were enriched by streptavidin-beads recognizing biotin-CD31 before sorting. Representative plots from three independent experiments are shown. d Immuno-phenotypes of pre-thymic progenitors in induced hematopoietic progenitor cells from OT1 - iR9 -iPSC-derived iHEC after ten-day maturation. Representative plots from three independent experiments are shown. Lin was defined as CD2 − CD3 − CD4 − CD8 − CD11b − Gr1 − Ter119 − CD19 − NK1.1 − TCRγδ − . pre-thymic progenitors were defined as Lin − c-kit + CD127 + /CD135 + . e TCRVα2 and TCRVβ5 expression of iT cells in PB of Rag1 −/− mice 6 weeks after transplantation of OT1 - iR9 -iPSC-derived iHPC. Three representative mice from three independent experiments were analyzed. f Tumor growth in Rag1 −/− and OT1-iT- Rag1 −/− mice. E.G7-OVA cells were transplanted into the groin of the Rag1 −/− (n = 8) or OT1-iT- Rag1 −/− mice (n = 8) by subcutaneous injection (0.2 million/mouse). The length and width of the tumors were measured every other day by a caliper, and each tumor size was calculated as length × width (mm 2 ). Mice with tumor size larger than 20 mm at the longest axis were euthanized for ethical consideration. *** P
    Figure Legend Snippet: OT1-iT cell therapy suppresses the solid tumor growth in mice transplanted with E.G7-OVA cells a Schematic diagram of OT1 engineered iT cells for anti-tumor therapy. Mouse MEF cells were isolated from CD45.2 + C57BL/6 mouse and reprogrammed into iPSC with Oct4, Klf4, and Sox2 retro-viruses. Then a rtTA-TRE-Runx1-Hoxa9-HygroR DNA cassette was inserted into the Rosa26 locus . Next, a CAG-OT1-IRES-GFP-PuroR expression element was inserted into the Hipp11 locus of iR9 -iPSC. OT1- iR9 -iPSC results in the production of CD8 + T cells carrying TCRVα2 and TCRVβ5 (MHC class I-restricted, ovalbumin-specific TCR). OT1- iR9 -iPSC-derived iHEC were induced into iHPC (OT1-iHPC) as described in material and method sections. The iHPC were injected into irradiated (4.5 Gy) Rag1 −/− recipient mice (3 million/mouse, 8-10-week-old C57BL/6 background). E.G7-OVA tumor cell line (C57BL/6 background) were transplanted into the groin of the Rag1 −/− (n = 8) or OT1-iT- Rag1 −/− (n = 8) by subcutaneous injection (0.2 million/mouse) six weeks after OT1-iHPC transplantation. b TCRVα2 and TCRVβ5 expression in OT1- iR9 -iPSC measured by intracellular staining. The iR9 -iPSC was used as negative control. c Sorting gates of the OT1 - iR9 -iPSC-derived iHEC population at day 11. The cells were enriched by streptavidin-beads recognizing biotin-CD31 before sorting. Representative plots from three independent experiments are shown. d Immuno-phenotypes of pre-thymic progenitors in induced hematopoietic progenitor cells from OT1 - iR9 -iPSC-derived iHEC after ten-day maturation. Representative plots from three independent experiments are shown. Lin was defined as CD2 − CD3 − CD4 − CD8 − CD11b − Gr1 − Ter119 − CD19 − NK1.1 − TCRγδ − . pre-thymic progenitors were defined as Lin − c-kit + CD127 + /CD135 + . e TCRVα2 and TCRVβ5 expression of iT cells in PB of Rag1 −/− mice 6 weeks after transplantation of OT1 - iR9 -iPSC-derived iHPC. Three representative mice from three independent experiments were analyzed. f Tumor growth in Rag1 −/− and OT1-iT- Rag1 −/− mice. E.G7-OVA cells were transplanted into the groin of the Rag1 −/− (n = 8) or OT1-iT- Rag1 −/− mice (n = 8) by subcutaneous injection (0.2 million/mouse). The length and width of the tumors were measured every other day by a caliper, and each tumor size was calculated as length × width (mm 2 ). Mice with tumor size larger than 20 mm at the longest axis were euthanized for ethical consideration. *** P

    Techniques Used: Mouse Assay, Isolation, Expressing, Derivative Assay, Injection, Irradiation, Transplantation Assay, Staining, Negative Control

    Single-cell transcriptomic characterization of iHEC and iHPC a Principal component analysis (PCA) of iHEC and developmental E11 AGM-derived EC, T1 pre-HSC, T2 pre-HSC, E12 HSC, E14 HSC, and adult HSC. The TPM values of iHEC (n = 70), natural E11 AGM-derived EC (n = 17), T1 pre-HSC (n = 28), T2 pre-HSC (n = 32), E12 HSC (n = 21), E14 HSC (n = 32) and adult HSC (n = 47) single-cell RNA-Seq data were calculated with Stringtie package. b The expression of the top 100 genes contributing most to PC2 (50 genes for each direction). The expression value (TPM) of each gene was converted by log2 and illustrated by pheatmap (R package). One column represents one cell repeat. c Violin plots show the expression profile of selected artery (A) and vein (V) related genes (A: Nrp1 , Efnb2 , and Hey1 ; V: Nrp2 , Nr2f2 , and Ephb4 ) in single iHEC. The expression value (TPM) of each gene was converted by log2 and illustrated by ggplot2 (R package). One point represents one cell. d Violin plots show the expression profile of selected surface markers ( Cdh5 , Esam , Tek , Procr , Cd47 , and Cd63 ) in single iHEC. The expression value (TPM) of each gene was converted by log2 and illustrated by ggplot2 (R package). One point represents one cell. e Violin plots show the expression profile of selected transcription factors ( Fli1 , Erg1 , Lmo2 , Lyl1 , Tal1 , Sox7 , Runx1 , Mycn , Gata2 , Bcl11a , Hoxa9 , and Hoxb5 ) related to hematopoietic development in single iHEC. The expression value (TPM) of each gene was converted by log2 and illustrated by ggplot2 (R package). One point represents one cell. f Two-dimensional tSNE analysis of iHEC and iHPC single-cell RNA-Seq. For single-cell RNA-Seq, the iHEC were collected on day 11, and the iHPC were collected at Day14, 17 and 21. Each dot represents one cell. The TPM values of iHEC (n = 65), iHPC at Day14 (n = 21), Day17 (n = 18) and Day21 (n = 56) from single-cell RNA-Seq data were calculated with Stringtie package. Cell types were defined as: iHEC CD31 + CD41 low CD45 − c-kit + CD201 high ; Day14 and Day17 iHPC, CD45 + Lin (Ter119/Gr1/F4-80/CD2/CD3/CD4/CD8/CD19/FcεRIα) − ; Day21 iHPC Ter119 − CD45 + c-kit + CD127 + . g tSNE analysis of the expression pattern of selected endothelia-related transcription factors ( Sox7 , Sox18 , and Ets1 ) in iHEC and iHPC. h tSNE analysis of the expression pattern of selected hematopoietic-related transcription factors ( Lyl1 , Etv6 , Prdm5 , Myb , Sfpi1 , and Meis1 ) in iHEC and iHPC. i tSNE analysis of the expression pattern of selected T cell development-related transcription factors ( Lmo2 , Bcl11a , Ikzf1 , Myc , Gata3 , and Tcf7 ) in iHEC and iHPC at Day14, Day17, and Day21. j tSNE analysis of the expression pattern of selected lymphopoiesis-related surface protein-coding genes ( Kit , Flt3 , Cd7 , Ccr9 , Ccr7 , and Cxcr4 ) in iHEC and iHPC at Day14, Day17, and Day21.
    Figure Legend Snippet: Single-cell transcriptomic characterization of iHEC and iHPC a Principal component analysis (PCA) of iHEC and developmental E11 AGM-derived EC, T1 pre-HSC, T2 pre-HSC, E12 HSC, E14 HSC, and adult HSC. The TPM values of iHEC (n = 70), natural E11 AGM-derived EC (n = 17), T1 pre-HSC (n = 28), T2 pre-HSC (n = 32), E12 HSC (n = 21), E14 HSC (n = 32) and adult HSC (n = 47) single-cell RNA-Seq data were calculated with Stringtie package. b The expression of the top 100 genes contributing most to PC2 (50 genes for each direction). The expression value (TPM) of each gene was converted by log2 and illustrated by pheatmap (R package). One column represents one cell repeat. c Violin plots show the expression profile of selected artery (A) and vein (V) related genes (A: Nrp1 , Efnb2 , and Hey1 ; V: Nrp2 , Nr2f2 , and Ephb4 ) in single iHEC. The expression value (TPM) of each gene was converted by log2 and illustrated by ggplot2 (R package). One point represents one cell. d Violin plots show the expression profile of selected surface markers ( Cdh5 , Esam , Tek , Procr , Cd47 , and Cd63 ) in single iHEC. The expression value (TPM) of each gene was converted by log2 and illustrated by ggplot2 (R package). One point represents one cell. e Violin plots show the expression profile of selected transcription factors ( Fli1 , Erg1 , Lmo2 , Lyl1 , Tal1 , Sox7 , Runx1 , Mycn , Gata2 , Bcl11a , Hoxa9 , and Hoxb5 ) related to hematopoietic development in single iHEC. The expression value (TPM) of each gene was converted by log2 and illustrated by ggplot2 (R package). One point represents one cell. f Two-dimensional tSNE analysis of iHEC and iHPC single-cell RNA-Seq. For single-cell RNA-Seq, the iHEC were collected on day 11, and the iHPC were collected at Day14, 17 and 21. Each dot represents one cell. The TPM values of iHEC (n = 65), iHPC at Day14 (n = 21), Day17 (n = 18) and Day21 (n = 56) from single-cell RNA-Seq data were calculated with Stringtie package. Cell types were defined as: iHEC CD31 + CD41 low CD45 − c-kit + CD201 high ; Day14 and Day17 iHPC, CD45 + Lin (Ter119/Gr1/F4-80/CD2/CD3/CD4/CD8/CD19/FcεRIα) − ; Day21 iHPC Ter119 − CD45 + c-kit + CD127 + . g tSNE analysis of the expression pattern of selected endothelia-related transcription factors ( Sox7 , Sox18 , and Ets1 ) in iHEC and iHPC. h tSNE analysis of the expression pattern of selected hematopoietic-related transcription factors ( Lyl1 , Etv6 , Prdm5 , Myb , Sfpi1 , and Meis1 ) in iHEC and iHPC. i tSNE analysis of the expression pattern of selected T cell development-related transcription factors ( Lmo2 , Bcl11a , Ikzf1 , Myc , Gata3 , and Tcf7 ) in iHEC and iHPC at Day14, Day17, and Day21. j tSNE analysis of the expression pattern of selected lymphopoiesis-related surface protein-coding genes ( Kit , Flt3 , Cd7 , Ccr9 , Ccr7 , and Cxcr4 ) in iHEC and iHPC at Day14, Day17, and Day21.

    Techniques Used: Derivative Assay, RNA Sequencing Assay, Expressing

    25) Product Images from "Clinical and imaging outcomes after intrathecal injection of umbilical cord tissue mesenchymal stem cells in cerebral palsy: a randomized double-blind sham-controlled clinical trial"

    Article Title: Clinical and imaging outcomes after intrathecal injection of umbilical cord tissue mesenchymal stem cells in cerebral palsy: a randomized double-blind sham-controlled clinical trial

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-021-02513-4

    The flow cytometry analysis of UCT-MSC-specific markers. The first three photos ( a , b , c ) shows the cell gating to select single live UCT-MSCs. Othe photos ( d - l ) shows that the cells were negative for CD11b, CD31, CD34, CD45, and HLA-DR and were positive for CD29, CD73, CD90, and CD105
    Figure Legend Snippet: The flow cytometry analysis of UCT-MSC-specific markers. The first three photos ( a , b , c ) shows the cell gating to select single live UCT-MSCs. Othe photos ( d - l ) shows that the cells were negative for CD11b, CD31, CD34, CD45, and HLA-DR and were positive for CD29, CD73, CD90, and CD105

    Techniques Used: Flow Cytometry

    26) Product Images from "Overexpression of Wnt5a Promotes Angiogenesis in NSCLC"

    Article Title: Overexpression of Wnt5a Promotes Angiogenesis in NSCLC

    Journal: BioMed Research International

    doi: 10.1155/2014/832562

    The angiogenesis status in NSCLC. (a) MVD staining for CD34 in NSCLC (immunohistochemical staining, ×200). A hotspot with high MVD was positively stained. (b) CD31/PAS double staining for VM (×400). The VM channel showed a positive expression for PAS but a negative expression for CD31 (red arrow). The endothelial channel showed positive expressions for both CD31 and PAS (yellow arrow).
    Figure Legend Snippet: The angiogenesis status in NSCLC. (a) MVD staining for CD34 in NSCLC (immunohistochemical staining, ×200). A hotspot with high MVD was positively stained. (b) CD31/PAS double staining for VM (×400). The VM channel showed a positive expression for PAS but a negative expression for CD31 (red arrow). The endothelial channel showed positive expressions for both CD31 and PAS (yellow arrow).

    Techniques Used: Staining, Immunohistochemistry, Double Staining, Expressing

    27) Product Images from "Tissue-Engineered Heart Valve with a Tubular Leaflet Design for Minimally Invasive Transcatheter Implantation"

    Article Title: Tissue-Engineered Heart Valve with a Tubular Leaflet Design for Minimally Invasive Transcatheter Implantation

    Journal: Tissue Engineering. Part C, Methods

    doi: 10.1089/ten.tec.2014.0214

    Tissue analysis by conventional hematoxylin and eosin (H E) (AA and AC) and Gomori's Trichrome stainings (AB and AD) and immunohistochemistry of tissue-engineered uncrimped and crimped valves. Immunohistochemical staining against alpha-smooth muscle actin (alpha-SMA) (A, G, and M) , collagen type I (B, H, and N) , collagen type III (C, I, and O) , elastin (D, J, and P) , fibronectin (E, K, and Q) , and chondroitin sulfate (F, L, and R) . A human aortic valve was used as a positive control (M–R, AE, and AF) . Endothelium analysis by SEM (AG and AJ , inner surface ) and immunohistochemical staining against CD31 (AH and AK) for uncrimped and crimped valves. White lines (A and G) indicate the reference for the cell alignment measurement. White arrows (AH and AK) indicate the inner surface of the valve. Black arrows (AA and AB) indicate the textile mesh. Negative controls (S–X, AI, and AL) for all markers reacted in the absence of the primary antibody showed undetectable levels of staining. Scale bars
    Figure Legend Snippet: Tissue analysis by conventional hematoxylin and eosin (H E) (AA and AC) and Gomori's Trichrome stainings (AB and AD) and immunohistochemistry of tissue-engineered uncrimped and crimped valves. Immunohistochemical staining against alpha-smooth muscle actin (alpha-SMA) (A, G, and M) , collagen type I (B, H, and N) , collagen type III (C, I, and O) , elastin (D, J, and P) , fibronectin (E, K, and Q) , and chondroitin sulfate (F, L, and R) . A human aortic valve was used as a positive control (M–R, AE, and AF) . Endothelium analysis by SEM (AG and AJ , inner surface ) and immunohistochemical staining against CD31 (AH and AK) for uncrimped and crimped valves. White lines (A and G) indicate the reference for the cell alignment measurement. White arrows (AH and AK) indicate the inner surface of the valve. Black arrows (AA and AB) indicate the textile mesh. Negative controls (S–X, AI, and AL) for all markers reacted in the absence of the primary antibody showed undetectable levels of staining. Scale bars

    Techniques Used: Immunohistochemistry, Staining, Positive Control

    28) Product Images from "Shear Stress Regulates Late EPC Differentiation via Mechanosensitive Molecule-Mediated Cytoskeletal Rearrangement"

    Article Title: Shear Stress Regulates Late EPC Differentiation via Mechanosensitive Molecule-Mediated Cytoskeletal Rearrangement

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067675

    The role of FAK in the shear stress-induced cytoskeletal rearrangement and differentiation in late EPCs. (A) Western blot was carried out with specific antibody for checking the phosphorylated FAK. The total FAK served as loading control. (B) Late EPCs were pretreated with PF-573,228 (2 µmol/l) for 1 h. The cells were then either exposed to shear stress (12 dyne/cm 2 ) for 60 min, or cultured in static condition. After this, F-actin was stained with FITC-Phalloidin. Bars: 100 µm. (C) Stress fibers were quantitated and normalized to the shear stress treated-EPCs. (D) Late EPCs were pretreated with PF-573,228 (2 µmol/l) for 1 h, and were then either exposed to shear stress (12 dyne/cm 2 ) for 3 h, or cultured in static condition. The gene expression of vWF and CD31 was determined by real time RT-PCR. (E) Late EPCs were pretreated with PF-573,228 (2 µmol/l) for 1 h, and the cells were then either exposed to shear stress (12 dyne/cm 2 ) for 24 h, or cultured in static condition for the same duration. The protein levels of vWF and CD31 were determined by FACS. The results represent the mean±SE from three independent experiments. **(P
    Figure Legend Snippet: The role of FAK in the shear stress-induced cytoskeletal rearrangement and differentiation in late EPCs. (A) Western blot was carried out with specific antibody for checking the phosphorylated FAK. The total FAK served as loading control. (B) Late EPCs were pretreated with PF-573,228 (2 µmol/l) for 1 h. The cells were then either exposed to shear stress (12 dyne/cm 2 ) for 60 min, or cultured in static condition. After this, F-actin was stained with FITC-Phalloidin. Bars: 100 µm. (C) Stress fibers were quantitated and normalized to the shear stress treated-EPCs. (D) Late EPCs were pretreated with PF-573,228 (2 µmol/l) for 1 h, and were then either exposed to shear stress (12 dyne/cm 2 ) for 3 h, or cultured in static condition. The gene expression of vWF and CD31 was determined by real time RT-PCR. (E) Late EPCs were pretreated with PF-573,228 (2 µmol/l) for 1 h, and the cells were then either exposed to shear stress (12 dyne/cm 2 ) for 24 h, or cultured in static condition for the same duration. The protein levels of vWF and CD31 were determined by FACS. The results represent the mean±SE from three independent experiments. **(P

    Techniques Used: Western Blot, Cell Culture, Staining, Expressing, Quantitative RT-PCR, FACS

    Ras was essential for the shear stress-induced cell differentiation associated with cytoskeletal rearrangement in late EPCs. (A) Late EPCs were transfected with RasN17 by the Lipofectamin 2000. The transfected late EPCs were then subjected to shear stress (12 dyne/cm 2 ) for 60 min. F-actin was stained with FITC-Phalloidin. Bars: 100 µm. (B) Stress fibers were quantitated and normalized to the shear stress-treated EPCs. (C) Late EPCs were transfected either with control vector or with RasN17. The transfected late EPCs were then subjected to shear stress (12 dyne/cm 2 ) for 3 h. The gene expression of vWF and CD31 was determined by real time RT-PCR. (D) Late EPCs were transfected either with control vector or with RasN17, and the transfected late EPCs were then subjected to shear stress (12 dyne/cm 2 ) for 24 h, or cultured in static condition. The protein levels of vWF and CD31 were determined by FACS. The results represent the mean±SE from three independent experiments. **(P
    Figure Legend Snippet: Ras was essential for the shear stress-induced cell differentiation associated with cytoskeletal rearrangement in late EPCs. (A) Late EPCs were transfected with RasN17 by the Lipofectamin 2000. The transfected late EPCs were then subjected to shear stress (12 dyne/cm 2 ) for 60 min. F-actin was stained with FITC-Phalloidin. Bars: 100 µm. (B) Stress fibers were quantitated and normalized to the shear stress-treated EPCs. (C) Late EPCs were transfected either with control vector or with RasN17. The transfected late EPCs were then subjected to shear stress (12 dyne/cm 2 ) for 3 h. The gene expression of vWF and CD31 was determined by real time RT-PCR. (D) Late EPCs were transfected either with control vector or with RasN17, and the transfected late EPCs were then subjected to shear stress (12 dyne/cm 2 ) for 24 h, or cultured in static condition. The protein levels of vWF and CD31 were determined by FACS. The results represent the mean±SE from three independent experiments. **(P

    Techniques Used: Cell Differentiation, Transfection, Staining, Plasmid Preparation, Expressing, Quantitative RT-PCR, Cell Culture, FACS

    The shear stress-induced endothelial marker expression was dependent on the cytoskeletal rearrangement in late EPCs. (A) Late EPCs were kept in static condition or exposed to shear stress at 12 dyne/cm 2 for 5, 30 or 60 min, and stained with FITC-Phalloidin to detect actin stress fibers. Bars: 100 µm. (B) Stress fibers were quantitated and normalized to the static control group. (C–D) Late EPCs were pretreated with Cyto D (1 µmol/l) for 30 min. The treated cells were then either subjected to shear stress (12 dyne/cm 2 ) for 24 h, or cultured in static conditions. The protein levels of vWF and CD31 were determined by FACS. (E) Late EPCs were pretreated with Cyto D (1 µmol/l) for 30 min. The treated cells were then either subjected to shear stress (12 dyne/cm 2 ) for 24 h, or cultured in static conditions. The protein expression of vWF and CD31 were determined by immunoreactivity. Bars: 200 µm. Data represent the mean±SE from three separate experiments. **(P
    Figure Legend Snippet: The shear stress-induced endothelial marker expression was dependent on the cytoskeletal rearrangement in late EPCs. (A) Late EPCs were kept in static condition or exposed to shear stress at 12 dyne/cm 2 for 5, 30 or 60 min, and stained with FITC-Phalloidin to detect actin stress fibers. Bars: 100 µm. (B) Stress fibers were quantitated and normalized to the static control group. (C–D) Late EPCs were pretreated with Cyto D (1 µmol/l) for 30 min. The treated cells were then either subjected to shear stress (12 dyne/cm 2 ) for 24 h, or cultured in static conditions. The protein levels of vWF and CD31 were determined by FACS. (E) Late EPCs were pretreated with Cyto D (1 µmol/l) for 30 min. The treated cells were then either subjected to shear stress (12 dyne/cm 2 ) for 24 h, or cultured in static conditions. The protein expression of vWF and CD31 were determined by immunoreactivity. Bars: 200 µm. Data represent the mean±SE from three separate experiments. **(P

    Techniques Used: Marker, Expressing, Staining, Cell Culture, FACS

    The shear stress-induced EPC differentiation associated with cytoskeletal rearrangement was mediated via the Ras/ERK1/2– dependent signal pathway. (A) Western blot was carried out with specific antibody for checking the phosphorylated ERK1/2. The total ERK1/2 served as loading control. (B) Late EPCs were transfected with RasN17. Transfected late EPCs were then subjected to shear stress (12 dyne/cm 2 ) for 5 min. The activation of ERK1/2 was analyzed by Western blot. (C) Late EPCs were pretreated with PD98059 (10 µmol/l) for 30 min. The cells were then either exposed to shear stress (12 dyne/cm 2 ) for 3 h, or cultured in static condition. After this, the vWF and CD31 mRNA expression was determined using real time RT-PCR. (D) Late EPCs were pretreated with PD98059 (10 µmol/l) for 30 min, and were then either exposed to shear stress (12 dyne/cm 2 ) for 24 h, or cultured in static condition. The protein levels of vWF and CD31 were determined by FACS. The results represent the mean±SE from three independent experiments. **(P
    Figure Legend Snippet: The shear stress-induced EPC differentiation associated with cytoskeletal rearrangement was mediated via the Ras/ERK1/2– dependent signal pathway. (A) Western blot was carried out with specific antibody for checking the phosphorylated ERK1/2. The total ERK1/2 served as loading control. (B) Late EPCs were transfected with RasN17. Transfected late EPCs were then subjected to shear stress (12 dyne/cm 2 ) for 5 min. The activation of ERK1/2 was analyzed by Western blot. (C) Late EPCs were pretreated with PD98059 (10 µmol/l) for 30 min. The cells were then either exposed to shear stress (12 dyne/cm 2 ) for 3 h, or cultured in static condition. After this, the vWF and CD31 mRNA expression was determined using real time RT-PCR. (D) Late EPCs were pretreated with PD98059 (10 µmol/l) for 30 min, and were then either exposed to shear stress (12 dyne/cm 2 ) for 24 h, or cultured in static condition. The protein levels of vWF and CD31 were determined by FACS. The results represent the mean±SE from three independent experiments. **(P

    Techniques Used: Western Blot, Transfection, Activation Assay, Cell Culture, Expressing, Quantitative RT-PCR, FACS

    Paxillin was necessary for the shear stress-induced differentiation associated with cytoskeletal rearrangement in late EPCs. (A) Western blot was carried out with specific antibody for checking the phosphorylated paxillin. The total paxillin served as loading control. (B) Late EPCs were kept in static condition or exposed to shear stress at 12 dyne/cm 2 for 60 min. Paxillin was stained with specific antibody. Bars: 50 µm. (C) Late EPCs were transfected either with scrambled siRNA or with paxillin siRNA by the Lipofectamin 2000. The cells were then either exposed to shear stress (12 dyne/cm 2 ) for 3 h, or cultured in static condition. The gene expression of vWF and CD31 was determined by real time RT-PCR. (D) The cells were either exposed to shear stress (12 dyne/cm 2 ) for 24 h, or cultured in static condition. The protein levels of vWF and CD31 were determined by FACS. (E) Late EPCs were transfected either with scrambled siRNA or paxillin siRNA by the Lipofectamin 2000. Transfected late EPCs were then subjected to shear stress (12 dyne/cm 2 ) for 60 min. F-actin was stained with FITC-Phalloidin. Bars: 100 µm. (F) Stress fibers were quantitated and normalized to the shear stress treated-EPCs. The results represent the mean±SE from three independent experiments. **(P
    Figure Legend Snippet: Paxillin was necessary for the shear stress-induced differentiation associated with cytoskeletal rearrangement in late EPCs. (A) Western blot was carried out with specific antibody for checking the phosphorylated paxillin. The total paxillin served as loading control. (B) Late EPCs were kept in static condition or exposed to shear stress at 12 dyne/cm 2 for 60 min. Paxillin was stained with specific antibody. Bars: 50 µm. (C) Late EPCs were transfected either with scrambled siRNA or with paxillin siRNA by the Lipofectamin 2000. The cells were then either exposed to shear stress (12 dyne/cm 2 ) for 3 h, or cultured in static condition. The gene expression of vWF and CD31 was determined by real time RT-PCR. (D) The cells were either exposed to shear stress (12 dyne/cm 2 ) for 24 h, or cultured in static condition. The protein levels of vWF and CD31 were determined by FACS. (E) Late EPCs were transfected either with scrambled siRNA or paxillin siRNA by the Lipofectamin 2000. Transfected late EPCs were then subjected to shear stress (12 dyne/cm 2 ) for 60 min. F-actin was stained with FITC-Phalloidin. Bars: 100 µm. (F) Stress fibers were quantitated and normalized to the shear stress treated-EPCs. The results represent the mean±SE from three independent experiments. **(P

    Techniques Used: Western Blot, Staining, Transfection, Cell Culture, Expressing, Quantitative RT-PCR, FACS

    29) Product Images from "Angiotensin Converting Enzyme 2 Priming Enhances the Function of Endothelial Progenitor Cells and their Therapeutic Efficacy"

    Article Title: Angiotensin Converting Enzyme 2 Priming Enhances the Function of Endothelial Progenitor Cells and their Therapeutic Efficacy

    Journal: Hypertension

    doi: 10.1161/HYPERTENSIONAHA.111.00202

    The phenotype of cultured EPCs. Representative flow plot showing the expression of CD34, VEGFR2, CD133, CD31, VE-Cadherin, vWF, CD45 and CD146 on the EPCs cultured from R-A- (A) and R+A+ mice (B).
    Figure Legend Snippet: The phenotype of cultured EPCs. Representative flow plot showing the expression of CD34, VEGFR2, CD133, CD31, VE-Cadherin, vWF, CD45 and CD146 on the EPCs cultured from R-A- (A) and R+A+ mice (B).

    Techniques Used: Cell Culture, Flow Cytometry, Expressing, Mouse Assay

    30) Product Images from "IL-7–dependent maintenance of ILC3s is required for normal entry of lymphocytes into lymph nodes"

    Article Title: IL-7–dependent maintenance of ILC3s is required for normal entry of lymphocytes into lymph nodes

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20170518

    Lymphocyte entry into lymph nodes is defective in the absence of ILC3s. (A–D) Mice specifically lacking ILC3s were generated by reconstitution of irradiated WT hosts with bone marrow from Rorc −/− ( n = 9) or WT ( n = 10) donor bone marrow as control. Both groups were also treated with anti-Thy1 for 2 wk after BMT. 12 wk after reconstitution, hosts were injected with 10 7 labeled WT lymphocytes from either the spleen or a mixture of the lymph nodes and spleen, and 1 or 3 h later cell numbers in the lymph nodes and spleen were determined by FACS. (A) Density plots are of IL-7R vs. Lin for total lymphocytes and Gata3 vs. RORgt by IL-7R + Lin − -gated cells. Bar charts show total numbers of ILC subpopulations in the indicated chimeras. (B) Bar charts are of total lymph node size in chimeras of WT hosts reconstituted with either Rorc −/− or WT bone marrow. (C) Bar charts show the recovery of donor T and B cells from the indicated Rorc −/− chimeras from either lymph nodes (LN) or spleen (SPN). (D) Frozen sections from lymph nodes from the indicated chimeras were analyzed for expression of PNAd (green), CD31 (red) and counterstained with nuclear DAPI staining. (E–H) CD45.1 hosts were irradiated and reconstituted with either WT or Rorc −/− bone marrow. WT chimeras were treated with either anti-IL-7R or isotype control for 1 wk after irradiation, whereas Rorc −/− chimeras all received anti-IL-7R mAb. (E) 2 wk after BMT, groups of anti-IL-7R (αIL-7R) and isotype-treated WT chimeras ( n = 3 per group) were injected with labeled lymphocytes. Bar charts show numbers of T and B cells in lymph nodes 1 h after transfer. (F) 12 wk after BMT, lymph nodes were analyzed by FACs. Bar chart shows total numbers of ILC populations in lymph nodes from isotype-treated WT chimeras (white bars), anti-IL-7R (αIL-7R)-treated WT chimeras (gray bars), and anti-IL-7R (αIL-7R)-treated Rorc −/− chimeras (black bars). (G) Density plots show CD45.1 host vs. CD45.2 donor cells among total lymphocytes, gated ILC2, CD4 + ILC3, and CD4 – ILC3 in the indicated chimeras. (H) 12 wk after BMT, chimeras ( n = 7 per group) were injected with labeled WT cells from mixture of the lymph nodes and spleen, and 1 h later the host lymph nodes and spleen were analyzed by FACS. Bar charts show the numbers of donor T and B cells recovered from the lymph nodes and spleen of the indicated chimeras. Data are pools of four (B and C) or two (A, D, and E–H) independent experiments. E, F, and H are also representative of one further experiment performed by using WT CD45.2 hosts. *, P
    Figure Legend Snippet: Lymphocyte entry into lymph nodes is defective in the absence of ILC3s. (A–D) Mice specifically lacking ILC3s were generated by reconstitution of irradiated WT hosts with bone marrow from Rorc −/− ( n = 9) or WT ( n = 10) donor bone marrow as control. Both groups were also treated with anti-Thy1 for 2 wk after BMT. 12 wk after reconstitution, hosts were injected with 10 7 labeled WT lymphocytes from either the spleen or a mixture of the lymph nodes and spleen, and 1 or 3 h later cell numbers in the lymph nodes and spleen were determined by FACS. (A) Density plots are of IL-7R vs. Lin for total lymphocytes and Gata3 vs. RORgt by IL-7R + Lin − -gated cells. Bar charts show total numbers of ILC subpopulations in the indicated chimeras. (B) Bar charts are of total lymph node size in chimeras of WT hosts reconstituted with either Rorc −/− or WT bone marrow. (C) Bar charts show the recovery of donor T and B cells from the indicated Rorc −/− chimeras from either lymph nodes (LN) or spleen (SPN). (D) Frozen sections from lymph nodes from the indicated chimeras were analyzed for expression of PNAd (green), CD31 (red) and counterstained with nuclear DAPI staining. (E–H) CD45.1 hosts were irradiated and reconstituted with either WT or Rorc −/− bone marrow. WT chimeras were treated with either anti-IL-7R or isotype control for 1 wk after irradiation, whereas Rorc −/− chimeras all received anti-IL-7R mAb. (E) 2 wk after BMT, groups of anti-IL-7R (αIL-7R) and isotype-treated WT chimeras ( n = 3 per group) were injected with labeled lymphocytes. Bar charts show numbers of T and B cells in lymph nodes 1 h after transfer. (F) 12 wk after BMT, lymph nodes were analyzed by FACs. Bar chart shows total numbers of ILC populations in lymph nodes from isotype-treated WT chimeras (white bars), anti-IL-7R (αIL-7R)-treated WT chimeras (gray bars), and anti-IL-7R (αIL-7R)-treated Rorc −/− chimeras (black bars). (G) Density plots show CD45.1 host vs. CD45.2 donor cells among total lymphocytes, gated ILC2, CD4 + ILC3, and CD4 – ILC3 in the indicated chimeras. (H) 12 wk after BMT, chimeras ( n = 7 per group) were injected with labeled WT cells from mixture of the lymph nodes and spleen, and 1 h later the host lymph nodes and spleen were analyzed by FACS. Bar charts show the numbers of donor T and B cells recovered from the lymph nodes and spleen of the indicated chimeras. Data are pools of four (B and C) or two (A, D, and E–H) independent experiments. E, F, and H are also representative of one further experiment performed by using WT CD45.2 hosts. *, P

    Techniques Used: Mouse Assay, Generated, Irradiation, Injection, Labeling, FACS, Expressing, Staining

    Normal numbers of stromal and dendritic subsets but reduced ILC populations in lymph nodes after IL-7 ablation. Il7 fx/KO R26 CreERT2 mice ( n = 4) and CreERT –ve littermate controls ( n = 4) were treated with tamoxifen for 5 d. 3 wk later, lymph nodes were recovered and cell composition analyzed by FACS. (A) Density plots are of CD45 vs. SSc on total live cells and show CD45 – gate used to display gp38 vs CD31 (PECAM-1) density plots used to identify FRCs, LECs, and BECs. Bar chart shows total numbers of these subsets recovered from R26 CreERT2 +ve (closed bars) and R26 CreERT2 –ve Il7 fx/KO mice (open bars). (B) Density plots are of Meca79 vs. anti-ICAM-1 staining by CD31 + gp38 – BECs from representative mice described in A. Meca79 was either stained ex vivo in cell suspensions (Total Meca79) or by injection of mice with Meca79 mAb before ex vivo staining and analysis (luminal Meca79). Histograms are of ICAM-1 and Meca79 staining (total vs. luminal) by cells from Cre+ (solid line) or Cre– litter mates (gray shading), gated on Meca79 + ICAM-1 + cells defined by the gate shown on density plots. (C) Frozen sections from lymph nodes from the indicated mice were analyzed for expression of PNAd (green), CD31 (red), and counterstained with nuclear DAPI staining. (D) Il7 fx/KO R26 CreERT2 mice ( n = 3) and CreERT –ve littermate controls ( n = 3) were treated with tamoxifen for 5 d. 3 wk later, total mRNA was isolated from total lymph nodes of individual mice and gene expression determined by RNAseq. Bar charts show mRNA expression level in transcripts per kilobase million (TPM) of the indicated genes. (E) Density plots are of CD11c vs. Class II MHC (MHC II) and PDCA-1 vs. CD11c, used to identify MHC II + CD11c Hi resident DC (Res), MHC II Hi CD11c + migratory DC (Mig), and PDCA-1 + CD11c + plasmacytoid DCs (pDC). Bar charts show total numbers of these DC subsets recovered from the lymph nodes and spleen of R26 CreERT2 +ve and R26 CreERT2 –ve Il7 fx/KO mice. (F) Density plots are of IL-7R vs. Lin; histograms are of Thy1 expression by IL-7R + lin – -gated cells. Density plots are of Gata3 vs. RORgt expression by Thy1 + IL-7R + lin – ILCs. Histograms are of CD4 expression by RORgt+ ILCs. Bar charts show total numbers of ILC subsets isolated from the lymph nodes and spleen of R26 CreERT2 +ve and R26 CreERT2 –ve Il7 fx/KO mice. Data are representative of three independent experiments (A–D), a pool of two experiments (E), or four independent experiments (F). *, P
    Figure Legend Snippet: Normal numbers of stromal and dendritic subsets but reduced ILC populations in lymph nodes after IL-7 ablation. Il7 fx/KO R26 CreERT2 mice ( n = 4) and CreERT –ve littermate controls ( n = 4) were treated with tamoxifen for 5 d. 3 wk later, lymph nodes were recovered and cell composition analyzed by FACS. (A) Density plots are of CD45 vs. SSc on total live cells and show CD45 – gate used to display gp38 vs CD31 (PECAM-1) density plots used to identify FRCs, LECs, and BECs. Bar chart shows total numbers of these subsets recovered from R26 CreERT2 +ve (closed bars) and R26 CreERT2 –ve Il7 fx/KO mice (open bars). (B) Density plots are of Meca79 vs. anti-ICAM-1 staining by CD31 + gp38 – BECs from representative mice described in A. Meca79 was either stained ex vivo in cell suspensions (Total Meca79) or by injection of mice with Meca79 mAb before ex vivo staining and analysis (luminal Meca79). Histograms are of ICAM-1 and Meca79 staining (total vs. luminal) by cells from Cre+ (solid line) or Cre– litter mates (gray shading), gated on Meca79 + ICAM-1 + cells defined by the gate shown on density plots. (C) Frozen sections from lymph nodes from the indicated mice were analyzed for expression of PNAd (green), CD31 (red), and counterstained with nuclear DAPI staining. (D) Il7 fx/KO R26 CreERT2 mice ( n = 3) and CreERT –ve littermate controls ( n = 3) were treated with tamoxifen for 5 d. 3 wk later, total mRNA was isolated from total lymph nodes of individual mice and gene expression determined by RNAseq. Bar charts show mRNA expression level in transcripts per kilobase million (TPM) of the indicated genes. (E) Density plots are of CD11c vs. Class II MHC (MHC II) and PDCA-1 vs. CD11c, used to identify MHC II + CD11c Hi resident DC (Res), MHC II Hi CD11c + migratory DC (Mig), and PDCA-1 + CD11c + plasmacytoid DCs (pDC). Bar charts show total numbers of these DC subsets recovered from the lymph nodes and spleen of R26 CreERT2 +ve and R26 CreERT2 –ve Il7 fx/KO mice. (F) Density plots are of IL-7R vs. Lin; histograms are of Thy1 expression by IL-7R + lin – -gated cells. Density plots are of Gata3 vs. RORgt expression by Thy1 + IL-7R + lin – ILCs. Histograms are of CD4 expression by RORgt+ ILCs. Bar charts show total numbers of ILC subsets isolated from the lymph nodes and spleen of R26 CreERT2 +ve and R26 CreERT2 –ve Il7 fx/KO mice. Data are representative of three independent experiments (A–D), a pool of two experiments (E), or four independent experiments (F). *, P

    Techniques Used: Mouse Assay, FACS, Staining, Ex Vivo, Injection, Expressing, Isolation

    31) Product Images from "Tumor-Derived Extracellular Vesicles Induce Abnormal Angiogenesis via TRPV4 Downregulation and Subsequent Activation of YAP and VEGFR2"

    Article Title: Tumor-Derived Extracellular Vesicles Induce Abnormal Angiogenesis via TRPV4 Downregulation and Subsequent Activation of YAP and VEGFR2

    Journal: Frontiers in Bioengineering and Biotechnology

    doi: 10.3389/fbioe.2021.790489

    t-EVs cause abnormal angiogenesis in vivo as evidenced by decreased pericyte coverage and increased VEGFR2 expression. (A) Representative immunofluorescence images (60x) of Matrigel sections stained with endothelial marker CD31 (red) and pericyte marker NG2 (green). Note, the reduced pericyte coverage and disorganized appearance of t-EV treated vessels. Scale bar = 100 μm. (B) Quantitative analysis revealing significantly decreased NG2/CD31 colocalization in t-EV Matrigel plugs ( n = 7) (*** p ≤ 0.001) compared with vehicle alone ( n = 4). (C) Representative immunofluorescence images (60X) taken from Matrigel plugs stained with endothelial marker CD31 (green) and VEGFR2 (red). Scale bar = 100 μm. (D) Quantitative analysis demonstrating significantly increased VEGFR2/CD31 colocalization (* p ≤ 0.05) in t-EV treated Matrigel plugs ( n = 7) compared to the vehicle ( n = 4).
    Figure Legend Snippet: t-EVs cause abnormal angiogenesis in vivo as evidenced by decreased pericyte coverage and increased VEGFR2 expression. (A) Representative immunofluorescence images (60x) of Matrigel sections stained with endothelial marker CD31 (red) and pericyte marker NG2 (green). Note, the reduced pericyte coverage and disorganized appearance of t-EV treated vessels. Scale bar = 100 μm. (B) Quantitative analysis revealing significantly decreased NG2/CD31 colocalization in t-EV Matrigel plugs ( n = 7) (*** p ≤ 0.001) compared with vehicle alone ( n = 4). (C) Representative immunofluorescence images (60X) taken from Matrigel plugs stained with endothelial marker CD31 (green) and VEGFR2 (red). Scale bar = 100 μm. (D) Quantitative analysis demonstrating significantly increased VEGFR2/CD31 colocalization (* p ≤ 0.05) in t-EV treated Matrigel plugs ( n = 7) compared to the vehicle ( n = 4).

    Techniques Used: In Vivo, Expressing, Immunofluorescence, Staining, Marker

    32) Product Images from "Cited4 is a sex‐biased mediator of the antidiabetic glitazone response in adipocyte progenitors"

    Article Title: Cited4 is a sex‐biased mediator of the antidiabetic glitazone response in adipocyte progenitors

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201708613

    Cited4 is a target of rosiglitazone in murine and human adipocyte progenitors promoting beige differentiation mRNA expression in FACS‐isolated Lin(Ter119/CD31/CD45) − Sca1 + progenitor cells from female mouse subcutaneous fat, differentiated in the presence of 1 μM cPGI 2 or vehicle for the indicated time, as determined by expression profiling ( n = 3, E‐MTAB‐3693). **** P = 3 × 10 −6 (Day 2), **** P = 4 × 10 −7 (Day 4), **** P = 1 × 10 −6 (Day 6) in 2 × 2 ANOVA with Bonferroni's posttests (cPGI 2 vs. vehicle). mRNA expression in MACS‐isolated Lin − Sca1 + progenitor cells from female mouse subcutaneous fat, differentiated in the presence of 100 nM Rosi or vehicle for the indicated time, as determined by qRT–PCR ( n = 4). **** P = 1 × 10 −10 (Days 1 and 2), ** P = 0.001, in 2 × 2 ANOVA with Bonferroni's posttests (Rosi vs. vehicle). mRNA expression in female Lin − Sca1 + cells, differentiated in the presence of 100 nM Rosi or vehicle for 8 days, as determined by qRT–PCR ( n = 3). t ‐test Cited4 −/− vs. Cited4 +/+ (Rosi) * P = 0.013 ( Ucp1 ), ** P = 0.004 ( Cpt1b ), * P = 0.026 ( Dio2 ). mRNA expression in primary SVF cells from human subcutaneous fat, differentiated in the presence of 100 nM Rosi (D) or vehicle (D–F), as determined by qRT–PCR at the indicated time points ( n = 5 patients). ♀/♂ represents individual data. (D) **** P = 3 × 10 −5 (Day 2), **** P = 3 × 10 −6 (Day 6), **** P = 4 × 10 −9 (Day 10), **** P = 3 × 10 −7 (Day 14), in 2 × 2 ANOVA with Bonferroni's posttests (Rosi vs. vehicle). (E, F) Pearson correlation coefficient ( r ) and P ‐value are shown. mRNA expression in primary SVF cells from human female subcutaneous fat transfected with the indicated siRNA prior to differentiation in the presence of 100 nM Rosi for 9 days, as determined by qRT–PCR ( n = 3). *** P = 0.0002 ( CITED4 ), ** P = 0.002 ( UCP1 ), * P = 0.02 ( UCP1 ), * P = 0.035/0.026 ( PPARG ), *** P = 0.0006 ( SLC2A4 ), ** P = 0.002 ( ADIPOQ ) in one‐way ANOVA with Tukey's posttests (vs. siCtrl ). Data information: Data are presented as mean ± SEM except for (D) ♀/♂, (E and F) individual data.
    Figure Legend Snippet: Cited4 is a target of rosiglitazone in murine and human adipocyte progenitors promoting beige differentiation mRNA expression in FACS‐isolated Lin(Ter119/CD31/CD45) − Sca1 + progenitor cells from female mouse subcutaneous fat, differentiated in the presence of 1 μM cPGI 2 or vehicle for the indicated time, as determined by expression profiling ( n = 3, E‐MTAB‐3693). **** P = 3 × 10 −6 (Day 2), **** P = 4 × 10 −7 (Day 4), **** P = 1 × 10 −6 (Day 6) in 2 × 2 ANOVA with Bonferroni's posttests (cPGI 2 vs. vehicle). mRNA expression in MACS‐isolated Lin − Sca1 + progenitor cells from female mouse subcutaneous fat, differentiated in the presence of 100 nM Rosi or vehicle for the indicated time, as determined by qRT–PCR ( n = 4). **** P = 1 × 10 −10 (Days 1 and 2), ** P = 0.001, in 2 × 2 ANOVA with Bonferroni's posttests (Rosi vs. vehicle). mRNA expression in female Lin − Sca1 + cells, differentiated in the presence of 100 nM Rosi or vehicle for 8 days, as determined by qRT–PCR ( n = 3). t ‐test Cited4 −/− vs. Cited4 +/+ (Rosi) * P = 0.013 ( Ucp1 ), ** P = 0.004 ( Cpt1b ), * P = 0.026 ( Dio2 ). mRNA expression in primary SVF cells from human subcutaneous fat, differentiated in the presence of 100 nM Rosi (D) or vehicle (D–F), as determined by qRT–PCR at the indicated time points ( n = 5 patients). ♀/♂ represents individual data. (D) **** P = 3 × 10 −5 (Day 2), **** P = 3 × 10 −6 (Day 6), **** P = 4 × 10 −9 (Day 10), **** P = 3 × 10 −7 (Day 14), in 2 × 2 ANOVA with Bonferroni's posttests (Rosi vs. vehicle). (E, F) Pearson correlation coefficient ( r ) and P ‐value are shown. mRNA expression in primary SVF cells from human female subcutaneous fat transfected with the indicated siRNA prior to differentiation in the presence of 100 nM Rosi for 9 days, as determined by qRT–PCR ( n = 3). *** P = 0.0002 ( CITED4 ), ** P = 0.002 ( UCP1 ), * P = 0.02 ( UCP1 ), * P = 0.035/0.026 ( PPARG ), *** P = 0.0006 ( SLC2A4 ), ** P = 0.002 ( ADIPOQ ) in one‐way ANOVA with Tukey's posttests (vs. siCtrl ). Data information: Data are presented as mean ± SEM except for (D) ♀/♂, (E and F) individual data.

    Techniques Used: Expressing, FACS, Isolation, Magnetic Cell Separation, Quantitative RT-PCR, Transfection

    33) Product Images from "Angiopoietin-2–Induced Arterial Stiffness in CKD"

    Article Title: Angiopoietin-2–Induced Arterial Stiffness in CKD

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2013050542

    Angpt2 induces discrete subpopulation of macrophages defined by Ly6C in aorta. (A) Immunofluorescence images showing that Tie-2 receptor is mainly expressed in CD31 + endothelial cells of the aorta from normal control mice. (B) FACS analysis showing that the Tie-2 receptor is also expressed in CD11b + macrophages of the aorta from normal control mice. (C) qPCR showing increased transcripts of chemokine and chemokine receptors in aorta of mice with AdAngpt2. (D and E) FACS analysis showing that Ly6C low subpopulation of CD11b + macrophages is induced in aorta of mice with AdAngpt2. (F) qPCR of M1- and M2-biased cytokines of sorted CD11b + macrophages defined by Ly6C expression. Expression levels are normalized by glyceraldehyde 3-phosphate dehydrogenase . * P
    Figure Legend Snippet: Angpt2 induces discrete subpopulation of macrophages defined by Ly6C in aorta. (A) Immunofluorescence images showing that Tie-2 receptor is mainly expressed in CD31 + endothelial cells of the aorta from normal control mice. (B) FACS analysis showing that the Tie-2 receptor is also expressed in CD11b + macrophages of the aorta from normal control mice. (C) qPCR showing increased transcripts of chemokine and chemokine receptors in aorta of mice with AdAngpt2. (D and E) FACS analysis showing that Ly6C low subpopulation of CD11b + macrophages is induced in aorta of mice with AdAngpt2. (F) qPCR of M1- and M2-biased cytokines of sorted CD11b + macrophages defined by Ly6C expression. Expression levels are normalized by glyceraldehyde 3-phosphate dehydrogenase . * P

    Techniques Used: Immunofluorescence, Mouse Assay, FACS, Real-time Polymerase Chain Reaction, Expressing

    34) Product Images from "Chemoattraction of bone marrow-derived stem cells towards human endometrial stromal cells is mediated by estradiol regulated CXCL12 and CXCR4 expression"

    Article Title: Chemoattraction of bone marrow-derived stem cells towards human endometrial stromal cells is mediated by estradiol regulated CXCL12 and CXCR4 expression

    Journal: Stem cell research

    doi: 10.1016/j.scr.2015.04.004

    Immunophenotypic characterization of mBMCs by FACS. Primary mBMCs were incubated with fluorescent-labeled CD90, CD105, CD34, CD31, and Flk-1 antibodies or with isotype-matched irrelevant antibody for FACS analysis. Experiments were performed 3 times with different samples and each time in duplicate. Results (% of cells) presented are the average of triplicates.
    Figure Legend Snippet: Immunophenotypic characterization of mBMCs by FACS. Primary mBMCs were incubated with fluorescent-labeled CD90, CD105, CD34, CD31, and Flk-1 antibodies or with isotype-matched irrelevant antibody for FACS analysis. Experiments were performed 3 times with different samples and each time in duplicate. Results (% of cells) presented are the average of triplicates.

    Techniques Used: FACS, Incubation, Labeling

    35) Product Images from "PDGFRα controls the balance of stromal and adipogenic cells during adipose tissue organogenesis"

    Article Title: PDGFRα controls the balance of stromal and adipogenic cells during adipose tissue organogenesis

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.135962

    PDGFRα activation inhibits preadipocyte commitment. (A,B,G,H) Fold change in adipocyte marker and adipogenic transcription factor mRNA levels in ingWAT at the indicated time points, as determined by quantitative RT-PCR. Results are shown as fold change in mutant over control. n =3 tissue samples per genotype. (C) Representative flow cytometry plots of the stromal-vascular fraction from ingWAT in control and Sox2-V561D mice at E18.5. Pseudocolor plots show Lin neg (CD45 − CD31 − ) cells. The Lin neg (CD29 + CD34 + ) adipocyte progenitor population and Lin neg (CD29 + CD34 − ) non-adipogenic populations are outlined. (D) Representative flow cytometry plots of Lin neg (CD29 + CD34 + ) cells from C. Pseudocolor plots show the Lin neg (CD29 + CD34 + Sca1 + CD24 + ) Q1 and Lin neg (CD29 + CD34 + Sca1 + CD24 − ) Q4 adipocyte precursor populations. (E,F) Quantification of data in C and D plus additional biological replicates (not shown). n =3 mice per genotype. All data are presented as mean±s.e.m. Red significance identifier refers to subpopulation shown in red; black significance identifier refers to the total cell population. * P
    Figure Legend Snippet: PDGFRα activation inhibits preadipocyte commitment. (A,B,G,H) Fold change in adipocyte marker and adipogenic transcription factor mRNA levels in ingWAT at the indicated time points, as determined by quantitative RT-PCR. Results are shown as fold change in mutant over control. n =3 tissue samples per genotype. (C) Representative flow cytometry plots of the stromal-vascular fraction from ingWAT in control and Sox2-V561D mice at E18.5. Pseudocolor plots show Lin neg (CD45 − CD31 − ) cells. The Lin neg (CD29 + CD34 + ) adipocyte progenitor population and Lin neg (CD29 + CD34 − ) non-adipogenic populations are outlined. (D) Representative flow cytometry plots of Lin neg (CD29 + CD34 + ) cells from C. Pseudocolor plots show the Lin neg (CD29 + CD34 + Sca1 + CD24 + ) Q1 and Lin neg (CD29 + CD34 + Sca1 + CD24 − ) Q4 adipocyte precursor populations. (E,F) Quantification of data in C and D plus additional biological replicates (not shown). n =3 mice per genotype. All data are presented as mean±s.e.m. Red significance identifier refers to subpopulation shown in red; black significance identifier refers to the total cell population. * P

    Techniques Used: Activation Assay, Marker, Quantitative RT-PCR, Mutagenesis, Flow Cytometry, Cytometry, Mouse Assay

    36) Product Images from "Simultaneous targeting of Eph receptors in glioblastoma"

    Article Title: Simultaneous targeting of Eph receptors in glioblastoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10978

    EphA3 co-stains with macrophage/leukocyte markers A. Immunofluorescent staining of CD68, GFAP and Von Willebrand factor in a GBM specimen. B. Immunofluorescent staining of EphA3 (red) and CD31, GFAP, CD68, CD163, and CD206 on consecutive frozen sections of BTCOE4443 human GBM specimen. Nuclei are stained with DAPI (blue). Selected areas were magnified (last column on the right). C. Confocal immunofluorescent staining of EphA3 and CD68 in a GBM specimen using stacked 2D images. D, E. -stacked 2D images, and F. Confocal microscopy of co-staining of EphA3 and CD45 in GBM specimens. G. CD4+ T cells do not express the EphA3 receptor. Western blot analysis of peripheral blood mononuclear cells (PBMC), CD4+ and CD4+ activated T cells were probed for EphA3 immunoreactivity.
    Figure Legend Snippet: EphA3 co-stains with macrophage/leukocyte markers A. Immunofluorescent staining of CD68, GFAP and Von Willebrand factor in a GBM specimen. B. Immunofluorescent staining of EphA3 (red) and CD31, GFAP, CD68, CD163, and CD206 on consecutive frozen sections of BTCOE4443 human GBM specimen. Nuclei are stained with DAPI (blue). Selected areas were magnified (last column on the right). C. Confocal immunofluorescent staining of EphA3 and CD68 in a GBM specimen using stacked 2D images. D, E. -stacked 2D images, and F. Confocal microscopy of co-staining of EphA3 and CD45 in GBM specimens. G. CD4+ T cells do not express the EphA3 receptor. Western blot analysis of peripheral blood mononuclear cells (PBMC), CD4+ and CD4+ activated T cells were probed for EphA3 immunoreactivity.

    Techniques Used: Staining, Confocal Microscopy, Western Blot

    Immunofluorescent staining of EphA3 in GBM A. Immunofluorescent staining of EphA3 (red) and NeuN (green) within the tumor, invading and contralateral areas in a patient who died from GBM (specimen G204). B. Immunofluorescent staining of EphA3 (red) and EphA2 (green) in a BTCOE4443 human GBM specimen. C. Confocal immunofluorescent staining of EphA3 (red) and CD31 (green) in two GBM specimens using stacked 2D images. Nuclei are stained with DAPI (blue).
    Figure Legend Snippet: Immunofluorescent staining of EphA3 in GBM A. Immunofluorescent staining of EphA3 (red) and NeuN (green) within the tumor, invading and contralateral areas in a patient who died from GBM (specimen G204). B. Immunofluorescent staining of EphA3 (red) and EphA2 (green) in a BTCOE4443 human GBM specimen. C. Confocal immunofluorescent staining of EphA3 (red) and CD31 (green) in two GBM specimens using stacked 2D images. Nuclei are stained with DAPI (blue).

    Techniques Used: Staining

    37) Product Images from "Efficient Reprogramming of Na?ve-Like Induced Pluripotent Stem Cells from Porcine Adipose-Derived Stem Cells with a Feeder-Independent and Serum-Free System"

    Article Title: Efficient Reprogramming of Na?ve-Like Induced Pluripotent Stem Cells from Porcine Adipose-Derived Stem Cells with a Feeder-Independent and Serum-Free System

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085089

    Identification of pADSCs. (A) Morphology of pADSCs at passage 3, scale bar = 100 µm. (B) Multi-lineage differentiation of pADSCs, mature adipocytes were detected by Oil Red O staining (a), scale bar = 50 µm; osteogenesis was analysis by Alizarin Red S staining (c), scale bar = 200 µm. Cells cultured in the corresponding proliferation medium served as negative controls, respectively (b, scale bar = 50 µm; d, scale bar = 200 µm). (C) Expression of cell surface markers in pADSCs at passage 3 including CD29, CD44, CD90, CD105,CD31, CD45 and HLA-DR. Positive cells were gated based on staining with isotype antibody controls.
    Figure Legend Snippet: Identification of pADSCs. (A) Morphology of pADSCs at passage 3, scale bar = 100 µm. (B) Multi-lineage differentiation of pADSCs, mature adipocytes were detected by Oil Red O staining (a), scale bar = 50 µm; osteogenesis was analysis by Alizarin Red S staining (c), scale bar = 200 µm. Cells cultured in the corresponding proliferation medium served as negative controls, respectively (b, scale bar = 50 µm; d, scale bar = 200 µm). (C) Expression of cell surface markers in pADSCs at passage 3 including CD29, CD44, CD90, CD105,CD31, CD45 and HLA-DR. Positive cells were gated based on staining with isotype antibody controls.

    Techniques Used: Staining, Cell Culture, Expressing

    38) Product Images from "Discovery of a periosteal stem cell mediating intramembranous bone formation"

    Article Title: Discovery of a periosteal stem cell mediating intramembranous bone formation

    Journal: Nature

    doi: 10.1038/s41586-018-0554-8

    FACS analysis on microdissected periosteal tissue and characterization of PSCs. a, Flow cytometry of CTSK-mGFP cells microdissected from the periosteum of mouse (P7) long bones showing the distribution of PSC, PP1 and PP2 cells. b-c, Flow cytometry showing the distribution of PSCs, PP1 and PP2 cells in mouse long bones at day 15 ( b ) and day 32 ( c ) (Lineage - = Ter119−, CD45− and CD31−). a-c, Representative FACS plots from 10 independent experiments. d, Schematic representation of the strategy used for flow cytometry for analysis of periosteal PSC, PP1 and PP2 cell populations. e, FACS plot displaying the distribution of CD146 (i) and CD140α (ii) expression in bone marrow stromal cells. f, FACS plots displaying the distribution of CD140α (i) and CD146 (ii) in mouse periosteum obtained through periosteal microdissection. (Lineage - = Ter119−, CD45− and CD31−). e-f, Representative FACS plots from 5 independent experiments. g, Mouse bone marrow immunostained for CD146 (cyan) and CD140α (magenta). CTSK-mGFP (green), DAPI (blue) and Tomato red (red) to visualize stromal cells. Scale bar 100 μm. Representative images from 3 independent experiments. h, Clonogenic cells detected in the periosteum (top two panels, white arrows) and perichondrium region ( bottom panel, white arrows) of mouse femur 2 weeks post induction of β-actin cre with tamoxifen. Enlarged view of areas marked by dotted white line are provided for each image. Scale bar 50 μm. Representative images from 3 independent experiments. i, FACS plots showing in vitro differentiation for PP1 (left plot) and PP2 (right plot) cells after 15 days of culture. Representative FACS plots from 3 independent experiments. j, Alizarin red staining (red) of bone 5 weeks after transplantation of non-CTSK MSCs (left) and PSCs (right) into the kidney capsule. Representative images from 5 independent experiments. k, Relative gene expression for bone ( Runx2 ) and cartilage specific genes ( Col2a1, Comp, Chad ) 5 weeks after trans plant of PSCs and non-CTSK MSCs. Non-CTSK MSC derived cells display significantly higher expression of cartilage specific genes than PSCs. (* p = 0.003 for Col2a1 , * p = 0.002 for Comp , * p = 0.002 for Chad , mean ± S.D, n=3, 2 tailed Student’s t-test). l, CTSK-mGFP positive PSCs (green) were immunostained for RUNX2 (magenta, top panel) and Osteocalcin (magenta, middle and bottom panels) 3, 4 or 5 weeks after transplantation into the kidney capsule. Nuclei were stained with DAPI. Scale bar 20 μm. Representative images from 3 independent experiments.
    Figure Legend Snippet: FACS analysis on microdissected periosteal tissue and characterization of PSCs. a, Flow cytometry of CTSK-mGFP cells microdissected from the periosteum of mouse (P7) long bones showing the distribution of PSC, PP1 and PP2 cells. b-c, Flow cytometry showing the distribution of PSCs, PP1 and PP2 cells in mouse long bones at day 15 ( b ) and day 32 ( c ) (Lineage - = Ter119−, CD45− and CD31−). a-c, Representative FACS plots from 10 independent experiments. d, Schematic representation of the strategy used for flow cytometry for analysis of periosteal PSC, PP1 and PP2 cell populations. e, FACS plot displaying the distribution of CD146 (i) and CD140α (ii) expression in bone marrow stromal cells. f, FACS plots displaying the distribution of CD140α (i) and CD146 (ii) in mouse periosteum obtained through periosteal microdissection. (Lineage - = Ter119−, CD45− and CD31−). e-f, Representative FACS plots from 5 independent experiments. g, Mouse bone marrow immunostained for CD146 (cyan) and CD140α (magenta). CTSK-mGFP (green), DAPI (blue) and Tomato red (red) to visualize stromal cells. Scale bar 100 μm. Representative images from 3 independent experiments. h, Clonogenic cells detected in the periosteum (top two panels, white arrows) and perichondrium region ( bottom panel, white arrows) of mouse femur 2 weeks post induction of β-actin cre with tamoxifen. Enlarged view of areas marked by dotted white line are provided for each image. Scale bar 50 μm. Representative images from 3 independent experiments. i, FACS plots showing in vitro differentiation for PP1 (left plot) and PP2 (right plot) cells after 15 days of culture. Representative FACS plots from 3 independent experiments. j, Alizarin red staining (red) of bone 5 weeks after transplantation of non-CTSK MSCs (left) and PSCs (right) into the kidney capsule. Representative images from 5 independent experiments. k, Relative gene expression for bone ( Runx2 ) and cartilage specific genes ( Col2a1, Comp, Chad ) 5 weeks after trans plant of PSCs and non-CTSK MSCs. Non-CTSK MSC derived cells display significantly higher expression of cartilage specific genes than PSCs. (* p = 0.003 for Col2a1 , * p = 0.002 for Comp , * p = 0.002 for Chad , mean ± S.D, n=3, 2 tailed Student’s t-test). l, CTSK-mGFP positive PSCs (green) were immunostained for RUNX2 (magenta, top panel) and Osteocalcin (magenta, middle and bottom panels) 3, 4 or 5 weeks after transplantation into the kidney capsule. Nuclei were stained with DAPI. Scale bar 20 μm. Representative images from 3 independent experiments.

    Techniques Used: FACS, Flow Cytometry, Cytometry, Expressing, Laser Capture Microdissection, In Vitro, Staining, Transplantation Assay, Derivative Assay

    Analysis of CTSK-mGFP cells in mouse femur. a, CTSK-mGFP mesenchymal cells (green) were visualized in the mouse long bones at E14.5. Scale bar 200 μm. Enlarged images of areas marked by the dotted white boxes are provided in i and ii. DAPI for nuclei. b, Immunostaining for CD200 (magenta) confirmed co-localization (shown by yellow arrows) with CTSK-mGFP cells (green) in the periosteum. A separate pool of CD200+ cells are detected at the future primary ossification site (marked by dotted orange line). Nuclei stained with DAPI. Scale bar 20 μm. a-b, 3 independent experiments. c, CTSK-mGFP mesenchymal cells in the long bones of mice were detected by FACS at embryonic day 16.5. d, Visualization of CTSK-mGFP (green) cells in mouse long bones at E16.5. Nuclei stained with DAPI. Scale bar 500 μm. An enlarged view of the areas marked by dotted yellow boxes are shown in i and ii. CTSK-mGFP positive cells (green) were detected in the mouse periosteum (i and ii). e, CD200 (magenta) immunostaining confirmed co-localization with CTSK-mGFP cells (green) in the periosteum (top panel). CTSK-mGFP cells in the primary spongiosa morphologically consistent with osteoclasts stained negative for CD200 (bottom panel). Scale bar 20μm. c-e, 3 independent experiments. f, Visualization of CTSK-mGFP (green) cells in the periosteum (dotted white line) of mouse femur at post-natal day 6 (top) and day 12 (bottom). Nuclei shown by DAPI staining. Scale bar 20 μm. g, An enlarged view of osteocytes within the dotted white box is provided (i). CTSK-mGFP (green), nuclei (DAPI). Scale bar 20μm. 3 independent experiments. (ii) Quantification of total CTSK-mGFP labelled periosteal cells and mGFP labelled osteocytes in the mouse femur (*** p = 6.95×10 −16 , mean ± S.E.M, n= 12 distinct areas of periosteum from 3 independent experiments. 2 tailed Student’s t-test). h, An enlarged view for main text Fig. 1e is provided. Representative images from 3 independent experiments. i, FACS plots showing expression of CD49f (left) and CD51 (right) in CTSK-mGFP cells isolated from long bones of 7-day old mice (Lineage - = Ter119−, CD45− and CD31−). 5 independent experiments. j, 8 week old Ctsk-cre mouse femurs were immunostained for RUNX2 ( a, magenta, top), Alkaline phosphatase (ALPL) ( b, magenta, middle), and Osteocalcin ( c, magenta, bottom). CTSK-mGFP (green), nuclei stained by DAPI, co-localization shown by yellow arrows. Scale bar 20 μm. Representative images from 3 independent experiments. k, 12 day old Ctsk-cre mouse femurs were immunostained for Gremlin 1 ( a, magenta, top) and Nestin ( b, magenta, bottom). CTSK-mGFP (green), nuclei stained by DAPI, dotted white line indicates periosteum. Scale bar 20 μm. Representative images from 3 independent experiments.
    Figure Legend Snippet: Analysis of CTSK-mGFP cells in mouse femur. a, CTSK-mGFP mesenchymal cells (green) were visualized in the mouse long bones at E14.5. Scale bar 200 μm. Enlarged images of areas marked by the dotted white boxes are provided in i and ii. DAPI for nuclei. b, Immunostaining for CD200 (magenta) confirmed co-localization (shown by yellow arrows) with CTSK-mGFP cells (green) in the periosteum. A separate pool of CD200+ cells are detected at the future primary ossification site (marked by dotted orange line). Nuclei stained with DAPI. Scale bar 20 μm. a-b, 3 independent experiments. c, CTSK-mGFP mesenchymal cells in the long bones of mice were detected by FACS at embryonic day 16.5. d, Visualization of CTSK-mGFP (green) cells in mouse long bones at E16.5. Nuclei stained with DAPI. Scale bar 500 μm. An enlarged view of the areas marked by dotted yellow boxes are shown in i and ii. CTSK-mGFP positive cells (green) were detected in the mouse periosteum (i and ii). e, CD200 (magenta) immunostaining confirmed co-localization with CTSK-mGFP cells (green) in the periosteum (top panel). CTSK-mGFP cells in the primary spongiosa morphologically consistent with osteoclasts stained negative for CD200 (bottom panel). Scale bar 20μm. c-e, 3 independent experiments. f, Visualization of CTSK-mGFP (green) cells in the periosteum (dotted white line) of mouse femur at post-natal day 6 (top) and day 12 (bottom). Nuclei shown by DAPI staining. Scale bar 20 μm. g, An enlarged view of osteocytes within the dotted white box is provided (i). CTSK-mGFP (green), nuclei (DAPI). Scale bar 20μm. 3 independent experiments. (ii) Quantification of total CTSK-mGFP labelled periosteal cells and mGFP labelled osteocytes in the mouse femur (*** p = 6.95×10 −16 , mean ± S.E.M, n= 12 distinct areas of periosteum from 3 independent experiments. 2 tailed Student’s t-test). h, An enlarged view for main text Fig. 1e is provided. Representative images from 3 independent experiments. i, FACS plots showing expression of CD49f (left) and CD51 (right) in CTSK-mGFP cells isolated from long bones of 7-day old mice (Lineage - = Ter119−, CD45− and CD31−). 5 independent experiments. j, 8 week old Ctsk-cre mouse femurs were immunostained for RUNX2 ( a, magenta, top), Alkaline phosphatase (ALPL) ( b, magenta, middle), and Osteocalcin ( c, magenta, bottom). CTSK-mGFP (green), nuclei stained by DAPI, co-localization shown by yellow arrows. Scale bar 20 μm. Representative images from 3 independent experiments. k, 12 day old Ctsk-cre mouse femurs were immunostained for Gremlin 1 ( a, magenta, top) and Nestin ( b, magenta, bottom). CTSK-mGFP (green), nuclei stained by DAPI, dotted white line indicates periosteum. Scale bar 20 μm. Representative images from 3 independent experiments.

    Techniques Used: Immunostaining, Staining, Mouse Assay, FACS, Expressing, Isolation

    Functional characterization of periosteal stem cells. a, Brightfield images, primary (left; scale bar 20μm) and secondary (right; scale bar 10μm) mesenspheres derived from PSCs (top), PP1s (middle) and PP2s (bottom). GFP (green) in insert. 3 independent experiments. b, % of cells able to form spheres (top) and cell number/sphere (bottom) in PSCs, PP1s and PP2s. ** p =0.0036 for % PSC tertiary mesenspheres, **** p =0.0001 for % PP1 secondary and tertiary mesenspheres, *** p = 0.0002 for % PP2 secondary, **** p = 0.0001 for % PP2 tertiary mesenspheres, (n=3 independent experiments). Dunnett’s multiple comparison test; mean ± S.D (top, bottom graphs) c, Immunostaining for CD200 (magenta) in primary (top) and secondary (bottom) PSC derived mesenspheres. DAPI (blue), scale bar 100μm. 3 independent experiments. d, Sorted PSCs cultured for 15 days and analyzed by flow (i, ii) 3 independent experiments. e, Single cell-derived PSC colonies were split for differentiation into osteoblasts (Alizarin red staining; left), adipocytes (Oil red O staining; middle; scale bar 50μm). Separately, chondrocyte differentiation potential was assayed (Alcian blue staining, right; scale bar 200μm). 3 independent experiments. f, Non-CTSK MSCs and CTSK-mGFP+ PSCs were transplanted into kidney capsule of secondary recipients (i, dotted black line). Donor origin of PSCs was confirmed with GFP retention (ii). Immunostaining for type 2 collagen (green in left box; magenta in right box) on 2 week non-CTSK MSC (red, left box) and PSC (green, right box)-derived organoids (iii). Safranin O (red) for cartilage and von Kossa (black) for mineralized bone on non-CTSK MSC (red box) and PSC (green box)-derived organoids 4 (top), 5 (middle) and 6 weeks (bottom) after transplantation (iv). Hematoxylin and eosin, non-CTSK MSC (top) and PSC (bottom)-derived organoids (v). 8 independent experiments. g, Schema of serial transplantation of PSCs into mouse mammary fat pad. h, FACS plots of PSC-derived cells after the first (i-iv) and second round of transplantation (v-viii). (Lineage- = Ter119−, CD45− and CD31−). Color coded boxes (green, magenta, orange) indicate parent/ daughter gates. 3 independent experiments.
    Figure Legend Snippet: Functional characterization of periosteal stem cells. a, Brightfield images, primary (left; scale bar 20μm) and secondary (right; scale bar 10μm) mesenspheres derived from PSCs (top), PP1s (middle) and PP2s (bottom). GFP (green) in insert. 3 independent experiments. b, % of cells able to form spheres (top) and cell number/sphere (bottom) in PSCs, PP1s and PP2s. ** p =0.0036 for % PSC tertiary mesenspheres, **** p =0.0001 for % PP1 secondary and tertiary mesenspheres, *** p = 0.0002 for % PP2 secondary, **** p = 0.0001 for % PP2 tertiary mesenspheres, (n=3 independent experiments). Dunnett’s multiple comparison test; mean ± S.D (top, bottom graphs) c, Immunostaining for CD200 (magenta) in primary (top) and secondary (bottom) PSC derived mesenspheres. DAPI (blue), scale bar 100μm. 3 independent experiments. d, Sorted PSCs cultured for 15 days and analyzed by flow (i, ii) 3 independent experiments. e, Single cell-derived PSC colonies were split for differentiation into osteoblasts (Alizarin red staining; left), adipocytes (Oil red O staining; middle; scale bar 50μm). Separately, chondrocyte differentiation potential was assayed (Alcian blue staining, right; scale bar 200μm). 3 independent experiments. f, Non-CTSK MSCs and CTSK-mGFP+ PSCs were transplanted into kidney capsule of secondary recipients (i, dotted black line). Donor origin of PSCs was confirmed with GFP retention (ii). Immunostaining for type 2 collagen (green in left box; magenta in right box) on 2 week non-CTSK MSC (red, left box) and PSC (green, right box)-derived organoids (iii). Safranin O (red) for cartilage and von Kossa (black) for mineralized bone on non-CTSK MSC (red box) and PSC (green box)-derived organoids 4 (top), 5 (middle) and 6 weeks (bottom) after transplantation (iv). Hematoxylin and eosin, non-CTSK MSC (top) and PSC (bottom)-derived organoids (v). 8 independent experiments. g, Schema of serial transplantation of PSCs into mouse mammary fat pad. h, FACS plots of PSC-derived cells after the first (i-iv) and second round of transplantation (v-viii). (Lineage- = Ter119−, CD45− and CD31−). Color coded boxes (green, magenta, orange) indicate parent/ daughter gates. 3 independent experiments.

    Techniques Used: Functional Assay, Derivative Assay, Immunostaining, Cell Culture, Flow Cytometry, Staining, Transplantation Assay, FACS

    Functional characterization of non-CTSK MSC cells, PSCs and its derivatives. a , Total numbers of PSCs and non-CTSK MSCs in mouse femurs at postnatal day 7, day 15 and day 32. Significant decreases in PSCs are observed at day 15 (** p =0.006) and day 32 (** p =0.009) when compared to day 7. Significant decreases in non-CTSK MSCs are observed at day 15 (*** p = 3.8×10 −5 ) and day 32 (*** p =0.0003) when compared to day 7 (mean ± S.D, n =3 independent experiments; 5 animals/group for day 7, day 15; 3 animals/group for day 32; 2-tailed Student’s t-test). b, μCT images of the bone formed by non-CTSK MSCs (left) and PSCs (right) 5 weeks after transplantation. Representative images from 5 independent experiments. c, Quantification of bone volume (BV) when equal numbers of non-CTSK MSCs and PSCs were transplanted into secondary hosts ( p = non-significant (ns), mean ± S.E.M, n= 3 independent experiments, 2-tailed Student’s t-test). d, Clonal non-CTSK MSC colonies were split for differentiation into osteoblasts (Alizarin red staining; left) and adipocytes (Oil red O staining; middle; scale bar 20μm). Separately, chondrocyte differentiation potential was assayed (Alcian blue staining, right; scale bar 100μm). Representative images from 4 independent experiments. e, Clonal differentiation capacity of 10 colonies isolated from PSCs and non-CTSK MSCs after subsequent culture under osteogenic (left) and adipogenic (right) differentiation conditions. All 10 colonies from each population were equally multipotent (mean ± S.D, n= 3 independent experiments. f, Bright field images of primary (left), secondary (middle) and tertiary mesenspheres (right, scale bar 20 μm) derived from non-CTSK MSCs. Tomato red (red) expression is shown in the inserts. Representative images from 3 independent experiments. g, Quantification of % of PSCs and non-CTSK MSCs able to form mesenspheres. * p =0.02, one way ANOVA, Dunnett’s multiple comparison test, mean ± S.D, n = 3 independent experiments. h, FACS analysis of in vitro differentiation of non-CTSK MSCs (right and left plots) after 15 days of culture. Color coded boxes (red) indicates parent/daughter gates. i, FACS plots of non-CTSK MSC-derived cells after the first round of mammary fat pad transplantation (Lineage- = Ter119−, CD45− and CD31−). Color coded boxes (red and green) indicates parent/daughter gates. h-i, Representative FACS plots from 3 independent experiments. j, FACS for CD140α (i) and CD146 (ii) in PSCs after transplantation into the mammary fat pad. k, FACS for expression of GFP (i),CD140α (ii), and CD146 (iii) in non-CTSK MSCs after mammary fat pad transplantation. l, PP1 cells were transplanted into the mammary fat pad of primary hosts for 2.5 weeks and then analyzed by FACS (i-iii). Color coded boxes (green and magenta) indicate parent/daughter gates. m-n, PP2 cells were isolated by FACS and implanted into the mammary fat-pad of primary recipients. The PP2-derived cells in primary recipients were analyzed by FACS ( m, i-iv), and PP2 cells were re-isolated for transplantation into secondary recipients. PP2-derived cells in secondary recipients were analyzed by FACS ( n, i-iv). Color coded boxes (green, magenta and orange) indicate parent/daughter gates. j-n, Representative FACS plots from 3 independent experiments.
    Figure Legend Snippet: Functional characterization of non-CTSK MSC cells, PSCs and its derivatives. a , Total numbers of PSCs and non-CTSK MSCs in mouse femurs at postnatal day 7, day 15 and day 32. Significant decreases in PSCs are observed at day 15 (** p =0.006) and day 32 (** p =0.009) when compared to day 7. Significant decreases in non-CTSK MSCs are observed at day 15 (*** p = 3.8×10 −5 ) and day 32 (*** p =0.0003) when compared to day 7 (mean ± S.D, n =3 independent experiments; 5 animals/group for day 7, day 15; 3 animals/group for day 32; 2-tailed Student’s t-test). b, μCT images of the bone formed by non-CTSK MSCs (left) and PSCs (right) 5 weeks after transplantation. Representative images from 5 independent experiments. c, Quantification of bone volume (BV) when equal numbers of non-CTSK MSCs and PSCs were transplanted into secondary hosts ( p = non-significant (ns), mean ± S.E.M, n= 3 independent experiments, 2-tailed Student’s t-test). d, Clonal non-CTSK MSC colonies were split for differentiation into osteoblasts (Alizarin red staining; left) and adipocytes (Oil red O staining; middle; scale bar 20μm). Separately, chondrocyte differentiation potential was assayed (Alcian blue staining, right; scale bar 100μm). Representative images from 4 independent experiments. e, Clonal differentiation capacity of 10 colonies isolated from PSCs and non-CTSK MSCs after subsequent culture under osteogenic (left) and adipogenic (right) differentiation conditions. All 10 colonies from each population were equally multipotent (mean ± S.D, n= 3 independent experiments. f, Bright field images of primary (left), secondary (middle) and tertiary mesenspheres (right, scale bar 20 μm) derived from non-CTSK MSCs. Tomato red (red) expression is shown in the inserts. Representative images from 3 independent experiments. g, Quantification of % of PSCs and non-CTSK MSCs able to form mesenspheres. * p =0.02, one way ANOVA, Dunnett’s multiple comparison test, mean ± S.D, n = 3 independent experiments. h, FACS analysis of in vitro differentiation of non-CTSK MSCs (right and left plots) after 15 days of culture. Color coded boxes (red) indicates parent/daughter gates. i, FACS plots of non-CTSK MSC-derived cells after the first round of mammary fat pad transplantation (Lineage- = Ter119−, CD45− and CD31−). Color coded boxes (red and green) indicates parent/daughter gates. h-i, Representative FACS plots from 3 independent experiments. j, FACS for CD140α (i) and CD146 (ii) in PSCs after transplantation into the mammary fat pad. k, FACS for expression of GFP (i),CD140α (ii), and CD146 (iii) in non-CTSK MSCs after mammary fat pad transplantation. l, PP1 cells were transplanted into the mammary fat pad of primary hosts for 2.5 weeks and then analyzed by FACS (i-iii). Color coded boxes (green and magenta) indicate parent/daughter gates. m-n, PP2 cells were isolated by FACS and implanted into the mammary fat-pad of primary recipients. The PP2-derived cells in primary recipients were analyzed by FACS ( m, i-iv), and PP2 cells were re-isolated for transplantation into secondary recipients. PP2-derived cells in secondary recipients were analyzed by FACS ( n, i-iv). Color coded boxes (green, magenta and orange) indicate parent/daughter gates. j-n, Representative FACS plots from 3 independent experiments.

    Techniques Used: Functional Assay, Transplantation Assay, Staining, Isolation, Derivative Assay, Expressing, FACS, In Vitro

    39) Product Images from "Integrated cytometry with machine learning applied to high-content imaging of human kidney tissue for in-situ cell classification and neighborhood analysis"

    Article Title: Integrated cytometry with machine learning applied to high-content imaging of human kidney tissue for in-situ cell classification and neighborhood analysis

    Journal: bioRxiv

    doi: 10.1101/2021.12.27.474025

    Separating masked populations of podocytes, leukocytes and endothelium by unsupervised analysis of cell image volumes in VTEA. A . Human nephrectomy tissue was fixed in and stained with DAPI and with antibodies against CD31 (yellow), CD45 (green) and Nestin (magenta) and imaged by confocal microscopy. Separated channels are given at right. All images are maximum intensity Z-projections, scale bar = 100 um. B . Nuclei segmented as fiduciaries of cell with connected components in 3D, ‘Connect 3D’, and associated intensities of CD31, CD45 and Nestin were measured. Ba . map of all segmented cells uniquely colorized. Bb-d Scatterplots of CD31, CD45 and Nestin demonstrate overlap in Nestin and CD31 signal in 2D plots. C . Unsupervised analysis of cell and marker intensity uncovers closely associated Nestin and CD31-positive cells. Ca-Cc . Mapping of cell associated intensity (every dot is a cell) to t -SNE projection calculated from the mean intensity of DAPI, CD31, CD45 and Nestin. Cd . Ward clustering dendogram using the same mean intensities with major bifurcation between labeled and unlabeled and at least 3 subclusters of labeled cells at k=5 (red line). Ce . Ward clusters mapped to t- SNE projection. Putative cell types are colored and labeled. Cf . Colorizing of segmented pixels for cells within CD45-positive, Nestin-positive and CD31-positive classes as given in Ce .
    Figure Legend Snippet: Separating masked populations of podocytes, leukocytes and endothelium by unsupervised analysis of cell image volumes in VTEA. A . Human nephrectomy tissue was fixed in and stained with DAPI and with antibodies against CD31 (yellow), CD45 (green) and Nestin (magenta) and imaged by confocal microscopy. Separated channels are given at right. All images are maximum intensity Z-projections, scale bar = 100 um. B . Nuclei segmented as fiduciaries of cell with connected components in 3D, ‘Connect 3D’, and associated intensities of CD31, CD45 and Nestin were measured. Ba . map of all segmented cells uniquely colorized. Bb-d Scatterplots of CD31, CD45 and Nestin demonstrate overlap in Nestin and CD31 signal in 2D plots. C . Unsupervised analysis of cell and marker intensity uncovers closely associated Nestin and CD31-positive cells. Ca-Cc . Mapping of cell associated intensity (every dot is a cell) to t -SNE projection calculated from the mean intensity of DAPI, CD31, CD45 and Nestin. Cd . Ward clustering dendogram using the same mean intensities with major bifurcation between labeled and unlabeled and at least 3 subclusters of labeled cells at k=5 (red line). Ce . Ward clusters mapped to t- SNE projection. Putative cell types are colored and labeled. Cf . Colorizing of segmented pixels for cells within CD45-positive, Nestin-positive and CD31-positive classes as given in Ce .

    Techniques Used: Staining, Confocal Microscopy, Marker, Labeling

    Automated glomerular census in mesoscale 3D tissue cytometry with machine learning classification and spatial analysis in VTEA. A . Human nephrectomy tissue was fixed in and stained with DAPI (gray) and with antibodies against CD31 (yellow), CD45 (green) and Nestin (magenta) and imaged by confocal microscopy. Scale bar = 500 um B . The whole 3D volume was analyzed, and 172,258 cells were segmented and processed by VTEA. Unsupervised analysis, G-means clustering and t -SNE mapping identified CD45-positive, CD31-positive, and Nestin-positive cells automatically. C . The cluster identities were confirmed by the plots generated by VTEA including average mean fluorescence intensity of each class, (heatmap, left panel) or by the distribution of mean fluorescence intensities for each cell (violin plots, right panels). D . Unsupervised classifications were used in combination (left panel) with regions-of-interest (ROIs) drawn with ImageJ during analysis to perform a glomerular census of the endothelium (CD31), leukocytes (CD45) and podocytes (Nestin). The census is presented as density of cells per mm 3 (right panels).
    Figure Legend Snippet: Automated glomerular census in mesoscale 3D tissue cytometry with machine learning classification and spatial analysis in VTEA. A . Human nephrectomy tissue was fixed in and stained with DAPI (gray) and with antibodies against CD31 (yellow), CD45 (green) and Nestin (magenta) and imaged by confocal microscopy. Scale bar = 500 um B . The whole 3D volume was analyzed, and 172,258 cells were segmented and processed by VTEA. Unsupervised analysis, G-means clustering and t -SNE mapping identified CD45-positive, CD31-positive, and Nestin-positive cells automatically. C . The cluster identities were confirmed by the plots generated by VTEA including average mean fluorescence intensity of each class, (heatmap, left panel) or by the distribution of mean fluorescence intensities for each cell (violin plots, right panels). D . Unsupervised classifications were used in combination (left panel) with regions-of-interest (ROIs) drawn with ImageJ during analysis to perform a glomerular census of the endothelium (CD31), leukocytes (CD45) and podocytes (Nestin). The census is presented as density of cells per mm 3 (right panels).

    Techniques Used: Cytometry, Staining, Confocal Microscopy, Generated, Fluorescence

    Using VTEA to classify leukocytes based on association with vascular endothelium. Using the image volume collected for Figure 4 , spatially defined neighborhoods with CD45-positive cells were generated in VTEA. A . Mesoscale maximum z-projection of human nephrectomy tissue stained with DAPI (gray) and with antibodies against [CD31 (yellow), CD45 (green) and Nestin (magenta) and presented as separate channels in gray. CD45-positive leukocytes localize in clusters that associate with CD31-positive structures including glomeruli and blood vessels ( Aa-Ad ). Scale bar = 500 um B . CD45-positive leukocytes and CD31+ endothelium was subgated from all cells ( Figure 4B ) and reclustered for k=5. Ba-Be , feature plots and VTEA generated heatmaps of mean signal intensity for CD45, Nestin and CD31 uncovers podocytes (Pod., green), Endothelium (Endo., red) and two putative populations of CD45-positive cells based on DAPI and CD45 intensity (p1 or pop1 vs. p2 or pop2, orange and blue respectively). Bf . Using cell-wise export with VTEA ground truth generation routine, 100 cells sampled from the two CD45-positive populations. Ca-Cb . Maximum projections of mapped CD45-p1 and CD45-p2 in the image volume identifies tissue regions of mixed, and uniquely CD45-p2 cells. Cg , neighborhood analysis, for every classified cell within r = 25 um and unsupervised clustering of neighborhoods based on cellular census, demonstrates CD45-p1 are found in neighborhoods with (N4, red) and without endothelium (Endo, N7-9, green e.g., Ca and Ce ) in neighborhoods with CD45-p2. CD45-p2 are also found alone without CD45-p1 nor endothelium (N10, orange, e.g., Cd and Cf ).
    Figure Legend Snippet: Using VTEA to classify leukocytes based on association with vascular endothelium. Using the image volume collected for Figure 4 , spatially defined neighborhoods with CD45-positive cells were generated in VTEA. A . Mesoscale maximum z-projection of human nephrectomy tissue stained with DAPI (gray) and with antibodies against [CD31 (yellow), CD45 (green) and Nestin (magenta) and presented as separate channels in gray. CD45-positive leukocytes localize in clusters that associate with CD31-positive structures including glomeruli and blood vessels ( Aa-Ad ). Scale bar = 500 um B . CD45-positive leukocytes and CD31+ endothelium was subgated from all cells ( Figure 4B ) and reclustered for k=5. Ba-Be , feature plots and VTEA generated heatmaps of mean signal intensity for CD45, Nestin and CD31 uncovers podocytes (Pod., green), Endothelium (Endo., red) and two putative populations of CD45-positive cells based on DAPI and CD45 intensity (p1 or pop1 vs. p2 or pop2, orange and blue respectively). Bf . Using cell-wise export with VTEA ground truth generation routine, 100 cells sampled from the two CD45-positive populations. Ca-Cb . Maximum projections of mapped CD45-p1 and CD45-p2 in the image volume identifies tissue regions of mixed, and uniquely CD45-p2 cells. Cg , neighborhood analysis, for every classified cell within r = 25 um and unsupervised clustering of neighborhoods based on cellular census, demonstrates CD45-p1 are found in neighborhoods with (N4, red) and without endothelium (Endo, N7-9, green e.g., Ca and Ce ) in neighborhoods with CD45-p2. CD45-p2 are also found alone without CD45-p1 nor endothelium (N10, orange, e.g., Cd and Cf ).

    Techniques Used: Generated, Staining

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    thermo fisher mouse specific cd31
    VEGF signaling and activation of endogenous ETV2 in the standard S1-S2 differentiation protocol. ( A-D ) Generation of h-iPSCs-KDR -/- and h-iPSCs-ETV2 -/- clones by CRISPR/Cas9. ( A ) Sanger sequencing of the two edited alleles encoding the 3 rd exon of KDR . ( B ) Flow cytometry showed the conversion of h-iPSCs-KDR -/- into <t>FLK1-/CD31+</t> h-iECs at 48 h using the early modETV2 protocol. ( C ) Sanger sequencing of the two edited alleles encoding the 4 th exon of ETV2 . ( D ) Immunofluorescence staining for ETV2 at 72 h using the S1-S2 differentiation protocol. Nuclei stained by DAPI. Scale bar, 200 μm. ( E ) Differences in differentiation efficiency between four alternative S1-S2 methodologies and the S1-modETV2 protocol for h-iPSC clones lacking either ETV2 and KDR (h-iPSC-ETV2 -/- and h-iPSC-KDR -/- ). Only S1-modETV2 protocol could successfully derive h-iECs from either h-iPSC-ETV2 -/- or h-iPSC-KDR -/- cell lines with high efficiency. In contrast, the four alternative S1-S2 methodologies failed to get any h-iECs. ( F-G ) Effect of VEGF-A concentration on h-iEC yield using the S1-S2 differentiation protocol. ( F ) Dose dependent conversion efficiency of h-iPSCs into CD31+ h-iECs by flow cytometry. ( G ) Immunofluorescence staining for ETV2 and VE-Cadherin at 72 h with different concentrations of VEGF-A. Nuclei stained by DAPI. Scale bar, 50 μm.
    Mouse Specific Cd31, supplied by thermo fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse specific cd31/product/thermo fisher
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    Thermo Fisher cd31 pe
    Bone-derived mesenchymal stem cell (MSC) isolation, identification, and injection. a Morphology of MSCs in cell culture. Cultured cells showed typically spindle-shaped morphology under phase-contrast microscopy. b Schematic diagram of cell injection into the striatum of the brain and the survival of MSCs after injection. c Green fluorescent (CFDA SE dye) cells were located in the ischemic hemisphere after 3 days of injection. Scale bar = 300 μm. d Cytometry analysis depicted that 99.99 + % cultured cells were positive for CD29 and CD90 and negative for <t>CD31</t> and CD45
    Cd31 Pe, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cd31 pe/product/Thermo Fisher
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    98
    Thermo Fisher antibodies against mouse cd3
    Deletion of Stim1 and Stim2 Prolongs the Survival of Mice with T-ALL (A and B) STIM1 (A) and STIM2 ); hematological malignancies are in red. (C) Experimental model of T-ALL used in (D)–(I). BM cells isolated either from poly(I:C)-treated wild-type (WT) or Stim1/2 fl/fl Mx1-Cre mice and untreated WT or Stim1/2 fl/fl Vav-Cre mice ( Stim1/2 −/− ) were transduced with ICN1-IRES-GFP and cultured for 3 days in vitro. 5 × 10 4 ICN1-transduced (GFP + ) lineage negative (lin – ) BM progenitor cells were injected i.v. (together with 5 × 10 5 bone marrow cells from WT mice) into lethally irradiated CD45.1 + WT host mice. (D) SOCE in c-kit + progenitor cells isolated from WT and Stim1/2 −/− mice. (E and F) Representative flow cytometry plots of surface (s) and cytoplasmic (cy) <t>CD3</t> expression (E) or CD4/CD8 expression (F) on cells isolated from the BMand spleen of healthy WT control mice (Ctrl) and mice with WT or Stim1/2 −/− leukemia (gated on GFP + ). (G) SOCE in WT and Stim1/2 −/− leukemic cells (GFP + ) isolated from the spleen at 21 days of disease. (H) Relative weight of mice with WT and Stim1/2 −/− leukemia. Values shown are mean ± SEM. Statistical analysis was performed using Student’s t test. *p
    Antibodies Against Mouse Cd3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    VEGF signaling and activation of endogenous ETV2 in the standard S1-S2 differentiation protocol. ( A-D ) Generation of h-iPSCs-KDR -/- and h-iPSCs-ETV2 -/- clones by CRISPR/Cas9. ( A ) Sanger sequencing of the two edited alleles encoding the 3 rd exon of KDR . ( B ) Flow cytometry showed the conversion of h-iPSCs-KDR -/- into FLK1-/CD31+ h-iECs at 48 h using the early modETV2 protocol. ( C ) Sanger sequencing of the two edited alleles encoding the 4 th exon of ETV2 . ( D ) Immunofluorescence staining for ETV2 at 72 h using the S1-S2 differentiation protocol. Nuclei stained by DAPI. Scale bar, 200 μm. ( E ) Differences in differentiation efficiency between four alternative S1-S2 methodologies and the S1-modETV2 protocol for h-iPSC clones lacking either ETV2 and KDR (h-iPSC-ETV2 -/- and h-iPSC-KDR -/- ). Only S1-modETV2 protocol could successfully derive h-iECs from either h-iPSC-ETV2 -/- or h-iPSC-KDR -/- cell lines with high efficiency. In contrast, the four alternative S1-S2 methodologies failed to get any h-iECs. ( F-G ) Effect of VEGF-A concentration on h-iEC yield using the S1-S2 differentiation protocol. ( F ) Dose dependent conversion efficiency of h-iPSCs into CD31+ h-iECs by flow cytometry. ( G ) Immunofluorescence staining for ETV2 and VE-Cadherin at 72 h with different concentrations of VEGF-A. Nuclei stained by DAPI. Scale bar, 50 μm.

    Journal: bioRxiv

    Article Title: Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA

    doi: 10.1101/2020.03.02.973289

    Figure Lengend Snippet: VEGF signaling and activation of endogenous ETV2 in the standard S1-S2 differentiation protocol. ( A-D ) Generation of h-iPSCs-KDR -/- and h-iPSCs-ETV2 -/- clones by CRISPR/Cas9. ( A ) Sanger sequencing of the two edited alleles encoding the 3 rd exon of KDR . ( B ) Flow cytometry showed the conversion of h-iPSCs-KDR -/- into FLK1-/CD31+ h-iECs at 48 h using the early modETV2 protocol. ( C ) Sanger sequencing of the two edited alleles encoding the 4 th exon of ETV2 . ( D ) Immunofluorescence staining for ETV2 at 72 h using the S1-S2 differentiation protocol. Nuclei stained by DAPI. Scale bar, 200 μm. ( E ) Differences in differentiation efficiency between four alternative S1-S2 methodologies and the S1-modETV2 protocol for h-iPSC clones lacking either ETV2 and KDR (h-iPSC-ETV2 -/- and h-iPSC-KDR -/- ). Only S1-modETV2 protocol could successfully derive h-iECs from either h-iPSC-ETV2 -/- or h-iPSC-KDR -/- cell lines with high efficiency. In contrast, the four alternative S1-S2 methodologies failed to get any h-iECs. ( F-G ) Effect of VEGF-A concentration on h-iEC yield using the S1-S2 differentiation protocol. ( F ) Dose dependent conversion efficiency of h-iPSCs into CD31+ h-iECs by flow cytometry. ( G ) Immunofluorescence staining for ETV2 and VE-Cadherin at 72 h with different concentrations of VEGF-A. Nuclei stained by DAPI. Scale bar, 50 μm.

    Article Snippet: Microvessel density was reported as the average number of erythrocyte-filled vessels (vessels/mm2) in H & E-stained sections from the middle of the implants as previously were deparaffinized and antigen retrieval was carried out with boiling citric buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 30 min. Proteinase K (Abcam, Cat No. ab64220) treatment for 15 min was only applied to retrieve the mouse-specific CD31 described.

    Techniques: Activation Assay, Clone Assay, CRISPR, Sequencing, Flow Cytometry, Immunofluorescence, Staining, Concentration Assay

    Generation of h-iECs with the early modETV2 differentiation protocol. ( A ) Schematic of the early modETV2 protocol. ( B ) Effect of modRNA(ETV2) concentration on h-iPSC-to-h-iEC conversion efficiency at 48 h and 96 h. Titration analysis by flow cytometry for electroporation-based delivery of modRNA. ( C ) Flow cytometry analysis of differentiation efficiency at 48 h (early modETV2 protocol) and 96 h (S1-S2 protocol) in 13 h-iPSC clones generated from dermal fibroblasts (dFB), umbilical cord blood-derived ECFCs (cbECFC), and urine-derived epithelial cells (uEP). ( D ) Differences in differentiation efficiency between early modETV2 and S1-S2 protocols for all 13 h-iPSC clones. Bars represent mean ± s.d. ( E ) Time course immunofluorescence staining for ETV2, CD31 and OCT4 during the early modETV2 differentiation protocol. Nuclei stained by DAPI. Scale bar, 100 μm. Phase contrast micrographs represent time course morphological changes of cells during the early modETV2 protocol. Scale bar, 200 μm. ( F ) Time course analysis of mRNA expression (qRT–PCR) of mesodermal markers ( TBXT and MIXL1 ), endothelial commitment transcription factors ( ETV2 and ERG ), endothelial markers ( PECAM1, CDH5, NOS3, VWF, TEK and KDR ), pluripotency marker ( POU5F1 ) and smooth muscle marker ( ACTA2 ) in h-iECs generated with the early modETV2 protocol. Data normalized to GAPDH expression.

    Journal: bioRxiv

    Article Title: Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA

    doi: 10.1101/2020.03.02.973289

    Figure Lengend Snippet: Generation of h-iECs with the early modETV2 differentiation protocol. ( A ) Schematic of the early modETV2 protocol. ( B ) Effect of modRNA(ETV2) concentration on h-iPSC-to-h-iEC conversion efficiency at 48 h and 96 h. Titration analysis by flow cytometry for electroporation-based delivery of modRNA. ( C ) Flow cytometry analysis of differentiation efficiency at 48 h (early modETV2 protocol) and 96 h (S1-S2 protocol) in 13 h-iPSC clones generated from dermal fibroblasts (dFB), umbilical cord blood-derived ECFCs (cbECFC), and urine-derived epithelial cells (uEP). ( D ) Differences in differentiation efficiency between early modETV2 and S1-S2 protocols for all 13 h-iPSC clones. Bars represent mean ± s.d. ( E ) Time course immunofluorescence staining for ETV2, CD31 and OCT4 during the early modETV2 differentiation protocol. Nuclei stained by DAPI. Scale bar, 100 μm. Phase contrast micrographs represent time course morphological changes of cells during the early modETV2 protocol. Scale bar, 200 μm. ( F ) Time course analysis of mRNA expression (qRT–PCR) of mesodermal markers ( TBXT and MIXL1 ), endothelial commitment transcription factors ( ETV2 and ERG ), endothelial markers ( PECAM1, CDH5, NOS3, VWF, TEK and KDR ), pluripotency marker ( POU5F1 ) and smooth muscle marker ( ACTA2 ) in h-iECs generated with the early modETV2 protocol. Data normalized to GAPDH expression.

    Article Snippet: Microvessel density was reported as the average number of erythrocyte-filled vessels (vessels/mm2) in H & E-stained sections from the middle of the implants as previously were deparaffinized and antigen retrieval was carried out with boiling citric buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 30 min. Proteinase K (Abcam, Cat No. ab64220) treatment for 15 min was only applied to retrieve the mouse-specific CD31 described.

    Techniques: Concentration Assay, Titration, Flow Cytometry, Electroporation, Clone Assay, Generated, Derivative Assay, Immunofluorescence, Staining, Expressing, Quantitative RT-PCR, Marker

    Inefficient activation of endogenous ETV2 in intermediate h-MPCs during the standard S1-S2 differentiation protocol. ( A ) Time course analysis of mRNA expression (qRT– PCR) of transcription factors TBXT (mesodermal commitment) and ETV2 (endothelial commitment) during the standard S1-S2 differentiation protocol. Relative fold change normalized to GAPDH expression. ( B ) Immunofluorescence staining for Brachyury in h-iPSCs at 48 h during the S1-S2 protocol. h-iPSCs lacking endogenous ETV2 (h-iPSCs-ETV2 -/- ) served as control. Nuclei stained by DAPI. Scale bar, 100 μm. Percentage of Brachyury+ cells at day 1. ( C ) Immunofluorescence staining for ETV2 in h-iPSCs at 72 h during the S1-S2 protocol. h-iPSCs-ETV2 -/- served as control. Nuclei stained by DAPI. Scale bar, 100 μm. Percentage of ETV2+ cells at day 3. ( D ) Effect of VEGF-A concentration on the percentages of ETV2+ cells at 72 h and CD31+ cells at 96 h during the S1-S2 protocol measured by immunofluorescence staining (ETV2) and flow cytometry (CD31). ( E ) Immunofluorescence staining for ETV2 in h-iPSCs during the optimized S1-modETV2 protocol. h-iPSCs-ETV2 -/- served as control. Nuclei stained by DAPI. Scale bar, 100 μm. Percentage of ETV2+ cells after transfection with modRNA. ( F ) Conversion efficiency of h-iPSCs into CD31+ h-iECs by flow cytometry. Comparison of the standard S1-S2 and the S1-modETV2 protocols. h-iPSCs-ETV2 -/- , h-iPSCs-KDR -/- , and h-iPSCs treated with the VEGFR2 inhibitor SU5416 served as controls. In panels b, c, and e, bars represent mean ± s.d.; n = 4; n.s. = no statistical differences and *** P

    Journal: bioRxiv

    Article Title: Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA

    doi: 10.1101/2020.03.02.973289

    Figure Lengend Snippet: Inefficient activation of endogenous ETV2 in intermediate h-MPCs during the standard S1-S2 differentiation protocol. ( A ) Time course analysis of mRNA expression (qRT– PCR) of transcription factors TBXT (mesodermal commitment) and ETV2 (endothelial commitment) during the standard S1-S2 differentiation protocol. Relative fold change normalized to GAPDH expression. ( B ) Immunofluorescence staining for Brachyury in h-iPSCs at 48 h during the S1-S2 protocol. h-iPSCs lacking endogenous ETV2 (h-iPSCs-ETV2 -/- ) served as control. Nuclei stained by DAPI. Scale bar, 100 μm. Percentage of Brachyury+ cells at day 1. ( C ) Immunofluorescence staining for ETV2 in h-iPSCs at 72 h during the S1-S2 protocol. h-iPSCs-ETV2 -/- served as control. Nuclei stained by DAPI. Scale bar, 100 μm. Percentage of ETV2+ cells at day 3. ( D ) Effect of VEGF-A concentration on the percentages of ETV2+ cells at 72 h and CD31+ cells at 96 h during the S1-S2 protocol measured by immunofluorescence staining (ETV2) and flow cytometry (CD31). ( E ) Immunofluorescence staining for ETV2 in h-iPSCs during the optimized S1-modETV2 protocol. h-iPSCs-ETV2 -/- served as control. Nuclei stained by DAPI. Scale bar, 100 μm. Percentage of ETV2+ cells after transfection with modRNA. ( F ) Conversion efficiency of h-iPSCs into CD31+ h-iECs by flow cytometry. Comparison of the standard S1-S2 and the S1-modETV2 protocols. h-iPSCs-ETV2 -/- , h-iPSCs-KDR -/- , and h-iPSCs treated with the VEGFR2 inhibitor SU5416 served as controls. In panels b, c, and e, bars represent mean ± s.d.; n = 4; n.s. = no statistical differences and *** P

    Article Snippet: Microvessel density was reported as the average number of erythrocyte-filled vessels (vessels/mm2) in H & E-stained sections from the middle of the implants as previously were deparaffinized and antigen retrieval was carried out with boiling citric buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 30 min. Proteinase K (Abcam, Cat No. ab64220) treatment for 15 min was only applied to retrieve the mouse-specific CD31 described.

    Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Concentration Assay, Flow Cytometry, Transfection

    Derivation of h-iECs from h-iPSCs with ETV2 modRNA. ( A ) Schematic of optimized two-stage endothelial differentiation protocol. Stage 1: conversion of h-iPSCs into h-MPCs mediated by the GSK-3 inhibitor CHIR99021. Stage 2: transfection of h-MPCs with modRNA encoding ETV2 and culture in chemically defined medium. A group that used modRNA:GFP served as control. ( B ) Conversion efficiency of h-iPSCs into VE-Cadherin+/CD31+ h-iECs measured by flow cytometry at day 4 for both the S1-S2 (left; no electroporation, no modRNA) and S1-modETV2 (right) protocols. Groups corresponding to electroporation without modRNA and electroporation with modRNA encoding GFP served as controls for the S1-modETV2 group. ( C-D ) Effect of modRNA(ETV2) concentration on h-iPSC-to-h-iEC conversion efficiency at 96 h using the S1-modETV2 differentiation protocol. ( C ) Titration analysis by flow cytometry for electroporation-based delivery of modRNA. ( D ) Titration analysis by flow cytometry for lipofection-based delivery of modRNA. (E) Immunofluorescence staining for VE-Cadherin and SM22 between S1-S2 and S1-modETV2 protocols at day 4. Nuclei stained by DAPI. Scale bar, 100 μm. Percentage of SM22+/VE-Cadherin-cells. Bars represent mean ± s.d.; n = 5. *** P

    Journal: bioRxiv

    Article Title: Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA

    doi: 10.1101/2020.03.02.973289

    Figure Lengend Snippet: Derivation of h-iECs from h-iPSCs with ETV2 modRNA. ( A ) Schematic of optimized two-stage endothelial differentiation protocol. Stage 1: conversion of h-iPSCs into h-MPCs mediated by the GSK-3 inhibitor CHIR99021. Stage 2: transfection of h-MPCs with modRNA encoding ETV2 and culture in chemically defined medium. A group that used modRNA:GFP served as control. ( B ) Conversion efficiency of h-iPSCs into VE-Cadherin+/CD31+ h-iECs measured by flow cytometry at day 4 for both the S1-S2 (left; no electroporation, no modRNA) and S1-modETV2 (right) protocols. Groups corresponding to electroporation without modRNA and electroporation with modRNA encoding GFP served as controls for the S1-modETV2 group. ( C-D ) Effect of modRNA(ETV2) concentration on h-iPSC-to-h-iEC conversion efficiency at 96 h using the S1-modETV2 differentiation protocol. ( C ) Titration analysis by flow cytometry for electroporation-based delivery of modRNA. ( D ) Titration analysis by flow cytometry for lipofection-based delivery of modRNA. (E) Immunofluorescence staining for VE-Cadherin and SM22 between S1-S2 and S1-modETV2 protocols at day 4. Nuclei stained by DAPI. Scale bar, 100 μm. Percentage of SM22+/VE-Cadherin-cells. Bars represent mean ± s.d.; n = 5. *** P

    Article Snippet: Microvessel density was reported as the average number of erythrocyte-filled vessels (vessels/mm2) in H & E-stained sections from the middle of the implants as previously were deparaffinized and antigen retrieval was carried out with boiling citric buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 30 min. Proteinase K (Abcam, Cat No. ab64220) treatment for 15 min was only applied to retrieve the mouse-specific CD31 described.

    Techniques: Transfection, Flow Cytometry, Electroporation, Concentration Assay, Titration, Immunofluorescence, Staining

    Transcriptional analysis of h-iECs obtained from various differentiation protocols. ( A ) Schematic of protocol for early transfection of h-iPSCs with modRNA encoding ETV2 . ( B ) Conversion efficiency h-iPSCs into CD31+ cells by flow cytometry using the early modETV2 protocol. ( C-H ) RNAseq analysis across multiple h-iECs samples generated from three independent h-iPSC lines using all three differentiation protocols. Human ECFCs and the parental undifferentiated h-iPSCs served as positive and negative controls, respectively. ( C ) Number of differentially expressed genes between h-iECs samples from each differentiation protocol. ( D ) Pairwise correlation based on Pearson coefficients between all samples. ( E ) Principal component analysis. ( F ) Heatmap and hierarchical clustering analysis of global differentially expressed genes. ( G ) Heatmap and hierarchical clustering analysis of selected EC-specific genes. ( H ) GO analysis between h-iECs generated with the S1-modETV2 and the early modETV2 differentiation protocols. Analysis carried out with differentially expressed genes from EC clusters #5 and #10 based on (f). Genes displayed correspond to positive enrichment for h-iECs generated with the S1-modETV2 protocol.

    Journal: bioRxiv

    Article Title: Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA

    doi: 10.1101/2020.03.02.973289

    Figure Lengend Snippet: Transcriptional analysis of h-iECs obtained from various differentiation protocols. ( A ) Schematic of protocol for early transfection of h-iPSCs with modRNA encoding ETV2 . ( B ) Conversion efficiency h-iPSCs into CD31+ cells by flow cytometry using the early modETV2 protocol. ( C-H ) RNAseq analysis across multiple h-iECs samples generated from three independent h-iPSC lines using all three differentiation protocols. Human ECFCs and the parental undifferentiated h-iPSCs served as positive and negative controls, respectively. ( C ) Number of differentially expressed genes between h-iECs samples from each differentiation protocol. ( D ) Pairwise correlation based on Pearson coefficients between all samples. ( E ) Principal component analysis. ( F ) Heatmap and hierarchical clustering analysis of global differentially expressed genes. ( G ) Heatmap and hierarchical clustering analysis of selected EC-specific genes. ( H ) GO analysis between h-iECs generated with the S1-modETV2 and the early modETV2 differentiation protocols. Analysis carried out with differentially expressed genes from EC clusters #5 and #10 based on (f). Genes displayed correspond to positive enrichment for h-iECs generated with the S1-modETV2 protocol.

    Article Snippet: Microvessel density was reported as the average number of erythrocyte-filled vessels (vessels/mm2) in H & E-stained sections from the middle of the implants as previously were deparaffinized and antigen retrieval was carried out with boiling citric buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 30 min. Proteinase K (Abcam, Cat No. ab64220) treatment for 15 min was only applied to retrieve the mouse-specific CD31 described.

    Techniques: Transfection, Flow Cytometry, Generated

    In vivo vascular network-forming ability of h-iECs without h-MSCs. ( A ) Grafts contained only h-iECs that were generated by the S1-modETV2 protocol and expanded until day 18. Images are macroscopic views of the explanted grafts from four mice at day 7. ( B ) Hematoxylin and eosin (H E) staining of explanted grafts after 7 days in vivo . Perfused vessels were identified as luminal structures containing red blood cells (yellow arrowheads). Scale bar, 400 μm. ( C ) Immunofluorescent staining of explanted grafts after 7 days in vivo . Human lumens stained by h-CD31. Perivascular coverage stained by α-SMA. Nuclei stained by DAPI. Scale bar, 100 μm. ( D ) Immunofluorescence staining of explanted grafts that contained h-iECs-GFP after 7 days in vivo . Human lumens stained by GFP (green). Perivascular cells stained by α-SMA (red). Nuclei stained by DAPI. Scale bar, 50 μm.

    Journal: bioRxiv

    Article Title: Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA

    doi: 10.1101/2020.03.02.973289

    Figure Lengend Snippet: In vivo vascular network-forming ability of h-iECs without h-MSCs. ( A ) Grafts contained only h-iECs that were generated by the S1-modETV2 protocol and expanded until day 18. Images are macroscopic views of the explanted grafts from four mice at day 7. ( B ) Hematoxylin and eosin (H E) staining of explanted grafts after 7 days in vivo . Perfused vessels were identified as luminal structures containing red blood cells (yellow arrowheads). Scale bar, 400 μm. ( C ) Immunofluorescent staining of explanted grafts after 7 days in vivo . Human lumens stained by h-CD31. Perivascular coverage stained by α-SMA. Nuclei stained by DAPI. Scale bar, 100 μm. ( D ) Immunofluorescence staining of explanted grafts that contained h-iECs-GFP after 7 days in vivo . Human lumens stained by GFP (green). Perivascular cells stained by α-SMA (red). Nuclei stained by DAPI. Scale bar, 50 μm.

    Article Snippet: Microvessel density was reported as the average number of erythrocyte-filled vessels (vessels/mm2) in H & E-stained sections from the middle of the implants as previously were deparaffinized and antigen retrieval was carried out with boiling citric buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 30 min. Proteinase K (Abcam, Cat No. ab64220) treatment for 15 min was only applied to retrieve the mouse-specific CD31 described.

    Techniques: In Vivo, Generated, Mouse Assay, Staining, Immunofluorescence

    Robust endothelial phenotype of h-iECs along their expansion in culture. h-iECs generated by the S1-modETV2 protocol maintained an endothelial phenotype during in vitro expansion. ( A ) Schematic of S1-modETV2 protocol to generate, purify, and expand h-iECs. h-iECs were analyzed at three different time points of expansion (namely, day 4, day 11, and day 21). (B) Flow cytometry analysis revealed that h-iECs remained fairly pure during expansion ( > 95% cells are VE-cadherin+/CD31+). Confluent h-iECs showed typical cobblestone morphology and UEA-I staining. ( C-D ) The expanded h-iECs maintained expression of EC markers at the mRNA (C) and protein ( D ) levels and remained negative for POU5F1 (OCT4) and α-Smooth muscle actin (α-SMA).

    Journal: bioRxiv

    Article Title: Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA

    doi: 10.1101/2020.03.02.973289

    Figure Lengend Snippet: Robust endothelial phenotype of h-iECs along their expansion in culture. h-iECs generated by the S1-modETV2 protocol maintained an endothelial phenotype during in vitro expansion. ( A ) Schematic of S1-modETV2 protocol to generate, purify, and expand h-iECs. h-iECs were analyzed at three different time points of expansion (namely, day 4, day 11, and day 21). (B) Flow cytometry analysis revealed that h-iECs remained fairly pure during expansion ( > 95% cells are VE-cadherin+/CD31+). Confluent h-iECs showed typical cobblestone morphology and UEA-I staining. ( C-D ) The expanded h-iECs maintained expression of EC markers at the mRNA (C) and protein ( D ) levels and remained negative for POU5F1 (OCT4) and α-Smooth muscle actin (α-SMA).

    Article Snippet: Microvessel density was reported as the average number of erythrocyte-filled vessels (vessels/mm2) in H & E-stained sections from the middle of the implants as previously were deparaffinized and antigen retrieval was carried out with boiling citric buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 30 min. Proteinase K (Abcam, Cat No. ab64220) treatment for 15 min was only applied to retrieve the mouse-specific CD31 described.

    Techniques: Generated, In Vitro, Flow Cytometry, Staining, Expressing

    Transcriptional analysis of h-iECs obtained from various differentiation protocols. ( A ) Schematic of protocol for early transfection of h-iPSCs with modRNA encoding ETV2 . ( B ) Conversion efficiency h-iPSCs into CD31 + cells by flow cytometry using the early modETV2 protocol. ( C to H ) RNA-seq analysis across multiple h-iEC samples generated from three independent h-iPSC lines using all three differentiation protocols. Human ECFCs and the parental undifferentiated h-iPSCs served as positive and negative controls, respectively. (C) Number of differentially expressed genes between h-iEC samples from each differentiation protocol. (D) Pairwise correlation based on Pearson coefficients between all samples. (E) Principal components (PC) analysis. (F) Heat map and hierarchical clustering analysis of global differentially expressed genes. (G) Heat map and hierarchical clustering analysis of selected EC-specific genes. (H) Gene ontology analysis between h-iECs generated with the S1-modETV2 and the early modETV2 differentiation protocols. Analysis carried out with differentially expressed genes from EC clusters 5 and 10 based on (F). Genes displayed correspond to positive enrichment for h-iECs generated with the S1-modETV2 protocol.

    Journal: Science Advances

    Article Title: Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA

    doi: 10.1126/sciadv.aba7606

    Figure Lengend Snippet: Transcriptional analysis of h-iECs obtained from various differentiation protocols. ( A ) Schematic of protocol for early transfection of h-iPSCs with modRNA encoding ETV2 . ( B ) Conversion efficiency h-iPSCs into CD31 + cells by flow cytometry using the early modETV2 protocol. ( C to H ) RNA-seq analysis across multiple h-iEC samples generated from three independent h-iPSC lines using all three differentiation protocols. Human ECFCs and the parental undifferentiated h-iPSCs served as positive and negative controls, respectively. (C) Number of differentially expressed genes between h-iEC samples from each differentiation protocol. (D) Pairwise correlation based on Pearson coefficients between all samples. (E) Principal components (PC) analysis. (F) Heat map and hierarchical clustering analysis of global differentially expressed genes. (G) Heat map and hierarchical clustering analysis of selected EC-specific genes. (H) Gene ontology analysis between h-iECs generated with the S1-modETV2 and the early modETV2 differentiation protocols. Analysis carried out with differentially expressed genes from EC clusters 5 and 10 based on (F). Genes displayed correspond to positive enrichment for h-iECs generated with the S1-modETV2 protocol.

    Article Snippet: For immunostaining, sections were deparaffinized, and antigen retrieval was carried out with boiling citric buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 30 min. Proteinase K (Abcam, catalog no. ab64220) treatment for 15 min was only applied to retrieve the mouse-specific CD31.

    Techniques: Transfection, Flow Cytometry, RNA Sequencing Assay, Generated

    Inefficient activation of endogenous ETV2 in intermediate h-MPCs during the standard S1-S2 differentiation protocol. ( A ) mRNA expression of TBXT and ETV2 during the S1-S2 differentiation protocol. Data normalized to GAPDH expression. ( B ) Brachyury expression during the S1-S2 protocol. h-iPSCs lacking endogenous ETV2 (h-iPSCs-ETV2 −/− ) served as control. Percentage of Brachyury + cells at 48 hours. ( C ) ETV2 expression during the S1-S2 protocol. Percentage of ETV2 + cells at 72 hours. ( D ) Effect of VEGF-A concentration on the percentages of ETV2 + cells at 72 hours (immunofluorescence staining) and CD31 + cells at 96 hours (flow cytometry) during the S1-S2 protocol. ( E ) Immunofluorescence staining for ETV2 in h-iPSCs during the S1-modETV2 protocol. h-iPSCs-ETV2 −/− served as control. Percentage of ETV2 + cells after transfection with modRNA. ( F ) Conversion efficiency of h-iPSCs into CD31 + h-iECs by flow cytometry. Comparison of the S1-S2 and the S1-modETV2 protocols. h-iPSCs-ETV2 −/− , h-iPSCs-KDR −/− , and h-iPSCs treated with the VEGF receptor 2 inhibitor SU5416 served as controls. SSC-H, side scatter height. In (B), (C), and (E), nuclei stained by DAPI. Scale bar, 100 μm. Bars represent means ± SD; n = 4; n.s., no statistical differences; and *** P

    Journal: Science Advances

    Article Title: Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA

    doi: 10.1126/sciadv.aba7606

    Figure Lengend Snippet: Inefficient activation of endogenous ETV2 in intermediate h-MPCs during the standard S1-S2 differentiation protocol. ( A ) mRNA expression of TBXT and ETV2 during the S1-S2 differentiation protocol. Data normalized to GAPDH expression. ( B ) Brachyury expression during the S1-S2 protocol. h-iPSCs lacking endogenous ETV2 (h-iPSCs-ETV2 −/− ) served as control. Percentage of Brachyury + cells at 48 hours. ( C ) ETV2 expression during the S1-S2 protocol. Percentage of ETV2 + cells at 72 hours. ( D ) Effect of VEGF-A concentration on the percentages of ETV2 + cells at 72 hours (immunofluorescence staining) and CD31 + cells at 96 hours (flow cytometry) during the S1-S2 protocol. ( E ) Immunofluorescence staining for ETV2 in h-iPSCs during the S1-modETV2 protocol. h-iPSCs-ETV2 −/− served as control. Percentage of ETV2 + cells after transfection with modRNA. ( F ) Conversion efficiency of h-iPSCs into CD31 + h-iECs by flow cytometry. Comparison of the S1-S2 and the S1-modETV2 protocols. h-iPSCs-ETV2 −/− , h-iPSCs-KDR −/− , and h-iPSCs treated with the VEGF receptor 2 inhibitor SU5416 served as controls. SSC-H, side scatter height. In (B), (C), and (E), nuclei stained by DAPI. Scale bar, 100 μm. Bars represent means ± SD; n = 4; n.s., no statistical differences; and *** P

    Article Snippet: For immunostaining, sections were deparaffinized, and antigen retrieval was carried out with boiling citric buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 30 min. Proteinase K (Abcam, catalog no. ab64220) treatment for 15 min was only applied to retrieve the mouse-specific CD31.

    Techniques: Activation Assay, Expressing, Concentration Assay, Immunofluorescence, Staining, Flow Cytometry, Transfection

    Robust endothelial differentiation of h-iPSCs. ( A ) Schematic of two-stage EC differentiation protocol. Stage 1, conversion of h-iPSCs into h-MPCs. Stage 2, differentiation of h-MPCs into h-iECs via modRNA(ETV2). ( B ) Time course conversion efficiency of h-iPSCs into VE-Cadherin + /CD31 + h-iECs by flow cytometry ( n = 3). ( C ) Effect of modRNA concentration on h-iPSC–to–h-iEC conversion at 96 hours. Analysis for both electroporation- and lipofection-based delivery of modRNA. ( D ) Western blot analysis of ETV2, CD31, and VE-Cadherin expression during EC differentiation. Lane 1 corresponds to cells at day 2 of the S1. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( E ) Time course immunofluorescence staining for ETV2 and CD31 in S1-S2 and S1-modETV2 protocols (insets: mean %; n = 3). Nuclei stained by 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 100 μm. ( F ) Flow cytometry analysis of differentiation efficiency at 96 hours in 13 h-iPSC clones generated from dermal FBs, umbilical cbECFCs, and uEPs. ( G ) Differences in differentiation efficiency between S1-S2 and S1-modETV2 protocols for all 13 h-iPSC clones. Data correspond to percentage of CD31 + cells by flow cytometry. ( H ) Differences in differentiation efficiency between four alternative S1-S2 methodologies and the S1-modETV2 protocol for three independent h-iPSC clones. Bars represent means ± SD; *** P

    Journal: Science Advances

    Article Title: Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA

    doi: 10.1126/sciadv.aba7606

    Figure Lengend Snippet: Robust endothelial differentiation of h-iPSCs. ( A ) Schematic of two-stage EC differentiation protocol. Stage 1, conversion of h-iPSCs into h-MPCs. Stage 2, differentiation of h-MPCs into h-iECs via modRNA(ETV2). ( B ) Time course conversion efficiency of h-iPSCs into VE-Cadherin + /CD31 + h-iECs by flow cytometry ( n = 3). ( C ) Effect of modRNA concentration on h-iPSC–to–h-iEC conversion at 96 hours. Analysis for both electroporation- and lipofection-based delivery of modRNA. ( D ) Western blot analysis of ETV2, CD31, and VE-Cadherin expression during EC differentiation. Lane 1 corresponds to cells at day 2 of the S1. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( E ) Time course immunofluorescence staining for ETV2 and CD31 in S1-S2 and S1-modETV2 protocols (insets: mean %; n = 3). Nuclei stained by 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 100 μm. ( F ) Flow cytometry analysis of differentiation efficiency at 96 hours in 13 h-iPSC clones generated from dermal FBs, umbilical cbECFCs, and uEPs. ( G ) Differences in differentiation efficiency between S1-S2 and S1-modETV2 protocols for all 13 h-iPSC clones. Data correspond to percentage of CD31 + cells by flow cytometry. ( H ) Differences in differentiation efficiency between four alternative S1-S2 methodologies and the S1-modETV2 protocol for three independent h-iPSC clones. Bars represent means ± SD; *** P

    Article Snippet: For immunostaining, sections were deparaffinized, and antigen retrieval was carried out with boiling citric buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 30 min. Proteinase K (Abcam, catalog no. ab64220) treatment for 15 min was only applied to retrieve the mouse-specific CD31.

    Techniques: Flow Cytometry, Concentration Assay, Electroporation, Western Blot, Expressing, Immunofluorescence, Staining, Clone Assay, Generated

    In vivo vascular network–forming ability of h-iECs. Comparison of grafts containing h-iECs generated by different protocols. ( A ) Macroscopic views of grafts explanted at day 7. ( B ) H E staining of grafts at day 7. ( C ) Microvascular density on day 7. ( D and E ) Immunofluorescence staining at day 7. Human lumens stained by (D) UEA-I and (E) h-CD31 antibody. Perivascular coverage stained by α-SMA antibody. ( F ) Human lumens with α-SMA + perivascular coverage at day 7. ( G ) Human and mouse vessels distinguished by m-CD31 and h-CD31 antibodies. (erythrocytes within the lumens had green autofluorescence). ( H ) Perfused human vessels detected by infused UEA-I and h-CD31 antibody. ( I ) Macroscopic views and immunofluorescence staining of grafts with h-iECs (S1-modETV2 protocol) at day 30. ( J ) TUNEL and h-CD31 staining of grafts at day 7. ( K ) Schematic of in vivo vascular network–forming ability of h-iECs. Bars represent means ± SD; n = 5. In (C) and (F), *** P

    Journal: Science Advances

    Article Title: Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA

    doi: 10.1126/sciadv.aba7606

    Figure Lengend Snippet: In vivo vascular network–forming ability of h-iECs. Comparison of grafts containing h-iECs generated by different protocols. ( A ) Macroscopic views of grafts explanted at day 7. ( B ) H E staining of grafts at day 7. ( C ) Microvascular density on day 7. ( D and E ) Immunofluorescence staining at day 7. Human lumens stained by (D) UEA-I and (E) h-CD31 antibody. Perivascular coverage stained by α-SMA antibody. ( F ) Human lumens with α-SMA + perivascular coverage at day 7. ( G ) Human and mouse vessels distinguished by m-CD31 and h-CD31 antibodies. (erythrocytes within the lumens had green autofluorescence). ( H ) Perfused human vessels detected by infused UEA-I and h-CD31 antibody. ( I ) Macroscopic views and immunofluorescence staining of grafts with h-iECs (S1-modETV2 protocol) at day 30. ( J ) TUNEL and h-CD31 staining of grafts at day 7. ( K ) Schematic of in vivo vascular network–forming ability of h-iECs. Bars represent means ± SD; n = 5. In (C) and (F), *** P

    Article Snippet: For immunostaining, sections were deparaffinized, and antigen retrieval was carried out with boiling citric buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 30 min. Proteinase K (Abcam, catalog no. ab64220) treatment for 15 min was only applied to retrieve the mouse-specific CD31.

    Techniques: In Vivo, Generated, Staining, Immunofluorescence, TUNEL Assay

    Bone-derived mesenchymal stem cell (MSC) isolation, identification, and injection. a Morphology of MSCs in cell culture. Cultured cells showed typically spindle-shaped morphology under phase-contrast microscopy. b Schematic diagram of cell injection into the striatum of the brain and the survival of MSCs after injection. c Green fluorescent (CFDA SE dye) cells were located in the ischemic hemisphere after 3 days of injection. Scale bar = 300 μm. d Cytometry analysis depicted that 99.99 + % cultured cells were positive for CD29 and CD90 and negative for CD31 and CD45

    Journal: Journal of Neuroinflammation

    Article Title: Mesenchymal stem cells attenuate blood-brain barrier leakage after cerebral ischemia in mice

    doi: 10.1186/s12974-018-1153-1

    Figure Lengend Snippet: Bone-derived mesenchymal stem cell (MSC) isolation, identification, and injection. a Morphology of MSCs in cell culture. Cultured cells showed typically spindle-shaped morphology under phase-contrast microscopy. b Schematic diagram of cell injection into the striatum of the brain and the survival of MSCs after injection. c Green fluorescent (CFDA SE dye) cells were located in the ischemic hemisphere after 3 days of injection. Scale bar = 300 μm. d Cytometry analysis depicted that 99.99 + % cultured cells were positive for CD29 and CD90 and negative for CD31 and CD45

    Article Snippet: Cells were incubated in 100 μl PBS with CD29-APC (1:100 dilution, BD Biosciences, Mississauga, ON), CD90-cy5.5 (1:100 dilution, BD Biosciences), CD31-PE (1:100 dilution, eBioscience, San Diego, CA), FITC-CD45 (1:100 dilution, eBioscience), and their isotype control antibodies for 20 min on ice.

    Techniques: Derivative Assay, Isolation, Injection, Cell Culture, Microscopy, Cytometry

    MSCs reversed gap formation of ZO-1, occludin, and claudin-5. Sections from ischemic penumbra were stained of ZO-1, occludin, and claudin-5 (red) and then co-stained with endothelial marker CD31 (green). Discontinuous labeling and gap formation (white arrows) were observed in ipsilateral brains following 3 days of tMCAO. Scale bar = 30 μm. Data are mean ± SD, n = 5–6 per group. * p

    Journal: Journal of Neuroinflammation

    Article Title: Mesenchymal stem cells attenuate blood-brain barrier leakage after cerebral ischemia in mice

    doi: 10.1186/s12974-018-1153-1

    Figure Lengend Snippet: MSCs reversed gap formation of ZO-1, occludin, and claudin-5. Sections from ischemic penumbra were stained of ZO-1, occludin, and claudin-5 (red) and then co-stained with endothelial marker CD31 (green). Discontinuous labeling and gap formation (white arrows) were observed in ipsilateral brains following 3 days of tMCAO. Scale bar = 30 μm. Data are mean ± SD, n = 5–6 per group. * p

    Article Snippet: Cells were incubated in 100 μl PBS with CD29-APC (1:100 dilution, BD Biosciences, Mississauga, ON), CD90-cy5.5 (1:100 dilution, BD Biosciences), CD31-PE (1:100 dilution, eBioscience, San Diego, CA), FITC-CD45 (1:100 dilution, eBioscience), and their isotype control antibodies for 20 min on ice.

    Techniques: Staining, Marker, Labeling

    Deletion of Stim1 and Stim2 Prolongs the Survival of Mice with T-ALL (A and B) STIM1 (A) and STIM2 ); hematological malignancies are in red. (C) Experimental model of T-ALL used in (D)–(I). BM cells isolated either from poly(I:C)-treated wild-type (WT) or Stim1/2 fl/fl Mx1-Cre mice and untreated WT or Stim1/2 fl/fl Vav-Cre mice ( Stim1/2 −/− ) were transduced with ICN1-IRES-GFP and cultured for 3 days in vitro. 5 × 10 4 ICN1-transduced (GFP + ) lineage negative (lin – ) BM progenitor cells were injected i.v. (together with 5 × 10 5 bone marrow cells from WT mice) into lethally irradiated CD45.1 + WT host mice. (D) SOCE in c-kit + progenitor cells isolated from WT and Stim1/2 −/− mice. (E and F) Representative flow cytometry plots of surface (s) and cytoplasmic (cy) CD3 expression (E) or CD4/CD8 expression (F) on cells isolated from the BMand spleen of healthy WT control mice (Ctrl) and mice with WT or Stim1/2 −/− leukemia (gated on GFP + ). (G) SOCE in WT and Stim1/2 −/− leukemic cells (GFP + ) isolated from the spleen at 21 days of disease. (H) Relative weight of mice with WT and Stim1/2 −/− leukemia. Values shown are mean ± SEM. Statistical analysis was performed using Student’s t test. *p

    Journal: Cell reports

    Article Title: STIM1 and STIM2 Mediate Cancer-Induced Inflammation in T Cell Acute Lymphoblastic Leukemia

    doi: 10.1016/j.celrep.2018.08.030

    Figure Lengend Snippet: Deletion of Stim1 and Stim2 Prolongs the Survival of Mice with T-ALL (A and B) STIM1 (A) and STIM2 ); hematological malignancies are in red. (C) Experimental model of T-ALL used in (D)–(I). BM cells isolated either from poly(I:C)-treated wild-type (WT) or Stim1/2 fl/fl Mx1-Cre mice and untreated WT or Stim1/2 fl/fl Vav-Cre mice ( Stim1/2 −/− ) were transduced with ICN1-IRES-GFP and cultured for 3 days in vitro. 5 × 10 4 ICN1-transduced (GFP + ) lineage negative (lin – ) BM progenitor cells were injected i.v. (together with 5 × 10 5 bone marrow cells from WT mice) into lethally irradiated CD45.1 + WT host mice. (D) SOCE in c-kit + progenitor cells isolated from WT and Stim1/2 −/− mice. (E and F) Representative flow cytometry plots of surface (s) and cytoplasmic (cy) CD3 expression (E) or CD4/CD8 expression (F) on cells isolated from the BMand spleen of healthy WT control mice (Ctrl) and mice with WT or Stim1/2 −/− leukemia (gated on GFP + ). (G) SOCE in WT and Stim1/2 −/− leukemic cells (GFP + ) isolated from the spleen at 21 days of disease. (H) Relative weight of mice with WT and Stim1/2 −/− leukemia. Values shown are mean ± SEM. Statistical analysis was performed using Student’s t test. *p

    Article Snippet: Fluorescently conjugated antibodies against mouse CD3 (17A2), CD4 (GK1.5), CD5 (53–7.3), CD8 (53–6.7), CD11b (M1/70), CD71 , B220 (RA3–6B2), Gr-1 (RB6–8C5) and Ter-119 (TER-119) (all from eBiosciences), F4/80 (BM8), Ki-67 (16A8) and Annexin V (all from Biolegend) were used.

    Techniques: Mouse Assay, Isolation, Transduction, Cell Culture, In Vitro, Injection, Irradiation, Flow Cytometry, Cytometry, Expressing

    STIM1/STIM2-Deficient Leukemic Cells Fail to Cause Cell Death and Necrosis of Leukemia-Infiltrated Organs (A) H E staining of BM (femur) and spleen from healthy WT control mice (Ctrl) and mice with WT and Stim1/2 −/− leukemia 24 days after T-ALL induction Magnification 4003; images in bottom row represent boxed areas in top row. Images are representative of 3–5 mice per group. (B and C) Viability of cells isolated from the BM (B) and spleen (C) of mice with WT and Stim1/2 −/− leukemia measured by flow cytometry. Data are representative of 3–5 mice per cohort. (D) Fluorescent confocal microscopy images of TUNEL staining and CD3 expression in the BM of mice with WT and Stim1/2 −/− leukemia. Scale bar, 50 μm. Data are representative of three mice per group and time point.

    Journal: Cell reports

    Article Title: STIM1 and STIM2 Mediate Cancer-Induced Inflammation in T Cell Acute Lymphoblastic Leukemia

    doi: 10.1016/j.celrep.2018.08.030

    Figure Lengend Snippet: STIM1/STIM2-Deficient Leukemic Cells Fail to Cause Cell Death and Necrosis of Leukemia-Infiltrated Organs (A) H E staining of BM (femur) and spleen from healthy WT control mice (Ctrl) and mice with WT and Stim1/2 −/− leukemia 24 days after T-ALL induction Magnification 4003; images in bottom row represent boxed areas in top row. Images are representative of 3–5 mice per group. (B and C) Viability of cells isolated from the BM (B) and spleen (C) of mice with WT and Stim1/2 −/− leukemia measured by flow cytometry. Data are representative of 3–5 mice per cohort. (D) Fluorescent confocal microscopy images of TUNEL staining and CD3 expression in the BM of mice with WT and Stim1/2 −/− leukemia. Scale bar, 50 μm. Data are representative of three mice per group and time point.

    Article Snippet: Fluorescently conjugated antibodies against mouse CD3 (17A2), CD4 (GK1.5), CD5 (53–7.3), CD8 (53–6.7), CD11b (M1/70), CD71 , B220 (RA3–6B2), Gr-1 (RB6–8C5) and Ter-119 (TER-119) (all from eBiosciences), F4/80 (BM8), Ki-67 (16A8) and Annexin V (all from Biolegend) were used.

    Techniques: Staining, Mouse Assay, Isolation, Flow Cytometry, Cytometry, Confocal Microscopy, TUNEL Assay, Expressing