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    Thermo Fisher succinimidyl ester
    Cardiac adherent proliferating cells decrease cardiac mononuclear cell activation and cardiac damage in Coxsackievirus B3-infected mice. A. Cardiac MNCs were isolated from control mice (open bars) and CVB3-infected mice (closed bars) injected with PBS or CAPs. Next, cardiac MNCs were labeled with 10 µM of carboxyfluorescein <t>succinimidyl</t> ester to be able to measure cell proliferation, and stimulated with phorbol myristate acetate (PMA) and ionomycin at a final concentration of 50 ng/ml and 500 ng/ml, respectively, for 72 h followed by flow cytometry and analysis with FlowJo 8.7. software. Bar graphs represent the division index of cardiac MNCs with n = 4/group. B. Representative hematoxylin and eosin stained heart sections of control mice receiving PBS (control+PBS; upper left panel) or CAPs (control+CAPs; lower left panel), or of CVB3-infected mice receiving PBS (CVB3+PBS; upper right panel) or CAPs (CVB3+CAPs; lower right panel), at 200× magnification. MNCs were isolated from the spleen of control mice (open bar graph) or CVB3-infected (closed bar graph) mice. Next, carboxyfluorescein succinimidyl ester-labeled MNCs were directly stimulated with PMA and ionomycin and cultured with or without CAPs (untreated or 24 h pre-treated with L-NAME), in the presence or absence of 1 µg/ml of anti-human IL-10 or anti-murine IFN-γ antibody for 72 h. Then, cells were stained with monoclonal anti-CD4 or anti-CD8 antibodies, followed by flow cytometry and analysis with FlowJo 8.7. software. Bar graphs represent the division index of C. CD4+ (upper panel) and of D. CD8+ T cells (lower panel) with n = 4/group.
    Succinimidyl Ester, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Human Cardiac-Derived Adherent Proliferating Cells Reduce Murine Acute Coxsackievirus B3-Induced Myocarditis"

    Article Title: Human Cardiac-Derived Adherent Proliferating Cells Reduce Murine Acute Coxsackievirus B3-Induced Myocarditis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028513

    Cardiac adherent proliferating cells decrease cardiac mononuclear cell activation and cardiac damage in Coxsackievirus B3-infected mice. A. Cardiac MNCs were isolated from control mice (open bars) and CVB3-infected mice (closed bars) injected with PBS or CAPs. Next, cardiac MNCs were labeled with 10 µM of carboxyfluorescein succinimidyl ester to be able to measure cell proliferation, and stimulated with phorbol myristate acetate (PMA) and ionomycin at a final concentration of 50 ng/ml and 500 ng/ml, respectively, for 72 h followed by flow cytometry and analysis with FlowJo 8.7. software. Bar graphs represent the division index of cardiac MNCs with n = 4/group. B. Representative hematoxylin and eosin stained heart sections of control mice receiving PBS (control+PBS; upper left panel) or CAPs (control+CAPs; lower left panel), or of CVB3-infected mice receiving PBS (CVB3+PBS; upper right panel) or CAPs (CVB3+CAPs; lower right panel), at 200× magnification. MNCs were isolated from the spleen of control mice (open bar graph) or CVB3-infected (closed bar graph) mice. Next, carboxyfluorescein succinimidyl ester-labeled MNCs were directly stimulated with PMA and ionomycin and cultured with or without CAPs (untreated or 24 h pre-treated with L-NAME), in the presence or absence of 1 µg/ml of anti-human IL-10 or anti-murine IFN-γ antibody for 72 h. Then, cells were stained with monoclonal anti-CD4 or anti-CD8 antibodies, followed by flow cytometry and analysis with FlowJo 8.7. software. Bar graphs represent the division index of C. CD4+ (upper panel) and of D. CD8+ T cells (lower panel) with n = 4/group.
    Figure Legend Snippet: Cardiac adherent proliferating cells decrease cardiac mononuclear cell activation and cardiac damage in Coxsackievirus B3-infected mice. A. Cardiac MNCs were isolated from control mice (open bars) and CVB3-infected mice (closed bars) injected with PBS or CAPs. Next, cardiac MNCs were labeled with 10 µM of carboxyfluorescein succinimidyl ester to be able to measure cell proliferation, and stimulated with phorbol myristate acetate (PMA) and ionomycin at a final concentration of 50 ng/ml and 500 ng/ml, respectively, for 72 h followed by flow cytometry and analysis with FlowJo 8.7. software. Bar graphs represent the division index of cardiac MNCs with n = 4/group. B. Representative hematoxylin and eosin stained heart sections of control mice receiving PBS (control+PBS; upper left panel) or CAPs (control+CAPs; lower left panel), or of CVB3-infected mice receiving PBS (CVB3+PBS; upper right panel) or CAPs (CVB3+CAPs; lower right panel), at 200× magnification. MNCs were isolated from the spleen of control mice (open bar graph) or CVB3-infected (closed bar graph) mice. Next, carboxyfluorescein succinimidyl ester-labeled MNCs were directly stimulated with PMA and ionomycin and cultured with or without CAPs (untreated or 24 h pre-treated with L-NAME), in the presence or absence of 1 µg/ml of anti-human IL-10 or anti-murine IFN-γ antibody for 72 h. Then, cells were stained with monoclonal anti-CD4 or anti-CD8 antibodies, followed by flow cytometry and analysis with FlowJo 8.7. software. Bar graphs represent the division index of C. CD4+ (upper panel) and of D. CD8+ T cells (lower panel) with n = 4/group.

    Techniques Used: Activation Assay, Infection, Mouse Assay, Isolation, Injection, Labeling, Concentration Assay, Flow Cytometry, Cytometry, Software, Staining, Cell Culture

    2) Product Images from "Identification of a sub-population of B cells that proliferates after infection with epstein-barr virus"

    Article Title: Identification of a sub-population of B cells that proliferates after infection with epstein-barr virus

    Journal: Virology Journal

    doi: 10.1186/1743-422X-8-84

    CD23 hi CD58 + cells proliferate and constitute the bulk of the population by day 30 . A. Carboxyfluorescein diacetate, succinimidyl ester (CFSE)-labeled B cells were exposed to EBV and harvested on day 5. CD58 + cells or CD58 - cells were examined for proliferation and intracellular expression of IL6 (using APC-anti-IL6 antibody). Percentages represent fraction of gated cells (CD58 + or CD58 - ) producing IL6. G0-3 represents non-proliferated cells (G0) and three generations of progeny (G1-3). Percentages above G0-3 indicate the fraction of CD58 + or CD58 - cells in each generation. B. EBV-exposed B cells were harvested on days 5, 6 and 7, and examined for expression of CD23 (PE) and CD58 (PE-Cy7). Percentages represent fractions of cells in regions R1 (CD23 lo CD58 - ), R2 (CD23 lo CD58 + ), and R3 (CD23 hi CD58 + ). C. In a separate experiment, EBV-exposed cells (top panels) were harvested on days 4, 5, 6, 7, 10, and 30 and un-infected cells (bottom five panels) were harvested on days 4, 5, 6, 7, and 10 and examined for expression of CD23 and CD58. Percentages of cells in regions R1, R2, and R3 are shown. EBV-exposed cells harvested on day 30 and stained with PE and PE-Cy7 isotype control antibodies are also shown. D. CFSE-labeled B cells were exposed to EBV, harvested on day 5, and examined for expression of CD23 (PE) and CD58 (PE-Cy7). Cells in region R1, R2, and R3 were examined for proliferation and expression of IL6 (APC) by flow cytometry. Percentages represent fraction of cells in regions R1, R2, or R3 that produced IL6. Percentages above G0-3 indicate non-proliferating cells (G0) and proliferating cells (G1-3).
    Figure Legend Snippet: CD23 hi CD58 + cells proliferate and constitute the bulk of the population by day 30 . A. Carboxyfluorescein diacetate, succinimidyl ester (CFSE)-labeled B cells were exposed to EBV and harvested on day 5. CD58 + cells or CD58 - cells were examined for proliferation and intracellular expression of IL6 (using APC-anti-IL6 antibody). Percentages represent fraction of gated cells (CD58 + or CD58 - ) producing IL6. G0-3 represents non-proliferated cells (G0) and three generations of progeny (G1-3). Percentages above G0-3 indicate the fraction of CD58 + or CD58 - cells in each generation. B. EBV-exposed B cells were harvested on days 5, 6 and 7, and examined for expression of CD23 (PE) and CD58 (PE-Cy7). Percentages represent fractions of cells in regions R1 (CD23 lo CD58 - ), R2 (CD23 lo CD58 + ), and R3 (CD23 hi CD58 + ). C. In a separate experiment, EBV-exposed cells (top panels) were harvested on days 4, 5, 6, 7, 10, and 30 and un-infected cells (bottom five panels) were harvested on days 4, 5, 6, 7, and 10 and examined for expression of CD23 and CD58. Percentages of cells in regions R1, R2, and R3 are shown. EBV-exposed cells harvested on day 30 and stained with PE and PE-Cy7 isotype control antibodies are also shown. D. CFSE-labeled B cells were exposed to EBV, harvested on day 5, and examined for expression of CD23 (PE) and CD58 (PE-Cy7). Cells in region R1, R2, and R3 were examined for proliferation and expression of IL6 (APC) by flow cytometry. Percentages represent fraction of cells in regions R1, R2, or R3 that produced IL6. Percentages above G0-3 indicate non-proliferating cells (G0) and proliferating cells (G1-3).

    Techniques Used: Labeling, Expressing, Infection, Staining, Flow Cytometry, Cytometry, Produced

    3) Product Images from "Graft Versus Leukemia Response Without Graft-versus-host Disease Elicited By Adoptively Transferred Multivirus-specific T-cells"

    Article Title: Graft Versus Leukemia Response Without Graft-versus-host Disease Elicited By Adoptively Transferred Multivirus-specific T-cells

    Journal: Molecular Therapy

    doi: 10.1038/mt.2014.192

    Recognition of patient leukemic cells by the virus-specific donor T cell line. The trivirus-specific T cell line was labeled with carboxyfluorescein diacetate, succinimidyl ester and incubated with patient phytohemagglutinin (PHA) blasts, patient bone marrow, containing 85% leukemia blasts, or with cytomegalovirus (CMV)-pulsed Epstein-Barr virus (EBV)-LCL (cognate Ag) for 6 hours in the presence of cytokine secretion inhibitors at 37 °C, 5% CO2, and subsequently examined for the production of granulocyte-macrophage colony stimulating factor, interleukin (IL)-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α within CD4 + ( a ) and CD8 + ( b ) T cells by intracellular cytokine detection as outlined in the Materials and Methods section. Indicated in each plot is the percentage of the respective subset producing cytokine in response to each separate stimulation. The data show a strong reactivity of donor CD4 + T cells with patient leukemic marrow but not with patient PHA blasts, while CD8 + T cells show only marginal antileukemia cross-reactivity. ( c ) Reactivity against known leukemia-associated tumor-associated antigens was tested in an IFN-γ ELISPOT assay using overlapping peptides for tumor-associated antigens. Cells alone indicate cells without any antigen, and actin represents an irrelevant mixture of overlapping peptides. Error bars indicate standard deviation from the mean.
    Figure Legend Snippet: Recognition of patient leukemic cells by the virus-specific donor T cell line. The trivirus-specific T cell line was labeled with carboxyfluorescein diacetate, succinimidyl ester and incubated with patient phytohemagglutinin (PHA) blasts, patient bone marrow, containing 85% leukemia blasts, or with cytomegalovirus (CMV)-pulsed Epstein-Barr virus (EBV)-LCL (cognate Ag) for 6 hours in the presence of cytokine secretion inhibitors at 37 °C, 5% CO2, and subsequently examined for the production of granulocyte-macrophage colony stimulating factor, interleukin (IL)-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α within CD4 + ( a ) and CD8 + ( b ) T cells by intracellular cytokine detection as outlined in the Materials and Methods section. Indicated in each plot is the percentage of the respective subset producing cytokine in response to each separate stimulation. The data show a strong reactivity of donor CD4 + T cells with patient leukemic marrow but not with patient PHA blasts, while CD8 + T cells show only marginal antileukemia cross-reactivity. ( c ) Reactivity against known leukemia-associated tumor-associated antigens was tested in an IFN-γ ELISPOT assay using overlapping peptides for tumor-associated antigens. Cells alone indicate cells without any antigen, and actin represents an irrelevant mixture of overlapping peptides. Error bars indicate standard deviation from the mean.

    Techniques Used: Labeling, Incubation, Enzyme-linked Immunospot, Standard Deviation

    4) Product Images from "The focal adhesion kinase inhibitor PF-562,271 impairs primary CD4+ T cell activation"

    Article Title: The focal adhesion kinase inhibitor PF-562,271 impairs primary CD4+ T cell activation

    Journal: Biochemical pharmacology

    doi: 10.1016/j.bcp.2013.07.024

    PF-562,271 inhibits T cell proliferation. OVA peptide expanded mouse CD4+ cells were stained with 0.25 μM carboxyfluorescein diacetate, succinimidyl ester and stimulated with anti-CD3/CD28 coated beads (A), irradiated splenocytes loaded 2.5 μg/mL
    Figure Legend Snippet: PF-562,271 inhibits T cell proliferation. OVA peptide expanded mouse CD4+ cells were stained with 0.25 μM carboxyfluorescein diacetate, succinimidyl ester and stimulated with anti-CD3/CD28 coated beads (A), irradiated splenocytes loaded 2.5 μg/mL

    Techniques Used: Staining, Irradiation

    5) Product Images from "Ability of the polysaccharide chitosan to inhibit proliferation of CD4+ lymphocytes from mucosal inductive sites, in vitro and in vivo"

    Article Title: Ability of the polysaccharide chitosan to inhibit proliferation of CD4+ lymphocytes from mucosal inductive sites, in vitro and in vivo

    Journal: Cell Proliferation

    doi: 10.1111/j.1365-2184.2009.00634.x

    Effect of chitosan on ConA‐induced T‐cell proliferation in vitro . Mesenteric lymph node (MLN) lymphocytes from normal rats labelled with succinimidyl ester of carboxyfluorescein diacetate (CFSE) were stimulated with 2.5 μg/ml of mitogen in presence or absence of chitosan (1–100 μg/ml) at 37 °C in RPMI 1640 medium supplemented with 10% foetal calf serum in a 95% air/5% CO 2 atmosphere for 72 h. After incubation, cells were labelled with anti‐CD4 and 20 000 cells were acquired by flow cytometry. Representative dot plots of CFSE‐labelled cells representing chitosan‐induced inhibition of ConA‐induced mitogenesis are shown. Percentage of daughter cells in the box is included. (a) Unstimulated control and ConA stimulated cells; (b) left: ConA + chitosan; middle: ConA for 24 h and then chitosan for further 72 h; right: chitosan for 24 h and then ConA for further 72 h.
    Figure Legend Snippet: Effect of chitosan on ConA‐induced T‐cell proliferation in vitro . Mesenteric lymph node (MLN) lymphocytes from normal rats labelled with succinimidyl ester of carboxyfluorescein diacetate (CFSE) were stimulated with 2.5 μg/ml of mitogen in presence or absence of chitosan (1–100 μg/ml) at 37 °C in RPMI 1640 medium supplemented with 10% foetal calf serum in a 95% air/5% CO 2 atmosphere for 72 h. After incubation, cells were labelled with anti‐CD4 and 20 000 cells were acquired by flow cytometry. Representative dot plots of CFSE‐labelled cells representing chitosan‐induced inhibition of ConA‐induced mitogenesis are shown. Percentage of daughter cells in the box is included. (a) Unstimulated control and ConA stimulated cells; (b) left: ConA + chitosan; middle: ConA for 24 h and then chitosan for further 72 h; right: chitosan for 24 h and then ConA for further 72 h.

    Techniques Used: In Vitro, Incubation, Flow Cytometry, Inhibition

    Effect of chitosan on ConA‐induced T‐cell proliferation ex vivo . Mesenteric lymph node (MLN) cells from rats fed a single (a) or five doses (b) of diluent (Dil) or of 1–5 mg chitosan (Ch1–5) were labelled with succinimidyl ester of carboxyfluorescein diacetate (CFSE) and stimulated with 2.5 μg/ml ConA as described in the Materials and methods section. After incubation, cells were labelled with anti‐CD4 and 20 000 cells were acquired by flow cytometry. (a) Representative dot plots of CFSE‐labelled cells representing chitosan‐induced inhibition of ConA‐induced mitogenesis are shown. The percentage of daughter cells in the box is included. Bars are means of data from 4–6 rats per treatment. In culture supernatants, the production of INF‐γ was evaluated by ELISA. (b) Representative dot plots of CFSE‐labelled cells representing inhibition of ConA‐induced mitogenesis after sustained administration of chitosan.
    Figure Legend Snippet: Effect of chitosan on ConA‐induced T‐cell proliferation ex vivo . Mesenteric lymph node (MLN) cells from rats fed a single (a) or five doses (b) of diluent (Dil) or of 1–5 mg chitosan (Ch1–5) were labelled with succinimidyl ester of carboxyfluorescein diacetate (CFSE) and stimulated with 2.5 μg/ml ConA as described in the Materials and methods section. After incubation, cells were labelled with anti‐CD4 and 20 000 cells were acquired by flow cytometry. (a) Representative dot plots of CFSE‐labelled cells representing chitosan‐induced inhibition of ConA‐induced mitogenesis are shown. The percentage of daughter cells in the box is included. Bars are means of data from 4–6 rats per treatment. In culture supernatants, the production of INF‐γ was evaluated by ELISA. (b) Representative dot plots of CFSE‐labelled cells representing inhibition of ConA‐induced mitogenesis after sustained administration of chitosan.

    Techniques Used: Ex Vivo, Incubation, Flow Cytometry, Inhibition, Enzyme-linked Immunosorbent Assay

    6) Product Images from "Effect of Spa Spring Water on Cytokine Expression in Human Keratinocyte HaCaT Cells and on Differentiation of CD4+ T Cells"

    Article Title: Effect of Spa Spring Water on Cytokine Expression in Human Keratinocyte HaCaT Cells and on Differentiation of CD4+ T Cells

    Journal: Annals of Dermatology

    doi: 10.5021/ad.2012.24.3.324

    Spa spring water inhibited the differentiation and proliferation of effector T cells and promoted T reg proliferation. The CD4 + naïve T cells were isolated, labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE, CellTrace™ CFSE Cell Prolifetration kit, Invitrogen, Paisley, UK), and cultured under skewing conditions for Th1, Th2, Th17, and T reg cells, respectively, in the presence or absence of spa spring water. Serially diluted (1 : 3) anti-CD3 antibodies were treated at a concentration starting from 3 µg/ml to monitor dose-dependent proliferation. On day 3, cells were harvested and proliferation was measured by flow cytometry. CD4 + IFN-γ + Th1 cells (A), CD4 + IL-4 + Th2 cells (B), and CD4 + IL-17 + Th17 cells (C) were proliferated along with the increase of anti-CD3 stimulation, whereas they were inhibited under spa spring water. In contrast, spa spring water promoted proliferation of CD4 + Foxp3 + T regs (D) relative to those of the HaCaT cells cultured alone. Percentages of proliferative cells were represented as precursor frequency via analysis using ModFit LT software (Verity Software House, Topsham, ME, USA) based on the reduction of CFSE positive cells. The data are expressed as the means±SEM ( * p
    Figure Legend Snippet: Spa spring water inhibited the differentiation and proliferation of effector T cells and promoted T reg proliferation. The CD4 + naïve T cells were isolated, labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE, CellTrace™ CFSE Cell Prolifetration kit, Invitrogen, Paisley, UK), and cultured under skewing conditions for Th1, Th2, Th17, and T reg cells, respectively, in the presence or absence of spa spring water. Serially diluted (1 : 3) anti-CD3 antibodies were treated at a concentration starting from 3 µg/ml to monitor dose-dependent proliferation. On day 3, cells were harvested and proliferation was measured by flow cytometry. CD4 + IFN-γ + Th1 cells (A), CD4 + IL-4 + Th2 cells (B), and CD4 + IL-17 + Th17 cells (C) were proliferated along with the increase of anti-CD3 stimulation, whereas they were inhibited under spa spring water. In contrast, spa spring water promoted proliferation of CD4 + Foxp3 + T regs (D) relative to those of the HaCaT cells cultured alone. Percentages of proliferative cells were represented as precursor frequency via analysis using ModFit LT software (Verity Software House, Topsham, ME, USA) based on the reduction of CFSE positive cells. The data are expressed as the means±SEM ( * p

    Techniques Used: Isolation, Labeling, Cell Culture, Concentration Assay, Flow Cytometry, Cytometry, Software

    7) Product Images from "Near-Term Anti-CD25 Monoclonal Antibody Administration Protects Murine Liver from Ischemia-Reperfusion Injury Due to Reduced Numbers of CD4+ T Cells"

    Article Title: Near-Term Anti-CD25 Monoclonal Antibody Administration Protects Murine Liver from Ischemia-Reperfusion Injury Due to Reduced Numbers of CD4+ T Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0106892

    Anti-CD25 mAb pretreatment mitigated intrahepatic inflammatory milieu. (A) TNF-α, IFN-γ, IL-2, and IL-6 mRNA expression among sham, PC61, and I/R group after hepatic ischemia and 60 min of reperfusion. Samples were analyzed by RT-PCR. Data are expressed as means±SE; n = 6 per group. (B) Splenocytes were labeled with 5 mM 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester and co-cultured with samples’ protein in 5% CO 2 at 37°C. After 72 hours proliferation was assessed by flow Cytometry. (*p
    Figure Legend Snippet: Anti-CD25 mAb pretreatment mitigated intrahepatic inflammatory milieu. (A) TNF-α, IFN-γ, IL-2, and IL-6 mRNA expression among sham, PC61, and I/R group after hepatic ischemia and 60 min of reperfusion. Samples were analyzed by RT-PCR. Data are expressed as means±SE; n = 6 per group. (B) Splenocytes were labeled with 5 mM 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester and co-cultured with samples’ protein in 5% CO 2 at 37°C. After 72 hours proliferation was assessed by flow Cytometry. (*p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Labeling, Cell Culture, Flow Cytometry, Cytometry

    8) Product Images from "Bi-Directional Exchange of Membrane Components Occurs during Co-Culture of Mesenchymal Stem Cells and Nucleus Pulposus Cells"

    Article Title: Bi-Directional Exchange of Membrane Components Occurs during Co-Culture of Mesenchymal Stem Cells and Nucleus Pulposus Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033739

    Cell fusion of MSCs and NP cells during direct co-culture. A–C: An exemplar flow cytometry analysis for CFDA labeled MSCs and SNARF labeled NP cells after 7 days: A) CFDA labeled MSCs alone; B) SNARF labeled NP cells alone; C) Co-culture of CFDA labeled MSCs and SNARF labeled NP cells. D–F: Fluorescence microscopy of sorted double labeled cells from region R4: D) Cells labeled with CFDA; E) Cells labeled with SNARF; F) Color combine; enlargement of a 100× magnification. G) Percentage of cell fusion after 1, 3 and 7 days calculated from flow cytometry data. Error bars indicate standard error of the mean. Abbreviations: MSC: mesenchymal stem cell; NP: nucleus pulposus; CFDA: 5,6 caboxyfluorescein diactetae, succinimidyl ester; SNARF: carboxylic acid, acetate, SE.
    Figure Legend Snippet: Cell fusion of MSCs and NP cells during direct co-culture. A–C: An exemplar flow cytometry analysis for CFDA labeled MSCs and SNARF labeled NP cells after 7 days: A) CFDA labeled MSCs alone; B) SNARF labeled NP cells alone; C) Co-culture of CFDA labeled MSCs and SNARF labeled NP cells. D–F: Fluorescence microscopy of sorted double labeled cells from region R4: D) Cells labeled with CFDA; E) Cells labeled with SNARF; F) Color combine; enlargement of a 100× magnification. G) Percentage of cell fusion after 1, 3 and 7 days calculated from flow cytometry data. Error bars indicate standard error of the mean. Abbreviations: MSC: mesenchymal stem cell; NP: nucleus pulposus; CFDA: 5,6 caboxyfluorescein diactetae, succinimidyl ester; SNARF: carboxylic acid, acetate, SE.

    Techniques Used: Co-Culture Assay, Flow Cytometry, Cytometry, Labeling, Fluorescence, Microscopy

    Transfer of membrane components during direct co-culture of MSC and NP cells. A–C: Exemplar flow cytometry to quantify transfer of membrane (DiL) components after 7 days. A) Dot plots for unlabeled MSCs and NP cells. B) Dot plots for CFDA and DiL labeled MSCs and NP cells. C) Dot plots for CFDA and DiL double labeled MSCs co-cultured with unlabeled NP cells; CFDA and DiL double labeled NP cells co-cultured with unlabeled MSCs. D) Percentages of DiL transfer after 1, 3 and 7 days from a labeled cell to an unlabeled cell during direct co-culture calculated from flow cytometry data. Error bars indicate standard error of the mean. Abbreviations: MSC: mesenchymal stem cell; NP: nucleus pulposus; CFDA: 5,6 carboxyfluorescein diactetae, succinimidyl ester; DiL: Vybrant CM-DiL cell-labeling solution.
    Figure Legend Snippet: Transfer of membrane components during direct co-culture of MSC and NP cells. A–C: Exemplar flow cytometry to quantify transfer of membrane (DiL) components after 7 days. A) Dot plots for unlabeled MSCs and NP cells. B) Dot plots for CFDA and DiL labeled MSCs and NP cells. C) Dot plots for CFDA and DiL double labeled MSCs co-cultured with unlabeled NP cells; CFDA and DiL double labeled NP cells co-cultured with unlabeled MSCs. D) Percentages of DiL transfer after 1, 3 and 7 days from a labeled cell to an unlabeled cell during direct co-culture calculated from flow cytometry data. Error bars indicate standard error of the mean. Abbreviations: MSC: mesenchymal stem cell; NP: nucleus pulposus; CFDA: 5,6 carboxyfluorescein diactetae, succinimidyl ester; DiL: Vybrant CM-DiL cell-labeling solution.

    Techniques Used: Co-Culture Assay, Flow Cytometry, Cytometry, Labeling, Cell Culture

    Incorporation of DiL-labeled microvesicles into MSC and NP cells during direct co-culture. Exemplar flow cytometry analysis. A) MVs derived from a direct co-culture of MSCs and NP cells (one cell population was DiL labeled) and incubated with the unlabeled cell population. B) MV-free supernatant after ultracentrifugation. C) Conditioned medium derived from a direct co-culture of MSCs and NP cells (one cell population was DiL labeled). Abbreviations: MSC: mesenchymal stem cell; NP: nucleus pulposus; MV: microvesicles; SN: supernatant; CM: conditioned medium; CFDA: 5,6 caboxyfluorescein diactetae, succinimidyl ester; DiL: Vybrant CM-DiL cell-labeling solution.
    Figure Legend Snippet: Incorporation of DiL-labeled microvesicles into MSC and NP cells during direct co-culture. Exemplar flow cytometry analysis. A) MVs derived from a direct co-culture of MSCs and NP cells (one cell population was DiL labeled) and incubated with the unlabeled cell population. B) MV-free supernatant after ultracentrifugation. C) Conditioned medium derived from a direct co-culture of MSCs and NP cells (one cell population was DiL labeled). Abbreviations: MSC: mesenchymal stem cell; NP: nucleus pulposus; MV: microvesicles; SN: supernatant; CM: conditioned medium; CFDA: 5,6 caboxyfluorescein diactetae, succinimidyl ester; DiL: Vybrant CM-DiL cell-labeling solution.

    Techniques Used: Labeling, Co-Culture Assay, Flow Cytometry, Cytometry, Derivative Assay, Incubation

    9) Product Images from "Systemic Immunization with CCL27/CTACK Modulates Immune Responses at Mucosal Sites in Mice and Macaques"

    Article Title: Systemic Immunization with CCL27/CTACK Modulates Immune Responses at Mucosal Sites in Mice and Macaques

    Journal: Vaccine

    doi: 10.1016/j.vaccine.2009.10.095

    Immunization with SIV antigenic constructs elicits potent IFN-γ secretion and highly proliferative CD8+T cells A) IFN-γ ELISpot was performed on lymphocytes isolated from peripheral blood of rhesus macaques 2 weeks post each immunization. Results are graphed as IFN-γ secreting cells/10 6 PBMCs in response to each antigen with values from media wells subtracted. Data are shown as averages for each group of macaques following each immunization. B,C) PBMCs from immunized macaques were stained with Carboxyfluorescein succinimidyl ester (CFDA-SE) and cultured for 5 days in the presence of media (negative control), SIV Gag, Env, or Pol peptides, or ConA (positive control). Cells were then stained for surface markers (CD3, CD4, CD8) and run on flow cytometry. B) An example of proliferation in an immunized macaque. C) The average proliferation of CD8+ T cells in response to each SIV antigen from groups of macaques 4 weeks after each vaccination.
    Figure Legend Snippet: Immunization with SIV antigenic constructs elicits potent IFN-γ secretion and highly proliferative CD8+T cells A) IFN-γ ELISpot was performed on lymphocytes isolated from peripheral blood of rhesus macaques 2 weeks post each immunization. Results are graphed as IFN-γ secreting cells/10 6 PBMCs in response to each antigen with values from media wells subtracted. Data are shown as averages for each group of macaques following each immunization. B,C) PBMCs from immunized macaques were stained with Carboxyfluorescein succinimidyl ester (CFDA-SE) and cultured for 5 days in the presence of media (negative control), SIV Gag, Env, or Pol peptides, or ConA (positive control). Cells were then stained for surface markers (CD3, CD4, CD8) and run on flow cytometry. B) An example of proliferation in an immunized macaque. C) The average proliferation of CD8+ T cells in response to each SIV antigen from groups of macaques 4 weeks after each vaccination.

    Techniques Used: Construct, Enzyme-linked Immunospot, Isolation, Staining, Cell Culture, Negative Control, Positive Control, Flow Cytometry, Cytometry

    10) Product Images from "Increased frequency of CD4+ CD25+ regulatory T cells in the cerebrospinal fluid but not in the blood of multiple sclerosis patients"

    Article Title: Increased frequency of CD4+ CD25+ regulatory T cells in the cerebrospinal fluid but not in the blood of multiple sclerosis patients

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/j.1365-2249.2006.03271.x

    Impaired suppressive function of nT reg in multiple sclerosis (MS) patients. Suppression assay with CD4 + CD25 + nT reg . CD4 + CD25 + nT reg were titrated into carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE)-labelled CD4 + CD25 – responder T
    Figure Legend Snippet: Impaired suppressive function of nT reg in multiple sclerosis (MS) patients. Suppression assay with CD4 + CD25 + nT reg . CD4 + CD25 + nT reg were titrated into carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE)-labelled CD4 + CD25 – responder T

    Techniques Used: Mass Spectrometry, Suppression Assay

    11) Product Images from "Different Effects of Human Umbilical Cord Mesenchymal Stem Cells on Glioblastoma Stem Cells by Direct Cell Interaction or Via Released Soluble Factors"

    Article Title: Different Effects of Human Umbilical Cord Mesenchymal Stem Cells on Glioblastoma Stem Cells by Direct Cell Interaction or Via Released Soluble Factors

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2017.00312

    UC-MSC co-culture reduces GBM CSC proliferation rate. The proliferation rate of CSC1, CSC2 and CSC3 was tracked by carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) dye dilution and analyzed by flow cytometry. The distribution of CSC1, CSC2 and CSC3 cells among generations after 72 h of co-culture with UC-MSCs (1:1) is depicted in histograms, each curve represents a new generation that occurred, starting from the parental cells (blue curve). The quantification of the data is summarized in the corresponding tables. Proliferation index (p.i.) is reported on the top of tables representing the sum of cells in all generations divided by the number of original parent cells. Representative data of three independent experiments using different UC-MSCs (1, 3 and 4) are shown.
    Figure Legend Snippet: UC-MSC co-culture reduces GBM CSC proliferation rate. The proliferation rate of CSC1, CSC2 and CSC3 was tracked by carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) dye dilution and analyzed by flow cytometry. The distribution of CSC1, CSC2 and CSC3 cells among generations after 72 h of co-culture with UC-MSCs (1:1) is depicted in histograms, each curve represents a new generation that occurred, starting from the parental cells (blue curve). The quantification of the data is summarized in the corresponding tables. Proliferation index (p.i.) is reported on the top of tables representing the sum of cells in all generations divided by the number of original parent cells. Representative data of three independent experiments using different UC-MSCs (1, 3 and 4) are shown.

    Techniques Used: Co-Culture Assay, Flow Cytometry, Cytometry

    12) Product Images from "Absence of Nkx2-3 Homeodomain Transcription Factor Induces the Formation of LYVE-1-Positive Endothelial Cysts without Lymphatic Commitment in the Spleen"

    Article Title: Absence of Nkx2-3 Homeodomain Transcription Factor Induces the Formation of LYVE-1-Positive Endothelial Cysts without Lymphatic Commitment in the Spleen

    Journal: Journal of Histochemistry and Cytochemistry

    doi: 10.1369/0022155411410061

    LYVE-1 + vessels do not contribute to the early splenic recirculation of lymphocytes. The ratio of carboxyfluorescein diacetate, succinimidyl ester (CFSE)–labeled cells within/outside the sacs was 0.023 at 20 minutes (A), whereas at 8 hr (B) the
    Figure Legend Snippet: LYVE-1 + vessels do not contribute to the early splenic recirculation of lymphocytes. The ratio of carboxyfluorescein diacetate, succinimidyl ester (CFSE)–labeled cells within/outside the sacs was 0.023 at 20 minutes (A), whereas at 8 hr (B) the

    Techniques Used: Labeling

    13) Product Images from "Bi-Directional Exchange of Membrane Components Occurs during Co-Culture of Mesenchymal Stem Cells and Nucleus Pulposus Cells"

    Article Title: Bi-Directional Exchange of Membrane Components Occurs during Co-Culture of Mesenchymal Stem Cells and Nucleus Pulposus Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033739

    Cell fusion of MSCs and NP cells during direct co-culture. A–C: An exemplar flow cytometry analysis for CFDA labeled MSCs and SNARF labeled NP cells after 7 days: A) CFDA labeled MSCs alone; B) SNARF labeled NP cells alone; C) Co-culture of CFDA labeled MSCs and SNARF labeled NP cells. D–F: Fluorescence microscopy of sorted double labeled cells from region R4: D) Cells labeled with CFDA; E) Cells labeled with SNARF; F) Color combine; enlargement of a 100× magnification. G) Percentage of cell fusion after 1, 3 and 7 days calculated from flow cytometry data. Error bars indicate standard error of the mean. Abbreviations: MSC: mesenchymal stem cell; NP: nucleus pulposus; CFDA: 5,6 caboxyfluorescein diactetae, succinimidyl ester; SNARF: carboxylic acid, acetate, SE.
    Figure Legend Snippet: Cell fusion of MSCs and NP cells during direct co-culture. A–C: An exemplar flow cytometry analysis for CFDA labeled MSCs and SNARF labeled NP cells after 7 days: A) CFDA labeled MSCs alone; B) SNARF labeled NP cells alone; C) Co-culture of CFDA labeled MSCs and SNARF labeled NP cells. D–F: Fluorescence microscopy of sorted double labeled cells from region R4: D) Cells labeled with CFDA; E) Cells labeled with SNARF; F) Color combine; enlargement of a 100× magnification. G) Percentage of cell fusion after 1, 3 and 7 days calculated from flow cytometry data. Error bars indicate standard error of the mean. Abbreviations: MSC: mesenchymal stem cell; NP: nucleus pulposus; CFDA: 5,6 caboxyfluorescein diactetae, succinimidyl ester; SNARF: carboxylic acid, acetate, SE.

    Techniques Used: Co-Culture Assay, Flow Cytometry, Cytometry, Labeling, Fluorescence, Microscopy

    Transfer of membrane components during direct co-culture of MSC and NP cells. A–C: Exemplar flow cytometry to quantify transfer of membrane (DiL) components after 7 days. A) Dot plots for unlabeled MSCs and NP cells. B) Dot plots for CFDA and DiL labeled MSCs and NP cells. C) Dot plots for CFDA and DiL double labeled MSCs co-cultured with unlabeled NP cells; CFDA and DiL double labeled NP cells co-cultured with unlabeled MSCs. D) Percentages of DiL transfer after 1, 3 and 7 days from a labeled cell to an unlabeled cell during direct co-culture calculated from flow cytometry data. Error bars indicate standard error of the mean. Abbreviations: MSC: mesenchymal stem cell; NP: nucleus pulposus; CFDA: 5,6 carboxyfluorescein diactetae, succinimidyl ester; DiL: Vybrant CM-DiL cell-labeling solution.
    Figure Legend Snippet: Transfer of membrane components during direct co-culture of MSC and NP cells. A–C: Exemplar flow cytometry to quantify transfer of membrane (DiL) components after 7 days. A) Dot plots for unlabeled MSCs and NP cells. B) Dot plots for CFDA and DiL labeled MSCs and NP cells. C) Dot plots for CFDA and DiL double labeled MSCs co-cultured with unlabeled NP cells; CFDA and DiL double labeled NP cells co-cultured with unlabeled MSCs. D) Percentages of DiL transfer after 1, 3 and 7 days from a labeled cell to an unlabeled cell during direct co-culture calculated from flow cytometry data. Error bars indicate standard error of the mean. Abbreviations: MSC: mesenchymal stem cell; NP: nucleus pulposus; CFDA: 5,6 carboxyfluorescein diactetae, succinimidyl ester; DiL: Vybrant CM-DiL cell-labeling solution.

    Techniques Used: Co-Culture Assay, Flow Cytometry, Cytometry, Labeling, Cell Culture

    Incorporation of DiL-labeled microvesicles into MSC and NP cells during direct co-culture. Exemplar flow cytometry analysis. A) MVs derived from a direct co-culture of MSCs and NP cells (one cell population was DiL labeled) and incubated with the unlabeled cell population. B) MV-free supernatant after ultracentrifugation. C) Conditioned medium derived from a direct co-culture of MSCs and NP cells (one cell population was DiL labeled). Abbreviations: MSC: mesenchymal stem cell; NP: nucleus pulposus; MV: microvesicles; SN: supernatant; CM: conditioned medium; CFDA: 5,6 caboxyfluorescein diactetae, succinimidyl ester; DiL: Vybrant CM-DiL cell-labeling solution.
    Figure Legend Snippet: Incorporation of DiL-labeled microvesicles into MSC and NP cells during direct co-culture. Exemplar flow cytometry analysis. A) MVs derived from a direct co-culture of MSCs and NP cells (one cell population was DiL labeled) and incubated with the unlabeled cell population. B) MV-free supernatant after ultracentrifugation. C) Conditioned medium derived from a direct co-culture of MSCs and NP cells (one cell population was DiL labeled). Abbreviations: MSC: mesenchymal stem cell; NP: nucleus pulposus; MV: microvesicles; SN: supernatant; CM: conditioned medium; CFDA: 5,6 caboxyfluorescein diactetae, succinimidyl ester; DiL: Vybrant CM-DiL cell-labeling solution.

    Techniques Used: Labeling, Co-Culture Assay, Flow Cytometry, Cytometry, Derivative Assay, Incubation

    14) Product Images from "MOLECULAR RESURFACING OF CARTILAGE WITH PROTEOGLYCAN 4 (PRG4)"

    Article Title: MOLECULAR RESURFACING OF CARTILAGE WITH PROTEOGLYCAN 4 (PRG4)

    Journal: Acta biomaterialia

    doi: 10.1016/j.actbio.2010.03.025

    Chemical modification of PRG4 with Oregon Green (OG) and aldehyde (CHO). Purified PRG4 was reacted with succinimidyl ester of Oregon Green 488 carboxylic acid, or succinimidyl-4-formylbenzamide (S-4FB), generating PRG4-OG or PRG4-CHO, respectively. A
    Figure Legend Snippet: Chemical modification of PRG4 with Oregon Green (OG) and aldehyde (CHO). Purified PRG4 was reacted with succinimidyl ester of Oregon Green 488 carboxylic acid, or succinimidyl-4-formylbenzamide (S-4FB), generating PRG4-OG or PRG4-CHO, respectively. A

    Techniques Used: Modification, Purification

    15) Product Images from "Multiple roles of filopodial dynamics in particle capture and phagocytosis and phenotypes of Cdc42 and Myo10 deletion"

    Article Title: Multiple roles of filopodial dynamics in particle capture and phagocytosis and phenotypes of Cdc42 and Myo10 deletion

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.766923

    Dectin-1 mediates zymosan binding and internalization. A , fluorescence emission spectra of pHrodo-conjugated zymosan particles suspended in buffers with different pH values, ranging from pH 4 to 8. Particles were labeled using the succinimidyl ester of pHrodo. B , top panel : DIC and fluorescence images of pH-sensitive pHrodo-conjugated zymosan particles superfused with media buffered to different pH values, as indicated. Bottom panel : time-lapse images showing the ingestion and acidification of pHrodo-conjugated zymosan particles. C , DIC image, with superimposed red fluorescence, of two wild-type (WT) macrophages, which have ingested multiple pHrodo-conjugated zymosan particles. Red fluorescence indicates acidification of phagosomes. Scale bar , 10 μm. D , box plots of the number of attached ( left ) and red fluorescent ( right ) pHrodo-conjugated zymosan particles in WT, Toll-like receptor 2/4 double knock-out (TLR2/4 dKO), mannose receptor knock-out ( MR −/− ), and Dectin-1 knock-out ( Dectin-1 −/− ) macrophages. Cells, seeded in fibronectin-coated μ-slide I chambers, were incubated with particles for 15 min before washing. *, p
    Figure Legend Snippet: Dectin-1 mediates zymosan binding and internalization. A , fluorescence emission spectra of pHrodo-conjugated zymosan particles suspended in buffers with different pH values, ranging from pH 4 to 8. Particles were labeled using the succinimidyl ester of pHrodo. B , top panel : DIC and fluorescence images of pH-sensitive pHrodo-conjugated zymosan particles superfused with media buffered to different pH values, as indicated. Bottom panel : time-lapse images showing the ingestion and acidification of pHrodo-conjugated zymosan particles. C , DIC image, with superimposed red fluorescence, of two wild-type (WT) macrophages, which have ingested multiple pHrodo-conjugated zymosan particles. Red fluorescence indicates acidification of phagosomes. Scale bar , 10 μm. D , box plots of the number of attached ( left ) and red fluorescent ( right ) pHrodo-conjugated zymosan particles in WT, Toll-like receptor 2/4 double knock-out (TLR2/4 dKO), mannose receptor knock-out ( MR −/− ), and Dectin-1 knock-out ( Dectin-1 −/− ) macrophages. Cells, seeded in fibronectin-coated μ-slide I chambers, were incubated with particles for 15 min before washing. *, p

    Techniques Used: Binding Assay, Fluorescence, Labeling, Knock-Out, Incubation

    16) Product Images from "The Pim kinases control rapamycin-resistant T cell survival and activation"

    Article Title: The Pim kinases control rapamycin-resistant T cell survival and activation

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20042020

    The Pim kinases confer rapamycin-resistant T cell blastogenesis. (A) C57BL/6 splenic T cells were activated by αCD3/αCD28 and lysates were prepared after 0, 1/2, 1, 2, 3, 6, and 12 h of stimulation and serially probed for the expression of actin, Pim-1, and Pim-2 proteins by Western blot. (B) Activation-induced p70S6K and S6 phosphorylation is rapamycin sensitive. C57BL/6 splenic T cells were preincubated for 1 h in the presence (+) or absence (−) of 50 nM RAPA and activated with αCD3/αCD28 for 0, 10, and 30 min. Lysates were probed for actin, phospho-serine 371-p70 S6 kinase (pSer371 p70S6K), total p70S6K, phospho-serine 235/serine 236-S6 (pSer235/236 S6), and total S6 expression. (C) In the experiments described in B, Western blots were also performed with lysates from rapamycin-treated and control cells after 3 and 12 h of activation and probed for actin, Pim-1, and Pim-2 expression. (D) Splenic T cells from Pim-1 +/+ 2 +/+ , Pim-1 −/− 2 +/+ , Pim-1 +/+ 2 −/− , and Pim-1 −/− 2 −/− mice were activated by αCD3/αCD28 in the absence (−) or presence (+) of 25 nM RAPA (key). Control cells were cultured with αCD28 only (αCD28). After 2 d of culture, forward scatter, and surface CD69, CD25, and CD62L expression was assessed. (E) Cell cycle was assessed in the Pim-1 +/+ 2 +/+ and Pim-1 −/− 2 −/− T cells described in D by BrdU incorporation analysis. The percentage of cells in the G 1 (2N-PI + BrdU − ; bottom left), S (PI + BrdU + ; top), and G 2 M (4N-PI + BrdU − ; bottom right) phases of the cell cycle is shown (insets). Results shown are representative of six experiments. (F)5-(and 6-)carboxyfluorescein diacetate, succinimidyl ester–labeled input T cells from Pim-1 +/+ 2 +/+ or Pim-1 −/− 2 −/− mice were activated for 4 d in the presence (+) or absence (−) of 25 nM RAPA. Control cells were cultured with αCD28 only for 2 d (αCD28) and showed no decay in fluorescence relative to input cells at day 0. Numbers above the leftmost brackets represent the percent of cells that had divided more than three generations. For Pim-1 −/− 2 −/− cells, the majority of the input population failed to divide (*). Data are representative of three experiments.
    Figure Legend Snippet: The Pim kinases confer rapamycin-resistant T cell blastogenesis. (A) C57BL/6 splenic T cells were activated by αCD3/αCD28 and lysates were prepared after 0, 1/2, 1, 2, 3, 6, and 12 h of stimulation and serially probed for the expression of actin, Pim-1, and Pim-2 proteins by Western blot. (B) Activation-induced p70S6K and S6 phosphorylation is rapamycin sensitive. C57BL/6 splenic T cells were preincubated for 1 h in the presence (+) or absence (−) of 50 nM RAPA and activated with αCD3/αCD28 for 0, 10, and 30 min. Lysates were probed for actin, phospho-serine 371-p70 S6 kinase (pSer371 p70S6K), total p70S6K, phospho-serine 235/serine 236-S6 (pSer235/236 S6), and total S6 expression. (C) In the experiments described in B, Western blots were also performed with lysates from rapamycin-treated and control cells after 3 and 12 h of activation and probed for actin, Pim-1, and Pim-2 expression. (D) Splenic T cells from Pim-1 +/+ 2 +/+ , Pim-1 −/− 2 +/+ , Pim-1 +/+ 2 −/− , and Pim-1 −/− 2 −/− mice were activated by αCD3/αCD28 in the absence (−) or presence (+) of 25 nM RAPA (key). Control cells were cultured with αCD28 only (αCD28). After 2 d of culture, forward scatter, and surface CD69, CD25, and CD62L expression was assessed. (E) Cell cycle was assessed in the Pim-1 +/+ 2 +/+ and Pim-1 −/− 2 −/− T cells described in D by BrdU incorporation analysis. The percentage of cells in the G 1 (2N-PI + BrdU − ; bottom left), S (PI + BrdU + ; top), and G 2 M (4N-PI + BrdU − ; bottom right) phases of the cell cycle is shown (insets). Results shown are representative of six experiments. (F)5-(and 6-)carboxyfluorescein diacetate, succinimidyl ester–labeled input T cells from Pim-1 +/+ 2 +/+ or Pim-1 −/− 2 −/− mice were activated for 4 d in the presence (+) or absence (−) of 25 nM RAPA. Control cells were cultured with αCD28 only for 2 d (αCD28) and showed no decay in fluorescence relative to input cells at day 0. Numbers above the leftmost brackets represent the percent of cells that had divided more than three generations. For Pim-1 −/− 2 −/− cells, the majority of the input population failed to divide (*). Data are representative of three experiments.

    Techniques Used: Expressing, Western Blot, Activation Assay, Mouse Assay, Cell Culture, BrdU Incorporation Assay, Labeling, Fluorescence

    17) Product Images from "Interaction of THP-1 Monocytes with Conidia and Hyphae of Different Curvularia Strains"

    Article Title: Interaction of THP-1 Monocytes with Conidia and Hyphae of Different Curvularia Strains

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01369

    Phagocytosis of Curvularia lunata (A) and Aspergillus fumigatus (B,C) conidia by THP-1 monocytes. THP-1 cells and conidia were stained with CellMask Deep Red Plasma Membrane Stain and Alexa Fluor 488 carboxylic acid, succinimidyl ester, respectively. Number of the Curvularia conidia and cells were set to maintain an E:T ratio of 20:1 (A) , while for A. fumigatus E:T ratio was 1:2 (B) or 20:1 (C) . Monocytes were identified by detecting fluorescence intensity on channel 11 (Intensity_MC_CH_11) while channel 2 (Intensity_MC_CH_2) was used to detect the conidia. Cells and conidia were co-incubated for 1 h. Fluorescent micrographs showing conidia (green border) and THP-1 cells alone (red border) and in interaction (i.e., phagocytosis or attachment) (yellow border) were recorded during the imaging flow cytometry.
    Figure Legend Snippet: Phagocytosis of Curvularia lunata (A) and Aspergillus fumigatus (B,C) conidia by THP-1 monocytes. THP-1 cells and conidia were stained with CellMask Deep Red Plasma Membrane Stain and Alexa Fluor 488 carboxylic acid, succinimidyl ester, respectively. Number of the Curvularia conidia and cells were set to maintain an E:T ratio of 20:1 (A) , while for A. fumigatus E:T ratio was 1:2 (B) or 20:1 (C) . Monocytes were identified by detecting fluorescence intensity on channel 11 (Intensity_MC_CH_11) while channel 2 (Intensity_MC_CH_2) was used to detect the conidia. Cells and conidia were co-incubated for 1 h. Fluorescent micrographs showing conidia (green border) and THP-1 cells alone (red border) and in interaction (i.e., phagocytosis or attachment) (yellow border) were recorded during the imaging flow cytometry.

    Techniques Used: Staining, Fluorescence, Incubation, Imaging, Flow Cytometry, Cytometry

    18) Product Images from "Association of Neisseria gonorrhoeae OpaCEA with Dendritic Cells Suppresses Their Ability to Elicit an HIV-1-Specific T Cell Memory Response"

    Article Title: Association of Neisseria gonorrhoeae OpaCEA with Dendritic Cells Suppresses Their Ability to Elicit an HIV-1-Specific T Cell Memory Response

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0056705

    Gonococcal binding and internalization to iMDDCs. A ) Immature MDDCs were incubated with FITC-labeled gonococcal strains N302, N303, N309, and N496, or E.coli DH5α bacteria at an MOI of 10 for 30 min at RT. Bacterial binding was determined by measuring the percentage of cells that bound FITC-labeled bacteria using flow cytometric analysis. The percentage of cells with bound FITC-labeled bacteria is indicated in each condition. B) Bacterial binding to iMDDCs is not mediated by DC-SIGN on the cell surface. Mannan blocking assays were carried out site-by-site with the bacterial binding tests. The percentage of cells with bound FITC-labeled bacteria is indicated in each condition in the presence of mannan at 20 µg/ml. C) Internalization of gonococcal strains and E.coli DH5α into iMDDCs. Immature MDDCs were allowed to adhere onto coverslips pre-coated with 0.2% gelatin. These cells were pulsed with gonococcal strains or E.coli DH5α (MOI = 100) prelabeled with Texas red-X-succinimidyl ester at 37 o C for 1 h. Extracellular bacteria were then labeled with the polyclonal anti-gonococcal serum, followed by a staining with a BODIPY-FL-conjugated secondary Ab. Immature MDDCs were then permeabilized with 0.4% Triton X-100 and stained with Phalloidin-FITC. Intracellular (red) versus extracellular (yellow) bacteria with MDDCs (green) were then distinguished by visualization with a Leica DM-IRBE inverted fluorescence microscope. The magnification for all conditions is 40.
    Figure Legend Snippet: Gonococcal binding and internalization to iMDDCs. A ) Immature MDDCs were incubated with FITC-labeled gonococcal strains N302, N303, N309, and N496, or E.coli DH5α bacteria at an MOI of 10 for 30 min at RT. Bacterial binding was determined by measuring the percentage of cells that bound FITC-labeled bacteria using flow cytometric analysis. The percentage of cells with bound FITC-labeled bacteria is indicated in each condition. B) Bacterial binding to iMDDCs is not mediated by DC-SIGN on the cell surface. Mannan blocking assays were carried out site-by-site with the bacterial binding tests. The percentage of cells with bound FITC-labeled bacteria is indicated in each condition in the presence of mannan at 20 µg/ml. C) Internalization of gonococcal strains and E.coli DH5α into iMDDCs. Immature MDDCs were allowed to adhere onto coverslips pre-coated with 0.2% gelatin. These cells were pulsed with gonococcal strains or E.coli DH5α (MOI = 100) prelabeled with Texas red-X-succinimidyl ester at 37 o C for 1 h. Extracellular bacteria were then labeled with the polyclonal anti-gonococcal serum, followed by a staining with a BODIPY-FL-conjugated secondary Ab. Immature MDDCs were then permeabilized with 0.4% Triton X-100 and stained with Phalloidin-FITC. Intracellular (red) versus extracellular (yellow) bacteria with MDDCs (green) were then distinguished by visualization with a Leica DM-IRBE inverted fluorescence microscope. The magnification for all conditions is 40.

    Techniques Used: Binding Assay, Incubation, Labeling, Flow Cytometry, Blocking Assay, Staining, Fluorescence, Microscopy

    19) Product Images from "Expression of Cutaneous Lymphocyte-Associated Antigen and E-selectin Ligand by Circulating Human Memory CD4+ T Lymphocytes Specific for Herpes Simplex Virus Type 2"

    Article Title: Expression of Cutaneous Lymphocyte-Associated Antigen and E-selectin Ligand by Circulating Human Memory CD4+ T Lymphocytes Specific for Herpes Simplex Virus Type 2

    Journal: The Journal of infectious diseases

    doi: 10.1086/426944

    Expression of cutaneous lymphocyte–associated antigen (CLA) by CD4 + lymphoblasts responding to whole viral antigens. 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE)–labeled peripheral-blood mononuclear cells (PBMCs) were
    Figure Legend Snippet: Expression of cutaneous lymphocyte–associated antigen (CLA) by CD4 + lymphoblasts responding to whole viral antigens. 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE)–labeled peripheral-blood mononuclear cells (PBMCs) were

    Techniques Used: Expressing, Labeling

    20) Product Images from "Cell size sensing in animal cells coordinates anabolic growth rates and cell cycle progression to maintain cell size uniformity"

    Article Title: Cell size sensing in animal cells coordinates anabolic growth rates and cell cycle progression to maintain cell size uniformity

    Journal: eLife

    doi: 10.7554/eLife.26957

    Protein mass quantification with SE-A647. To measure the total protein content of single cells, we used the amine-reactive fluorescent dye AlexaFluor 647-Succinimidyl Ester (SE-A647) to covalently label lysine residues, as described previously ( Kafri et al., 2013 ). The integrated fluorescence intensity of an SE-A647-stained cell reflects the amount of protein it contains and correlates well with its total dry mass measured by quantitative phase microscopy ( Kafri et al., 2013 ). To demonstrate the utility of this method, we labeled the S-phase HeLa cells in an unsynchronized population with a 20 minute-long pulse of EdU and then used SE-A647 staining to measure the protein content of the EdU-positive cells at various time intervals after pulse-labeling. Our measurement is sensitive enough to detect the change in protein mass that occurs in less than three hours of growth. Six hours after labeling, most labelled cells are still in S-phase or G2. Nine hours after labeling, labeled cells have begun to divide, so the distribution is bimodal with its first peak (representing newborn cells) at approximately half the size of its second peak (representing older cells). Between twelve and twenty-four hours after labeling, all labeled cells have divided, and the size distribution gradually shifts to the right again as the cells grow.
    Figure Legend Snippet: Protein mass quantification with SE-A647. To measure the total protein content of single cells, we used the amine-reactive fluorescent dye AlexaFluor 647-Succinimidyl Ester (SE-A647) to covalently label lysine residues, as described previously ( Kafri et al., 2013 ). The integrated fluorescence intensity of an SE-A647-stained cell reflects the amount of protein it contains and correlates well with its total dry mass measured by quantitative phase microscopy ( Kafri et al., 2013 ). To demonstrate the utility of this method, we labeled the S-phase HeLa cells in an unsynchronized population with a 20 minute-long pulse of EdU and then used SE-A647 staining to measure the protein content of the EdU-positive cells at various time intervals after pulse-labeling. Our measurement is sensitive enough to detect the change in protein mass that occurs in less than three hours of growth. Six hours after labeling, most labelled cells are still in S-phase or G2. Nine hours after labeling, labeled cells have begun to divide, so the distribution is bimodal with its first peak (representing newborn cells) at approximately half the size of its second peak (representing older cells). Between twelve and twenty-four hours after labeling, all labeled cells have divided, and the size distribution gradually shifts to the right again as the cells grow.

    Techniques Used: Fluorescence, Staining, Microscopy, Labeling

    21) Product Images from "Different Effects of Human Umbilical Cord Mesenchymal Stem Cells on Glioblastoma Stem Cells by Direct Cell Interaction or Via Released Soluble Factors"

    Article Title: Different Effects of Human Umbilical Cord Mesenchymal Stem Cells on Glioblastoma Stem Cells by Direct Cell Interaction or Via Released Soluble Factors

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2017.00312

    UC-MSC co-culture reduces GBM CSC proliferation rate. The proliferation rate of CSC1, CSC2 and CSC3 was tracked by carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) dye dilution and analyzed by flow cytometry. The distribution of CSC1, CSC2 and
    Figure Legend Snippet: UC-MSC co-culture reduces GBM CSC proliferation rate. The proliferation rate of CSC1, CSC2 and CSC3 was tracked by carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) dye dilution and analyzed by flow cytometry. The distribution of CSC1, CSC2 and

    Techniques Used: Co-Culture Assay, Flow Cytometry, Cytometry

    22) Product Images from "Trogocytosis of CD80 and CD86 by induced regulatory T cells"

    Article Title: Trogocytosis of CD80 and CD86 by induced regulatory T cells

    Journal: Cellular and Molecular Immunology

    doi: 10.1038/cmi.2011.62

    In vitro generation of CD4 + CD25 + Foxp3 + T cells from murine naive T cells. ( a ) The expression of Foxp3 and CD25 was evaluated in CD4 + CD25 − cells isolated from either C57BL/6 (WT) mice or from CD80 −/− CD86 −/− DKO mice, activated for 5 days with plate-bound anti-CD3 Ab, soluble anti-CD28 Ab and IL-2 in the presence of TGF-β. Left: WT iTreg. Right: CD80 −/− CD86 −/− DKO iTreg. ( b ) Dose-dependent suppression of T-cell proliferation by CD4 + CD25 + Foxp3 + iTreg cells. CD4 + CD25 − T cells (Thy1.1 + ) were purified from naive B6.PL-Th1a/CyJ mice, labeled with CFSE, and cultured (1×10 5 cells/well) with 1 µg/ml anti-CD3 Ab in the presence of irradiated syngeneic splenocytes (2×10 4 cells/well). Varying numbers of CD4 + CD25 + Foxp3 + iTreg from WT or DKO C57BL/6 mice were added to the culture at suppressor to responder ratios 1∶0.5, 1∶1, 1∶2, 1∶4 and 1∶8. After 4 days of culture, proliferation of responder cells was measured by gating on Thy1.1 + cells and assessing their CFSE signal by flow cytometry. Numbers represent the percentage of undivided cells. (−) control: CFSE labeled CD4 + CD25 − T cells without any stimulation or CD4 + CD25 + Foxp3 + T cells. (+) contol: CFSE labeled CD4 + CD25 − T cells were cultured with anti-CD3 Ab in the presence of irradiated APCs without CD4 + CD25 + Foxp3 + T cells. ( c ) Undivided populations were analyzed by determining the suppression ratio obtained with the formula: suppression ratio=(experiment group−positive control)/experiment group×100%. The data shown are representative of at least three different experiments. Ab, antibody; APC, antigen-presenting cell; CFSE, carboxyfluorescein diacetate, succinimidyl ester; DKO, double knockout; iTreg, induced regulatory T cell; WT, wild-type.
    Figure Legend Snippet: In vitro generation of CD4 + CD25 + Foxp3 + T cells from murine naive T cells. ( a ) The expression of Foxp3 and CD25 was evaluated in CD4 + CD25 − cells isolated from either C57BL/6 (WT) mice or from CD80 −/− CD86 −/− DKO mice, activated for 5 days with plate-bound anti-CD3 Ab, soluble anti-CD28 Ab and IL-2 in the presence of TGF-β. Left: WT iTreg. Right: CD80 −/− CD86 −/− DKO iTreg. ( b ) Dose-dependent suppression of T-cell proliferation by CD4 + CD25 + Foxp3 + iTreg cells. CD4 + CD25 − T cells (Thy1.1 + ) were purified from naive B6.PL-Th1a/CyJ mice, labeled with CFSE, and cultured (1×10 5 cells/well) with 1 µg/ml anti-CD3 Ab in the presence of irradiated syngeneic splenocytes (2×10 4 cells/well). Varying numbers of CD4 + CD25 + Foxp3 + iTreg from WT or DKO C57BL/6 mice were added to the culture at suppressor to responder ratios 1∶0.5, 1∶1, 1∶2, 1∶4 and 1∶8. After 4 days of culture, proliferation of responder cells was measured by gating on Thy1.1 + cells and assessing their CFSE signal by flow cytometry. Numbers represent the percentage of undivided cells. (−) control: CFSE labeled CD4 + CD25 − T cells without any stimulation or CD4 + CD25 + Foxp3 + T cells. (+) contol: CFSE labeled CD4 + CD25 − T cells were cultured with anti-CD3 Ab in the presence of irradiated APCs without CD4 + CD25 + Foxp3 + T cells. ( c ) Undivided populations were analyzed by determining the suppression ratio obtained with the formula: suppression ratio=(experiment group−positive control)/experiment group×100%. The data shown are representative of at least three different experiments. Ab, antibody; APC, antigen-presenting cell; CFSE, carboxyfluorescein diacetate, succinimidyl ester; DKO, double knockout; iTreg, induced regulatory T cell; WT, wild-type.

    Techniques Used: In Vitro, Expressing, Isolation, Mouse Assay, Purification, Labeling, Cell Culture, Irradiation, Flow Cytometry, Cytometry, Positive Control, Double Knockout

    23) Product Images from "Plasma membrane calcium ATPase deficiency causes neuronal pathology in the spinal cord: a potential mechanism for neurodegeneration in multiple sclerosis and spinal cord injury"

    Article Title: Plasma membrane calcium ATPase deficiency causes neuronal pathology in the spinal cord: a potential mechanism for neurodegeneration in multiple sclerosis and spinal cord injury

    Journal: The FASEB journal : official publication of the Federation of American Societies for Experimental Biology

    doi: 10.1096/fj.04-2549fje

    Effects of PMCA and SERCA inhibitors on spinal cord neurons, in vitro A ) RT-PCR showing expression of PMCA2 in neuronal cultures. Lane 1: molecular weight marker; bp, base pairs; lane 2: RT-PCR product (arrow) at the predicted molecular weight (1014 bp). The identity of the band was further verified by sequence analysis. B ) PMCA2 immunoreactivity in spinal cord neurons. Arrows and arrowheads point at immunopositive cells and processes, respectively. C ) SMI-32 positive cell number after exposure of cultures to 10, 30, and 100 μM Na 3 VO 4 . C: control. D ) Effects of SERCA inhibitors on SMI-32 positive cell number. CPA: cyclopiazonic acid. E ) SMI-32 positive cells after exposure of cultures to 5 μM 5-(and-6)-carboxyeosin diacetate, succinimidyl ester (CE). F ) Effects of CE on cell survival. G ) Induction of activated caspase-3 in cells treated with CE. The experiments were repeated at least twice and yielded similar results. Values are presented as means ± SEM. Significantly different from controls * P
    Figure Legend Snippet: Effects of PMCA and SERCA inhibitors on spinal cord neurons, in vitro A ) RT-PCR showing expression of PMCA2 in neuronal cultures. Lane 1: molecular weight marker; bp, base pairs; lane 2: RT-PCR product (arrow) at the predicted molecular weight (1014 bp). The identity of the band was further verified by sequence analysis. B ) PMCA2 immunoreactivity in spinal cord neurons. Arrows and arrowheads point at immunopositive cells and processes, respectively. C ) SMI-32 positive cell number after exposure of cultures to 10, 30, and 100 μM Na 3 VO 4 . C: control. D ) Effects of SERCA inhibitors on SMI-32 positive cell number. CPA: cyclopiazonic acid. E ) SMI-32 positive cells after exposure of cultures to 5 μM 5-(and-6)-carboxyeosin diacetate, succinimidyl ester (CE). F ) Effects of CE on cell survival. G ) Induction of activated caspase-3 in cells treated with CE. The experiments were repeated at least twice and yielded similar results. Values are presented as means ± SEM. Significantly different from controls * P

    Techniques Used: In Vitro, Reverse Transcription Polymerase Chain Reaction, Expressing, Molecular Weight, Marker, Sequencing

    24) Product Images from "Hemagglutinin-based polyanhydride nanovaccines against H5N1 influenza elicit protective virus neutralizing titers and cell-mediated immunity"

    Article Title: Hemagglutinin-based polyanhydride nanovaccines against H5N1 influenza elicit protective virus neutralizing titers and cell-mediated immunity

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S72264

    Enhanced CD4 + T cell memory with poly I:C polyanhydride nanovaccine. Notes: Draining lymph nodes were harvested from prime/boost/boost immunized mice 63 days after the primary immunization. Ex vivo antigen stimulation and CFSE-labeling demonstrated enhanced CD4 + T cell proliferation of mice immunized with poly I:C nanovaccines. Error bars represent the standard error of the mean. Different letters indicate statistical significance among treatments. P ≤0.0147. Abbreviations: CFSE, 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester; MPLA, monophosphoryl lipid A; sH5 3 , soluble H5 hemagglutinin trimer; poly I:C, polyinosinic-polycytidylic acid..
    Figure Legend Snippet: Enhanced CD4 + T cell memory with poly I:C polyanhydride nanovaccine. Notes: Draining lymph nodes were harvested from prime/boost/boost immunized mice 63 days after the primary immunization. Ex vivo antigen stimulation and CFSE-labeling demonstrated enhanced CD4 + T cell proliferation of mice immunized with poly I:C nanovaccines. Error bars represent the standard error of the mean. Different letters indicate statistical significance among treatments. P ≤0.0147. Abbreviations: CFSE, 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester; MPLA, monophosphoryl lipid A; sH5 3 , soluble H5 hemagglutinin trimer; poly I:C, polyinosinic-polycytidylic acid..

    Techniques Used: Mouse Assay, Ex Vivo, Labeling

    25) Product Images from "A Role for the Eph Ligand Ephrin-A3 in Entorhino-Hippocampal Axon Targeting"

    Article Title: A Role for the Eph Ligand Ephrin-A3 in Entorhino-Hippocampal Axon Targeting

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.19-20-08885.1999

    Inhibition of neurite outgrowth from entorhinal explants by ephrin-A3. Entorhinal explants were cultivated on a confluent membrane layer obtained from 3T3 mock-transfected ( n = 41) ( A ), ephrin-A3- ( n = 62) ( C ), and ephrin-A5- ( n = 31) ( E ) transfected cells. Neurites were visualized with a fluorescent vital dye, 5- and 6-carboxyfluorescein diacetate, succinimidyl ester. D , PI-PLC treatment of the obtained membranes ( n = 36) significantly prevents ephrin-A3 inhibition of entorhinal neurite outgrowth, whereas the effect of PI-PLC treatment on neurite outgrowth inhibition ( n = 25) was not significant in the case of ephrin-A5 ( F ). B , Quantification of entorhinal neurite length under the different conditions (±SEM). The differences of entorhinal outgrowth length were significant between ephrin-A3 treatment and all other experimental conditions (* p
    Figure Legend Snippet: Inhibition of neurite outgrowth from entorhinal explants by ephrin-A3. Entorhinal explants were cultivated on a confluent membrane layer obtained from 3T3 mock-transfected ( n = 41) ( A ), ephrin-A3- ( n = 62) ( C ), and ephrin-A5- ( n = 31) ( E ) transfected cells. Neurites were visualized with a fluorescent vital dye, 5- and 6-carboxyfluorescein diacetate, succinimidyl ester. D , PI-PLC treatment of the obtained membranes ( n = 36) significantly prevents ephrin-A3 inhibition of entorhinal neurite outgrowth, whereas the effect of PI-PLC treatment on neurite outgrowth inhibition ( n = 25) was not significant in the case of ephrin-A5 ( F ). B , Quantification of entorhinal neurite length under the different conditions (±SEM). The differences of entorhinal outgrowth length were significant between ephrin-A3 treatment and all other experimental conditions (* p

    Techniques Used: Inhibition, Transfection, Planar Chromatography

    26) Product Images from "Addition of an aminopeptidase N‐binding sequence to human endostatin improves inhibition of ovarian carcinoma growth"

    Article Title: Addition of an aminopeptidase N‐binding sequence to human endostatin improves inhibition of ovarian carcinoma growth

    Journal: Cancer

    doi: 10.1002/cncr.21149

    Characterization of NGR sequence‐modified endostatin (NGR‐endostatin). (A) Aminopeptidase N (APN)‐inhibitory activity. APN was extracted from human umbilical vein endothelial cell (HUVEC) cultures. Endostatin preparations, bestatin (positive control), and leupeptin (negative control) were used at a concentration of 5 μM. Endostatin‐RGD: endostatin containing an RGD sequence. (B) Cell attachment assay. HUVEC or WM35 cells that were prelabeled with 5‐(and‐6)‐carboxy fluorescein diacetate, succinimidyl ester, were added into triplicate wells coated with either endostatin or NGR‐endostatin at a concentration of 1 nmole per well. Wells coated with 0.2% gelatin were used as maximum attachment (100%). The attached cells were quantified by a fluorescence plate reader. Values represent the mean of two independent experiments. (C) The effect of endostatin and NGR‐endostatin on endothelial cell proliferation: Endostatin and NGR‐endostatin were used at a concentration of 2.5 μg/mL. Basic fibroblast growth factor (5 ng/mL) was used to induce proliferation of endothelial cells. The proliferation was determined by 5′‐bromo‐2′‐deoxyuridine uptake. (D) The effect of endostatin (open bar) and NGR‐endostatin (solid bar) on HUVEC migration. NGR‐containing peptide (SR‐1) plus endostatin (hatched bar) did not enhance the basal level of inhibition seen with endostatin alone (open bar). (E) Tumor localization: Human colon carcinoma cells (LS174T) were injected subcutaneously into female athymic nude mice. When the tumors reached a size of ≈ 500 mm 3 (10 days after inoculation), endostatin (open bars) or NGR‐endostatin (solid bars) was injected at a dose of 20 mg/kg subcutaneously. Tumor, liver, and lung tissues were resected and homogenized. Endostatin levels in the tissues and the sera were determined by enzyme‐linked immunoadsorbent assay in the soluble fraction. Endostatin levels are expressed as a relative concentration to serum levels of endostatin. Error bars indicate the standard error. Statistical significance was determined with a Student t test. An asterisk indicates P
    Figure Legend Snippet: Characterization of NGR sequence‐modified endostatin (NGR‐endostatin). (A) Aminopeptidase N (APN)‐inhibitory activity. APN was extracted from human umbilical vein endothelial cell (HUVEC) cultures. Endostatin preparations, bestatin (positive control), and leupeptin (negative control) were used at a concentration of 5 μM. Endostatin‐RGD: endostatin containing an RGD sequence. (B) Cell attachment assay. HUVEC or WM35 cells that were prelabeled with 5‐(and‐6)‐carboxy fluorescein diacetate, succinimidyl ester, were added into triplicate wells coated with either endostatin or NGR‐endostatin at a concentration of 1 nmole per well. Wells coated with 0.2% gelatin were used as maximum attachment (100%). The attached cells were quantified by a fluorescence plate reader. Values represent the mean of two independent experiments. (C) The effect of endostatin and NGR‐endostatin on endothelial cell proliferation: Endostatin and NGR‐endostatin were used at a concentration of 2.5 μg/mL. Basic fibroblast growth factor (5 ng/mL) was used to induce proliferation of endothelial cells. The proliferation was determined by 5′‐bromo‐2′‐deoxyuridine uptake. (D) The effect of endostatin (open bar) and NGR‐endostatin (solid bar) on HUVEC migration. NGR‐containing peptide (SR‐1) plus endostatin (hatched bar) did not enhance the basal level of inhibition seen with endostatin alone (open bar). (E) Tumor localization: Human colon carcinoma cells (LS174T) were injected subcutaneously into female athymic nude mice. When the tumors reached a size of ≈ 500 mm 3 (10 days after inoculation), endostatin (open bars) or NGR‐endostatin (solid bars) was injected at a dose of 20 mg/kg subcutaneously. Tumor, liver, and lung tissues were resected and homogenized. Endostatin levels in the tissues and the sera were determined by enzyme‐linked immunoadsorbent assay in the soluble fraction. Endostatin levels are expressed as a relative concentration to serum levels of endostatin. Error bars indicate the standard error. Statistical significance was determined with a Student t test. An asterisk indicates P

    Techniques Used: Sequencing, Modification, Activity Assay, Positive Control, Negative Control, Concentration Assay, Cell Attachment Assay, Fluorescence, Migration, Inhibition, Injection, Mouse Assay

    27) Product Images from "Turning the Old Adjuvant from Gel to Nanoparticles to Amplify CD8+ T Cell Responses"

    Article Title: Turning the Old Adjuvant from Gel to Nanoparticles to Amplify CD8+ T Cell Responses

    Journal: Advanced Science

    doi: 10.1002/advs.201700426

    APN‐CpG induces potent cellular immune responses. A) Schematic of APN‐CpG induces antibody and T cell responses. C57BL/6 mice were vaccinated in the footpad on day 0 with APNs (with or without 0.45 µg CpG), 1.5 µg OVA (with or without 0.45 µg CpG), or Algel2% with 0.45 µg CpG. A boost injection was performed on day 7 with the same doses of OVA and CpG. B) Mice were sacrificed on day 14, serum was collected, diluted 10 5 times, and analyzed for total anti‐OVA IgG. Popliteal lymph nodes and spleens of vaccinated mice were harvested and homogenized into cell suspensions. C,D) To restimulate CD4 + and CD8 + T cells, splenocytes were incubated for 6 h at 37 °C with, respectively, 100 µg mL −1 OVA or 2 µg mL −1 SIINFEKL. Brefeldin A (5 µg mL −1 ) was added for the last 5 h of culture. Percentages of IFN‐γ‐expressing C) CD4 + and D) CD8 + T cells were determined using flow cytometry. E) Lymph node cells and splenocytes were cultured for 60 h in the presence of 2 µg mL −1 SIINFEKL, and production of IFN‐γ was determined using ELISA. Panels (D) and (E) show that APN‐CpG activated CD8 + T cells more strongly than OVA + CpG or Algel2% + CpG. F) Schematic of the carboxy fluorescein succinimidyl ester (CFSE) labeling method to evaluate the OVA‐specific CTL response induced by APN‐CpG. C57BL/6 mice were vaccinated in the footpad on day 0 and boosted on day 7. On day 7 (for prime only) or 14 (for prime and boost), vaccinated and naive mice were injected with a 1:1 mixture of splenocytes, half of which had been incubated with SIINFEKL peptide and stained with a high level of CFSE, and the other half of which had been stained with a tenfold lower level of CFSE. Spleens were harvested after 18 h, and splenocytes were analyzed by flow cytometry. G) APN‐CpG triggered a potent CTL response. Box plots in panels (B)–(D) show medians and 95% confidence intervals, while the data in other panels are mean ± SEM. In panels (E) and (G), asterisks indicate P values associated with comparisons of APN‐CpG and OVA + CpG; pound signs indicate the same P ranges for comparisons between APN‐CpG and Algel2% + CpG. * P
    Figure Legend Snippet: APN‐CpG induces potent cellular immune responses. A) Schematic of APN‐CpG induces antibody and T cell responses. C57BL/6 mice were vaccinated in the footpad on day 0 with APNs (with or without 0.45 µg CpG), 1.5 µg OVA (with or without 0.45 µg CpG), or Algel2% with 0.45 µg CpG. A boost injection was performed on day 7 with the same doses of OVA and CpG. B) Mice were sacrificed on day 14, serum was collected, diluted 10 5 times, and analyzed for total anti‐OVA IgG. Popliteal lymph nodes and spleens of vaccinated mice were harvested and homogenized into cell suspensions. C,D) To restimulate CD4 + and CD8 + T cells, splenocytes were incubated for 6 h at 37 °C with, respectively, 100 µg mL −1 OVA or 2 µg mL −1 SIINFEKL. Brefeldin A (5 µg mL −1 ) was added for the last 5 h of culture. Percentages of IFN‐γ‐expressing C) CD4 + and D) CD8 + T cells were determined using flow cytometry. E) Lymph node cells and splenocytes were cultured for 60 h in the presence of 2 µg mL −1 SIINFEKL, and production of IFN‐γ was determined using ELISA. Panels (D) and (E) show that APN‐CpG activated CD8 + T cells more strongly than OVA + CpG or Algel2% + CpG. F) Schematic of the carboxy fluorescein succinimidyl ester (CFSE) labeling method to evaluate the OVA‐specific CTL response induced by APN‐CpG. C57BL/6 mice were vaccinated in the footpad on day 0 and boosted on day 7. On day 7 (for prime only) or 14 (for prime and boost), vaccinated and naive mice were injected with a 1:1 mixture of splenocytes, half of which had been incubated with SIINFEKL peptide and stained with a high level of CFSE, and the other half of which had been stained with a tenfold lower level of CFSE. Spleens were harvested after 18 h, and splenocytes were analyzed by flow cytometry. G) APN‐CpG triggered a potent CTL response. Box plots in panels (B)–(D) show medians and 95% confidence intervals, while the data in other panels are mean ± SEM. In panels (E) and (G), asterisks indicate P values associated with comparisons of APN‐CpG and OVA + CpG; pound signs indicate the same P ranges for comparisons between APN‐CpG and Algel2% + CpG. * P

    Techniques Used: Mouse Assay, Injection, Incubation, Expressing, Flow Cytometry, Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Labeling, CTL Assay, Staining

    28) Product Images from "Transfer of a TCR Gene Derived from a Patient with a Marked Antitumor Response Conveys Highly Active T-Cell Effector Functions"

    Article Title: Transfer of a TCR Gene Derived from a Patient with a Marked Antitumor Response Conveys Highly Active T-Cell Effector Functions

    Journal:

    doi: 10.1089/hum.2005.16.457

    Proliferation of TCR-transduced peripheral blood lymphocytes (PBL) in melanoma cocultures. Shown are resultant fluorescence-activated cell sorter (FACS) histograms of carboxy-fluorescein diacetate, succinimidyl ester (CFSE)-labeled PBL cocultured with
    Figure Legend Snippet: Proliferation of TCR-transduced peripheral blood lymphocytes (PBL) in melanoma cocultures. Shown are resultant fluorescence-activated cell sorter (FACS) histograms of carboxy-fluorescein diacetate, succinimidyl ester (CFSE)-labeled PBL cocultured with

    Techniques Used: Fluorescence, FACS, Labeling

    29) Product Images from "Phosphatidylserine and FVa Regulate FXa Structure"

    Article Title: Phosphatidylserine and FVa Regulate FXa Structure

    Journal: The Biochemical journal

    doi: 10.1042/BJ20131099

    Schematic representation of preparation of fluorophore-labeled FXa. FX was first labeled with donor (Alexa Fluor 555 succinimidyl ester; A555-NHS) followed by activation to remove the activation peptide at its N-terminus. The acceptor (fluorescein-labeled
    Figure Legend Snippet: Schematic representation of preparation of fluorophore-labeled FXa. FX was first labeled with donor (Alexa Fluor 555 succinimidyl ester; A555-NHS) followed by activation to remove the activation peptide at its N-terminus. The acceptor (fluorescein-labeled

    Techniques Used: Labeling, Activation Assay

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    Labeling:

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    Transgenic Assay:

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    Article Snippet: .. OT-II transgenic CD4+ T cells were stained with CFSE (7.5 µM, Invitrogen) , for 10 min at 37°C. .. An amount of 2.5×106 of CFSE-labelled T cells were injected into the tail vein of each recipient mouse.

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    Staining:

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    Article Title: DNA methyltransferase inhibition overcomes diphthamide pathway deficiencies underlying CD123-targeted treatment resistance
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    Thermo Fisher cfse
    c-Myb is indispensable for <t>CD8</t> + T cell stemness. ( a ) Experimental design assessing c-Myb function in long-term memory. ( b ) Flow cytometry of splenic CD8 + T cells after transfer of 3×10 5 pmel-1 Thy1.1 Myb fl / fl or pmel-1 Thy1.1 Myb Δ / Δ CD8 + T cells into wild-type mice infected with gp100-VV, assessed 90d after infection ( n = 3 mice per group). ( c ) Numbers of total (left) and CD62L + KLRG1 − (right) pmel-1 T cells after transfer as in b . ( d ) Experimental design testing c-Myb impact on secondary memory. Middle, flow cytometry exemplifying Thy1.1 + T cell frequencies 45d after transfer as in b . ( e , f ) Flow cytometry of splenocytes ( e ) and numbers of splenic pmel-1 Thy1.1 CD8 + T cells ( f ) after transfer of 5 ×10 4 primary memory pmel-1 Thy1.1 Myb fl / fl or pmel-1 Thy1.1 Myb Δ / Δ CD8 + T cells, assessed 30d after gp100-adV infection ( n = 3 mice per group). ( g ) Flow cytometry of splenic pmel-1 T cells 30d after transfer as in e , f . ( h ) Numbers of splenic CD62L + KLRG1 − pmel1 T cells obtained as in g . ( i ) Experimental design evaluating self-renewal of stem cell–like T CM cells. Middle, flow cytometry exemplifying the sorting strategy for isolation of CD62L + pmel-1 memory T cells from spleens and lymph nodes 45d after transfer of 10 6 pmel-1 Thy1.1 Myb fl/fl or pmel-1 Thy1.1 Myb Δ/Δ CD8 + T cells into wild-type mice infected with gp100-VV. ( j ) Flow cytometry of pmel-1 Thy1.1 CD8 + T cells 28d after transfer of 10 5 <t>CFSE-labeled</t> CD62L + pmel-1 Thy1.1 Myb fl / fl or pmel-1 Thy1.1 Myb Δ / Δ CD8 + T cells into sub-lethally irradiated mice ( n =2 mice per group, data shown after concatenating). Data are shown after gating on live ( e ) live, CD8 + ( b, d ) and live, CD8 + Thy1.1 + cells ( g , j ). Data in c , f , h are shown as mean ± s.e.m.; shapes represent individual mice ( c , f , h ). *= P
    Cfse, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2332 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher rtpal sata
    Isoelectric focusing (IEF) gel electrophoresis (panel a ) of intermediate products in <t>RtPAL-BPM6</t> stepwise conjugation: RtPAL (lane 1), <t>RtPAL-SATA</t> (lane 2), RtPAL-BPM6 (lane 3), RtPAL-BPM6 treated with alkaline phosphatase (lane 4), and IEF Marker (lane
    Rtpal Sata, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher efficacy sata 8505
    <t>SATA-8505</t> Improves MRSA Skin Infection at Low MOI. Mice were injected intraperitoneally (I.P.) with 10 7 plaque-forming units (PFU) of SATA-8505 immediately prior to subcutaneous injections of 10 7 CFU of USA300. (a) Lesion size progression over following six days. On day 6, skin biopsies were homogenized for culture, total bacterial (b) and phage burden (c) were calculated for individual mice. (d) mRNA transcript levels for IL-1ß, IL-6, and IL-17A in individual CGD mice relative to wild type controls, standardized to GAPDH. (e-g) mRNA transcript levels for IL-1ß, IL-6, and IL-17A in CGD and wild type mice with and without SATA-8505 treatment relative to wild type controls injected with diluent, standardized to GAPDH. Data shown are representative of 2–3 independent experiments using 5 or more mice per group, and displayed as mean + s.e.m. Differences were calculated by ANOVA with Bonferroni correction and depict differences from diluent treated wild type unless otherwise noted. ns = not significant, * = p
    Efficacy Sata 8505, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    c-Myb is indispensable for CD8 + T cell stemness. ( a ) Experimental design assessing c-Myb function in long-term memory. ( b ) Flow cytometry of splenic CD8 + T cells after transfer of 3×10 5 pmel-1 Thy1.1 Myb fl / fl or pmel-1 Thy1.1 Myb Δ / Δ CD8 + T cells into wild-type mice infected with gp100-VV, assessed 90d after infection ( n = 3 mice per group). ( c ) Numbers of total (left) and CD62L + KLRG1 − (right) pmel-1 T cells after transfer as in b . ( d ) Experimental design testing c-Myb impact on secondary memory. Middle, flow cytometry exemplifying Thy1.1 + T cell frequencies 45d after transfer as in b . ( e , f ) Flow cytometry of splenocytes ( e ) and numbers of splenic pmel-1 Thy1.1 CD8 + T cells ( f ) after transfer of 5 ×10 4 primary memory pmel-1 Thy1.1 Myb fl / fl or pmel-1 Thy1.1 Myb Δ / Δ CD8 + T cells, assessed 30d after gp100-adV infection ( n = 3 mice per group). ( g ) Flow cytometry of splenic pmel-1 T cells 30d after transfer as in e , f . ( h ) Numbers of splenic CD62L + KLRG1 − pmel1 T cells obtained as in g . ( i ) Experimental design evaluating self-renewal of stem cell–like T CM cells. Middle, flow cytometry exemplifying the sorting strategy for isolation of CD62L + pmel-1 memory T cells from spleens and lymph nodes 45d after transfer of 10 6 pmel-1 Thy1.1 Myb fl/fl or pmel-1 Thy1.1 Myb Δ/Δ CD8 + T cells into wild-type mice infected with gp100-VV. ( j ) Flow cytometry of pmel-1 Thy1.1 CD8 + T cells 28d after transfer of 10 5 CFSE-labeled CD62L + pmel-1 Thy1.1 Myb fl / fl or pmel-1 Thy1.1 Myb Δ / Δ CD8 + T cells into sub-lethally irradiated mice ( n =2 mice per group, data shown after concatenating). Data are shown after gating on live ( e ) live, CD8 + ( b, d ) and live, CD8 + Thy1.1 + cells ( g , j ). Data in c , f , h are shown as mean ± s.e.m.; shapes represent individual mice ( c , f , h ). *= P

    Journal: Nature immunology

    Article Title: The transcription factor c-Myb regulates CD8+ T cell stemness and antitumor immunity

    doi: 10.1038/s41590-018-0311-z

    Figure Lengend Snippet: c-Myb is indispensable for CD8 + T cell stemness. ( a ) Experimental design assessing c-Myb function in long-term memory. ( b ) Flow cytometry of splenic CD8 + T cells after transfer of 3×10 5 pmel-1 Thy1.1 Myb fl / fl or pmel-1 Thy1.1 Myb Δ / Δ CD8 + T cells into wild-type mice infected with gp100-VV, assessed 90d after infection ( n = 3 mice per group). ( c ) Numbers of total (left) and CD62L + KLRG1 − (right) pmel-1 T cells after transfer as in b . ( d ) Experimental design testing c-Myb impact on secondary memory. Middle, flow cytometry exemplifying Thy1.1 + T cell frequencies 45d after transfer as in b . ( e , f ) Flow cytometry of splenocytes ( e ) and numbers of splenic pmel-1 Thy1.1 CD8 + T cells ( f ) after transfer of 5 ×10 4 primary memory pmel-1 Thy1.1 Myb fl / fl or pmel-1 Thy1.1 Myb Δ / Δ CD8 + T cells, assessed 30d after gp100-adV infection ( n = 3 mice per group). ( g ) Flow cytometry of splenic pmel-1 T cells 30d after transfer as in e , f . ( h ) Numbers of splenic CD62L + KLRG1 − pmel1 T cells obtained as in g . ( i ) Experimental design evaluating self-renewal of stem cell–like T CM cells. Middle, flow cytometry exemplifying the sorting strategy for isolation of CD62L + pmel-1 memory T cells from spleens and lymph nodes 45d after transfer of 10 6 pmel-1 Thy1.1 Myb fl/fl or pmel-1 Thy1.1 Myb Δ/Δ CD8 + T cells into wild-type mice infected with gp100-VV. ( j ) Flow cytometry of pmel-1 Thy1.1 CD8 + T cells 28d after transfer of 10 5 CFSE-labeled CD62L + pmel-1 Thy1.1 Myb fl / fl or pmel-1 Thy1.1 Myb Δ / Δ CD8 + T cells into sub-lethally irradiated mice ( n =2 mice per group, data shown after concatenating). Data are shown after gating on live ( e ) live, CD8 + ( b, d ) and live, CD8 + Thy1.1 + cells ( g , j ). Data in c , f , h are shown as mean ± s.e.m.; shapes represent individual mice ( c , f , h ). *= P

    Article Snippet: CFSE and MitoTracker Green labeling CD8+ T cells were incubated with 1 μL of CFSE (Thermo Fischer # C34554) in 1ml protein-free PBS for 20 minutes at 37°C with agitation followed.

    Techniques: Flow Cytometry, Cytometry, Mouse Assay, Infection, Isolation, Labeling, Irradiation

    Modulation in cell cycle and proliferation pattern of neuro-2a under varied serum conditions with presence/absence of retinoic acid. Neuro-2a cells were subjected to culture conditions, namely DMEM + 10% FBS (GpI), DMEM + 10% FBS + 20 μM Retinoic acid (GpII), DMEM + O.1% FBS (GpIII), DMEM + 0.1% FBS + 20 μM Retinoic acid (GpIV) for 24 h, processed as per respective protocols and then scanned using FACS Calibur™. Cell cycle data are represented as the histogram (A–D) and CFSE assay data is represented in bar graphs (E, F) showing mean fluorescence intensity values (MFI values) and cell counts (cells/9.6 cm 2 ) respectively. Values are mean ± S.E. P ≤ 0.05 ( α , β , γ ), P ≤ 0.01 (αα, ββ, γγ), P ≤ 0.001 ( ααα , βββ , γγγ ) were considered to be statistically significant. α , Compared to GpI; β , Compared to GpII; γ , Compared to GpIII. Additionally, mean data values, S.E., ANOVA statistics and P . Values from Tukey׳s multiple comparisons (post hoc) are represented besides respective graphical representations.

    Journal: Data in Brief

    Article Title: Data on retinoic acid and reduced serum concentration induced differentiation of Neuro-2a neuroblastoma cells

    doi: 10.1016/j.dib.2018.11.097

    Figure Lengend Snippet: Modulation in cell cycle and proliferation pattern of neuro-2a under varied serum conditions with presence/absence of retinoic acid. Neuro-2a cells were subjected to culture conditions, namely DMEM + 10% FBS (GpI), DMEM + 10% FBS + 20 μM Retinoic acid (GpII), DMEM + O.1% FBS (GpIII), DMEM + 0.1% FBS + 20 μM Retinoic acid (GpIV) for 24 h, processed as per respective protocols and then scanned using FACS Calibur™. Cell cycle data are represented as the histogram (A–D) and CFSE assay data is represented in bar graphs (E, F) showing mean fluorescence intensity values (MFI values) and cell counts (cells/9.6 cm 2 ) respectively. Values are mean ± S.E. P ≤ 0.05 ( α , β , γ ), P ≤ 0.01 (αα, ββ, γγ), P ≤ 0.001 ( ααα , βββ , γγγ ) were considered to be statistically significant. α , Compared to GpI; β , Compared to GpII; γ , Compared to GpIII. Additionally, mean data values, S.E., ANOVA statistics and P . Values from Tukey׳s multiple comparisons (post hoc) are represented besides respective graphical representations.

    Article Snippet: Before starting the process of neuro-2a cellular differentiation, cells were processed for CFSE as per manufacturer protocol (CellTrace™, Thermofisher, C34554).

    Techniques: FACS, CFSE Assay, Fluorescence

    Isoelectric focusing (IEF) gel electrophoresis (panel a ) of intermediate products in RtPAL-BPM6 stepwise conjugation: RtPAL (lane 1), RtPAL-SATA (lane 2), RtPAL-BPM6 (lane 3), RtPAL-BPM6 treated with alkaline phosphatase (lane 4), and IEF Marker (lane

    Journal: JIMD Reports

    Article Title: Mannose 6-Phosphate Conjugation Is Not Sufficient to Allow Induction of Immune Tolerance to Phenylalanine Ammonia-Lyase in Dogs

    doi: 10.1007/8904_2012_162

    Figure Lengend Snippet: Isoelectric focusing (IEF) gel electrophoresis (panel a ) of intermediate products in RtPAL-BPM6 stepwise conjugation: RtPAL (lane 1), RtPAL-SATA (lane 2), RtPAL-BPM6 (lane 3), RtPAL-BPM6 treated with alkaline phosphatase (lane 4), and IEF Marker (lane

    Article Snippet: The purified product was then deacetylated to produce a deprotected sulfhydryl group on SATA by addition of hydroxylamine hydrochloride (Cl- H3 N+ OH) (Thermo Fisher Scientific Inc.) to 2.3 mg/mL of RtPAL-SATA in a 100:1 mole ratio (Cl- H3 N+ OH:RtPAL-SATA) and incubated at room temperature for 75 min. BPM6, a proprietarily synthesized molecule consisting of two carbohydrate chains terminated by mannose 6-phosphate groups and connected to a primary amine backbone, was prepared for conjugation to RtPAL-SATA by treatment with succinimidyl-4-( N -maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (Thermo Fisher Scientific Inc.) in DMSO in a 15:1 mole ratio (SMCC:BPM6) and incubated at room temperature for 45 min to form BPM6-SMCC.

    Techniques: Electrofocusing, Nucleic Acid Electrophoresis, Conjugation Assay, Marker

    Mannose 6-phosphate receptor (MPR)-coated ELISA plate wells incubated with RtPAL-BPM6 ( solid circles ), RtPAL ( open circles ), or RtPAL-SATA ( open triangles ), showing saturable binding of RtPAL-BPM6 with a calculated Kd of 0.4 nM

    Journal: JIMD Reports

    Article Title: Mannose 6-Phosphate Conjugation Is Not Sufficient to Allow Induction of Immune Tolerance to Phenylalanine Ammonia-Lyase in Dogs

    doi: 10.1007/8904_2012_162

    Figure Lengend Snippet: Mannose 6-phosphate receptor (MPR)-coated ELISA plate wells incubated with RtPAL-BPM6 ( solid circles ), RtPAL ( open circles ), or RtPAL-SATA ( open triangles ), showing saturable binding of RtPAL-BPM6 with a calculated Kd of 0.4 nM

    Article Snippet: The purified product was then deacetylated to produce a deprotected sulfhydryl group on SATA by addition of hydroxylamine hydrochloride (Cl- H3 N+ OH) (Thermo Fisher Scientific Inc.) to 2.3 mg/mL of RtPAL-SATA in a 100:1 mole ratio (Cl- H3 N+ OH:RtPAL-SATA) and incubated at room temperature for 75 min. BPM6, a proprietarily synthesized molecule consisting of two carbohydrate chains terminated by mannose 6-phosphate groups and connected to a primary amine backbone, was prepared for conjugation to RtPAL-SATA by treatment with succinimidyl-4-( N -maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (Thermo Fisher Scientific Inc.) in DMSO in a 15:1 mole ratio (SMCC:BPM6) and incubated at room temperature for 45 min to form BPM6-SMCC.

    Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay

    RtPAL enzymatic specific activity (percentage of unconjugated RtPAL activity) measured as a function of the amount of SATA added (SATA:RtPAL molar ratio) in the first step of the RtPAL conjugation scheme. The SATA:RtPAL molar ratio used in the conjugation

    Journal: JIMD Reports

    Article Title: Mannose 6-Phosphate Conjugation Is Not Sufficient to Allow Induction of Immune Tolerance to Phenylalanine Ammonia-Lyase in Dogs

    doi: 10.1007/8904_2012_162

    Figure Lengend Snippet: RtPAL enzymatic specific activity (percentage of unconjugated RtPAL activity) measured as a function of the amount of SATA added (SATA:RtPAL molar ratio) in the first step of the RtPAL conjugation scheme. The SATA:RtPAL molar ratio used in the conjugation

    Article Snippet: The purified product was then deacetylated to produce a deprotected sulfhydryl group on SATA by addition of hydroxylamine hydrochloride (Cl- H3 N+ OH) (Thermo Fisher Scientific Inc.) to 2.3 mg/mL of RtPAL-SATA in a 100:1 mole ratio (Cl- H3 N+ OH:RtPAL-SATA) and incubated at room temperature for 75 min. BPM6, a proprietarily synthesized molecule consisting of two carbohydrate chains terminated by mannose 6-phosphate groups and connected to a primary amine backbone, was prepared for conjugation to RtPAL-SATA by treatment with succinimidyl-4-( N -maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (Thermo Fisher Scientific Inc.) in DMSO in a 15:1 mole ratio (SMCC:BPM6) and incubated at room temperature for 45 min to form BPM6-SMCC.

    Techniques: Activity Assay, Conjugation Assay

    SATA-8505 Improves MRSA Skin Infection at Low MOI. Mice were injected intraperitoneally (I.P.) with 10 7 plaque-forming units (PFU) of SATA-8505 immediately prior to subcutaneous injections of 10 7 CFU of USA300. (a) Lesion size progression over following six days. On day 6, skin biopsies were homogenized for culture, total bacterial (b) and phage burden (c) were calculated for individual mice. (d) mRNA transcript levels for IL-1ß, IL-6, and IL-17A in individual CGD mice relative to wild type controls, standardized to GAPDH. (e-g) mRNA transcript levels for IL-1ß, IL-6, and IL-17A in CGD and wild type mice with and without SATA-8505 treatment relative to wild type controls injected with diluent, standardized to GAPDH. Data shown are representative of 2–3 independent experiments using 5 or more mice per group, and displayed as mean + s.e.m. Differences were calculated by ANOVA with Bonferroni correction and depict differences from diluent treated wild type unless otherwise noted. ns = not significant, * = p

    Journal: PLoS ONE

    Article Title: Strain Specific Phage Treatment for Staphylococcus aureus Infection Is Influenced by Host Immunity and Site of Infection

    doi: 10.1371/journal.pone.0124280

    Figure Lengend Snippet: SATA-8505 Improves MRSA Skin Infection at Low MOI. Mice were injected intraperitoneally (I.P.) with 10 7 plaque-forming units (PFU) of SATA-8505 immediately prior to subcutaneous injections of 10 7 CFU of USA300. (a) Lesion size progression over following six days. On day 6, skin biopsies were homogenized for culture, total bacterial (b) and phage burden (c) were calculated for individual mice. (d) mRNA transcript levels for IL-1ß, IL-6, and IL-17A in individual CGD mice relative to wild type controls, standardized to GAPDH. (e-g) mRNA transcript levels for IL-1ß, IL-6, and IL-17A in CGD and wild type mice with and without SATA-8505 treatment relative to wild type controls injected with diluent, standardized to GAPDH. Data shown are representative of 2–3 independent experiments using 5 or more mice per group, and displayed as mean + s.e.m. Differences were calculated by ANOVA with Bonferroni correction and depict differences from diluent treated wild type unless otherwise noted. ns = not significant, * = p

    Article Snippet: Phage propagation and selection for efficacy SATA-8505 was propagated by inoculating 10 mL BHI (Thermo Fisher Scientific) with 100μL of SATA-8505 stock and 100μL of an overnight culture (ONC) of USA300 and incubating at 37°C overnight with shaking.

    Techniques: Infection, Mouse Assay, Injection

    SATA-8505 Fails to Improve MRSA Skin Infection at High MOI. Lesion size (a), CFU (b), PFU (c), and transcript data (d-f) for mice injected with 10 9 plaque-forming units (PFU) of SATA-8505 immediately prior to subcutaneous injections of 10 7 CFU of USA300 processed in an identical manner as MOI of 1 experiments. Data shown are representative of 2–3 independent experiments using 5 or more mice per group, and displayed as mean + s.e.m. Differences were calculated by ANOVA with Bonferroni correction and depict differences from diluent treated wild type unless otherwise noted. ns = not significant, * = p

    Journal: PLoS ONE

    Article Title: Strain Specific Phage Treatment for Staphylococcus aureus Infection Is Influenced by Host Immunity and Site of Infection

    doi: 10.1371/journal.pone.0124280

    Figure Lengend Snippet: SATA-8505 Fails to Improve MRSA Skin Infection at High MOI. Lesion size (a), CFU (b), PFU (c), and transcript data (d-f) for mice injected with 10 9 plaque-forming units (PFU) of SATA-8505 immediately prior to subcutaneous injections of 10 7 CFU of USA300 processed in an identical manner as MOI of 1 experiments. Data shown are representative of 2–3 independent experiments using 5 or more mice per group, and displayed as mean + s.e.m. Differences were calculated by ANOVA with Bonferroni correction and depict differences from diluent treated wild type unless otherwise noted. ns = not significant, * = p

    Article Snippet: Phage propagation and selection for efficacy SATA-8505 was propagated by inoculating 10 mL BHI (Thermo Fisher Scientific) with 100μL of SATA-8505 stock and 100μL of an overnight culture (ONC) of USA300 and incubating at 37°C overnight with shaking.

    Techniques: Infection, Mouse Assay, Injection

    SATA-8505 Does Not Impact USA300 Growth in Human Blood. (a) Hourly quantification of starting culture of 10 6 CFU USA300 in three parts TSB and one part whole blood from healthy donor or a patient with CGD, grown with or without 10 8 PFU SATA-8505 in duplicate. (b) Regrowth of surviving colonies from whole blood culture run in triplicate. One colony of S . aureus previously grown in 3:1 TSB:Whole blood without phage was subsequently grown in TSB with SATA-8505 (TSB/Phage) or without phage (TSB/TSB); one colony of the surviving S . aureus previously grown in the presence of SATA-8505 was subsequently grown in TSB with SATA-8505 (Phage/Phage) or without phage (Phage/TSB). (c) Average quantification of starting culture of 10 10 CFU of USA300 grown in either 3:1 TSB:Whole blood or 3:1 TSB:HBSS, with or without 10 7 PFU SATA-8505. (d) Average quantification of starting culture of 10 7 CFU of USA300 grown in three parts TSB with either one part whole blood, HBSS, serum, or peripheral mononuclear cells (PBMC) in equivalent volume HBSS. Data shown are representative of 2–3 independent experiments using 3 or more different healthy volunteers and 2 patients with CGD. Data is displayed as mean + s.e.m. ** = p

    Journal: PLoS ONE

    Article Title: Strain Specific Phage Treatment for Staphylococcus aureus Infection Is Influenced by Host Immunity and Site of Infection

    doi: 10.1371/journal.pone.0124280

    Figure Lengend Snippet: SATA-8505 Does Not Impact USA300 Growth in Human Blood. (a) Hourly quantification of starting culture of 10 6 CFU USA300 in three parts TSB and one part whole blood from healthy donor or a patient with CGD, grown with or without 10 8 PFU SATA-8505 in duplicate. (b) Regrowth of surviving colonies from whole blood culture run in triplicate. One colony of S . aureus previously grown in 3:1 TSB:Whole blood without phage was subsequently grown in TSB with SATA-8505 (TSB/Phage) or without phage (TSB/TSB); one colony of the surviving S . aureus previously grown in the presence of SATA-8505 was subsequently grown in TSB with SATA-8505 (Phage/Phage) or without phage (Phage/TSB). (c) Average quantification of starting culture of 10 10 CFU of USA300 grown in either 3:1 TSB:Whole blood or 3:1 TSB:HBSS, with or without 10 7 PFU SATA-8505. (d) Average quantification of starting culture of 10 7 CFU of USA300 grown in three parts TSB with either one part whole blood, HBSS, serum, or peripheral mononuclear cells (PBMC) in equivalent volume HBSS. Data shown are representative of 2–3 independent experiments using 3 or more different healthy volunteers and 2 patients with CGD. Data is displayed as mean + s.e.m. ** = p

    Article Snippet: Phage propagation and selection for efficacy SATA-8505 was propagated by inoculating 10 mL BHI (Thermo Fisher Scientific) with 100μL of SATA-8505 stock and 100μL of an overnight culture (ONC) of USA300 and incubating at 37°C overnight with shaking.

    Techniques:

    SATA-8505 Exhibits Commercial Viability but Significant Strain Limitations. (a) SATA-8505 quantified over up to sixty days after storage at either room temperature (RT), 4 degrees Celsius (4 ° C), or frozen at -80 ° C. (b) Average hourly quantification of 10 8 –10 9 CFU of USA100 S . aureus or a vancomycin resistant variant of USA100 (VRSA) cultured with up to 10 11 PFU of SATA-8505. Data shown are a representative of 2 independent experiments.

    Journal: PLoS ONE

    Article Title: Strain Specific Phage Treatment for Staphylococcus aureus Infection Is Influenced by Host Immunity and Site of Infection

    doi: 10.1371/journal.pone.0124280

    Figure Lengend Snippet: SATA-8505 Exhibits Commercial Viability but Significant Strain Limitations. (a) SATA-8505 quantified over up to sixty days after storage at either room temperature (RT), 4 degrees Celsius (4 ° C), or frozen at -80 ° C. (b) Average hourly quantification of 10 8 –10 9 CFU of USA100 S . aureus or a vancomycin resistant variant of USA100 (VRSA) cultured with up to 10 11 PFU of SATA-8505. Data shown are a representative of 2 independent experiments.

    Article Snippet: Phage propagation and selection for efficacy SATA-8505 was propagated by inoculating 10 mL BHI (Thermo Fisher Scientific) with 100μL of SATA-8505 stock and 100μL of an overnight culture (ONC) of USA300 and incubating at 37°C overnight with shaking.

    Techniques: Variant Assay, Cell Culture

    SATA-8505 Induces Interferon Gamma in Primary Human Keratinocytes. Human peripheral mononuclear cells (PBMC) were cultured in triplicate at 2 6 /mL with SATA-8505 at 1 PFU/mL to 10 8 PFU/mL at ten-fold increments or diluent derived from the supernatant of an overnight culture of SA that had been pelleted and filter sterilized in a manner similar to the phage-containing media. At 72 hours supernatants were harvested and analyzed for IL-1ß (a), IL-6 (b), IL-17A (c), and IFN gamma (d). Human keratinocytes from primary foreskins (foreskin keratinocytes; FSKC) or the HaCaT cell line were cultured to confluence on 6-well plates and incubated in triplicate with SATA-8505 at 10 4 PFU/mL to 10 8 PFU/mL at ten-fold increments or TSB diluent. At 24 hours supernatants were harvested and analyzed for IL-1ß (e), IL-6 (f), and IFN gamma (g). Phage for all experiments was diluted in TSB from overnight culture of USA300 that was centrifuged at 5000rpm for 12 minutes and filter-sterilized through a 0.44 micrometer filter. Data shown are representative of 3 independent experiments using 3 different healthy volunteers (a-d) or a pool of 5 or more foreskin samples (e-g) and displayed as mean + s.e.m. * = p

    Journal: PLoS ONE

    Article Title: Strain Specific Phage Treatment for Staphylococcus aureus Infection Is Influenced by Host Immunity and Site of Infection

    doi: 10.1371/journal.pone.0124280

    Figure Lengend Snippet: SATA-8505 Induces Interferon Gamma in Primary Human Keratinocytes. Human peripheral mononuclear cells (PBMC) were cultured in triplicate at 2 6 /mL with SATA-8505 at 1 PFU/mL to 10 8 PFU/mL at ten-fold increments or diluent derived from the supernatant of an overnight culture of SA that had been pelleted and filter sterilized in a manner similar to the phage-containing media. At 72 hours supernatants were harvested and analyzed for IL-1ß (a), IL-6 (b), IL-17A (c), and IFN gamma (d). Human keratinocytes from primary foreskins (foreskin keratinocytes; FSKC) or the HaCaT cell line were cultured to confluence on 6-well plates and incubated in triplicate with SATA-8505 at 10 4 PFU/mL to 10 8 PFU/mL at ten-fold increments or TSB diluent. At 24 hours supernatants were harvested and analyzed for IL-1ß (e), IL-6 (f), and IFN gamma (g). Phage for all experiments was diluted in TSB from overnight culture of USA300 that was centrifuged at 5000rpm for 12 minutes and filter-sterilized through a 0.44 micrometer filter. Data shown are representative of 3 independent experiments using 3 different healthy volunteers (a-d) or a pool of 5 or more foreskin samples (e-g) and displayed as mean + s.e.m. * = p

    Article Snippet: Phage propagation and selection for efficacy SATA-8505 was propagated by inoculating 10 mL BHI (Thermo Fisher Scientific) with 100μL of SATA-8505 stock and 100μL of an overnight culture (ONC) of USA300 and incubating at 37°C overnight with shaking.

    Techniques: Cell Culture, Derivative Assay, Incubation

    SATA 8505 Effectively Kills USA 300 and Reduces its Viability in vitro. (a) Average colony forming unit (CFU) counts for USA300 cultured with SATA-8505 for up to four hours at ratios of S . aureus :Phage of 1:1, 1:10, or 1:100 (MOI 1, 10, 100 respectively). (b) Images of surviving colony morphology of USA300 grown in TSB after exposure to BHI (diluent) or SATA-8505. (c) Regrowth of surviving colonies pictured in panel b, S . aureus grown in TSB after prior exposure to BHI (diluent) or SATA-8505 run in triplicate culture. Data shown are representative of 3 or more independent experiments and displayed as mean + s.e.m. **** = p

    Journal: PLoS ONE

    Article Title: Strain Specific Phage Treatment for Staphylococcus aureus Infection Is Influenced by Host Immunity and Site of Infection

    doi: 10.1371/journal.pone.0124280

    Figure Lengend Snippet: SATA 8505 Effectively Kills USA 300 and Reduces its Viability in vitro. (a) Average colony forming unit (CFU) counts for USA300 cultured with SATA-8505 for up to four hours at ratios of S . aureus :Phage of 1:1, 1:10, or 1:100 (MOI 1, 10, 100 respectively). (b) Images of surviving colony morphology of USA300 grown in TSB after exposure to BHI (diluent) or SATA-8505. (c) Regrowth of surviving colonies pictured in panel b, S . aureus grown in TSB after prior exposure to BHI (diluent) or SATA-8505 run in triplicate culture. Data shown are representative of 3 or more independent experiments and displayed as mean + s.e.m. **** = p

    Article Snippet: Phage propagation and selection for efficacy SATA-8505 was propagated by inoculating 10 mL BHI (Thermo Fisher Scientific) with 100μL of SATA-8505 stock and 100μL of an overnight culture (ONC) of USA300 and incubating at 37°C overnight with shaking.

    Techniques: In Vitro, Cell Culture