brefeldin a  (Millipore)


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    Name:
    Brefeldin A
    Description:
    Chemical structure macrolide
    Catalog Number:
    b6542
    Price:
    None
    Applications:
    Suitable for molecular biology studies of secretory proteins and mechanisms of intracellular transport
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    Structured Review

    Millipore brefeldin a
    Brefeldin A
    Chemical structure macrolide
    https://www.bioz.com/result/brefeldin a/product/Millipore
    Average 99 stars, based on 4643 article reviews
    Price from $9.99 to $1999.99
    brefeldin a - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "HIV-1 T cell epitopes targeted to Rhesus macaque CD40 and DCIR: A comparative study of prototype dendritic cell targeting therapeutic vaccine candidates"

    Article Title: HIV-1 T cell epitopes targeted to Rhesus macaque CD40 and DCIR: A comparative study of prototype dendritic cell targeting therapeutic vaccine candidates

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0207794

    Analysis of HIV-1 epitope-specific CD4 + and CD8 + T cell responses elicited in naïve NHPs by αDCIR.HIV5pep and αCD40.HIV5pep vaccines. Intracellular cytokine staining analysis of HIV-1 antigen-specific CD154 + CD4 + and CD8 + T cells elicited by αDCIR.HIV5pep and αCD40.HIV5pep vaccines. PBMCs were collected from individual animals at week 14 (two weeks post DC-targeting vaccines) and week 24 (two weeks post MVA boost) for G3 αDCIR MVA and G4 αCD40 MVA. PBMC were stimulated in the presence of Brefeldin A for 6 h with pools of HIV-1 peptides corresponding to the indicated gene regions and analyzed by flow cytometry. Each dot is the background-subtracted value for individual animals of (A) CD154 + CD4 + and (B) CD8 + T cells secreting IFNγ, TNFα, IL-2, or combinations thereof when stimulated with Gag p17, Gag p24, Nef and Pol peptides. Negative background subtracted values were set to zero. Responses from individual animals in the indicated groups are presented. The mid-line of the box denotes the median, and the ends of the box denote the 25 th and 75 th shows the data corresponding to this figure.
    Figure Legend Snippet: Analysis of HIV-1 epitope-specific CD4 + and CD8 + T cell responses elicited in naïve NHPs by αDCIR.HIV5pep and αCD40.HIV5pep vaccines. Intracellular cytokine staining analysis of HIV-1 antigen-specific CD154 + CD4 + and CD8 + T cells elicited by αDCIR.HIV5pep and αCD40.HIV5pep vaccines. PBMCs were collected from individual animals at week 14 (two weeks post DC-targeting vaccines) and week 24 (two weeks post MVA boost) for G3 αDCIR MVA and G4 αCD40 MVA. PBMC were stimulated in the presence of Brefeldin A for 6 h with pools of HIV-1 peptides corresponding to the indicated gene regions and analyzed by flow cytometry. Each dot is the background-subtracted value for individual animals of (A) CD154 + CD4 + and (B) CD8 + T cells secreting IFNγ, TNFα, IL-2, or combinations thereof when stimulated with Gag p17, Gag p24, Nef and Pol peptides. Negative background subtracted values were set to zero. Responses from individual animals in the indicated groups are presented. The mid-line of the box denotes the median, and the ends of the box denote the 25 th and 75 th shows the data corresponding to this figure.

    Techniques Used: Staining, Flow Cytometry, Cytometry

    Analysis of HIV-1 epitope-specific CD4 + and CD8 + T cell responses elicited in MVA-primed NHPs by αDCIR.HIV5pep and αCD40.HIV5pep vaccines. PBMCs were collected from individual animals at week 10 (two weeks post MVA) and at peak response times of week 26 (2 weeks post DC-targeting vaccination) for G1 MVA αDCIR and G2 MVA αCD40. PBMC were stimulated in the presence of Brefeldin A for 6 h with pools of HIV-1 peptides corresponding to the four indicated gene regions and analyzed by flow cytometry. Each dot is the background-subtracted value for individual animals of (A) CD154 + CD4 + or (B) CD8 + T cells secreting IFNγ, TNFα, IL-2, or combinations thereof when stimulated with Gag p17, Gag p24, Nef and Pol peptides. Negative background subtracted values were set to zero. Responses from individual animals in the indicated groups are presented. The mid-line of the box denotes the median, and the ends of the box denote the 25 th and 75 th percentiles. The whiskers are the minimum/maximum value higher/lower than 1.5* Inter-Quartile Interval. One animal had a value (0.9%) outside the plotted scale for the CD4 + shows the data corresponding to this figure.
    Figure Legend Snippet: Analysis of HIV-1 epitope-specific CD4 + and CD8 + T cell responses elicited in MVA-primed NHPs by αDCIR.HIV5pep and αCD40.HIV5pep vaccines. PBMCs were collected from individual animals at week 10 (two weeks post MVA) and at peak response times of week 26 (2 weeks post DC-targeting vaccination) for G1 MVA αDCIR and G2 MVA αCD40. PBMC were stimulated in the presence of Brefeldin A for 6 h with pools of HIV-1 peptides corresponding to the four indicated gene regions and analyzed by flow cytometry. Each dot is the background-subtracted value for individual animals of (A) CD154 + CD4 + or (B) CD8 + T cells secreting IFNγ, TNFα, IL-2, or combinations thereof when stimulated with Gag p17, Gag p24, Nef and Pol peptides. Negative background subtracted values were set to zero. Responses from individual animals in the indicated groups are presented. The mid-line of the box denotes the median, and the ends of the box denote the 25 th and 75 th percentiles. The whiskers are the minimum/maximum value higher/lower than 1.5* Inter-Quartile Interval. One animal had a value (0.9%) outside the plotted scale for the CD4 + shows the data corresponding to this figure.

    Techniques Used: Flow Cytometry, Cytometry

    2) Product Images from "Human Macrophages Escape Inhibition of Major Histocompatibility Complex-Dependent Antigen Presentation by Cytomegalovirus and Drive Proliferation and Activation of Memory CD4+ and CD8+ T Cells"

    Article Title: Human Macrophages Escape Inhibition of Major Histocompatibility Complex-Dependent Antigen Presentation by Cytomegalovirus and Drive Proliferation and Activation of Memory CD4+ and CD8+ T Cells

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01129

    HCMV-infected Mφ induce functional activation of CD4 +  and CD8 +  T cells. M1- and M2-Mφ derived from HCMV-seropositive blood donors were infected with an MOI of 5 of the wild-type TB40E, the mutant ΔUS2–11 or left untreated (Mock).  (A,B)  After 1 day, Mφ were co-cultured with autologous PBMC for 18 h prior to addition of 1 µg/ml Brefeldin A. Four hours later, PBMC were harvested, permeabilized, and stained with antibodies directed against CD4, CD8, CD69, and IFN-γ. Percentages of CD4 + (A)  and CD8 + (B)  T cells expressing CD69 +  and IFN-γ +  are depicted. Cells derived from the same donor are indicated by a line.  (C)  After 1 day, Mφ were co-cultured with autologous PBMC for 48 hours. Cells were then incubated with monensin and a fluorescently labeled antibody directed against CD107a for 4 h. Subsequently, PBMC were stained with antibodies directed against CD8. Percentages of CD107a +  cells among the CD8 +  T cells are depicted. Cells derived from the same donor are connected by a line. * p
    Figure Legend Snippet: HCMV-infected Mφ induce functional activation of CD4 + and CD8 + T cells. M1- and M2-Mφ derived from HCMV-seropositive blood donors were infected with an MOI of 5 of the wild-type TB40E, the mutant ΔUS2–11 or left untreated (Mock). (A,B) After 1 day, Mφ were co-cultured with autologous PBMC for 18 h prior to addition of 1 µg/ml Brefeldin A. Four hours later, PBMC were harvested, permeabilized, and stained with antibodies directed against CD4, CD8, CD69, and IFN-γ. Percentages of CD4 + (A) and CD8 + (B) T cells expressing CD69 + and IFN-γ + are depicted. Cells derived from the same donor are indicated by a line. (C) After 1 day, Mφ were co-cultured with autologous PBMC for 48 hours. Cells were then incubated with monensin and a fluorescently labeled antibody directed against CD107a for 4 h. Subsequently, PBMC were stained with antibodies directed against CD8. Percentages of CD107a + cells among the CD8 + T cells are depicted. Cells derived from the same donor are connected by a line. * p

    Techniques Used: Infection, Functional Assay, Activation Assay, Derivative Assay, Mutagenesis, Cell Culture, Staining, Expressing, Incubation, Labeling

    3) Product Images from "The deletion of the ORF1 and ORF71 genes reduces virulence of the neuropathogenic EHV-1 strain Ab4 without compromising host immunity in horses"

    Article Title: The deletion of the ORF1 and ORF71 genes reduces virulence of the neuropathogenic EHV-1 strain Ab4 without compromising host immunity in horses

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0206679

    EHV-1 specific IFN-γ producing lymphocytes in PBMC. EHV-1 naïve horses (n = 5 per group) were infected with the EHV-1 strain Ab4 or the deletion mutant virus Ab4ΔORF1/71. Non-infected horses were kept as controls. The arrow marks the time of infection. PBMC were isolated at different times post infection and re-stimulated with EHV-1 strain Ab4 ex vivo or were kept in cell culture medium for control. The secretion inhibitor Brefeldin A was added to the cultures for the last 24 hours. Cells were harvested at 48 hours, fixed, stained for intracellular IFN-γ production and cell surface CD4 and CD8, and measured by flow cytometry. The analyzing gate was set on small lymphocytes. Mean and standard errors for the percentages of (A) total IFN-γ + lymphocytes, (B) CD8 + /IFN-γ + lymphocytes, and (C) CD4 + /IFN-γ + lymphocytes are displayed over time. All percentages for IFN-γ + cells were corrected by the respective medium control values. The dotted horizontal line represents a suggested cutoff of 0.05% IFN-γ + lymphocytes. Values below this cutoff were typically reached for IFN-γ + cells in the non-infected control group prior to correction by medium control values. Significant differences between groups are marked: a = Ab4 vs. controls, b = Ab4ΔORF1/71 vs. controls, and c = Ab4 vs. Ab4ΔORF1/71.
    Figure Legend Snippet: EHV-1 specific IFN-γ producing lymphocytes in PBMC. EHV-1 naïve horses (n = 5 per group) were infected with the EHV-1 strain Ab4 or the deletion mutant virus Ab4ΔORF1/71. Non-infected horses were kept as controls. The arrow marks the time of infection. PBMC were isolated at different times post infection and re-stimulated with EHV-1 strain Ab4 ex vivo or were kept in cell culture medium for control. The secretion inhibitor Brefeldin A was added to the cultures for the last 24 hours. Cells were harvested at 48 hours, fixed, stained for intracellular IFN-γ production and cell surface CD4 and CD8, and measured by flow cytometry. The analyzing gate was set on small lymphocytes. Mean and standard errors for the percentages of (A) total IFN-γ + lymphocytes, (B) CD8 + /IFN-γ + lymphocytes, and (C) CD4 + /IFN-γ + lymphocytes are displayed over time. All percentages for IFN-γ + cells were corrected by the respective medium control values. The dotted horizontal line represents a suggested cutoff of 0.05% IFN-γ + lymphocytes. Values below this cutoff were typically reached for IFN-γ + cells in the non-infected control group prior to correction by medium control values. Significant differences between groups are marked: a = Ab4 vs. controls, b = Ab4ΔORF1/71 vs. controls, and c = Ab4 vs. Ab4ΔORF1/71.

    Techniques Used: Infection, Mutagenesis, Isolation, Ex Vivo, Cell Culture, Staining, Flow Cytometry, Cytometry

    4) Product Images from "The Sheep Tetherin Paralog oBST2B Blocks Envelope Glycoprotein Incorporation into Nascent Retroviral Virions"

    Article Title: The Sheep Tetherin Paralog oBST2B Blocks Envelope Glycoprotein Incorporation into Nascent Retroviral Virions

    Journal: Journal of Virology

    doi: 10.1128/JVI.02751-14

    oBST2B localization is altered by treatment with brefeldin A. (A) CPT-Tert cells were transfected with oBST2B-HA expression plasmids. Eighteen hours after transfection, cells were treated or not treated with 200 ng/ml of brefeldin A for 90 min, fixed, and analyzed by confocal microscopy using antibodies to the giantin Golgi marker and the HA epitope as indicated. Two different staining patterns were observed: (i) dispersed within the cytoplasm and (ii) concentrated in a perinuclear region. Scale bars in all panels represent 10 μm. (B) Graph representing the number (%) of cells in which oBST2B-HA and giantin staining were observed to be concentrated as opposed to dispersed. At least 100 cells from two independent experiments were evaluated randomly.
    Figure Legend Snippet: oBST2B localization is altered by treatment with brefeldin A. (A) CPT-Tert cells were transfected with oBST2B-HA expression plasmids. Eighteen hours after transfection, cells were treated or not treated with 200 ng/ml of brefeldin A for 90 min, fixed, and analyzed by confocal microscopy using antibodies to the giantin Golgi marker and the HA epitope as indicated. Two different staining patterns were observed: (i) dispersed within the cytoplasm and (ii) concentrated in a perinuclear region. Scale bars in all panels represent 10 μm. (B) Graph representing the number (%) of cells in which oBST2B-HA and giantin staining were observed to be concentrated as opposed to dispersed. At least 100 cells from two independent experiments were evaluated randomly.

    Techniques Used: Cycling Probe Technology, Transfection, Expressing, Confocal Microscopy, Marker, Staining

    5) Product Images from "Phosphocholine‐containing ligands direct CRP induction of M2 macrophage polarization independent of T cell polarization: Implication for chronic inflammatory states"

    Article Title: Phosphocholine‐containing ligands direct CRP induction of M2 macrophage polarization independent of T cell polarization: Implication for chronic inflammatory states

    Journal: Immunity, Inflammation and Disease

    doi: 10.1002/iid3.112

    Levels of IL‐1RA in cells post TEM treated with liposomes (lipid) ± CRP measured by protein array. Integrated pixel density of the spots is shown in (a) and the data expressed as a percent of an untreated control is shown in (b). Cells were treated with Brefeldin A for 6 h before lysis to prevent secretion of the protein ( n = 4, * p
    Figure Legend Snippet: Levels of IL‐1RA in cells post TEM treated with liposomes (lipid) ± CRP measured by protein array. Integrated pixel density of the spots is shown in (a) and the data expressed as a percent of an untreated control is shown in (b). Cells were treated with Brefeldin A for 6 h before lysis to prevent secretion of the protein ( n = 4, * p

    Techniques Used: Transmission Electron Microscopy, Protein Array, Lysis

    6) Product Images from "IL-27 promotes T cell-dependent colitis through multiple mechanisms"

    Article Title: IL-27 promotes T cell-dependent colitis through multiple mechanisms

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20100410

    Reduced TH1 and enhanced TH17 polarization after transfer of  Il27ra −/−  CD45Rb hi  cells.  (A) Representative IFN-γ and IL-22 intracellular cytokine staining of lamina propria isolates from CB17-SCID mice injected with WT or  Il27ra −/−  (KO) CD45Rb hi  CD4 +  T cells stimulated with PMA, ionomycin, and brefeldin A. Samples are gated on CD4 +  cells. (B–E) Quantitative analysis of intracellular IFN-γ (B), IL-17A (C), IL-22 (D), and IL-13 (E) staining in splenocytes, mLNs, and colonic lamina propria lymphocytes from CB17-SCID mice transferred with WT or  Il27ra −/−  CD45Rb hi  cells. Data are representative of three individual experiments. *, P
    Figure Legend Snippet: Reduced TH1 and enhanced TH17 polarization after transfer of Il27ra −/− CD45Rb hi cells. (A) Representative IFN-γ and IL-22 intracellular cytokine staining of lamina propria isolates from CB17-SCID mice injected with WT or Il27ra −/− (KO) CD45Rb hi CD4 + T cells stimulated with PMA, ionomycin, and brefeldin A. Samples are gated on CD4 + cells. (B–E) Quantitative analysis of intracellular IFN-γ (B), IL-17A (C), IL-22 (D), and IL-13 (E) staining in splenocytes, mLNs, and colonic lamina propria lymphocytes from CB17-SCID mice transferred with WT or Il27ra −/− CD45Rb hi cells. Data are representative of three individual experiments. *, P

    Techniques Used: Staining, Mouse Assay, Injection

    7) Product Images from "Effect of Invariant Chain on Major Histocompatibility Complex Class I Molecule Expression and Stability on Human Breast Tumor Cell Lines"

    Article Title: Effect of Invariant Chain on Major Histocompatibility Complex Class I Molecule Expression and Stability on Human Breast Tumor Cell Lines

    Journal: Cancer immunology, immunotherapy : CII

    doi: 10.1007/s00262-008-0595-1

    The expression of HLA class I molecules on the surface of T47D was elevated by Ii transfection, and HLA class I molecule expression on T47D+Ii was more unstable than on T47D. (A) Western blot of lysates of T47D and T47D+Ii, probed with the HC10 monoclonal antibody to identify the HLA class I heavy chain (HC). (B) Ii immunoprecipitation was performed on a lysate of T47D+Ii (or T47D as a control), and after transfer of the electrophoresed immunoprecipitates to a membrane, the blot was probed with the HC10 monoclonal antibody to identify the co-immunoprecipitated HLA class I heavy chain (HC). The two upper bands that appear in both lanes are non-specific background bands. (C) The expression of folded HLA class I molecules was more inducible by incubation at 25°C on T47D+Ii than on T47D. T47D and T47D+Ii were incubated overnight at 25°C or at 37°C and compared by flow cytometry with W6/32. Bars display the average of the mean fluorescence intensity values obtained with duplicate samples. (D,E) The HLA class I molecules at the surface of T47D+Ii have a relatively rapid turnover rate. T47D+Ii and T47D were cultured in the presence of brefeldin A, and the cells were analyzed by flow cytometry using monoclonal antibody W6/32. Results from two assays, using different time courses, are shown (0, 1, 2, 3, and 4 hours in D, and 0, 3, and 6 hours in E). The percentage of folded HLA class I molecules remaining at each time point was calculated by the formula [(mean fluorescence after brefeldin A treatment after the specified number of hours/mean fluorescence after brefeldin A for 0 hours) × 100] and the percentages are shown on the graphs.
    Figure Legend Snippet: The expression of HLA class I molecules on the surface of T47D was elevated by Ii transfection, and HLA class I molecule expression on T47D+Ii was more unstable than on T47D. (A) Western blot of lysates of T47D and T47D+Ii, probed with the HC10 monoclonal antibody to identify the HLA class I heavy chain (HC). (B) Ii immunoprecipitation was performed on a lysate of T47D+Ii (or T47D as a control), and after transfer of the electrophoresed immunoprecipitates to a membrane, the blot was probed with the HC10 monoclonal antibody to identify the co-immunoprecipitated HLA class I heavy chain (HC). The two upper bands that appear in both lanes are non-specific background bands. (C) The expression of folded HLA class I molecules was more inducible by incubation at 25°C on T47D+Ii than on T47D. T47D and T47D+Ii were incubated overnight at 25°C or at 37°C and compared by flow cytometry with W6/32. Bars display the average of the mean fluorescence intensity values obtained with duplicate samples. (D,E) The HLA class I molecules at the surface of T47D+Ii have a relatively rapid turnover rate. T47D+Ii and T47D were cultured in the presence of brefeldin A, and the cells were analyzed by flow cytometry using monoclonal antibody W6/32. Results from two assays, using different time courses, are shown (0, 1, 2, 3, and 4 hours in D, and 0, 3, and 6 hours in E). The percentage of folded HLA class I molecules remaining at each time point was calculated by the formula [(mean fluorescence after brefeldin A treatment after the specified number of hours/mean fluorescence after brefeldin A for 0 hours) × 100] and the percentages are shown on the graphs.

    Techniques Used: Expressing, Transfection, Western Blot, Immunoprecipitation, Incubation, Flow Cytometry, Cytometry, Fluorescence, Cell Culture

    8) Product Images from "CpG-oligodeoxynucleotides enhance T-cell receptor-triggered interferon-? production and up-regulation of CD69 via induction of antigen-presenting cell-derived interferon type I and interleukin-12"

    Article Title: CpG-oligodeoxynucleotides enhance T-cell receptor-triggered interferon-? production and up-regulation of CD69 via induction of antigen-presenting cell-derived interferon type I and interleukin-12

    Journal: Immunology

    doi: 10.1046/j.1365-2567.2000.00964.x

    Detection of intracellular interferon-γ (IFN-γ) after stimulation of peripheral blood mononuclear cells (PBMC) with α-CD3 and CpG-oligodeoxynucleotides (ODN). PBMC were stimulated as described in the Material and methods, and Brefeldin A was added 8 hr after onset of culture. After an additional 12 hr of incubation, cells were harvested and double stained for surface lineage markers and intracellular IFN-γ, as outlined in the Materials and methods. (a) A representative experiment showing IFN-γ-producing cells in T cells (fluorescein isothiocyanate [FITC])-positive CD8 + cells have a higher intensity than CD4 + cells) or non-T cells (FITC-negative). Numbers indicate the percentage of IFN-γ + cells within T cells and non-T cells (FITC-positive and FITC-negative, respectively). (b) Mean and SEM of IFN-γ-producing CD4 + and CD8 + T cells and non-T cells from four (CD4 + and CD8 + ) and two (non-T cells) experiments.
    Figure Legend Snippet: Detection of intracellular interferon-γ (IFN-γ) after stimulation of peripheral blood mononuclear cells (PBMC) with α-CD3 and CpG-oligodeoxynucleotides (ODN). PBMC were stimulated as described in the Material and methods, and Brefeldin A was added 8 hr after onset of culture. After an additional 12 hr of incubation, cells were harvested and double stained for surface lineage markers and intracellular IFN-γ, as outlined in the Materials and methods. (a) A representative experiment showing IFN-γ-producing cells in T cells (fluorescein isothiocyanate [FITC])-positive CD8 + cells have a higher intensity than CD4 + cells) or non-T cells (FITC-negative). Numbers indicate the percentage of IFN-γ + cells within T cells and non-T cells (FITC-positive and FITC-negative, respectively). (b) Mean and SEM of IFN-γ-producing CD4 + and CD8 + T cells and non-T cells from four (CD4 + and CD8 + ) and two (non-T cells) experiments.

    Techniques Used: Incubation, Staining

    9) Product Images from "A novel, cellulose synthesis inhibitory action of ancymidol impairs plant cell expansion"

    Article Title: A novel, cellulose synthesis inhibitory action of ancymidol impairs plant cell expansion

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ern250

    Taxol and brefeldin A effects on the incidence of cell malformations induced by ANC and ISX in 3-d-old tobacco BY-2 cells. (A) Viability (via) and malformations (mal) of BY-2 cells treated with 100 μM ANC, 50 nM ISX, and 10 μM taxol for 36 h. (B) Viability and malformations of BY-2 cells treated with 100 μM ANC, 50 nM isoxaben, and 1 μM brefeldin A. (C–E) Nomarski DIC: BY-2 cells treated with 1 μM brefeldin A (C), 1 μM brefeldin A and 100 μM ANC (D), and 1 μM brefeldin A and 50 nM ISX (E) for 24 h. Scale bars 20 μm. Error bars=SE, n =3, at least 400 cells were counted in each variant.
    Figure Legend Snippet: Taxol and brefeldin A effects on the incidence of cell malformations induced by ANC and ISX in 3-d-old tobacco BY-2 cells. (A) Viability (via) and malformations (mal) of BY-2 cells treated with 100 μM ANC, 50 nM ISX, and 10 μM taxol for 36 h. (B) Viability and malformations of BY-2 cells treated with 100 μM ANC, 50 nM isoxaben, and 1 μM brefeldin A. (C–E) Nomarski DIC: BY-2 cells treated with 1 μM brefeldin A (C), 1 μM brefeldin A and 100 μM ANC (D), and 1 μM brefeldin A and 50 nM ISX (E) for 24 h. Scale bars 20 μm. Error bars=SE, n =3, at least 400 cells were counted in each variant.

    Techniques Used: Variant Assay

    10) Product Images from "Mycobacterial Hsp65 antigen upregulates the cellular immune response of healthy individuals compared with tuberculosis patients"

    Article Title: Mycobacterial Hsp65 antigen upregulates the cellular immune response of healthy individuals compared with tuberculosis patients

    Journal: Human Vaccines & Immunotherapeutics

    doi: 10.1080/21645515.2016.1264547

    Frequency of IFN-γ- or IL-4-producing CD4 + or CD8 + cells after M. tuberculosis antigen stimulation. Peripheral blood from healthy individuals or TB patients was cultured with Mtb antigens for 24 hours. For the final 4 hours, brefeldin A was added, and intracellular staining was performed. Cells gated as lymphocytes by FSC and SSC and dot plots for double positive cells were analyzed. (A and B) Frequency of IFN-γ -producing CD4 + and CD8 + cells. (C and D) Frequency of IL-4-producing CD4 + and CD8 + cells. Horizontal lines represent the mean value of seven healthy individuals (white squares) and ten TB patients (black squares). *p
    Figure Legend Snippet: Frequency of IFN-γ- or IL-4-producing CD4 + or CD8 + cells after M. tuberculosis antigen stimulation. Peripheral blood from healthy individuals or TB patients was cultured with Mtb antigens for 24 hours. For the final 4 hours, brefeldin A was added, and intracellular staining was performed. Cells gated as lymphocytes by FSC and SSC and dot plots for double positive cells were analyzed. (A and B) Frequency of IFN-γ -producing CD4 + and CD8 + cells. (C and D) Frequency of IL-4-producing CD4 + and CD8 + cells. Horizontal lines represent the mean value of seven healthy individuals (white squares) and ten TB patients (black squares). *p

    Techniques Used: Cell Culture, Staining

    11) Product Images from "A2B adenosine receptor activation switches differentiation of bone marrow cells to a CD11c+Gr‐1+ dendritic cell subset that promotes the Th17 response"

    Article Title: A2B adenosine receptor activation switches differentiation of bone marrow cells to a CD11c+Gr‐1+ dendritic cell subset that promotes the Th17 response

    Journal: Immunity, Inflammation and Disease

    doi: 10.1002/iid3.74

    Differentiation of bone marrow cells into BMDCs in the presence of the non‐specific AR agonist NECA results in increased Th17‐stimulating ability. A: Stimulatory effect of mouse BMDCs under non‐polarizing conditions. Bone marrow cells were cultured for 5 days in medium continuing GM‐CSF (10 ng/mL) in the absence or presence of NECA (100 nM), then were detached, washed, and seeded (5 × 10 4 /well) into 24‐well plates Responder CD3 T cells, isolated from immunized B6 mice (10 6  cells/well) were added to the plates (T cell/DC ratio 20:1), then the cells were incubated in the presence of graded doses of the immunizing peptide for 48 h and T cell proliferation was assessed by  3 H‐thymidine incorporation. The results shown are the mean ± SD for one study doing in triplicated wells and the experiment was repeated 3 times with similar results. B: Stimulatory effect of mouse BMDCs on Th1 and Th17 autoreactive T cells under polarizing conditions. Responder T cells were co‐cultured for 48 h with each of the two DC preparations (generated in the absence or presence of NECA) and the immunizing peptide under polarizing conditions favoring Th1 cell proliferation (culture medium containing IL‐12) or Th17 cell proliferation (culture medium containing IL‐23), then the culture supernatants were assayed for IL‐17 (top panel) or IFN‐γ (bottom panel). C: Intracellular staining of the proliferating T cells for IL‐17 or IFN‐γ expression. The activated T cells generated in (B) using DCs generated in the absence (top panels) or presence of NECA (bottom panels) were separated on day 2 and cultured for 3 days, then the separated, activated T cells were treated for 4 h with 50 ng/mL of phorbol myristic acetate, 1 μg/mL of ionomycin, and 1 μg/mL of brefeldin A, fixed, permeabilized overnight with Cytofix/Cytoperm buffer, and stained intracellularly with antibodies against IL‐17 (left panels) or IFN‐γ (right panels) and analyzed on a FACScalibur.
    Figure Legend Snippet: Differentiation of bone marrow cells into BMDCs in the presence of the non‐specific AR agonist NECA results in increased Th17‐stimulating ability. A: Stimulatory effect of mouse BMDCs under non‐polarizing conditions. Bone marrow cells were cultured for 5 days in medium continuing GM‐CSF (10 ng/mL) in the absence or presence of NECA (100 nM), then were detached, washed, and seeded (5 × 10 4 /well) into 24‐well plates Responder CD3 T cells, isolated from immunized B6 mice (10 6  cells/well) were added to the plates (T cell/DC ratio 20:1), then the cells were incubated in the presence of graded doses of the immunizing peptide for 48 h and T cell proliferation was assessed by 3 H‐thymidine incorporation. The results shown are the mean ± SD for one study doing in triplicated wells and the experiment was repeated 3 times with similar results. B: Stimulatory effect of mouse BMDCs on Th1 and Th17 autoreactive T cells under polarizing conditions. Responder T cells were co‐cultured for 48 h with each of the two DC preparations (generated in the absence or presence of NECA) and the immunizing peptide under polarizing conditions favoring Th1 cell proliferation (culture medium containing IL‐12) or Th17 cell proliferation (culture medium containing IL‐23), then the culture supernatants were assayed for IL‐17 (top panel) or IFN‐γ (bottom panel). C: Intracellular staining of the proliferating T cells for IL‐17 or IFN‐γ expression. The activated T cells generated in (B) using DCs generated in the absence (top panels) or presence of NECA (bottom panels) were separated on day 2 and cultured for 3 days, then the separated, activated T cells were treated for 4 h with 50 ng/mL of phorbol myristic acetate, 1 μg/mL of ionomycin, and 1 μg/mL of brefeldin A, fixed, permeabilized overnight with Cytofix/Cytoperm buffer, and stained intracellularly with antibodies against IL‐17 (left panels) or IFN‐γ (right panels) and analyzed on a FACScalibur.

    Techniques Used: Cell Culture, Isolation, Mouse Assay, Incubation, Generated, Staining, Expressing

    12) Product Images from "CD8+ Treg Cells Associated with Decreasing Disease Activity after Intravenous Methylprednisolone Pulse Therapy in Lupus Nephritis with Heavy Proteinuria"

    Article Title: CD8+ Treg Cells Associated with Decreasing Disease Activity after Intravenous Methylprednisolone Pulse Therapy in Lupus Nephritis with Heavy Proteinuria

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0081344

    Intracellular IL-10 and granzyme B levels in CD8 + CD25 + Treg cells before and after IVMP. PBMCs were stimulated with anti-CD3 mAb for five days, followed by stimulation of PMA (10 ng/ml) plus ionomycin (1 µg/ml) for the last five hours, with addition of brefeldin A (10 µg/ml) for the final hour. Intracellular expression of IL-10 and granzyme B was measured by gating in CD8 + CD25 + T cells, using flow cytometry. Isotype control (dotted line). (a) Results of 30 paired experiments for IL-10 (b) and granzyme B (c) production by PBMCs (* p
    Figure Legend Snippet: Intracellular IL-10 and granzyme B levels in CD8 + CD25 + Treg cells before and after IVMP. PBMCs were stimulated with anti-CD3 mAb for five days, followed by stimulation of PMA (10 ng/ml) plus ionomycin (1 µg/ml) for the last five hours, with addition of brefeldin A (10 µg/ml) for the final hour. Intracellular expression of IL-10 and granzyme B was measured by gating in CD8 + CD25 + T cells, using flow cytometry. Isotype control (dotted line). (a) Results of 30 paired experiments for IL-10 (b) and granzyme B (c) production by PBMCs (* p

    Techniques Used: Expressing, Flow Cytometry, Cytometry

    13) Product Images from "Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins - towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation"

    Article Title: Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins - towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-10-62

    Localization of human GalNAc-T2 expressed in  N. benthamiana  L. by confocal microscopy . Image of leaf (guard cells) stably expressing GalNAc-T2:GFP fusion protein:  A  - image without chloroplasts,  B  - image with chloroplasts. Image of leaf (guard cells) stably expressing GalNAc-T2:GFP fusion protein after 1 h treatment with 10 μg/ml Brefeldin A:  C  - image without chloroplasts,  D  - image with chloroplasts. The green signal represents fluorescence of GalNAc-T2:GFP fusion protein, the red signal represents the autofluorescence of chloroplasts.
    Figure Legend Snippet: Localization of human GalNAc-T2 expressed in N. benthamiana L. by confocal microscopy . Image of leaf (guard cells) stably expressing GalNAc-T2:GFP fusion protein: A - image without chloroplasts, B - image with chloroplasts. Image of leaf (guard cells) stably expressing GalNAc-T2:GFP fusion protein after 1 h treatment with 10 μg/ml Brefeldin A: C - image without chloroplasts, D - image with chloroplasts. The green signal represents fluorescence of GalNAc-T2:GFP fusion protein, the red signal represents the autofluorescence of chloroplasts.

    Techniques Used: Confocal Microscopy, Stable Transfection, Expressing, Fluorescence

    14) Product Images from "NKG2D is Required for NK Cell Activation and Function in Response to E1-deleted Adenovirus"

    Article Title: NKG2D is Required for NK Cell Activation and Function in Response to E1-deleted Adenovirus

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1002771

    NKG2D is critical for NK cell activation in response to adenovirus in vitro. Purified DX5 + CD3 −  NK cells were co-cultured with macrophages, and with stimulated Ad-LacZ for 18 h (Ad), or left unstimulated (Medium). In some cultures, anti-NKG2D (+Anti-NKG2D), anti-CD226 (+Anti-CD226), NKp46-Fc (+NKp46-Fc), or a control rat IgG (+Rat IgG) was added. The cultures were incubated for additional 4 h in the presence of Brefeldin A. (A) CD69 expression on DX5 + CD3 −  NK cells. MFI of CD69 staining is indicated. (B-C) IFN-γ and granzyme B production by DX5+CD3 −  NK cells. FACS plots of IFN-γ and granzyme B production with the percentage of IFN-γ and granzyme B positive cells indicated (B). The mean percentage ± SD of IFN-γ or granzyme B positive cells is indicated (n = 5 per group) (C). The data shown are representative of three independent experiments.
    Figure Legend Snippet: NKG2D is critical for NK cell activation in response to adenovirus in vitro. Purified DX5 + CD3 − NK cells were co-cultured with macrophages, and with stimulated Ad-LacZ for 18 h (Ad), or left unstimulated (Medium). In some cultures, anti-NKG2D (+Anti-NKG2D), anti-CD226 (+Anti-CD226), NKp46-Fc (+NKp46-Fc), or a control rat IgG (+Rat IgG) was added. The cultures were incubated for additional 4 h in the presence of Brefeldin A. (A) CD69 expression on DX5 + CD3 − NK cells. MFI of CD69 staining is indicated. (B-C) IFN-γ and granzyme B production by DX5+CD3 − NK cells. FACS plots of IFN-γ and granzyme B production with the percentage of IFN-γ and granzyme B positive cells indicated (B). The mean percentage ± SD of IFN-γ or granzyme B positive cells is indicated (n = 5 per group) (C). The data shown are representative of three independent experiments.

    Techniques Used: Activation Assay, In Vitro, Purification, Cell Culture, Incubation, Expressing, Staining, FACS

    Accessory cell-NK cell contact is necessary for NK cell activation in vitro. Purified NK cells (DX5 + CD3 − ) were co-cultured with macrophages either mixed together (Mixed) or separated by a transwell system (Transwell), followed by stimulation with Ad-LacZ for 18 h (Ad), or left unstimulated (Medium). Brefeldin A was added into each well and incubated for additional 4 h. (A) DX5 + CD3 − NK cells were analyzed for the expression of CD69. MFI of CD69 staining is indicated. (B-C) DX5 + CD3 − NK cells were analyzed for intracellular IFN-γ and granzyme B production. FACS plots of IFN-γ and granzyme B production with the percentage of IFN-γ and granzyme B positive cells indicated (B). The mean percentage ± SD of IFN-γ or granzyme B positive cells is indicated (n = 5 per group) (C). The data shown are representative of three independent experiments.
    Figure Legend Snippet: Accessory cell-NK cell contact is necessary for NK cell activation in vitro. Purified NK cells (DX5 + CD3 − ) were co-cultured with macrophages either mixed together (Mixed) or separated by a transwell system (Transwell), followed by stimulation with Ad-LacZ for 18 h (Ad), or left unstimulated (Medium). Brefeldin A was added into each well and incubated for additional 4 h. (A) DX5 + CD3 − NK cells were analyzed for the expression of CD69. MFI of CD69 staining is indicated. (B-C) DX5 + CD3 − NK cells were analyzed for intracellular IFN-γ and granzyme B production. FACS plots of IFN-γ and granzyme B production with the percentage of IFN-γ and granzyme B positive cells indicated (B). The mean percentage ± SD of IFN-γ or granzyme B positive cells is indicated (n = 5 per group) (C). The data shown are representative of three independent experiments.

    Techniques Used: Activation Assay, In Vitro, Purification, Cell Culture, Incubation, Expressing, Staining, FACS

    NKG2D is required for NK cell activation upon adenoviral infection in vivo. (A-B) C57BL/6 mice were treated with 100 μg of anti-NKG2D mAb (+Anti-NKG2D) or the control rat IgG (+Rat IgG) 24 h and 6 h before intravenous injection of Ad-lacZ (Ad). Some mice were left uninfected as controls (Naïve). 24 h after infection, splenocytes were harvested and restimulated with PMA/Inomycin in the presence of Brefeldin A for 4 h. Intracellular IFN-γ and granzyme B was determined by flow cytometry gated on DX5 + CD3 − NK cells. FACS plots of IFN-γ and granzyme B production with the percentage of IFN-γ and granzyme B positive cells indicated (A). The mean percentage ± SD of IFN-γ or granzyme B positive cells is indicated (n = 4 per group) (B). The data shown are representative of three independent experiments.
    Figure Legend Snippet: NKG2D is required for NK cell activation upon adenoviral infection in vivo. (A-B) C57BL/6 mice were treated with 100 μg of anti-NKG2D mAb (+Anti-NKG2D) or the control rat IgG (+Rat IgG) 24 h and 6 h before intravenous injection of Ad-lacZ (Ad). Some mice were left uninfected as controls (Naïve). 24 h after infection, splenocytes were harvested and restimulated with PMA/Inomycin in the presence of Brefeldin A for 4 h. Intracellular IFN-γ and granzyme B was determined by flow cytometry gated on DX5 + CD3 − NK cells. FACS plots of IFN-γ and granzyme B production with the percentage of IFN-γ and granzyme B positive cells indicated (A). The mean percentage ± SD of IFN-γ or granzyme B positive cells is indicated (n = 4 per group) (B). The data shown are representative of three independent experiments.

    Techniques Used: Activation Assay, Infection, In Vivo, Mouse Assay, Injection, Flow Cytometry, Cytometry, FACS

    Accessory cells are required for NK cell activation in response to adenoviral infection. Purified splenic NK cells (DX5 + CD3 − ) were either cultured alone (NK alone), or co-cultured with peritoneal macrophages (+Mφ), pDCs (+pDCs), cDCs (+cDCs) or B cells (+B cells) in the presence (Ad) or absence (Medium) of Ad-LacZ. 18 h later, brefeldin A was added into each well and incubated for additional 4 h. (A) NK cells were analyzed for the expression of the early activation marker, CD69, by flow cytometry. The mean fluorescent intensity (MFI) of CD69 staining is indicated. Events were gated on DX5 + CD3 − NK cells. (B-C) NK cells were stained intracellularly for IFN-γ and granzyme B and analyzed by flow cytometry. FACS plots of granzyme B production by NK cells with the percentage of granzyme B positive cells among DX5 + CD3 − NK cells indicated (B). The mean percentage ± SD of granzyme B or IFN-γ positive cells among DX5 + CD3 − cells is indicated (n = 5 per group) (C). The data shown are representative of four independent experiments.
    Figure Legend Snippet: Accessory cells are required for NK cell activation in response to adenoviral infection. Purified splenic NK cells (DX5 + CD3 − ) were either cultured alone (NK alone), or co-cultured with peritoneal macrophages (+Mφ), pDCs (+pDCs), cDCs (+cDCs) or B cells (+B cells) in the presence (Ad) or absence (Medium) of Ad-LacZ. 18 h later, brefeldin A was added into each well and incubated for additional 4 h. (A) NK cells were analyzed for the expression of the early activation marker, CD69, by flow cytometry. The mean fluorescent intensity (MFI) of CD69 staining is indicated. Events were gated on DX5 + CD3 − NK cells. (B-C) NK cells were stained intracellularly for IFN-γ and granzyme B and analyzed by flow cytometry. FACS plots of granzyme B production by NK cells with the percentage of granzyme B positive cells among DX5 + CD3 − NK cells indicated (B). The mean percentage ± SD of granzyme B or IFN-γ positive cells among DX5 + CD3 − cells is indicated (n = 5 per group) (C). The data shown are representative of four independent experiments.

    Techniques Used: Activation Assay, Infection, Purification, Cell Culture, Incubation, Expressing, Marker, Flow Cytometry, Cytometry, Staining, FACS

    15) Product Images from "Integrated Cross-Species Analysis Identifies a Conserved Transitional Dendritic Cell Population"

    Article Title: Integrated Cross-Species Analysis Identifies a Conserved Transitional Dendritic Cell Population

    Journal: Cell reports

    doi: 10.1016/j.celrep.2019.11.042

    Mouse and Human tDCs Display Similar Functional Capabilities (A) Splenic DCs were analyzed 6 h after intravenous (i.v.) inoculation of CpG-A (filled bars) or PBS control (empty bars). gMFI of activation markers is shown as the mean ± SD (n = 2–4 in 2–4 exp). See Figure S6 for histograms of activation markers. (B) Human DCs were sorted from PBMCs and analyzed at time 0 (empty bars) or after 24 h of culture with 5 μg/mL of CpG-A (filled bars). gMFI of activation markers is shown as the mean ± SD (n = 2–4 in 2–4 exp). (C) IFNα and IL-12p70 measured by ELISA in supernatants from sorted mouse DCs stimulated with CpG-A for 16–18 h (n = 4 in 4 exp). Frequency of IL-12p40-positive cells was measured by intracellular cytokine staining after 4 h of stimulation with CpG-A in the presence of brefeldin A (BFA). (D) IFNα measured by ELISA and IL-12p70 measured by cytometric bead array in the supernatants from sorted human DCs stimulated as in (B) or with an adjuvant cocktail (lipopolysaccharide-poly(I:C)-R848 [LPR]). (E) Frequency of CellTrace violet (CTV) low mouse CD4 + T cells in mixed leukocyte reactions. DCs were sorted from B6 mice and cocultured with CTV-labeled CD4 + T cells from BALB/c mice for 5 days (n = 3 in 3 exp). (F) Frequency of carboxyfluorescein succinimidyl ester (CFSE) low human CD4 + T cells in mixed leukocyte reactions. Human DC populations were sorted and cocultured with allogeneic CFSE-labeled T cells for 5–6 days (n = 3–4 in 3 exp). Bar graphs indicate mean ± SD; nd, not detected. Statistics determined by one-way ANOVA with Tukey’s multiple comparison test. *p
    Figure Legend Snippet: Mouse and Human tDCs Display Similar Functional Capabilities (A) Splenic DCs were analyzed 6 h after intravenous (i.v.) inoculation of CpG-A (filled bars) or PBS control (empty bars). gMFI of activation markers is shown as the mean ± SD (n = 2–4 in 2–4 exp). See Figure S6 for histograms of activation markers. (B) Human DCs were sorted from PBMCs and analyzed at time 0 (empty bars) or after 24 h of culture with 5 μg/mL of CpG-A (filled bars). gMFI of activation markers is shown as the mean ± SD (n = 2–4 in 2–4 exp). (C) IFNα and IL-12p70 measured by ELISA in supernatants from sorted mouse DCs stimulated with CpG-A for 16–18 h (n = 4 in 4 exp). Frequency of IL-12p40-positive cells was measured by intracellular cytokine staining after 4 h of stimulation with CpG-A in the presence of brefeldin A (BFA). (D) IFNα measured by ELISA and IL-12p70 measured by cytometric bead array in the supernatants from sorted human DCs stimulated as in (B) or with an adjuvant cocktail (lipopolysaccharide-poly(I:C)-R848 [LPR]). (E) Frequency of CellTrace violet (CTV) low mouse CD4 + T cells in mixed leukocyte reactions. DCs were sorted from B6 mice and cocultured with CTV-labeled CD4 + T cells from BALB/c mice for 5 days (n = 3 in 3 exp). (F) Frequency of carboxyfluorescein succinimidyl ester (CFSE) low human CD4 + T cells in mixed leukocyte reactions. Human DC populations were sorted and cocultured with allogeneic CFSE-labeled T cells for 5–6 days (n = 3–4 in 3 exp). Bar graphs indicate mean ± SD; nd, not detected. Statistics determined by one-way ANOVA with Tukey’s multiple comparison test. *p

    Techniques Used: Functional Assay, Activation Assay, Enzyme-linked Immunosorbent Assay, Staining, Mouse Assay, Labeling

    16) Product Images from "Post-ER Stress Biogenesis of Golgi Is Governed by Giantin"

    Article Title: Post-ER Stress Biogenesis of Golgi Is Governed by Giantin

    Journal: Cells

    doi: 10.3390/cells8121631

    Giantin dimerization in Brefeldin A (BFA) and BFA-washout (WO) cells. ( A ) HeLa cells were treated with 36 μM BFA for 1 h or with a corresponding amount of DMSO (control). The Golgi fractions isolated from cells were subjected to sucrose sedimentation analysis on a 5–25% sucrose gradient. The 25% and 5% fractions were collected and analyzed by 4–15% SDS-PAGE. The samples were prepared under low (1%) concentrations of β-mercaptoethanol, and the same amount of proteins were loaded. Giantin-dimer and monomer are indicated by arrows. The size marker indicates 800 kDa protein Nesprin, which corresponds to the giantin dimer. Densitometry analysis of ratio monomer/dimer is presented below. ( B ) Giantin W-B of the lysates of HeLa cells treated with DMSO and BFA for 1 h. The lysates were prepared in the presence (+) or absence (−) of 2 mM NEM followed by 1% β-mercaptoethanol and resolved by 8% SDS-PAGE. ( C ) Giantin W-B of the lysates of HeLa cells treated with DMSO, BFA for 1 h, and BFA-WO for another 60 min. The lysates were prepared under 2 mM NEM followed by 5% β-mercaptoethanol and resolved by 10% SDS-PAGE. Bottom panel: β-actin as a loading control. ( D ) Quantification of the band’s intensity monomer/dimer from three independent experiments presented in ( C ). Data are presented as a mean ± SD; * p
    Figure Legend Snippet: Giantin dimerization in Brefeldin A (BFA) and BFA-washout (WO) cells. ( A ) HeLa cells were treated with 36 μM BFA for 1 h or with a corresponding amount of DMSO (control). The Golgi fractions isolated from cells were subjected to sucrose sedimentation analysis on a 5–25% sucrose gradient. The 25% and 5% fractions were collected and analyzed by 4–15% SDS-PAGE. The samples were prepared under low (1%) concentrations of β-mercaptoethanol, and the same amount of proteins were loaded. Giantin-dimer and monomer are indicated by arrows. The size marker indicates 800 kDa protein Nesprin, which corresponds to the giantin dimer. Densitometry analysis of ratio monomer/dimer is presented below. ( B ) Giantin W-B of the lysates of HeLa cells treated with DMSO and BFA for 1 h. The lysates were prepared in the presence (+) or absence (−) of 2 mM NEM followed by 1% β-mercaptoethanol and resolved by 8% SDS-PAGE. ( C ) Giantin W-B of the lysates of HeLa cells treated with DMSO, BFA for 1 h, and BFA-WO for another 60 min. The lysates were prepared under 2 mM NEM followed by 5% β-mercaptoethanol and resolved by 10% SDS-PAGE. Bottom panel: β-actin as a loading control. ( D ) Quantification of the band’s intensity monomer/dimer from three independent experiments presented in ( C ). Data are presented as a mean ± SD; * p

    Techniques Used: Isolation, Sedimentation, SDS Page, Marker

    17) Product Images from "Interferon-λ modulates dendritic cells to facilitate T cell immunity during infection with influenza A virus"

    Article Title: Interferon-λ modulates dendritic cells to facilitate T cell immunity during infection with influenza A virus

    Journal: Nature immunology

    doi: 10.1038/s41590-019-0408-z

    IFN-λ signaling is required for viral control and generation of the CD8 +  T cell response during IAV infection. a-d . WT and  Ifnlr1 –/–  mice were mock- or IAV-infected i.n. with PBS or 40 PFU PR8 expressing a 2W epitope, respectively.  a . Lungs were harvested on day 9 p.i. and frequency (% of CD8 +  T cells) and numbers IAV-specific CD8 +  T cells were determined by flow cytometry.  b-d . On day 9 p.i., whole lung homogenates were stimulated with media, IAV (NP 366 ) peptide, or PMA + ionomycin for 6 hrs in the presence of brefeldin A. Following stimulation, IFN-γ  (b)  and TNF  (c)  single- or co-production  (d)  was determined by flow cytometry. For  a - b , two pooled independent experiments are shown. For ( a ) n=2 ( Ifnlr1 –/–  Mock), 3 (WT Mock), 5 (WT-IAV), or 7 ( Ifnlr1 –/– -IAV) mice/group. For ( b ) n=6 (WT-IAV), or 8 ( Ifnlr1 –/– -IAV) mice/group. For  a-d  error bars represent mean ± SEM. Significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test between WT-IAV and  Ifnlr1 –/– -IAV.
    Figure Legend Snippet: IFN-λ signaling is required for viral control and generation of the CD8 + T cell response during IAV infection. a-d . WT and Ifnlr1 –/– mice were mock- or IAV-infected i.n. with PBS or 40 PFU PR8 expressing a 2W epitope, respectively. a . Lungs were harvested on day 9 p.i. and frequency (% of CD8 + T cells) and numbers IAV-specific CD8 + T cells were determined by flow cytometry. b-d . On day 9 p.i., whole lung homogenates were stimulated with media, IAV (NP 366 ) peptide, or PMA + ionomycin for 6 hrs in the presence of brefeldin A. Following stimulation, IFN-γ (b) and TNF (c) single- or co-production (d) was determined by flow cytometry. For a - b , two pooled independent experiments are shown. For ( a ) n=2 ( Ifnlr1 –/– Mock), 3 (WT Mock), 5 (WT-IAV), or 7 ( Ifnlr1 –/– -IAV) mice/group. For ( b ) n=6 (WT-IAV), or 8 ( Ifnlr1 –/– -IAV) mice/group. For a-d error bars represent mean ± SEM. Significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test between WT-IAV and Ifnlr1 –/– -IAV.

    Techniques Used: Infection, Mouse Assay, Expressing, Flow Cytometry, Cytometry

    18) Product Images from "Natural killer cells expanded in vivo or ex vivo with IL-15 overcomes the inherent susceptibility of CAST mice to lethal infection with orthopoxviruses"

    Article Title: Natural killer cells expanded in vivo or ex vivo with IL-15 overcomes the inherent susceptibility of CAST mice to lethal infection with orthopoxviruses

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008505

    Proliferation of NK cells following IL-15 treatment of CAST mice. (A) NK cell numbers determined by flow cytometry. Mice were inoculated IP with PBS or PBS containing 5 μg of IL-15 daily for four days. On the following day, cells from spleens, livers and peritoneal washes were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD3 and allophycocyanin (APC)-conjugated anti-CD335 antibodies and analyzed by flow cytometry. To identify NK cells, PBS-treated samples were stained with anti-CD3 alone and gating tools were used to partition CD335 + cells in the upper-right quadrant of the scatter plot (first column). The established gates were then applied to all IL-15- and PBS-treated samples stained with both anti-CD3 and anti-CD335. (B) Decrease in NK cell numbers with time. NK cells were analyzed as in panel A at 1, 3 and 5 days after cessation of IL-15 inoculations and the numbers for individual mice are shown. Bars indicate geometric mean. Comparison of NK cell numbers in spleen and liver of PBS- and IL-15-treated mice on days 1, 3 and 5 and in peritoneal washes on days 1 and 5 were significant (p = 0.057). (C) Enumeration of IFN-γ expressing NK and T cells. Mice were inoculated IP with PBS or PBS containing 5 μg of IL-15 daily for four days and splenic cells were isolated and stimulated with PMA/Ionomycin for 4 h, treated with Brefeldin A and stained intracellularly with mouse anti-IFN-γ -APC antibody. Surface staining was accomplished with anti-CD3-FITC, anti-CD4-PE, anti-CD8-PerCP and anti-CD335-PerCP. Increases in NK cells following IL-15 were shown in three independent experiments.
    Figure Legend Snippet: Proliferation of NK cells following IL-15 treatment of CAST mice. (A) NK cell numbers determined by flow cytometry. Mice were inoculated IP with PBS or PBS containing 5 μg of IL-15 daily for four days. On the following day, cells from spleens, livers and peritoneal washes were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD3 and allophycocyanin (APC)-conjugated anti-CD335 antibodies and analyzed by flow cytometry. To identify NK cells, PBS-treated samples were stained with anti-CD3 alone and gating tools were used to partition CD335 + cells in the upper-right quadrant of the scatter plot (first column). The established gates were then applied to all IL-15- and PBS-treated samples stained with both anti-CD3 and anti-CD335. (B) Decrease in NK cell numbers with time. NK cells were analyzed as in panel A at 1, 3 and 5 days after cessation of IL-15 inoculations and the numbers for individual mice are shown. Bars indicate geometric mean. Comparison of NK cell numbers in spleen and liver of PBS- and IL-15-treated mice on days 1, 3 and 5 and in peritoneal washes on days 1 and 5 were significant (p = 0.057). (C) Enumeration of IFN-γ expressing NK and T cells. Mice were inoculated IP with PBS or PBS containing 5 μg of IL-15 daily for four days and splenic cells were isolated and stimulated with PMA/Ionomycin for 4 h, treated with Brefeldin A and stained intracellularly with mouse anti-IFN-γ -APC antibody. Surface staining was accomplished with anti-CD3-FITC, anti-CD4-PE, anti-CD8-PerCP and anti-CD335-PerCP. Increases in NK cells following IL-15 were shown in three independent experiments.

    Techniques Used: Mouse Assay, Flow Cytometry, Staining, Expressing, Isolation

    19) Product Images from "Circulating Th17, Th22, and Th1 Cells Are Elevated in the Guillain-Barr? Syndrome and Downregulated by IVIg Treatments"

    Article Title: Circulating Th17, Th22, and Th1 Cells Are Elevated in the Guillain-Barr? Syndrome and Downregulated by IVIg Treatments

    Journal: Mediators of Inflammation

    doi: 10.1155/2014/740947

    Quantification of circulating CD4 + IFN- γ + , Th17 (CD4 + IL-17A + ), and CD4 + IL-22 + cells in each group. (a) Representative two-color dot plot analyses of CD4 + IFN- γ + , CD4 + IL-17A + , and CD4 + IL-22 + cells after stimulation with PMA and ionomycin in the presence of Brefeldin A from a GBS patient. (b) Frequency of CD4 + IFN- γ + , CD4 + IL-17A + , and CD4 + IL-22 + cells on the gated lymphocytes in the FSC/SSC plot in each group: GBS ( n = 29), RR-MS/R ( n = 15), VEM ( n = 17), and HC ( n = 20). Results are represented as the mean ± SD. Statistical significance was indicated: * P
    Figure Legend Snippet: Quantification of circulating CD4 + IFN- γ + , Th17 (CD4 + IL-17A + ), and CD4 + IL-22 + cells in each group. (a) Representative two-color dot plot analyses of CD4 + IFN- γ + , CD4 + IL-17A + , and CD4 + IL-22 + cells after stimulation with PMA and ionomycin in the presence of Brefeldin A from a GBS patient. (b) Frequency of CD4 + IFN- γ + , CD4 + IL-17A + , and CD4 + IL-22 + cells on the gated lymphocytes in the FSC/SSC plot in each group: GBS ( n = 29), RR-MS/R ( n = 15), VEM ( n = 17), and HC ( n = 20). Results are represented as the mean ± SD. Statistical significance was indicated: * P

    Techniques Used: Mass Spectrometry

    20) Product Images from "Tumour-induced polarization of tumour vaccine-draining lymph node T cells to a type 1 cytokine profile predicts inherent strong immunogenicity of the tumour and correlates with therapeutic efficacy in adoptive transfer studies"

    Article Title: Tumour-induced polarization of tumour vaccine-draining lymph node T cells to a type 1 cytokine profile predicts inherent strong immunogenicity of the tumour and correlates with therapeutic efficacy in adoptive transfer studies

    Journal: Immunology

    doi: 10.1046/j.1365-2567.2003.01596.x

    Effector T cells from both wt and GKO mice express tumour-specific TNF-α. Effector T cells generated from D5-G6-vaccinated wt and GKO mice were stimulated with D5, syngeneic fibrosarcoma MCA 310, anti-CD3, or left alone for 12 hr in the presence of Brefeldin A. Cells were harvested, permeabilized and stained with PE-labelled anti-IFN-γ and -TNF-α mAbs and analysed for intracellular expression of IFN-γ and TNF-α. The number in the top-right quarter indicates the percentage of CD3 +  CD8 +  T cells producing IFN-γ or TNF-α.
    Figure Legend Snippet: Effector T cells from both wt and GKO mice express tumour-specific TNF-α. Effector T cells generated from D5-G6-vaccinated wt and GKO mice were stimulated with D5, syngeneic fibrosarcoma MCA 310, anti-CD3, or left alone for 12 hr in the presence of Brefeldin A. Cells were harvested, permeabilized and stained with PE-labelled anti-IFN-γ and -TNF-α mAbs and analysed for intracellular expression of IFN-γ and TNF-α. The number in the top-right quarter indicates the percentage of CD3 + CD8 + T cells producing IFN-γ or TNF-α.

    Techniques Used: Mouse Assay, Generated, Staining, Expressing

    21) Product Images from "Sterols regulate cycling of SREBP cleavage-activating protein (SCAP) between endoplasmic reticulum and Golgi"

    Article Title: Sterols regulate cycling of SREBP cleavage-activating protein (SCAP) between endoplasmic reticulum and Golgi

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

    doi:

    Effect of brefeldin A on sterol-regulated acyl-CoA:cholesterol acyltransferase activity. On day 0, CHO-7 cells were set up at 2.5 × 10 5 cells per 60-mm dish in medium A supplemented with 5% FCS. On day 2, cells were switched to medium A containing 5% newborn calf lipoprotein-deficient serum, 50 μM compactin, 50 μM sodium mevalonate, and 0.2% ethanol in the absence or presence (+ sterols) of 1 μg/ml 25-hydroxycholesterol plus 10 μg/ml cholesterol as indicated. After incubation at 37°C for 11 hr, the cells were incubated for an additional 5 hr with 0.1% methanol in the absence or presence of 10 μg/ml brefeldin A as indicated. During the final 2 hr, each monolayer was pulse-labeled with 0.2 mM sodium [ 14 C]oleate (7,600 dpm/pmol), after which the cells were harvested for measurement of the rate of [ 14 C]oleate incorporation into cholesteryl [ 14 ). Error bars denote the SD of triplicate values.
    Figure Legend Snippet: Effect of brefeldin A on sterol-regulated acyl-CoA:cholesterol acyltransferase activity. On day 0, CHO-7 cells were set up at 2.5 × 10 5 cells per 60-mm dish in medium A supplemented with 5% FCS. On day 2, cells were switched to medium A containing 5% newborn calf lipoprotein-deficient serum, 50 μM compactin, 50 μM sodium mevalonate, and 0.2% ethanol in the absence or presence (+ sterols) of 1 μg/ml 25-hydroxycholesterol plus 10 μg/ml cholesterol as indicated. After incubation at 37°C for 11 hr, the cells were incubated for an additional 5 hr with 0.1% methanol in the absence or presence of 10 μg/ml brefeldin A as indicated. During the final 2 hr, each monolayer was pulse-labeled with 0.2 mM sodium [ 14 C]oleate (7,600 dpm/pmol), after which the cells were harvested for measurement of the rate of [ 14 C]oleate incorporation into cholesteryl [ 14 ). Error bars denote the SD of triplicate values.

    Techniques Used: Activity Assay, Incubation, Labeling

    Brefeldin A induces endo H-resistance of SCAP in the presence of sterols. On day 0, CHO-7 cells were set up in medium A supplemented with 10% FCS. On day 2, cells were switched to medium A containing 10% newborn calf lipoprotein-deficient serum, 50 μM compactin, 50 μM sodium mevalonate, and 0.2% ethanol in the absence or presence (+ sterols) of 1 μg/ml 25-hydroxycholesterol plus 10 μg/ml cholesterol as indicated. After incubation at 37°C for 10.5 hr, cultures received either no addition (lanes 1–16) or 5 μg/ml swainsonine (lane 17). After a 30-min preincubation, cells were incubated for an additional 5 hr with 0.1% (vol/vol) methanol in the absence or presence of 10 μg/ml brefeldin A as indicated. Cells were harvested, and membrane fractions were prepared as described in Materials and Methods . Aliquots (54 μg of protein) were incubated in the absence (lanes 1–4) or presence (lanes 5–17) of trypsin. Proteolysis was stopped, and the samples were incubated either in the absence (lanes 1–8) or presence (lanes 9–17) of the indicated glycosidase, subjected to SDS/PAGE (5–12% gel), and transferred to nitrocellulose. The filter was blotted with 10 μg/ml IgG-9D5 (anti-SCAP) and exposed to film for 2 min.
    Figure Legend Snippet: Brefeldin A induces endo H-resistance of SCAP in the presence of sterols. On day 0, CHO-7 cells were set up in medium A supplemented with 10% FCS. On day 2, cells were switched to medium A containing 10% newborn calf lipoprotein-deficient serum, 50 μM compactin, 50 μM sodium mevalonate, and 0.2% ethanol in the absence or presence (+ sterols) of 1 μg/ml 25-hydroxycholesterol plus 10 μg/ml cholesterol as indicated. After incubation at 37°C for 10.5 hr, cultures received either no addition (lanes 1–16) or 5 μg/ml swainsonine (lane 17). After a 30-min preincubation, cells were incubated for an additional 5 hr with 0.1% (vol/vol) methanol in the absence or presence of 10 μg/ml brefeldin A as indicated. Cells were harvested, and membrane fractions were prepared as described in Materials and Methods . Aliquots (54 μg of protein) were incubated in the absence (lanes 1–4) or presence (lanes 5–17) of trypsin. Proteolysis was stopped, and the samples were incubated either in the absence (lanes 1–8) or presence (lanes 9–17) of the indicated glycosidase, subjected to SDS/PAGE (5–12% gel), and transferred to nitrocellulose. The filter was blotted with 10 μg/ml IgG-9D5 (anti-SCAP) and exposed to film for 2 min.

    Techniques Used: Incubation, SDS Page

    22) Product Images from "Sterols regulate cycling of SREBP cleavage-activating protein (SCAP) between endoplasmic reticulum and Golgi"

    Article Title: Sterols regulate cycling of SREBP cleavage-activating protein (SCAP) between endoplasmic reticulum and Golgi

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

    doi:

    Effect of brefeldin A on sterol-regulated acyl-CoA:cholesterol acyltransferase activity. On day 0, CHO-7 cells were set up at 2.5 × 10 5 cells per 60-mm dish in medium A supplemented with 5% FCS. On day 2, cells were switched to medium A containing 5% newborn calf lipoprotein-deficient serum, 50 μM compactin, 50 μM sodium mevalonate, and 0.2% ethanol in the absence or presence (+ sterols) of 1 μg/ml 25-hydroxycholesterol plus 10 μg/ml cholesterol as indicated. After incubation at 37°C for 11 hr, the cells were incubated for an additional 5 hr with 0.1% methanol in the absence or presence of 10 μg/ml brefeldin A as indicated. During the final 2 hr, each monolayer was pulse-labeled with 0.2 mM sodium [ 14 C]oleate (7,600 dpm/pmol), after which the cells were harvested for measurement of the rate of [ 14 C]oleate incorporation into cholesteryl [ 14 ). Error bars denote the SD of triplicate values.
    Figure Legend Snippet: Effect of brefeldin A on sterol-regulated acyl-CoA:cholesterol acyltransferase activity. On day 0, CHO-7 cells were set up at 2.5 × 10 5 cells per 60-mm dish in medium A supplemented with 5% FCS. On day 2, cells were switched to medium A containing 5% newborn calf lipoprotein-deficient serum, 50 μM compactin, 50 μM sodium mevalonate, and 0.2% ethanol in the absence or presence (+ sterols) of 1 μg/ml 25-hydroxycholesterol plus 10 μg/ml cholesterol as indicated. After incubation at 37°C for 11 hr, the cells were incubated for an additional 5 hr with 0.1% methanol in the absence or presence of 10 μg/ml brefeldin A as indicated. During the final 2 hr, each monolayer was pulse-labeled with 0.2 mM sodium [ 14 C]oleate (7,600 dpm/pmol), after which the cells were harvested for measurement of the rate of [ 14 C]oleate incorporation into cholesteryl [ 14 ). Error bars denote the SD of triplicate values.

    Techniques Used: Activity Assay, Incubation, Labeling

    Brefeldin A induces endo H-resistance of SCAP in the presence of sterols. On day 0, CHO-7 cells were set up in medium A supplemented with 10% FCS. On day 2, cells were switched to medium A containing 10% newborn calf lipoprotein-deficient serum, 50 μM compactin, 50 μM sodium mevalonate, and 0.2% ethanol in the absence or presence (+ sterols) of 1 μg/ml 25-hydroxycholesterol plus 10 μg/ml cholesterol as indicated. After incubation at 37°C for 10.5 hr, cultures received either no addition (lanes 1–16) or 5 μg/ml swainsonine (lane 17). After a 30-min preincubation, cells were incubated for an additional 5 hr with 0.1% (vol/vol) methanol in the absence or presence of 10 μg/ml brefeldin A as indicated. Cells were harvested, and membrane fractions were prepared as described in Materials and Methods . Aliquots (54 μg of protein) were incubated in the absence (lanes 1–4) or presence (lanes 5–17) of trypsin. Proteolysis was stopped, and the samples were incubated either in the absence (lanes 1–8) or presence (lanes 9–17) of the indicated glycosidase, subjected to SDS/PAGE (5–12% gel), and transferred to nitrocellulose. The filter was blotted with 10 μg/ml IgG-9D5 (anti-SCAP) and exposed to film for 2 min.
    Figure Legend Snippet: Brefeldin A induces endo H-resistance of SCAP in the presence of sterols. On day 0, CHO-7 cells were set up in medium A supplemented with 10% FCS. On day 2, cells were switched to medium A containing 10% newborn calf lipoprotein-deficient serum, 50 μM compactin, 50 μM sodium mevalonate, and 0.2% ethanol in the absence or presence (+ sterols) of 1 μg/ml 25-hydroxycholesterol plus 10 μg/ml cholesterol as indicated. After incubation at 37°C for 10.5 hr, cultures received either no addition (lanes 1–16) or 5 μg/ml swainsonine (lane 17). After a 30-min preincubation, cells were incubated for an additional 5 hr with 0.1% (vol/vol) methanol in the absence or presence of 10 μg/ml brefeldin A as indicated. Cells were harvested, and membrane fractions were prepared as described in Materials and Methods . Aliquots (54 μg of protein) were incubated in the absence (lanes 1–4) or presence (lanes 5–17) of trypsin. Proteolysis was stopped, and the samples were incubated either in the absence (lanes 1–8) or presence (lanes 9–17) of the indicated glycosidase, subjected to SDS/PAGE (5–12% gel), and transferred to nitrocellulose. The filter was blotted with 10 μg/ml IgG-9D5 (anti-SCAP) and exposed to film for 2 min.

    Techniques Used: Incubation, SDS Page

    23) Product Images from "A Novel, Live-Attenuated Vesicular Stomatitis Virus Vector Displaying Conformationally Intact, Functional HIV-1 Envelope Trimers That Elicits Potent Cellular and Humoral Responses in Mice"

    Article Title: A Novel, Live-Attenuated Vesicular Stomatitis Virus Vector Displaying Conformationally Intact, Functional HIV-1 Envelope Trimers That Elicits Potent Cellular and Humoral Responses in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0106597

    Frequency of JR-FL Env-specific CD4+ T cell responses in the lung and spleen. 1.5×10 6 leukocytes isolated from the lungs (a, b) andspleen (c, d) at the end of the study were stimulated ex vivo with JR-FL gp140, anti-CD28 and brefeldin A before being analyzed by flow cytometry for production of IFNg, IL-2 and TNFa. The total frequency of cytokine secreting CD4+ T cells (%) producing IFNγ, IL-2 or TNFα are shown on the left (a, c) and the frequency of multifunctional CD4+ T cells producing any combination of IFN γ +, IL-2+, and/or TNFα+ on the right (b, d). *p
    Figure Legend Snippet: Frequency of JR-FL Env-specific CD4+ T cell responses in the lung and spleen. 1.5×10 6 leukocytes isolated from the lungs (a, b) andspleen (c, d) at the end of the study were stimulated ex vivo with JR-FL gp140, anti-CD28 and brefeldin A before being analyzed by flow cytometry for production of IFNg, IL-2 and TNFa. The total frequency of cytokine secreting CD4+ T cells (%) producing IFNγ, IL-2 or TNFα are shown on the left (a, c) and the frequency of multifunctional CD4+ T cells producing any combination of IFN γ +, IL-2+, and/or TNFα+ on the right (b, d). *p

    Techniques Used: Isolation, Ex Vivo, Flow Cytometry, Cytometry

    24) Product Images from "The Aryl Hydrocarbon Receptor: Differential Contribution to T Helper 17 and T Cytotoxic 17 Cell Development"

    Article Title: The Aryl Hydrocarbon Receptor: Differential Contribution to T Helper 17 and T Cytotoxic 17 Cell Development

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0106955

    The effect of AhR modulation on Th17 and Tc17 cell frequency. Naïve CD4 +  cells from AhR +/−  (A and E) or AhR −/−  mice (B and F) and naïve CD8 +  cells from AhR +/−  (C and G) or AhR −/−  mice (D and H) were polarised under Th17/Tc17 conditions for 5 days. Cells were then stimulated with PdBU and ionomycin in the presence of brefeldin A. The polarised cells were then permeabilised and stained using fluorescent anti-IL-17A (PE [Y-axis]) and -IFN-γ (APC [X-axis]) labelled antibodies. Cells (25000) were analysed by flow cytometry. The cells were cultured in the presence of AhR antagonist (CH-223191) (white bar) or AhR agonist (FICZ) (grey bar) both formulated in DMSO or with an equivalent amount of DMSO alone (black bar). Representative quadrant analyses are illustrated (A–D). The percentage of cells positive for IL-17, IL-17 and IFN-γ or IFN-γ alone was calculated by subtracting the isotype controls from the positive cells in each quadrant and are illustrated as % positive cells (E-H; mean ± SE; n = 3 independent experiments). Statistical significance of differences between DMSO control and AhR antagonist/agonist treated cells was analysed by one-way ANOVA. ***,  p
    Figure Legend Snippet: The effect of AhR modulation on Th17 and Tc17 cell frequency. Naïve CD4 + cells from AhR +/− (A and E) or AhR −/− mice (B and F) and naïve CD8 + cells from AhR +/− (C and G) or AhR −/− mice (D and H) were polarised under Th17/Tc17 conditions for 5 days. Cells were then stimulated with PdBU and ionomycin in the presence of brefeldin A. The polarised cells were then permeabilised and stained using fluorescent anti-IL-17A (PE [Y-axis]) and -IFN-γ (APC [X-axis]) labelled antibodies. Cells (25000) were analysed by flow cytometry. The cells were cultured in the presence of AhR antagonist (CH-223191) (white bar) or AhR agonist (FICZ) (grey bar) both formulated in DMSO or with an equivalent amount of DMSO alone (black bar). Representative quadrant analyses are illustrated (A–D). The percentage of cells positive for IL-17, IL-17 and IFN-γ or IFN-γ alone was calculated by subtracting the isotype controls from the positive cells in each quadrant and are illustrated as % positive cells (E-H; mean ± SE; n = 3 independent experiments). Statistical significance of differences between DMSO control and AhR antagonist/agonist treated cells was analysed by one-way ANOVA. ***, p

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

    25) Product Images from "RAC1-Dependent ORAI1 Translocation to the Leading Edge Supports Lamellipodia Formation and Directional Persistence"

    Article Title: RAC1-Dependent ORAI1 Translocation to the Leading Edge Supports Lamellipodia Formation and Directional Persistence

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-63353-5

    RAC1 T17N blocked the transport of ORAI1 to the cell surface. Panel A : U2OS cells stably expressing Flag-RAC1 T17N were transfected for the transient expression of ORAI1-GFP. Cells were fixed 24 h after transfection, and used for the immunolocalization of GM130, TGN46, EEA1, LAMP1, and Sec13a. Secondary antibodies were labelled with Alexa Fluor 594. Images are representative of 2 independent experiments ( > 20 cells per condition). Fluorescence of both channels was measured over the arrow depicted in the image, which was placed over the intracellularly trapped GFP- signal. Panel B : U2OS cells were treated with 50 μM NSC 23766 for 8 h, fixed, and the immunolocalization of GM130 was performed as in panel A . Images are representative of 26 cells from 2 independent experiments. Bar = 10 μm. Panel C: Left : Cells were cultured on 96-well plates in DMEM + 10% FBS medium. The secreted and the intracellular luciferase activity were measured after 28 h of culture. The treatment with 50 μM NSC 23766 was performed during the last 8 h of culture. The overexpression of Flag-RAC1 T17N was triggered with doxycycline during the last 22 h of culture. Data from n = 15 wells and 3 independent experiments are shown as bar chart. Middle : Intracellular Gluc-YFP protein was evaluated from cell lysates by immunoblot using an anti-GFP antibody. Right : Cells were treated with 5 μg/ml brefeldin A for 2–4 h to assess the inhibition of the secretory pathway, as a control of the experiment.
    Figure Legend Snippet: RAC1 T17N blocked the transport of ORAI1 to the cell surface. Panel A : U2OS cells stably expressing Flag-RAC1 T17N were transfected for the transient expression of ORAI1-GFP. Cells were fixed 24 h after transfection, and used for the immunolocalization of GM130, TGN46, EEA1, LAMP1, and Sec13a. Secondary antibodies were labelled with Alexa Fluor 594. Images are representative of 2 independent experiments ( > 20 cells per condition). Fluorescence of both channels was measured over the arrow depicted in the image, which was placed over the intracellularly trapped GFP- signal. Panel B : U2OS cells were treated with 50 μM NSC 23766 for 8 h, fixed, and the immunolocalization of GM130 was performed as in panel A . Images are representative of 26 cells from 2 independent experiments. Bar = 10 μm. Panel C: Left : Cells were cultured on 96-well plates in DMEM + 10% FBS medium. The secreted and the intracellular luciferase activity were measured after 28 h of culture. The treatment with 50 μM NSC 23766 was performed during the last 8 h of culture. The overexpression of Flag-RAC1 T17N was triggered with doxycycline during the last 22 h of culture. Data from n = 15 wells and 3 independent experiments are shown as bar chart. Middle : Intracellular Gluc-YFP protein was evaluated from cell lysates by immunoblot using an anti-GFP antibody. Right : Cells were treated with 5 μg/ml brefeldin A for 2–4 h to assess the inhibition of the secretory pathway, as a control of the experiment.

    Techniques Used: Stable Transfection, Expressing, Transfection, Fluorescence, Cell Culture, Luciferase, Activity Assay, Over Expression, Inhibition

    26) Product Images from "Latrophilin fragments behave as independent proteins that associate and signal on binding of LTXN4C"

    Article Title: Latrophilin fragments behave as independent proteins that associate and signal on binding of LTXN4C

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7600443

    Cellular processing of latrophilin. ( A ) A typical LNB GPCR. Constitutive cleavage (arrow) creates two fragments corresponding to the cell-adhesion domain and GPCR domain. ( B ) Analysis of latrophilin expression in COS7 cells. Solubilised receptors from rat brain and COS7 cells transfected with vector or LPH were enriched by α-latrotoxin chromatography and analysed by WB. N-terminal sequences of the native and recombinant CTFs extracted from the gel are shown. ( C ) Analysis of post-translational modification of latrophilin. Cells were either cultured in the presence of brefeldin A or solubilised and treated with glycosidases, as indicated. For abbreviations of enzymes, see Materials and methods. ( D ) Identification of surface-exposed species of latrophilin. Live cells were biotinylated, solubilised, enriched on α-latrotoxin column and analysed by WB. Only αNTF is labelled with biotin. Molecular masses are shown on the right (B, C). FS, full-size latrophilin.
    Figure Legend Snippet: Cellular processing of latrophilin. ( A ) A typical LNB GPCR. Constitutive cleavage (arrow) creates two fragments corresponding to the cell-adhesion domain and GPCR domain. ( B ) Analysis of latrophilin expression in COS7 cells. Solubilised receptors from rat brain and COS7 cells transfected with vector or LPH were enriched by α-latrotoxin chromatography and analysed by WB. N-terminal sequences of the native and recombinant CTFs extracted from the gel are shown. ( C ) Analysis of post-translational modification of latrophilin. Cells were either cultured in the presence of brefeldin A or solubilised and treated with glycosidases, as indicated. For abbreviations of enzymes, see Materials and methods. ( D ) Identification of surface-exposed species of latrophilin. Live cells were biotinylated, solubilised, enriched on α-latrotoxin column and analysed by WB. Only αNTF is labelled with biotin. Molecular masses are shown on the right (B, C). FS, full-size latrophilin.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Chromatography, Western Blot, Recombinant, Modification, Cell Culture

    27) Product Images from "The ATP-binding Cassette Transporter-2 (ABCA2) Promotes Amyloidogenic Processing of Amyloid Precursor Protein by Glu11 Site Cleavage"

    Article Title: The ATP-binding Cassette Transporter-2 (ABCA2) Promotes Amyloidogenic Processing of Amyloid Precursor Protein by Glu11 Site Cleavage

    Journal: Current Alzheimer research

    doi:

    Effect of protein trafficking inhibitors on APP processing A. Cells were treated with 10 µg/ml Brefeldin A for 24 hours before protein extraction and Western blot for APP and CTF with the 0443 antibody. B. Cells were treated with 10 µg/ ml monensin for 24 hours before protein extraction and Western blot as described above. The data are expressed as fold-change relative to untreated cells.
    Figure Legend Snippet: Effect of protein trafficking inhibitors on APP processing A. Cells were treated with 10 µg/ml Brefeldin A for 24 hours before protein extraction and Western blot for APP and CTF with the 0443 antibody. B. Cells were treated with 10 µg/ ml monensin for 24 hours before protein extraction and Western blot as described above. The data are expressed as fold-change relative to untreated cells.

    Techniques Used: Protein Extraction, Western Blot

    28) Product Images from "Cross-presentation of viral antigens in dribbles leads to efficient activation of virus-specific human memory t cells"

    Article Title: Cross-presentation of viral antigens in dribbles leads to efficient activation of virus-specific human memory t cells

    Journal: Journal of Translational Medicine

    doi: 10.1186/1479-5876-12-100

    The kinetics of CD4 and CD8 T-cell activation following exposure to DRibble-pulsed APC. (A,B) PBMCs were separated into monocytes and lymphocytes by Elutra apheresis. Monocytes were pulsed with CEF DRibbles for 6 hours. After that time autologous T cells were added into wells at a ratio of 5:1 (T cells:monocytes). Brefeldin A was added to the culture after the time periods specified in the figure. All cells were harvested at the same time and prepared for flow cytometry. (C,D) PBMCs (monocytes and T cells) were cultured with pp65 DRibbles and then Brefeldin A was added to the culture after the time periods specified in the figure. Cells were harvested at the same time and prepared for flow cytometry.
    Figure Legend Snippet: The kinetics of CD4 and CD8 T-cell activation following exposure to DRibble-pulsed APC. (A,B) PBMCs were separated into monocytes and lymphocytes by Elutra apheresis. Monocytes were pulsed with CEF DRibbles for 6 hours. After that time autologous T cells were added into wells at a ratio of 5:1 (T cells:monocytes). Brefeldin A was added to the culture after the time periods specified in the figure. All cells were harvested at the same time and prepared for flow cytometry. (C,D) PBMCs (monocytes and T cells) were cultured with pp65 DRibbles and then Brefeldin A was added to the culture after the time periods specified in the figure. Cells were harvested at the same time and prepared for flow cytometry.

    Techniques Used: Activation Assay, Flow Cytometry, Cytometry, Cell Culture

    29) Product Images from "Immunological Synapse Predicts Effectiveness of Chimeric Antigen Receptor Cells"

    Article Title: Immunological Synapse Predicts Effectiveness of Chimeric Antigen Receptor Cells

    Journal: Molecular Therapy

    doi: 10.1016/j.ymthe.2018.01.020

    Neither Standard Killing nor Intracellular Cytokine and Ratio of CD4/CD8 Can Distinguish the Difference between 4-1BB-CAR and CD28-CAR (A) Cytotoxicity of Kappa-CAR T cells (left) and CD19-CAR T cells (right) from the same healthy donor was measured using a standard 4-hr 51 Cr-release assay. Daudi cells (the Kappa-positive B cell lymphoma cell line) were used as the Kappa-CAR T cell’s target cells. Raji cells (the CD19-positive B cell lymphoma cell line) were used as the CD19-CAR T cell’s target cells. PBMCs from five individuals were transduced with 4-1BB construct (red dots) or CD28 construct (black dots) retrovirus. (B and C) Production of TNF-α and IFN-γ by (B) Kappa-CAR and (C) CD19-CAR from five healthy donors. CAR T cells were stimulated with target cells for 6 hr in the presence of GolgiStop and brefeldin A treatment. The percentages of TNF-α or IFN-γ-positive cells were measured by flow cytometry. (D and E) Ratio of CD4/CD8 by (D) Kappa-CAR and (E) CD19-CAR from five individuals. CAR T cell stimulation with corresponding target cells for 6-hr treatment is shown. Surface staining of CD4- and CD8-positive cells was measured by flow cytometry. The ratio of CD4/CD8 and CD8 MFI was calculated by FlowJo software. Error bars show ±SD. The p value is for unpaired t-test.
    Figure Legend Snippet: Neither Standard Killing nor Intracellular Cytokine and Ratio of CD4/CD8 Can Distinguish the Difference between 4-1BB-CAR and CD28-CAR (A) Cytotoxicity of Kappa-CAR T cells (left) and CD19-CAR T cells (right) from the same healthy donor was measured using a standard 4-hr 51 Cr-release assay. Daudi cells (the Kappa-positive B cell lymphoma cell line) were used as the Kappa-CAR T cell’s target cells. Raji cells (the CD19-positive B cell lymphoma cell line) were used as the CD19-CAR T cell’s target cells. PBMCs from five individuals were transduced with 4-1BB construct (red dots) or CD28 construct (black dots) retrovirus. (B and C) Production of TNF-α and IFN-γ by (B) Kappa-CAR and (C) CD19-CAR from five healthy donors. CAR T cells were stimulated with target cells for 6 hr in the presence of GolgiStop and brefeldin A treatment. The percentages of TNF-α or IFN-γ-positive cells were measured by flow cytometry. (D and E) Ratio of CD4/CD8 by (D) Kappa-CAR and (E) CD19-CAR from five individuals. CAR T cell stimulation with corresponding target cells for 6-hr treatment is shown. Surface staining of CD4- and CD8-positive cells was measured by flow cytometry. The ratio of CD4/CD8 and CD8 MFI was calculated by FlowJo software. Error bars show ±SD. The p value is for unpaired t-test.

    Techniques Used: Release Assay, Transduction, Construct, Flow Cytometry, Cytometry, Cell Stimulation, Staining, Software

    30) Product Images from "Heavy Cannabis Use Associated With Reduction in Activated and Inflammatory Immune Cell Frequencies in Antiretroviral Therapy–Treated Human Immunodeficiency Virus–Infected Individuals"

    Article Title: Heavy Cannabis Use Associated With Reduction in Activated and Inflammatory Immune Cell Frequencies in Antiretroviral Therapy–Treated Human Immunodeficiency Virus–Infected Individuals

    Journal: Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America

    doi: 10.1093/cid/cix1116

    Lower frequency of cytokine-producing antigen-presenting cells (APCs) in cannabis-using individuals as compared to noncannabis users. Multiparameter flow cytometry was used to identify the frequency of APCs within total peripheral blood mononuclear cells of cannabis-using and nonusing individuals that expressed interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 23 (IL-23), and tumor necrosis factor alpha (TNF-α) after overnight culture in the presence of phorbol myistate acetate, ionomycin, and brefeldin A . Cells were identified by first excluding doublets using forward and side scatter properties, gating on total leukocytes as determined by CD45 expression and removing dead cells with an Aqua Live/Dead viability dye. Total APCs were then identified by gating on CD3 – CD20 – HLA-DR + cells. Expression of the indicated cytokine was then determined within this population. Pooled data is accompanied by a representative flow plot showing gating for the indicated cytokine. In all plots, individuals are classified as noncannabis users or cannabis users stratified by moderate or heavy cannabis use as determined by plasma quantities of 11-nor-carboxy-tetrahydrocannabinol. Each individual is represented by a single point. Horizontal bars indicate the median value. The statistical significance of differences between each of the cannabis-using groups and the noncannabis users was determined using the Mann-Whitney test. Abbreviations: SSC, side scatter; SSChi, side scatter high.
    Figure Legend Snippet: Lower frequency of cytokine-producing antigen-presenting cells (APCs) in cannabis-using individuals as compared to noncannabis users. Multiparameter flow cytometry was used to identify the frequency of APCs within total peripheral blood mononuclear cells of cannabis-using and nonusing individuals that expressed interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 23 (IL-23), and tumor necrosis factor alpha (TNF-α) after overnight culture in the presence of phorbol myistate acetate, ionomycin, and brefeldin A . Cells were identified by first excluding doublets using forward and side scatter properties, gating on total leukocytes as determined by CD45 expression and removing dead cells with an Aqua Live/Dead viability dye. Total APCs were then identified by gating on CD3 – CD20 – HLA-DR + cells. Expression of the indicated cytokine was then determined within this population. Pooled data is accompanied by a representative flow plot showing gating for the indicated cytokine. In all plots, individuals are classified as noncannabis users or cannabis users stratified by moderate or heavy cannabis use as determined by plasma quantities of 11-nor-carboxy-tetrahydrocannabinol. Each individual is represented by a single point. Horizontal bars indicate the median value. The statistical significance of differences between each of the cannabis-using groups and the noncannabis users was determined using the Mann-Whitney test. Abbreviations: SSC, side scatter; SSChi, side scatter high.

    Techniques Used: Flow Cytometry, Cytometry, Expressing, MANN-WHITNEY

    31) Product Images from "Establishment and validation of an endoplasmic reticulum stress reporter to monitor zebrafish ATF6 activity in development and disease"

    Article Title: Establishment and validation of an endoplasmic reticulum stress reporter to monitor zebrafish ATF6 activity in development and disease

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.041426

    Chemical ER stressors activate reporter expression. (A) Zebrafish embryos were treated with ER stressors at 1 dpf. Quantification (dashed line) revealed that 5XATF6RE :d2GFP expression was significantly higher at 8 hpt with thapsigargin ( P =0.0004) and brefeldin A ( P =0.0003) treatment, at 24 hpt with tunicamycin ( P =0.0001) and brefeldin A ( P =0.0001) treatment, and at 48 hpt with tunicamycin ( P =0.0001) and brefeldin A ( P =0.0065) treatment. ** P ≤0.01; *** P ≤0.001; **** P ≤0.0001; unpaired one-way ANOVA with Dunnett's post-hoc test with respect to DMSO control group. All error bars are s.e.m. (B) Representative images showing increased 5XATF6RE :d2GFP expression in the spinal cord of embryos (at 48 hpt) treated with tunicamycin or brefeldin A compared to the DMSO-treated control. Scale bars: 1 mm (A); 0.1 mm (B).
    Figure Legend Snippet: Chemical ER stressors activate reporter expression. (A) Zebrafish embryos were treated with ER stressors at 1 dpf. Quantification (dashed line) revealed that 5XATF6RE :d2GFP expression was significantly higher at 8 hpt with thapsigargin ( P =0.0004) and brefeldin A ( P =0.0003) treatment, at 24 hpt with tunicamycin ( P =0.0001) and brefeldin A ( P =0.0001) treatment, and at 48 hpt with tunicamycin ( P =0.0001) and brefeldin A ( P =0.0065) treatment. ** P ≤0.01; *** P ≤0.001; **** P ≤0.0001; unpaired one-way ANOVA with Dunnett's post-hoc test with respect to DMSO control group. All error bars are s.e.m. (B) Representative images showing increased 5XATF6RE :d2GFP expression in the spinal cord of embryos (at 48 hpt) treated with tunicamycin or brefeldin A compared to the DMSO-treated control. Scale bars: 1 mm (A); 0.1 mm (B).

    Techniques Used: Expressing

    32) Product Images from "IL-10 deficiency reveals a role for TLR2-dependent bystander activation of T cells in Lyme arthritis"

    Article Title: IL-10 deficiency reveals a role for TLR2-dependent bystander activation of T cells in Lyme arthritis

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1701248

    B. burgdorferi  infection of CD8 +  TCR transgenic mice results in arthritis and IFN-γ production IL-10 −/−  and CD8 +  TCR transgenic OT-I/Rag2 −/− /IL-10 −/−  mice were infected with  B. burgdorferi  for 4 weeks. (A) Rear ankles of mice were measured before infection and 4 weeks post-infection, and change in ankle measurement is shown. (B) The most swollen ankle was subjected to histopathologic evaluation in a blind fashion. Scores 0–5, with 5 being most severe, were assigned to each sample. (C) The total number of CD8 +  were quantified from the popliteal and inguinal lymph nodes. (D) Lymph node cells were stimulated with PMA/Ionomycin in presence of Brefeldin A, and the number of CD8 + IFN-γ +  T cells were quantified using flow cytometry. (E–I) Quantification of  B. burgdorferi -specific 16S rRNA ,  Ifng ,  Cxcl9 , and Cxcl10  was normalized to 1,000  β-actin  in the joint using qRT-PCR. (J) Unfractionated naive OT-I/Rag2 −/− /IL-10 −/−  splenocytes were isolated and stimulated in the presence of  B. burgdorferi (10µg/mL) or Ova 257–264  (1µM) for 3 hours. OT-I CD8 +  T cells were analyzed by flow cytometry for phosphorylated ZAP-70. Error bars indicate the SEM (n≥5 per group). Significant differences between infected and uninfected groups for each genotype are indicated by Student’s t-test (*p
    Figure Legend Snippet: B. burgdorferi infection of CD8 + TCR transgenic mice results in arthritis and IFN-γ production IL-10 −/− and CD8 + TCR transgenic OT-I/Rag2 −/− /IL-10 −/− mice were infected with B. burgdorferi for 4 weeks. (A) Rear ankles of mice were measured before infection and 4 weeks post-infection, and change in ankle measurement is shown. (B) The most swollen ankle was subjected to histopathologic evaluation in a blind fashion. Scores 0–5, with 5 being most severe, were assigned to each sample. (C) The total number of CD8 + were quantified from the popliteal and inguinal lymph nodes. (D) Lymph node cells were stimulated with PMA/Ionomycin in presence of Brefeldin A, and the number of CD8 + IFN-γ + T cells were quantified using flow cytometry. (E–I) Quantification of B. burgdorferi -specific 16S rRNA , Ifng , Cxcl9 , and Cxcl10 was normalized to 1,000 β-actin in the joint using qRT-PCR. (J) Unfractionated naive OT-I/Rag2 −/− /IL-10 −/− splenocytes were isolated and stimulated in the presence of B. burgdorferi (10µg/mL) or Ova 257–264 (1µM) for 3 hours. OT-I CD8 + T cells were analyzed by flow cytometry for phosphorylated ZAP-70. Error bars indicate the SEM (n≥5 per group). Significant differences between infected and uninfected groups for each genotype are indicated by Student’s t-test (*p

    Techniques Used: Infection, Transgenic Assay, Mouse Assay, Flow Cytometry, Cytometry, Quantitative RT-PCR, Isolation

    B. burgdorferi  infection results in antigen-independent expansion and activation of TCR transgenic CD4 +  T cells IL-10 −/−  and CD4 +  TCR transgenic SMARTA/TCRα −/− /IL-10 −/− mice were infected with  B. burgdorferi  for 4 weeks. (A) Measurements of rear ankles of mice were taken before infection and 4 weeks post-infection, and change in ankle measurement is shown. (B) Representative images of H  E-stained tibiotarsal joints from mock-infected (control) and 4 week-infected SMARTA/TCRα −/− /IL-10 −/− mice used for histopathology scoring t= tendon, * = tendon space, ts= tendon sheath, arrow = tenocytes, arrowhead = synoviocytes, a = arteriole. scale bar = 100 µm. (C) The most swollen ankle was subjected to blinded histopathologic evaluation in a blind fashion. Scores 0–5, with 5 being most severe, were assigned to each sample. (D) The total number of CD4 +  T cells were quantified from the popliteal and inguinal lymph nodes. (E) Lymph node cells were stimulated with PMA/Ionomycin in presence of Brefeldin A, and the number of CD4 + IFN-γ +  T cells were quantified using flow cytometry. (F–I) Quantification of  B. burgdorferi -specific 16S rRNA ,  Ifng ,  Cxcl9 , and Cxcl10  was normalized to 1,000  β-actin  in the joint using qRT-PCR. (J–K) Serum was obtained from mice two weeks post-infection and assessed for  B. burgdorferi -specific IgM (J) and  B. burgdorferi -specific IgG (K) using sonicated  B. burgdorferi bound to ELISA plates ( Methods ). (L) Unfractionated naive SMARTA/TCRα −/− /IL-10 −/− splenocytes were isolated and stimulated in the presence of  B. burgdorferi  (10µg/mL) or GP 61–80  (1µM) for 3 hours. SMARTA CD4 +  T cells were analyzed by flow cytometry for phosphorylated ZAP-70. Error bars indicate the SEM (n≥8 per group) Significant differences between mock and  B. burgdorferi  infected groups for each genotype were determined by Student’s t-test, with Mann-Whitney  U  test used for categorically accessed lesion scores (C) (*p
    Figure Legend Snippet: B. burgdorferi infection results in antigen-independent expansion and activation of TCR transgenic CD4 + T cells IL-10 −/− and CD4 + TCR transgenic SMARTA/TCRα −/− /IL-10 −/− mice were infected with B. burgdorferi for 4 weeks. (A) Measurements of rear ankles of mice were taken before infection and 4 weeks post-infection, and change in ankle measurement is shown. (B) Representative images of H E-stained tibiotarsal joints from mock-infected (control) and 4 week-infected SMARTA/TCRα −/− /IL-10 −/− mice used for histopathology scoring t= tendon, * = tendon space, ts= tendon sheath, arrow = tenocytes, arrowhead = synoviocytes, a = arteriole. scale bar = 100 µm. (C) The most swollen ankle was subjected to blinded histopathologic evaluation in a blind fashion. Scores 0–5, with 5 being most severe, were assigned to each sample. (D) The total number of CD4 + T cells were quantified from the popliteal and inguinal lymph nodes. (E) Lymph node cells were stimulated with PMA/Ionomycin in presence of Brefeldin A, and the number of CD4 + IFN-γ + T cells were quantified using flow cytometry. (F–I) Quantification of B. burgdorferi -specific 16S rRNA , Ifng , Cxcl9 , and Cxcl10 was normalized to 1,000 β-actin in the joint using qRT-PCR. (J–K) Serum was obtained from mice two weeks post-infection and assessed for B. burgdorferi -specific IgM (J) and B. burgdorferi -specific IgG (K) using sonicated B. burgdorferi bound to ELISA plates ( Methods ). (L) Unfractionated naive SMARTA/TCRα −/− /IL-10 −/− splenocytes were isolated and stimulated in the presence of B. burgdorferi (10µg/mL) or GP 61–80 (1µM) for 3 hours. SMARTA CD4 + T cells were analyzed by flow cytometry for phosphorylated ZAP-70. Error bars indicate the SEM (n≥8 per group) Significant differences between mock and B. burgdorferi infected groups for each genotype were determined by Student’s t-test, with Mann-Whitney U test used for categorically accessed lesion scores (C) (*p

    Techniques Used: Infection, Activation Assay, Transgenic Assay, Mouse Assay, Staining, Histopathology, Flow Cytometry, Cytometry, Quantitative RT-PCR, Sonication, Enzyme-linked Immunosorbent Assay, Isolation, MANN-WHITNEY

    CD4 + and CD8 + T cells produce IFN-γ throughout B. burgdorferi infection IL-10 −/− mice were infected with B. burgdorferi , after which popliteal and inguinal lymph nodes were collected for analysis of T cells and IFN-γ production. (A) B. burgdorferi infected or mock-infected IL-10 −/− mice were sacrificed after 4 weeks of infection. Representative flow plots of IFN-γ production by CD4 + and CD8 + T cells in after 4 hours of stimulation with PMA/Ionomycin in presence of Brefeldin A. Numbers in upper left corner of boxes are %IFN-γ + cells. (B) The total number of IFN-γ + CD4 + and IFN-γ + CD8 + T by flow cytometry and the mean fluorescence intensity (MFI) of IFN-γ. (C–F) B. burgdorferi infected or mock-infected IL-10 −/− mice were injected with Brefeldin A 6 hours before sacrifice at 1 week time intervals for 4 weeks. (C) Representative flow plots of IFN-γ production by CD4 + and CD8 + T cells in B. burgdorferi infected or mock-infected IL-10 −/− mice at 3 weeks of infection. Numbers in upper left corner of boxes are %IFN-γ + cells. (D) Total number of CD4 + IFN-γ + and CD8 + IFN-γ + T cells from infected and mock-infected IL-10 −/− mice. (E) Representative flow plots of CD4 + CD44 hi and CD8 + CD44 hi T cells in B. burgdorferi infected or mock-infected IL-10 −/− mice. Numbers in upper left corner of boxes are %CD44 hi cells. (F) Total number of CD4 + CD44 hi and CD8 + CD44 hi T cells from infected and mock-infected IL-10 −/− mice. Error bars indicate the SEM (n≥3 per group). Statistically significant differences between groups by Student’s t-test are indicated (*p
    Figure Legend Snippet: CD4 + and CD8 + T cells produce IFN-γ throughout B. burgdorferi infection IL-10 −/− mice were infected with B. burgdorferi , after which popliteal and inguinal lymph nodes were collected for analysis of T cells and IFN-γ production. (A) B. burgdorferi infected or mock-infected IL-10 −/− mice were sacrificed after 4 weeks of infection. Representative flow plots of IFN-γ production by CD4 + and CD8 + T cells in after 4 hours of stimulation with PMA/Ionomycin in presence of Brefeldin A. Numbers in upper left corner of boxes are %IFN-γ + cells. (B) The total number of IFN-γ + CD4 + and IFN-γ + CD8 + T by flow cytometry and the mean fluorescence intensity (MFI) of IFN-γ. (C–F) B. burgdorferi infected or mock-infected IL-10 −/− mice were injected with Brefeldin A 6 hours before sacrifice at 1 week time intervals for 4 weeks. (C) Representative flow plots of IFN-γ production by CD4 + and CD8 + T cells in B. burgdorferi infected or mock-infected IL-10 −/− mice at 3 weeks of infection. Numbers in upper left corner of boxes are %IFN-γ + cells. (D) Total number of CD4 + IFN-γ + and CD8 + IFN-γ + T cells from infected and mock-infected IL-10 −/− mice. (E) Representative flow plots of CD4 + CD44 hi and CD8 + CD44 hi T cells in B. burgdorferi infected or mock-infected IL-10 −/− mice. Numbers in upper left corner of boxes are %CD44 hi cells. (F) Total number of CD4 + CD44 hi and CD8 + CD44 hi T cells from infected and mock-infected IL-10 −/− mice. Error bars indicate the SEM (n≥3 per group). Statistically significant differences between groups by Student’s t-test are indicated (*p

    Techniques Used: Infection, Mouse Assay, Flow Cytometry, Cytometry, Fluorescence, Injection

    33) Product Images from "Adipocyte Piezo1 mediates obesogenic adipogenesis through the FGF1/FGFR1 signaling pathway in mice"

    Article Title: Adipocyte Piezo1 mediates obesogenic adipogenesis through the FGF1/FGFR1 signaling pathway in mice

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16026-w

    FGF1 mediates Piezo1-dependent regulation of adipocyte differentiation. a Yoda1 (10 μM) effect on release of protein mediators (red) and lipids (black) from mouse adipocytes (ratio of release in the presence and absence of Yoda-1 ( n = 5 mice)). b , c Effect of Yoda1 (10 µM) or hypotonic buffer (hypo; 210 mOsm/kg) on FGF1 release from vWAT adipocytes from wild-type (WT) and Ad-Piezo1-KO mice (KO) ( b ) or from human vWAT ( c ) ( n = 3 mice ( b ), n = 4 samples ( c )). d , e Effect of Yoda1 (10 μM; d ) or hypotonic buffer (hypo; 210 mOsm/kg; e ) on FGF1 release from mouse adipocytes pretreated in the absence or presence of brefeldin A (bref. A) or BAPTA-AM ( n = 3 mice (control and bref. A in d as well as control and control + bref. A in e ); n = 5 (control + hypo in e ); n = 4 mice (BAPTA in d and all other groups)). f PD173074 (PD; 10 μM) effect on 3T3-F442A cell differentiation induced by conditioned medium of vWAT adipocytes from wild-type mice fed HFD for 16 weeks and effect of FGF1 (10 ng/ml) and FGF1 + PD173074 on differentiation of 3T3-F442A cells exposed to conditioned medium from vWAT adipocytes from KO animals. Control: non-conditioned medium. Cells were stained with Oil-red-O. g Differentiation of SVF cells from wild-type or Pdgfrα-CreERT2;Fgfr1 flox/flox (Pα-Fgfr1-KO) mice exposed to conditioned medium from wild-type adipocytes from HFD-fed mice (Oil-red-O staining; n = 4 mice per condition). h H E-stained vWAT sections from wild-type (WT) and Pα-Fgfr1-KO mice fed a HFD for 12 weeks (one representative of three independent experiments). i , j Average diameter ( i ) and total number ( j ) of adipocytes in vWAT from wild-type (WT) and Pα-Fgfr1-KO mice fed a HFD ( n = 5 mice each). k Wild-type (WT) and Pα-Fgfr1-KO mice received BrdU during the first week of an 8-week HFD feeding period. Representative images and quantifications of immunofluorescence staining for BrdU in adipocyte nuclei of vWAT after HFD feeding ( n = 5 mice (≥15 sections per mouse)). Bar lengths in f , g , h , k : 50 μm. Shown are mean values ± s.e.m.; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; n.s., not significant (two-tailed non-parametric Mann–Whitney U -test). Source data are provided as a Source data file.
    Figure Legend Snippet: FGF1 mediates Piezo1-dependent regulation of adipocyte differentiation. a Yoda1 (10 μM) effect on release of protein mediators (red) and lipids (black) from mouse adipocytes (ratio of release in the presence and absence of Yoda-1 ( n = 5 mice)). b , c Effect of Yoda1 (10 µM) or hypotonic buffer (hypo; 210 mOsm/kg) on FGF1 release from vWAT adipocytes from wild-type (WT) and Ad-Piezo1-KO mice (KO) ( b ) or from human vWAT ( c ) ( n = 3 mice ( b ), n = 4 samples ( c )). d , e Effect of Yoda1 (10 μM; d ) or hypotonic buffer (hypo; 210 mOsm/kg; e ) on FGF1 release from mouse adipocytes pretreated in the absence or presence of brefeldin A (bref. A) or BAPTA-AM ( n = 3 mice (control and bref. A in d as well as control and control + bref. A in e ); n = 5 (control + hypo in e ); n = 4 mice (BAPTA in d and all other groups)). f PD173074 (PD; 10 μM) effect on 3T3-F442A cell differentiation induced by conditioned medium of vWAT adipocytes from wild-type mice fed HFD for 16 weeks and effect of FGF1 (10 ng/ml) and FGF1 + PD173074 on differentiation of 3T3-F442A cells exposed to conditioned medium from vWAT adipocytes from KO animals. Control: non-conditioned medium. Cells were stained with Oil-red-O. g Differentiation of SVF cells from wild-type or Pdgfrα-CreERT2;Fgfr1 flox/flox (Pα-Fgfr1-KO) mice exposed to conditioned medium from wild-type adipocytes from HFD-fed mice (Oil-red-O staining; n = 4 mice per condition). h H E-stained vWAT sections from wild-type (WT) and Pα-Fgfr1-KO mice fed a HFD for 12 weeks (one representative of three independent experiments). i , j Average diameter ( i ) and total number ( j ) of adipocytes in vWAT from wild-type (WT) and Pα-Fgfr1-KO mice fed a HFD ( n = 5 mice each). k Wild-type (WT) and Pα-Fgfr1-KO mice received BrdU during the first week of an 8-week HFD feeding period. Representative images and quantifications of immunofluorescence staining for BrdU in adipocyte nuclei of vWAT after HFD feeding ( n = 5 mice (≥15 sections per mouse)). Bar lengths in f , g , h , k : 50 μm. Shown are mean values ± s.e.m.; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; n.s., not significant (two-tailed non-parametric Mann–Whitney U -test). Source data are provided as a Source data file.

    Techniques Used: Mouse Assay, Cell Differentiation, Staining, Immunofluorescence, Two Tailed Test, MANN-WHITNEY

    34) Product Images from "N-terminal cleavage of proTGF? occurs at the cell surface by a TACE-independent activity"

    Article Title: N-terminal cleavage of proTGF? occurs at the cell surface by a TACE-independent activity

    Journal: Biochemical Journal

    doi: 10.1042/BJ20041128

    ProTGFα N-terminal shedding occurs outside the ER ( A ) Action of brefeldin A on proTGFα forms. CHO TGFα cells were incubated overnight with brefeldin A (10 μg/ml), and/or BB3103 (10 μM). Where indicated, proteinase K was added to the cells (200 μg/ml) for 30 min at room temperature. The different forms of proTGFα were analysed by Western blotting. The arrow indicates the position of the ≈20 kDa form. ( B ) CHO cells expressing HA-tagged proTGFα were labelled with 500 μCi/ml of [ 35 S]cysteine for 20 min and then chased in complete medium for 30 min. Cells were then washed with cold PBS and incubated at 4 °C with 10 μg/ml of anti-HA for 1 h. Unbound anti-HA antibodies were washed, and the monolayers were incubated in DMEM at 37 °C for the indicated time points. Immune complexes were recovered and analysed by SDS/PAGE, followed by autoradiography. ( C ) CHO TGFα cells were incubated overnight with brefeldin A (10 μg/ml), and then chased from this compound for the indicated times. ProTGFα forms were analysed by Western blotting. Molecular masses are indicated in kDa in all panels.
    Figure Legend Snippet: ProTGFα N-terminal shedding occurs outside the ER ( A ) Action of brefeldin A on proTGFα forms. CHO TGFα cells were incubated overnight with brefeldin A (10 μg/ml), and/or BB3103 (10 μM). Where indicated, proteinase K was added to the cells (200 μg/ml) for 30 min at room temperature. The different forms of proTGFα were analysed by Western blotting. The arrow indicates the position of the ≈20 kDa form. ( B ) CHO cells expressing HA-tagged proTGFα were labelled with 500 μCi/ml of [ 35 S]cysteine for 20 min and then chased in complete medium for 30 min. Cells were then washed with cold PBS and incubated at 4 °C with 10 μg/ml of anti-HA for 1 h. Unbound anti-HA antibodies were washed, and the monolayers were incubated in DMEM at 37 °C for the indicated time points. Immune complexes were recovered and analysed by SDS/PAGE, followed by autoradiography. ( C ) CHO TGFα cells were incubated overnight with brefeldin A (10 μg/ml), and then chased from this compound for the indicated times. ProTGFα forms were analysed by Western blotting. Molecular masses are indicated in kDa in all panels.

    Techniques Used: Incubation, Western Blot, Expressing, SDS Page, Autoradiography

    35) Product Images from "Regulation of T-type Ca2+ channel expression by herpes simplex virus-1 infection in sensory-like ND7 cells"

    Article Title: Regulation of T-type Ca2+ channel expression by herpes simplex virus-1 infection in sensory-like ND7 cells

    Journal: Journal of neurovirology

    doi: 10.1007/s13365-017-0545-9

    Effect of cycloheximide, acyclovir, brefeldin-A, chloroquine, and lactacystin on T-type Ca 2+ channel expression following infection of differentiated ND7-23 cells with HSV-1. ND7-23 cells were cultured for ~4 days with differentiation media, supplemented
    Figure Legend Snippet: Effect of cycloheximide, acyclovir, brefeldin-A, chloroquine, and lactacystin on T-type Ca 2+ channel expression following infection of differentiated ND7-23 cells with HSV-1. ND7-23 cells were cultured for ~4 days with differentiation media, supplemented

    Techniques Used: Expressing, Infection, Cell Culture

    Effect of cycloheximide, acyclovir, brefeldin-A, chloroquine, and lactacystin on viral replication following infection of differentiated ND7-23 cells with HSV-1. a Cycloheximide, acyclovir, brefeldin-A, chloroquine, and lactacystin evoke a considerable
    Figure Legend Snippet: Effect of cycloheximide, acyclovir, brefeldin-A, chloroquine, and lactacystin on viral replication following infection of differentiated ND7-23 cells with HSV-1. a Cycloheximide, acyclovir, brefeldin-A, chloroquine, and lactacystin evoke a considerable

    Techniques Used: Infection

    36) Product Images from "Contribution of Herpesvirus Specific CD8 T Cells to Anti-Viral T Cell Response in Humans"

    Article Title: Contribution of Herpesvirus Specific CD8 T Cells to Anti-Viral T Cell Response in Humans

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1001051

    Functional analysis of HCMV and EBV-specific CD8 T cells during acute heterologous viral infections. A) Schematic representation of experimental design: PBMC from healthy volunteers were incubated for 48 h in presence or absence of IL-15. Cells were then collected, incubated with pentamer or in PBS for 15 min, washed and incubated for 5 h in presence of Brefeldin A. Following the 5 h incubation the cells were collected and the unstimulated cells, those that have not been stained with pentamer prior to BFA incubation, were stained with pentamer for 15 min. Then the cells were washed and ICS performed. The expression of IFN-γ in EBV-pentamer+ (EBNA-1 407–417, +EBNA4, 416–424), HCMV-pentamer+ (pp65 495–504) and Flu-pentamer+ (MP, 58–66) CD8 T cells is shown. Histogram plots gated on CD3+ CD8+ pentamer + cells (contour plots) of one representative healthy individual are shown. B) Percent of IFN-γ+ MHC/peptide pentamer stimulated HCMV-, EBV- and Flu-specific CD8 T cells is shown based on gating for unstimulated cells cultured without cytokine. C) Histogram plots representing IFN-γ production of unstimulated and MHC/peptide pentamer stimulated HCMV-pentamer+ (pp65 495–504 + pp65 123–131) CD8 T cells in a representative HBV acute patient. D) Percent increase of IFN-γ production by MHC-peptide pentamer stimulated HCMV-, EBV- and Flu-specific CD8 T cells during the onset of acute HBV infection compared to the resolution.
    Figure Legend Snippet: Functional analysis of HCMV and EBV-specific CD8 T cells during acute heterologous viral infections. A) Schematic representation of experimental design: PBMC from healthy volunteers were incubated for 48 h in presence or absence of IL-15. Cells were then collected, incubated with pentamer or in PBS for 15 min, washed and incubated for 5 h in presence of Brefeldin A. Following the 5 h incubation the cells were collected and the unstimulated cells, those that have not been stained with pentamer prior to BFA incubation, were stained with pentamer for 15 min. Then the cells were washed and ICS performed. The expression of IFN-γ in EBV-pentamer+ (EBNA-1 407–417, +EBNA4, 416–424), HCMV-pentamer+ (pp65 495–504) and Flu-pentamer+ (MP, 58–66) CD8 T cells is shown. Histogram plots gated on CD3+ CD8+ pentamer + cells (contour plots) of one representative healthy individual are shown. B) Percent of IFN-γ+ MHC/peptide pentamer stimulated HCMV-, EBV- and Flu-specific CD8 T cells is shown based on gating for unstimulated cells cultured without cytokine. C) Histogram plots representing IFN-γ production of unstimulated and MHC/peptide pentamer stimulated HCMV-pentamer+ (pp65 495–504 + pp65 123–131) CD8 T cells in a representative HBV acute patient. D) Percent increase of IFN-γ production by MHC-peptide pentamer stimulated HCMV-, EBV- and Flu-specific CD8 T cells during the onset of acute HBV infection compared to the resolution.

    Techniques Used: Functional Assay, Incubation, Staining, Expressing, Cell Culture, Infection

    37) Product Images from "Phagocytosis and LPS alter the maturation state of ?-amyloid precursor protein and induce different A? peptide release signatures in human mononuclear phagocytes"

    Article Title: Phagocytosis and LPS alter the maturation state of ?-amyloid precursor protein and induce different A? peptide release signatures in human mononuclear phagocytes

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-7-59

    Shifts in full length APP patterns induced by the glycosylation inhibitors tunicamycin and brefeldin A in mononuclear phagocyte cultures . Human mononuclear phagocytes were isolated as indicated and left unstimulated on ultra-low attachment plates for 3 days. 6 h, 4 h, 2 h or 0.5 h prior to the lysis of the cells, tunicamycin or brefeldin A were added in a concentration of 10 μg/ml each. Cells were lysed in RIPA buffer and APP expression was analysed by separation on 7.5% SDS-PAGE, subsequent blotting on PVDF-membranes and staining with the 1E8 monoclonal antibody. Staining of β-actin served as a loading control. The right hand side shows a molecular weight standard. Note the slight shift in molecular weight after brefeldin A treatment and the decrease of band APP1 corresponding to mature APP and the increased amounts of the bands APP2 and APP3 after treatment with Tunicamycin.
    Figure Legend Snippet: Shifts in full length APP patterns induced by the glycosylation inhibitors tunicamycin and brefeldin A in mononuclear phagocyte cultures . Human mononuclear phagocytes were isolated as indicated and left unstimulated on ultra-low attachment plates for 3 days. 6 h, 4 h, 2 h or 0.5 h prior to the lysis of the cells, tunicamycin or brefeldin A were added in a concentration of 10 μg/ml each. Cells were lysed in RIPA buffer and APP expression was analysed by separation on 7.5% SDS-PAGE, subsequent blotting on PVDF-membranes and staining with the 1E8 monoclonal antibody. Staining of β-actin served as a loading control. The right hand side shows a molecular weight standard. Note the slight shift in molecular weight after brefeldin A treatment and the decrease of band APP1 corresponding to mature APP and the increased amounts of the bands APP2 and APP3 after treatment with Tunicamycin.

    Techniques Used: Isolation, Lysis, Concentration Assay, Expressing, SDS Page, Staining, Molecular Weight

    38) Product Images from "Shared Molecular Mechanisms in Alzheimer’s Disease and Amyotrophic Lateral Sclerosis: Neurofilament-dependent Transport of sAPP, FUS, TDP-43, and SOD1, with Endoplasmic Reticulum-like Tubules"

    Article Title: Shared Molecular Mechanisms in Alzheimer’s Disease and Amyotrophic Lateral Sclerosis: Neurofilament-dependent Transport of sAPP, FUS, TDP-43, and SOD1, with Endoplasmic Reticulum-like Tubules

    Journal: Neuro-degenerative diseases

    doi: 10.1159/000439256

    Transport into neurites of sAPP is not affected by Brefeldin A (BFA) treatment. CAD cells were subjected to BFA treatment for 22 hrs, and probed for the distribution of GM130 (a Golgi marker), APP C-terminal (APP C , detected with antibody Y188; a ) and
    Figure Legend Snippet: Transport into neurites of sAPP is not affected by Brefeldin A (BFA) treatment. CAD cells were subjected to BFA treatment for 22 hrs, and probed for the distribution of GM130 (a Golgi marker), APP C-terminal (APP C , detected with antibody Y188; a ) and

    Techniques Used: Marker

    39) Product Images from "The role of TLR9 on Leishmania amazonensis infection and its influence on intranasal LaAg vaccine efficacy"

    Article Title: The role of TLR9 on Leishmania amazonensis infection and its influence on intranasal LaAg vaccine efficacy

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0007146

    IFN-γ produced by CD4 +  T cells present in the draining lymph node. Lymph node cells isolated from mice after 67 days of infection were cultured for 4 h in a solution with 20 ng/ml of PMA, 1μg/ml Ionomycin and brefeldin A. Cells were surface stained with anti-CD3-PerCP and anti-CD4-PE-Cy7 and evaluated by flow cytometry (FACSCanto II BD).  A.  CD4 +  T IFN-γ + B.  CD4 +  T IL-10 + C.  Dot plot of CD4 +  T staining gated on. Mean ± SD (n = 4–5); (*) P ≤ 0.05. Results representative of two independent experiments.
    Figure Legend Snippet: IFN-γ produced by CD4 + T cells present in the draining lymph node. Lymph node cells isolated from mice after 67 days of infection were cultured for 4 h in a solution with 20 ng/ml of PMA, 1μg/ml Ionomycin and brefeldin A. Cells were surface stained with anti-CD3-PerCP and anti-CD4-PE-Cy7 and evaluated by flow cytometry (FACSCanto II BD). A. CD4 + T IFN-γ + B. CD4 + T IL-10 + C. Dot plot of CD4 + T staining gated on. Mean ± SD (n = 4–5); (*) P ≤ 0.05. Results representative of two independent experiments.

    Techniques Used: Produced, Isolation, Mouse Assay, Infection, Cell Culture, Staining, Flow Cytometry, Cytometry

    IFN-γ produced by CD8 +  T cells present in the draining lymph node. Lymph node cells isolated from mice after 67 days of infection were cultured for 4 h in a solution with 20 ng/ml of PMA, 1μg/ml Ionomycin and brefeldin A. Cells were surface stained with anti-CD3-PerCP and anti-CD8-APC-CY7 and evaluated by flow cytometry (FACSCanto II BD). CD8 +  T producing IFNγ +  A. percentage B. number of cells. CD8 +  T producing IL-10 +  C. percentage D. number of cells. Dot plot of CD8 +  T cell gating strategy. (Mean ± SD; n = 4–5); (*) P ≤ 0.05. Results representative of two independent experiments.
    Figure Legend Snippet: IFN-γ produced by CD8 + T cells present in the draining lymph node. Lymph node cells isolated from mice after 67 days of infection were cultured for 4 h in a solution with 20 ng/ml of PMA, 1μg/ml Ionomycin and brefeldin A. Cells were surface stained with anti-CD3-PerCP and anti-CD8-APC-CY7 and evaluated by flow cytometry (FACSCanto II BD). CD8 + T producing IFNγ + A. percentage B. number of cells. CD8 + T producing IL-10 + C. percentage D. number of cells. Dot plot of CD8 + T cell gating strategy. (Mean ± SD; n = 4–5); (*) P ≤ 0.05. Results representative of two independent experiments.

    Techniques Used: Produced, Isolation, Mouse Assay, Infection, Cell Culture, Staining, Flow Cytometry, Cytometry

    40) Product Images from "High Throughput Analysis of Golgi Structure by Imaging Flow Cytometry"

    Article Title: High Throughput Analysis of Golgi Structure by Imaging Flow Cytometry

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-00909-y

    Brefeldin A induced rapid Golgi fragmentation. HeLa cells were treated with 0.2 μM BFA for the indicated times, then fixed and stained for GM130 and the Draq5. (A) Intact, Partially and fully fragmented populations were analyzed by IFC at each time point. (B) Representative IFC images of cells from non-treated and 60 min BFA treatment. (C) HeLa cells were treated with 0.2 μM BFA or vehicle for 60 min or left untreated. Cells were fixed and stained for GM130 and DAPI. Cells were imaged by confocal spinning disc microscope, manually counted and classified into the 3 Golgi populations. More than 150 cells were counted for each treatment, n = 2. (D) HeLa cells were treated with 0.2 μM BFA for 60 min or left untreated. Representative histograms of PulSA values (the width of the GM130 signal) for GM130 of two independent experiments.
    Figure Legend Snippet: Brefeldin A induced rapid Golgi fragmentation. HeLa cells were treated with 0.2 μM BFA for the indicated times, then fixed and stained for GM130 and the Draq5. (A) Intact, Partially and fully fragmented populations were analyzed by IFC at each time point. (B) Representative IFC images of cells from non-treated and 60 min BFA treatment. (C) HeLa cells were treated with 0.2 μM BFA or vehicle for 60 min or left untreated. Cells were fixed and stained for GM130 and DAPI. Cells were imaged by confocal spinning disc microscope, manually counted and classified into the 3 Golgi populations. More than 150 cells were counted for each treatment, n = 2. (D) HeLa cells were treated with 0.2 μM BFA for 60 min or left untreated. Representative histograms of PulSA values (the width of the GM130 signal) for GM130 of two independent experiments.

    Techniques Used: Staining, Microscopy

    Related Articles

    Flow Cytometry:

    Article Title: The deletion of the ORF1 and ORF71 genes reduces virulence of the neuropathogenic EHV-1 strain Ab4 without compromising host immunity in horses
    Article Snippet: .. For intracellular staining of cytokines and flow cytometric analysis, the secretion of proteins from the cells was blocked by adding Brefeldin A (10 μg/ml; Sigma, St. Louis, MO) to the plates after 24 hours of culturing. ..

    Concentration Assay:

    Article Title: HIV-1 T cell epitopes targeted to Rhesus macaque CD40 and DCIR: A comparative study of prototype dendritic cell targeting therapeutic vaccine candidates
    Article Snippet: .. The following morning, cells were stimulated with peptide pools (2 μg/ml) in the presence of Brefeldin A (10 μg/ml; Sigma-Aldrich) for 6 h. Negative controls received an equal concentration of DMSO peptide solvent without peptides. .. Five groups of pooled peptides covering Gag p2.p6.p7 (non-HIV5pep), Gag p17, Gag p24, Nef and Pol, as well as Staphylococcal Enterotoxin B (1 μg/ml) as a positive control, were used for the stimulations [ ].

    Co-Culture Assay:

    Article Title: Human Macrophages Escape Inhibition of Major Histocompatibility Complex-Dependent Antigen Presentation by Cytomegalovirus and Drive Proliferation and Activation of Memory CD4+ and CD8+ T Cells
    Article Snippet: .. After 24 h, Mφ were co-cultured with autologous PBMC at a ratio 1:10 for further 18 h. For T-cell activation assays, 1 µg/ml brefeldin A (Sigma-Aldrich Chemie GmbH) was added after 18 h of Mφ/PBMC co-culture to all conditions for 4 h to prevent cytokine release. .. PBMC were then collected, permeabilized, stained with antibodies directed against CD4, CD8, CD69, and IFN-γ, and analyzed by flow cytometry as described above.

    Activation Assay:

    Article Title: Human Macrophages Escape Inhibition of Major Histocompatibility Complex-Dependent Antigen Presentation by Cytomegalovirus and Drive Proliferation and Activation of Memory CD4+ and CD8+ T Cells
    Article Snippet: .. After 24 h, Mφ were co-cultured with autologous PBMC at a ratio 1:10 for further 18 h. For T-cell activation assays, 1 µg/ml brefeldin A (Sigma-Aldrich Chemie GmbH) was added after 18 h of Mφ/PBMC co-culture to all conditions for 4 h to prevent cytokine release. .. PBMC were then collected, permeabilized, stained with antibodies directed against CD4, CD8, CD69, and IFN-γ, and analyzed by flow cytometry as described above.

    other:

    Article Title: A novel, cellulose synthesis inhibitory action of ancymidol impairs plant cell expansion
    Article Snippet: Chemicals Stock solutions of 100 mM ancymidol (α-cyclopropyl-α-[4-methoxyphenyl]-5-pyrimidine-methanol; Sigma), 1 mM isoxaben (Pestanal; Sigma), 100 mM DCB (Sigma), 2.53 mM latrunculin B (Sigma), 10 mM taxol (Paclitaxel; MP Biomedicals, Irvine, California, USA), 10 mM oryzalin (Surflan; Elanco Products Co., USA) in DMSO and stock solution of 20 mM brefeldin A (Sigma) in ethanol were prepared and appropriate volumes were added directly to the growth media to obtain the final concentrations required.

    Mass Spectrometry:

    Article Title: The Inhibitor Endosidin 4 Targets SEC7 Domain-Type ARF GTPase Exchange Factors and Interferes with Subcellular Trafficking in Eukaryotes [OPEN]
    Article Snippet: .. Stock solutions of BFA (Sigma-Aldrich), ES4 (Chembridge ID 6938485), and FM4-64 (Invitrogen) were prepared in DMSO and diluted in liquid half-strength MS (or growth) medium for treatments with the indicated concentrations and times. ..

    Staining:

    Article Title: The deletion of the ORF1 and ORF71 genes reduces virulence of the neuropathogenic EHV-1 strain Ab4 without compromising host immunity in horses
    Article Snippet: .. For intracellular staining of cytokines and flow cytometric analysis, the secretion of proteins from the cells was blocked by adding Brefeldin A (10 μg/ml; Sigma, St. Louis, MO) to the plates after 24 hours of culturing. ..

    Article Title: Blockade of LAG3 enhances responses of tumor-infiltrating T cells in mismatch repair-proficient liver metastases of colorectal cancer
    Article Snippet: .. For intracellular cytokine staining, cells were stimulated with 40 ng/mL PMA (Sigma, Zwijndrecht, The Netherlands) and 1 µg/mL ionomycin (Sigma) at 37°C for five hours in the presence of 5 µg/mL brefeldin (Sigma) for the last four hours, followed by staining of IFN-γ and TNF-α upon fixation and permeabilization using the Foxp3 staining buffer set. .. Dead cells were excluded by using a LIVE/DEAD fixable dead cell stain kit with aqua fluorescent reactive dye (Invitrogen, Paisley, UK).

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  • 99
    Millipore brefeldin a
    Csp accelerates turnover of CFTR. A , HEK293 cells were transfected with CFTR and Sar1-GTP, or CFTR and CspWT, or CFTR and CspH43Q plasmids and the next day treated with 100 μg/ml cycloheximide and 10 μg/ml <t>brefeldin</t> A for 0, 1, 2, or
    Brefeldin A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4643 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore cyanine 5 reactions
    Comparison of the labeling bias between conventional cDNA labeling and the new mRNA labeling technologies. RNA from HL-60 cells was labeled both with Cyanine 3 and <t>Cyanine</t> 5 (same versus same assay). Microarray data scatter plots of the intensity of the Cyanine 3 signals versus those observed for Cyanine 5 should ideally lie in a perfectly straight 45° line. Deviations from this line indicate both the inherent noise in the assay as well as any preference to incorporate one Cyanine dye over the other. The amount of labeling bias can be quantified by determining the percentage of the genes spots on the scatter plot that lie between the differential cut-off values. This figure shows that the percentage of non-differential genes drops off significantly for targets that were labeled enzymatically with the labeled cDNA assay as the differential cut-off intervals were narrowed. This indicates that there is significantly less (∼80% less) labeling bias observed with the labeled mRNA assay. Note that the differential expression axes are set at 1.75× in the labeled mRNA assays as compared with the traditional 2× in labeled cDNA assays. It shows that 97.3% of all genes are within the 1.75 differential axes (99.3% fall within the 2× differential axes). In comparison, only 95.9% genes fall within the 2× differential axes in a labeled cDNA assay.
    Cyanine 5 Reactions, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Csp accelerates turnover of CFTR. A , HEK293 cells were transfected with CFTR and Sar1-GTP, or CFTR and CspWT, or CFTR and CspH43Q plasmids and the next day treated with 100 μg/ml cycloheximide and 10 μg/ml brefeldin A for 0, 1, 2, or

    Journal: The Journal of Biological Chemistry

    Article Title: Cysteine String Protein Promotes Proteasomal Degradation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Increasing Its Interaction with the C Terminus of Hsp70-interacting Protein and Promoting CFTR Ubiquitylation

    doi: 10.1074/jbc.M806485200

    Figure Lengend Snippet: Csp accelerates turnover of CFTR. A , HEK293 cells were transfected with CFTR and Sar1-GTP, or CFTR and CspWT, or CFTR and CspH43Q plasmids and the next day treated with 100 μg/ml cycloheximide and 10 μg/ml brefeldin A for 0, 1, 2, or

    Article Snippet: Cycloheximide was from Calbiochem; Brefeldin A and other reagents were from Sigma.

    Techniques: Transfection

    Brefeldin A (BfA) blocks the exit of ErbB3 from the ER. (A) HEK-293T cells transfected with ErbB3 were treated without or with BfA; surface proteins were biotinylated; and cleared lysates (left) were immunoprecipitated (IP) with anti-ErbB3 antibodies

    Journal: Molecular and Cellular Biology

    Article Title: Quantity Control of the ErbB3 Receptor Tyrosine Kinase at the Endoplasmic Reticulum ▿

    doi: 10.1128/MCB.05105-11

    Figure Lengend Snippet: Brefeldin A (BfA) blocks the exit of ErbB3 from the ER. (A) HEK-293T cells transfected with ErbB3 were treated without or with BfA; surface proteins were biotinylated; and cleared lysates (left) were immunoprecipitated (IP) with anti-ErbB3 antibodies

    Article Snippet: HEK-293T cells in 10-cm-diameter plates were transfected with 5 μg ErbB3, and after 48 h, cells were treated with either a vehicle control or 1 μg/ml brefeldin A for 6 h. Rinsed cells were then incubated for 45 min with 0.5 mg/ml biotin-X- N -hydroxysuccinimide (NHS) (Calbiochem) in borate buffer (10 mM boric acid, 150 mM NaCl [pH 8.0]) at 4°C.

    Techniques: Transfection, Immunoprecipitation

    Nrdp1 acts on the nascent form of endogenous ErbB3. (A) MCF7 breast cancer cells stably transduced with scrambled or Nrdp1-directed shRNAs were treated for various times with brefeldin A. (Left) Lysates were immunoblotted with antibodies to ErbB3 and

    Journal: Molecular and Cellular Biology

    Article Title: Quantity Control of the ErbB3 Receptor Tyrosine Kinase at the Endoplasmic Reticulum ▿

    doi: 10.1128/MCB.05105-11

    Figure Lengend Snippet: Nrdp1 acts on the nascent form of endogenous ErbB3. (A) MCF7 breast cancer cells stably transduced with scrambled or Nrdp1-directed shRNAs were treated for various times with brefeldin A. (Left) Lysates were immunoblotted with antibodies to ErbB3 and

    Article Snippet: HEK-293T cells in 10-cm-diameter plates were transfected with 5 μg ErbB3, and after 48 h, cells were treated with either a vehicle control or 1 μg/ml brefeldin A for 6 h. Rinsed cells were then incubated for 45 min with 0.5 mg/ml biotin-X- N -hydroxysuccinimide (NHS) (Calbiochem) in borate buffer (10 mM boric acid, 150 mM NaCl [pH 8.0]) at 4°C.

    Techniques: Stable Transfection, Transduction

    Atovaquone specifically and rapidly downregulates cell-surface expression of gp130. (A) U266 cells were treated with atovaquone (20 μM), JAK inhibitor 1 (1 μM), or brefeldin A (3 μg/mL) for 2.5 hours, then analyzed by flow cytometry

    Journal: Blood

    Article Title: Gene expression–based discovery of atovaquone as a STAT3 inhibitor and anticancer agent

    doi: 10.1182/blood-2015-07-660506

    Figure Lengend Snippet: Atovaquone specifically and rapidly downregulates cell-surface expression of gp130. (A) U266 cells were treated with atovaquone (20 μM), JAK inhibitor 1 (1 μM), or brefeldin A (3 μg/mL) for 2.5 hours, then analyzed by flow cytometry

    Article Snippet: Lastly, brefeldin A, a compound that inhibits transport of all proteins from endoplasmic reticulum to Golgi, reduced the cell-surface expression of both gp130 and IL-6 receptor, suggesting atovaquone was not inhibiting protein transport in a nonspecific fashion.

    Techniques: Expressing, Flow Cytometry, Cytometry

    Comparison of the labeling bias between conventional cDNA labeling and the new mRNA labeling technologies. RNA from HL-60 cells was labeled both with Cyanine 3 and Cyanine 5 (same versus same assay). Microarray data scatter plots of the intensity of the Cyanine 3 signals versus those observed for Cyanine 5 should ideally lie in a perfectly straight 45° line. Deviations from this line indicate both the inherent noise in the assay as well as any preference to incorporate one Cyanine dye over the other. The amount of labeling bias can be quantified by determining the percentage of the genes spots on the scatter plot that lie between the differential cut-off values. This figure shows that the percentage of non-differential genes drops off significantly for targets that were labeled enzymatically with the labeled cDNA assay as the differential cut-off intervals were narrowed. This indicates that there is significantly less (∼80% less) labeling bias observed with the labeled mRNA assay. Note that the differential expression axes are set at 1.75× in the labeled mRNA assays as compared with the traditional 2× in labeled cDNA assays. It shows that 97.3% of all genes are within the 1.75 differential axes (99.3% fall within the 2× differential axes). In comparison, only 95.9% genes fall within the 2× differential axes in a labeled cDNA assay.

    Journal: Nucleic Acids Research

    Article Title: Directly labeled mRNA produces highly precise and unbiased differential gene expression data

    doi:

    Figure Lengend Snippet: Comparison of the labeling bias between conventional cDNA labeling and the new mRNA labeling technologies. RNA from HL-60 cells was labeled both with Cyanine 3 and Cyanine 5 (same versus same assay). Microarray data scatter plots of the intensity of the Cyanine 3 signals versus those observed for Cyanine 5 should ideally lie in a perfectly straight 45° line. Deviations from this line indicate both the inherent noise in the assay as well as any preference to incorporate one Cyanine dye over the other. The amount of labeling bias can be quantified by determining the percentage of the genes spots on the scatter plot that lie between the differential cut-off values. This figure shows that the percentage of non-differential genes drops off significantly for targets that were labeled enzymatically with the labeled cDNA assay as the differential cut-off intervals were narrowed. This indicates that there is significantly less (∼80% less) labeling bias observed with the labeled mRNA assay. Note that the differential expression axes are set at 1.75× in the labeled mRNA assays as compared with the traditional 2× in labeled cDNA assays. It shows that 97.3% of all genes are within the 1.75 differential axes (99.3% fall within the 2× differential axes). In comparison, only 95.9% genes fall within the 2× differential axes in a labeled cDNA assay.

    Article Snippet: Combined Cyanine 3 and Cyanine 5 reactions were purified using Microcon YM-100 columns (Millipore, Bedford, MA).

    Techniques: Labeling, Microarray, Expressing

    (Next page) ( A ) Comparison of microarray results obtained with the conventional cDNA labeling and the new mRNA chemical labeling methodologies. Two-color differential expression assay overlay images of Cyanine 3 and Cyanine 5 scans are shown along with their respective scatter-plots. In both experiments, the target from HL-60 cells was labeled with Cyanine 3 and the target from Jurkat cells was labeled with Cyanine 5 and hybridized to 4800-element cDNA arrays. It shows that the two methods produce very similar data. (i) Total RNA (50 µg) was used to produce labeled cDNA in a standard cDNA labeling assay with each Cyanine dye. Median S/N was 2.6 for Cyanine 3 and 2.4 for Cyanine 5. (ii) Total RNA (10 µg) was used to produce labeled mRNA in the new labeling assay with each Cyanine dye. Median S/N was 3.1 for Cyanine 3 and 4.4 for Cyanine 5. ( B ) Overlay scans (at same laser power and PMT settings 780 and 710 for the Cyanine 3 and Cyanine 5 channels respectively) from one quadrant of a microarray comparing expression patterns obtained from 2 µg of total RNA versus 10 µg of total RNA in a RNA labeling assay. It shows that 2 µg of total RNA produces data (median S/N was 3.2 for Cyanine 3 and 4.0 for Cyanine 5) similar to 10 µg of total RNA.

    Journal: Nucleic Acids Research

    Article Title: Directly labeled mRNA produces highly precise and unbiased differential gene expression data

    doi:

    Figure Lengend Snippet: (Next page) ( A ) Comparison of microarray results obtained with the conventional cDNA labeling and the new mRNA chemical labeling methodologies. Two-color differential expression assay overlay images of Cyanine 3 and Cyanine 5 scans are shown along with their respective scatter-plots. In both experiments, the target from HL-60 cells was labeled with Cyanine 3 and the target from Jurkat cells was labeled with Cyanine 5 and hybridized to 4800-element cDNA arrays. It shows that the two methods produce very similar data. (i) Total RNA (50 µg) was used to produce labeled cDNA in a standard cDNA labeling assay with each Cyanine dye. Median S/N was 2.6 for Cyanine 3 and 2.4 for Cyanine 5. (ii) Total RNA (10 µg) was used to produce labeled mRNA in the new labeling assay with each Cyanine dye. Median S/N was 3.1 for Cyanine 3 and 4.4 for Cyanine 5. ( B ) Overlay scans (at same laser power and PMT settings 780 and 710 for the Cyanine 3 and Cyanine 5 channels respectively) from one quadrant of a microarray comparing expression patterns obtained from 2 µg of total RNA versus 10 µg of total RNA in a RNA labeling assay. It shows that 2 µg of total RNA produces data (median S/N was 3.2 for Cyanine 3 and 4.0 for Cyanine 5) similar to 10 µg of total RNA.

    Article Snippet: Combined Cyanine 3 and Cyanine 5 reactions were purified using Microcon YM-100 columns (Millipore, Bedford, MA).

    Techniques: Polyacrylamide Gel Electrophoresis, Microarray, Labeling, Expressing