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 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    brefeldin a - by Bioz Stars, 2021-06
    97/100 stars

    Images

    1) Product Images from "Lymphocytes are a major source of circulating soluble dipeptidyl peptidase 4"

    Article Title: Lymphocytes are a major source of circulating soluble dipeptidyl peptidase 4

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/cei.13163

    Peripheral blood mononuclear cell (PBMC) stimulation by anti‐CD3 and superantigen toxic shock syndrome toxin 1 (TSST1) induces release of soluble dipeptidyl peptidase 4 (sDPP4). PBMCs were isolated from healthy donors and stimulated as indicated and treated or not with brefeldin A (BFA). (a) sDPP4 concentration and (b) activity were measured in supernatants after 20 or 44 h of culture. Bars represent errors of experimental duplicates. A representative experiment of seven independent experiments with different healthy donors is shown.
    Figure Legend Snippet: Peripheral blood mononuclear cell (PBMC) stimulation by anti‐CD3 and superantigen toxic shock syndrome toxin 1 (TSST1) induces release of soluble dipeptidyl peptidase 4 (sDPP4). PBMCs were isolated from healthy donors and stimulated as indicated and treated or not with brefeldin A (BFA). (a) sDPP4 concentration and (b) activity were measured in supernatants after 20 or 44 h of culture. Bars represent errors of experimental duplicates. A representative experiment of seven independent experiments with different healthy donors is shown.

    Techniques Used: Isolation, Concentration Assay, Activity Assay

    2) Product Images from "Protein kinase C regulates FLT1 abundance and stimulates its cleavage in vascular endothelial cells with the release of a soluble PlGF/VEGF antagonist"

    Article Title: Protein kinase C regulates FLT1 abundance and stimulates its cleavage in vascular endothelial cells with the release of a soluble PlGF/VEGF antagonist

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2013.07.005

    Role of glycosylation and effect of trafficking inhibitors on FLT1 cleavage and on sFLT1 secretion Panel A: FLT1 expressing vector or an empty vector transfected into HEK293 cells and then conditioned media digested with PNGase F and then immunoblotted with anti-HA and anti-sFLT1 (AF321). On deglycosylation with PNGase F, the ~130 kDa cleaved fragment undergoes a shift in molecular mass and appears as ~90 kDa peptide. Also seen with AF321 are native sFLT1 isoforms that also undergo a shift in mass. Panel B: FLT1 transfected HEK293 cells treated with vehicle, monensin, brefeldin A or tunicamycin and then subject to immunoblotting. Tunicamycin reduces the size of full length FLT1 (FL FLT1). All agents reduce cleavage of N and C-term FLT1 fragments. Panel C . sFLT1-i13 and sFLT1-e15a transfected HEK293 cells treated with vehicle, monensin, brefeldin A or tunicamycin and then lysates and conditioned media subject to immunoblotting with an anti-V5 antibody. Tunicamycin reduces the size of each sFLT1 isoform and all agents inhibit secretion of these isoforms.
    Figure Legend Snippet: Role of glycosylation and effect of trafficking inhibitors on FLT1 cleavage and on sFLT1 secretion Panel A: FLT1 expressing vector or an empty vector transfected into HEK293 cells and then conditioned media digested with PNGase F and then immunoblotted with anti-HA and anti-sFLT1 (AF321). On deglycosylation with PNGase F, the ~130 kDa cleaved fragment undergoes a shift in molecular mass and appears as ~90 kDa peptide. Also seen with AF321 are native sFLT1 isoforms that also undergo a shift in mass. Panel B: FLT1 transfected HEK293 cells treated with vehicle, monensin, brefeldin A or tunicamycin and then subject to immunoblotting. Tunicamycin reduces the size of full length FLT1 (FL FLT1). All agents reduce cleavage of N and C-term FLT1 fragments. Panel C . sFLT1-i13 and sFLT1-e15a transfected HEK293 cells treated with vehicle, monensin, brefeldin A or tunicamycin and then lysates and conditioned media subject to immunoblotting with an anti-V5 antibody. Tunicamycin reduces the size of each sFLT1 isoform and all agents inhibit secretion of these isoforms.

    Techniques Used: Expressing, Plasmid Preparation, Transfection

    3) Product Images from "Reolysin is a novel reovirus-based agent that induces endoplasmic reticular stress-mediated apoptosis in pancreatic cancer"

    Article Title: Reolysin is a novel reovirus-based agent that induces endoplasmic reticular stress-mediated apoptosis in pancreatic cancer

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.259

    Reolysin promotes caspase-4 processing and apoptosis and sensitizes cells to ER stress-mediated apoptosis. ( a ) Reolysin decreases cell viability in a panel of pancreatic cancer cell lines. Cells were treated with the indicated concentrations of Reolysin for 72 h. Cell viability was measured by MTT assay. Mean±S.D.,  n =3. ( b ) Reolysin promotes cleavage of caspase-4 and caspase-3. Panc-1 cells were treated with the indicated concentrations of Reolysin for 48 h, and caspase cleavage was measured by immunoblotting. Arrows denote cleaved caspase-4 fragments. ( c ) Reolysin induces apoptosis. Panc-1 and CFPAC-1 cells were treated with Reolysin for 48 h. Apoptosis was measured by PI-FACS analysis. Mean±S.D.,  n =3. *Indicates a significant difference compared with controls. ( d ) Reolysin augments ER stress-mediated apoptosis. Panc-1 cells were treated for 48 h with 300 PFU/cell Reolysin, 5  μ g/ml tunicamycin, 5  μ M brefeldin A, and combinations. Mean±S.D.,  n =3. *Indicates a significant difference compared with controls; **indicates a significant difference compared with either single-agent treatment  P
    Figure Legend Snippet: Reolysin promotes caspase-4 processing and apoptosis and sensitizes cells to ER stress-mediated apoptosis. ( a ) Reolysin decreases cell viability in a panel of pancreatic cancer cell lines. Cells were treated with the indicated concentrations of Reolysin for 72 h. Cell viability was measured by MTT assay. Mean±S.D., n =3. ( b ) Reolysin promotes cleavage of caspase-4 and caspase-3. Panc-1 cells were treated with the indicated concentrations of Reolysin for 48 h, and caspase cleavage was measured by immunoblotting. Arrows denote cleaved caspase-4 fragments. ( c ) Reolysin induces apoptosis. Panc-1 and CFPAC-1 cells were treated with Reolysin for 48 h. Apoptosis was measured by PI-FACS analysis. Mean±S.D., n =3. *Indicates a significant difference compared with controls. ( d ) Reolysin augments ER stress-mediated apoptosis. Panc-1 cells were treated for 48 h with 300 PFU/cell Reolysin, 5  μ g/ml tunicamycin, 5  μ M brefeldin A, and combinations. Mean±S.D., n =3. *Indicates a significant difference compared with controls; **indicates a significant difference compared with either single-agent treatment P

    Techniques Used: MTT Assay, FACS

    4) Product Images from "Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells"

    Article Title: Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0145217

    TNFα stimulates secretion of S100 proteins without causing cell death. THP-1 cells were cultured in triplicate either in the presence or absence of 10 ng/ml TNFα and TNFα + IL-10 (10 ng/ml). Culture supernatant was collected after 48h of incubation and S100A8/S100A9 dimer concentration was determined with ELISA following manufacturer’s protocol (A). Percent cell death was determined using LDH assay (B). In some experiments, THP-1 cells were stimulated with TNFα for 48 h in presence of methylamine (30 μM). In other experiments, cells were stimulated 12 h with TNFα without methylamine, followed by 36 h of culture with the drug. Alternatively, THP-1 cells were stimulated with TNFα; after 36 h, brefeldin A (5 μg/ml) was added and cells were cultured for another 12 h. Culture supernatant was collected after completion of incubation and S100A8/A9 heterodimer (C) and S100A9 (D) concentration was determined with ELISA following manufacturer’s protocol. Values are means of triplicates ± SD. Differences between various treatment groups are significant at **P
    Figure Legend Snippet: TNFα stimulates secretion of S100 proteins without causing cell death. THP-1 cells were cultured in triplicate either in the presence or absence of 10 ng/ml TNFα and TNFα + IL-10 (10 ng/ml). Culture supernatant was collected after 48h of incubation and S100A8/S100A9 dimer concentration was determined with ELISA following manufacturer’s protocol (A). Percent cell death was determined using LDH assay (B). In some experiments, THP-1 cells were stimulated with TNFα for 48 h in presence of methylamine (30 μM). In other experiments, cells were stimulated 12 h with TNFα without methylamine, followed by 36 h of culture with the drug. Alternatively, THP-1 cells were stimulated with TNFα; after 36 h, brefeldin A (5 μg/ml) was added and cells were cultured for another 12 h. Culture supernatant was collected after completion of incubation and S100A8/A9 heterodimer (C) and S100A9 (D) concentration was determined with ELISA following manufacturer’s protocol. Values are means of triplicates ± SD. Differences between various treatment groups are significant at **P

    Techniques Used: Cell Culture, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay

    5) Product Images from "Identification of novel HIV-1 dependency factors in primary CCR4+CCR6+Th17 cells via a genome-wide transcriptional approach"

    Article Title: Identification of novel HIV-1 dependency factors in primary CCR4+CCR6+Th17 cells via a genome-wide transcriptional approach

    Journal: Retrovirology

    doi: 10.1186/s12977-015-0226-9

    Proliferation of CD4 + T-cells expressing IL-17A versus IFN-γ in response to low TCR triggering. Memory CD4 + T-cells were isolated from PBMCs of four different HIV-uninfected donors by negative selection using magnetic beads. Cells were loaded with CFSE (0.5 µM) and cultured in the presence of different doses of immobilized CD3 and soluble CD28 Abs (0.1, 0.25, or 0.5 µg/ml) for 3 days. Cells were then stimulated with PMA/Ionomycin in the presence of Brefeldin A for 18 h. Intracellular staining was performed with IL-17A and IFN-γ Abs. Based on the expression of IL-17A and/or IFN-γ, three cell subsets were identified as follows: Th1 (IL-17A − IFN-γ + ), Th17 (IL-17A + IFN-γ − ), and Th1Th17 (IL-17A + IFN-γ + ). The frequency of proliferating cells (CFSE low ) was analyzed in each of the three cytokine-expressing subsets. a Shown are flow cytometry results in one representative donor out of four. Shown are statistical analyses of absolute (b) and relative (Th1Th17 proliferation levels were considered 100 %) c proliferation (CFSE low ) levels in Th1, Th17, and Th1Th17 cells from four different donors. Paired t t est p values are indicated on the graphs
    Figure Legend Snippet: Proliferation of CD4 + T-cells expressing IL-17A versus IFN-γ in response to low TCR triggering. Memory CD4 + T-cells were isolated from PBMCs of four different HIV-uninfected donors by negative selection using magnetic beads. Cells were loaded with CFSE (0.5 µM) and cultured in the presence of different doses of immobilized CD3 and soluble CD28 Abs (0.1, 0.25, or 0.5 µg/ml) for 3 days. Cells were then stimulated with PMA/Ionomycin in the presence of Brefeldin A for 18 h. Intracellular staining was performed with IL-17A and IFN-γ Abs. Based on the expression of IL-17A and/or IFN-γ, three cell subsets were identified as follows: Th1 (IL-17A − IFN-γ + ), Th17 (IL-17A + IFN-γ − ), and Th1Th17 (IL-17A + IFN-γ + ). The frequency of proliferating cells (CFSE low ) was analyzed in each of the three cytokine-expressing subsets. a Shown are flow cytometry results in one representative donor out of four. Shown are statistical analyses of absolute (b) and relative (Th1Th17 proliferation levels were considered 100 %) c proliferation (CFSE low ) levels in Th1, Th17, and Th1Th17 cells from four different donors. Paired t t est p values are indicated on the graphs

    Techniques Used: Expressing, Isolation, Selection, Magnetic Beads, Cell Culture, Staining, Flow Cytometry, Cytometry

    6) Product Images from "Aspirin metabolite sodium salicylate selectively inhibits transcriptional activity of ATF6α and downstream target genes"

    Article Title: Aspirin metabolite sodium salicylate selectively inhibits transcriptional activity of ATF6α and downstream target genes

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-09500-x

    Salicylate inhibits ATF6-mediated ER stress. ( a ) Effect of NaSal on ATF6α transcriptional activity induced by ER stress inducers. Twenty-four hours post-transfection with p5xATF6-GL3 and pRL-TK plasmids, MEFs were left untreated or pretreated for one hour with NaSal 20 mM, followed by treatment or not with tunicamycin (Tm, 3 µg/mL), DTT (1 mM), brefeldin A (BFA, 5 µg/mL) or thapsigargin (Tg, 1 µM) for six hours. Cell lysates were harvested and assayed for Firefly and Renilla luciferase activities. Results are presented as the fold increase of luciferase activity in treated cells compared to not treated ones. HEK293 ( b ), A549 ( c ), HepG2 ( d ) cells, and pEGFP-ATF6α-complemented Atf6α knockout MEFs ( e ) were transfected and treated with Tm in the presence or absence of NaSal as described in a . As additional control, cells were also left in the presence of the NaSal 20 mM only over the full time of treatment, i.e., seven hours. The medium remained unchanged between treatments. * , ** or *** indicate statistical significant differences between groups (p
    Figure Legend Snippet: Salicylate inhibits ATF6-mediated ER stress. ( a ) Effect of NaSal on ATF6α transcriptional activity induced by ER stress inducers. Twenty-four hours post-transfection with p5xATF6-GL3 and pRL-TK plasmids, MEFs were left untreated or pretreated for one hour with NaSal 20 mM, followed by treatment or not with tunicamycin (Tm, 3 µg/mL), DTT (1 mM), brefeldin A (BFA, 5 µg/mL) or thapsigargin (Tg, 1 µM) for six hours. Cell lysates were harvested and assayed for Firefly and Renilla luciferase activities. Results are presented as the fold increase of luciferase activity in treated cells compared to not treated ones. HEK293 ( b ), A549 ( c ), HepG2 ( d ) cells, and pEGFP-ATF6α-complemented Atf6α knockout MEFs ( e ) were transfected and treated with Tm in the presence or absence of NaSal as described in a . As additional control, cells were also left in the presence of the NaSal 20 mM only over the full time of treatment, i.e., seven hours. The medium remained unchanged between treatments. * , ** or *** indicate statistical significant differences between groups (p

    Techniques Used: Activity Assay, Transfection, Luciferase, Knock-Out

    Salicylate inhibits ATF6α translocation to the nucleus. ( a ) MEFs were plated on coverslips and transfected with 1 µg of pCMVshortEGFP-ATF6. Eight hours after transfection cells were treated either with 20 mM NaSal, 1 mM DTT or a combination of both for the indicated times. Cells were then fixed with 4% PFA, counterstained with DAPI and the coverslips were mounted on glass slides. Cells were visualized under 600 x magnification in the fluorescence microscope. White scale bars indicate 50 µm. ( b ) HEK293 cells were transfected with pEGFP-ATF6 treated either with 20 mM NaSal, 1 mM DTT or pre-treated with NaSal and later with DTT. Cells were carefully collected and lysed with nuclei flow cytometry buffer containing 1% Triton X-100. Nuclei obtained after centrifugation were washed and analysed for green fluorescence in a FACSCan flow cytometer. 10.000 events were collected inside the gate determined for nuclei size and granulosity and results were plotted as histograms for green fluorescence intensity versus cell counts. ( c ) Nuclei from HEK293 cells transfected pEGFP-ATF6 and treated with 20 mM NaSal and/or 1 mM DTT were collected following flow cytometry protocol and were lysed for nuclear extract obtaining. Extracts were analysed with anti-ATF6α and anti-lamin B antibodies after western blotting. Asterisk indicates a nonspecific band. ( d ) HEK293 cells were transfected with V5-CREBH-HA for 24 hours where indicated. Cell culture medium was replaced with fresh medium containing 10 µM bortezomib and cells were left untreated, treated with 5 µg/mL brefeldin A (BFA) for six hours or pre-treated with 20 mM NaSal for one hour and later with BFA for six hours. Whole cell extracts were obtained after lysis with RIPA buffer and analysed with anti-V5 and anti-β-actin after western blotting. ( e ) HEK293 cells were transfected with V5-OASIS-HA where indicated. Twenty four hours after transfection, cell medium was replaced with medium containing 10 µM bortezomib, followed by treatment with 5 µg/mL tunicamycin (Tm) for six hours or pre-treated with 20 mM NaSal for one hour and later with Tm for six hours. Cell nuclei were collected and processed as described in ( c ) Samples were analysed using anti-V5 and anti-lamin B antibodies. ( f ) HEK293 cells transfected with pEGFP-ATF6 were treated with 20 mM for the indicated times and/or with 5 µg/mL Tm for six hours. Cytoplasmatic cell extract was collected after cell lysis and nuclei separation and analysed with anti-ATF6α and anti-β-actin antibodies after western blotting. Results are representative of two independent experiments. Images of full-length western blots can be found in Supplementary Fig. 3 .
    Figure Legend Snippet: Salicylate inhibits ATF6α translocation to the nucleus. ( a ) MEFs were plated on coverslips and transfected with 1 µg of pCMVshortEGFP-ATF6. Eight hours after transfection cells were treated either with 20 mM NaSal, 1 mM DTT or a combination of both for the indicated times. Cells were then fixed with 4% PFA, counterstained with DAPI and the coverslips were mounted on glass slides. Cells were visualized under 600 x magnification in the fluorescence microscope. White scale bars indicate 50 µm. ( b ) HEK293 cells were transfected with pEGFP-ATF6 treated either with 20 mM NaSal, 1 mM DTT or pre-treated with NaSal and later with DTT. Cells were carefully collected and lysed with nuclei flow cytometry buffer containing 1% Triton X-100. Nuclei obtained after centrifugation were washed and analysed for green fluorescence in a FACSCan flow cytometer. 10.000 events were collected inside the gate determined for nuclei size and granulosity and results were plotted as histograms for green fluorescence intensity versus cell counts. ( c ) Nuclei from HEK293 cells transfected pEGFP-ATF6 and treated with 20 mM NaSal and/or 1 mM DTT were collected following flow cytometry protocol and were lysed for nuclear extract obtaining. Extracts were analysed with anti-ATF6α and anti-lamin B antibodies after western blotting. Asterisk indicates a nonspecific band. ( d ) HEK293 cells were transfected with V5-CREBH-HA for 24 hours where indicated. Cell culture medium was replaced with fresh medium containing 10 µM bortezomib and cells were left untreated, treated with 5 µg/mL brefeldin A (BFA) for six hours or pre-treated with 20 mM NaSal for one hour and later with BFA for six hours. Whole cell extracts were obtained after lysis with RIPA buffer and analysed with anti-V5 and anti-β-actin after western blotting. ( e ) HEK293 cells were transfected with V5-OASIS-HA where indicated. Twenty four hours after transfection, cell medium was replaced with medium containing 10 µM bortezomib, followed by treatment with 5 µg/mL tunicamycin (Tm) for six hours or pre-treated with 20 mM NaSal for one hour and later with Tm for six hours. Cell nuclei were collected and processed as described in ( c ) Samples were analysed using anti-V5 and anti-lamin B antibodies. ( f ) HEK293 cells transfected with pEGFP-ATF6 were treated with 20 mM for the indicated times and/or with 5 µg/mL Tm for six hours. Cytoplasmatic cell extract was collected after cell lysis and nuclei separation and analysed with anti-ATF6α and anti-β-actin antibodies after western blotting. Results are representative of two independent experiments. Images of full-length western blots can be found in Supplementary Fig. 3 .

    Techniques Used: Translocation Assay, Transfection, Fluorescence, Microscopy, Flow Cytometry, Cytometry, Centrifugation, Western Blot, Cell Culture, Lysis

    7) 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

    8) Product Images from "Adenovirus-Specific Human T cells are Pervasive, Polyfunctional, and Cross Reactive"

    Article Title: Adenovirus-Specific Human T cells are Pervasive, Polyfunctional, and Cross Reactive

    Journal: Vaccine

    doi: 10.1016/j.vaccine.2009.10.091

    Measuring Adenovirus Specific T-cell responses following whole vector stimulation. PBMCs from healthy adults were cultured overnight at 37°C in 5% CO 2 with Ad5 vector and costimulatory antibodies αCD28 and αCD49d. The following morning golgi secretion inhibitor brefeldin A and monensin were added for 6 hours before standard intracellular cytokine staining. A) PBMCs were incubated with 1×10 9 -1×10 11 vp Ad5 expressing green fluorescent protein (GFP). The upper graphs show GFP expression in CD14 + monocytes and CD19 + B-cells, the lower graph shows expression in CD3 + T-cells. B) PBMCs were stimulated with 1×10 9 -1×10 11 vp Ad5 vector and CD4 + and CD8 + T-cell responses were measured by IFN-γ production. C) Ad-specific IFN- γ + CD4 and CD8 T-cell responses were measured in seven donors following stimulation with 1×10 9 -1×10 11 vp Ad5.
    Figure Legend Snippet: Measuring Adenovirus Specific T-cell responses following whole vector stimulation. PBMCs from healthy adults were cultured overnight at 37°C in 5% CO 2 with Ad5 vector and costimulatory antibodies αCD28 and αCD49d. The following morning golgi secretion inhibitor brefeldin A and monensin were added for 6 hours before standard intracellular cytokine staining. A) PBMCs were incubated with 1×10 9 -1×10 11 vp Ad5 expressing green fluorescent protein (GFP). The upper graphs show GFP expression in CD14 + monocytes and CD19 + B-cells, the lower graph shows expression in CD3 + T-cells. B) PBMCs were stimulated with 1×10 9 -1×10 11 vp Ad5 vector and CD4 + and CD8 + T-cell responses were measured by IFN-γ production. C) Ad-specific IFN- γ + CD4 and CD8 T-cell responses were measured in seven donors following stimulation with 1×10 9 -1×10 11 vp Ad5.

    Techniques Used: Plasmid Preparation, Cell Culture, Staining, Incubation, Expressing

    9) Product Images from "Cellular and Molecular Mechanisms Underlying the Strong Neonatal IL-12 Response of Lamb Mesenteric Lymph Node Cells to R-848"

    Article Title: Cellular and Molecular Mechanisms Underlying the Strong Neonatal IL-12 Response of Lamb Mesenteric Lymph Node Cells to R-848

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013705

    Main cell population producing IL-12 in response to R-848. Neonatal MLN cells were cultured in vitro with or without 0.5 µg/ml R-848 and, at various time points, supernatants were harvested and ELISA was carried out for IL-12. Mean ± SEM IL-12 secretion is shown for each time point ( A ). Neonatal MLN cells were cultured in vitro with (black bars) or without (hatched bars) 0.5 µg/ml R-848 for 8 h, with brefeldin A added for the last 5 h. Cells were harvested for IL-12 intracellular staining combined with labelling of CD11b, CD14, MHC class II and CD205. Three independent experiments were carried out and the data shown are the mean proportions of IL-12 + cells after gating on CD11b + , CD14 + , MHC class II + or CD205 + MLN cells ( B ).
    Figure Legend Snippet: Main cell population producing IL-12 in response to R-848. Neonatal MLN cells were cultured in vitro with or without 0.5 µg/ml R-848 and, at various time points, supernatants were harvested and ELISA was carried out for IL-12. Mean ± SEM IL-12 secretion is shown for each time point ( A ). Neonatal MLN cells were cultured in vitro with (black bars) or without (hatched bars) 0.5 µg/ml R-848 for 8 h, with brefeldin A added for the last 5 h. Cells were harvested for IL-12 intracellular staining combined with labelling of CD11b, CD14, MHC class II and CD205. Three independent experiments were carried out and the data shown are the mean proportions of IL-12 + cells after gating on CD11b + , CD14 + , MHC class II + or CD205 + MLN cells ( B ).

    Techniques Used: Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay, Staining

    10) Product Images from "Acute SARS-CoV-2 Infection Impairs Dendritic Cell and T Cell Responses"

    Article Title: Acute SARS-CoV-2 Infection Impairs Dendritic Cell and T Cell Responses

    Journal: Immunity

    doi: 10.1016/j.immuni.2020.07.026

    Peripheral T Cells Display Functional Loss during Acute SARS-CoV-2 Infection (A) Frequencies of Ki67 + cells on CD4 and CD8 T cells were determined by flow cytometry. Fresh PBMCs from 13 APs and 9 CPs were collected at a median of 9 (range, 1–20 days) and 31 days (range, 23–54 days) after symptoms onset, respectively. Frequencies of CD38 + HLA-DR + and PD-1 + cells on CD4 T cells (left) and CD8 T cells (right) were also determined by flow cytometry. Samples of 17 APs and 20 CPs were collected at a median of 13 (range, 1–42 days) and 29.5 days (range, 21–54 days) after symptoms onset, respectively. Samples of 17 HDs were included as controls. Severe patients in the AP and CP groups were presented as black symbols. (B) Proliferation ability of T cells from COVID-19 patients was determined by flow cytometry. Fresh PBMCs from 6 APs (1 severe and 5 mild patients) and 6 mild CPs were obtained at a median of 12 (range, 2–25 days) and 32 days (range, 23–39 days) after symptoms onset, respectively. PBMCs were labeled with CFSE and then were cultured in the presence or absences of anti-CD3 and anti-CD28 mAbs for 3 days before the flow cytometry. PBMCs of 6 HDs were included as controls. Representative histograms (top left) and quantified results (top right) depict the CFSE profiles of CD4 and CD8 T cells, respectively. The presence of IFN-γ, TNF-α, and IL-2 in culture supernatants after anti-CD3/CD28 stimulation was also quantified by using the bead-based cytokine assays (bottom). (C) T cell responses to non-specific stimulation. Fresh PBMCs (same samples from Figure 3 B) were stimulated with PMA/Ionomycin activation cocktail in the presence of brefeldin A (BFA) for 6 h. Expression of IFN-γ and TNF-α in T cells were determined by intracellular cytokine staining analysis. Representative plots showing IFN-γ and TNF-α expression in CD4 and CD8 T cells (top). Frequencies of IFN-γ + and TNF-α + cells were gated on CD45RA − CCR7 + CM and CD45RA − CCR7 − EM CD4 T cells (middle), as well as on EM and CD45RA + CCR7 − (CD45RA + effector memory, EMRA) CD8 T cells (bottom). (D) Expression of granzyme B and perforin in unstimulated EM and EMRA CD8 T cells (same samples from Figure 3 B) was determined by intracellular staining. Representative plots (top) and quantified results (bottom) are shown. Each symbol represents an individual donor. Error bars indicate standard deviation. Statistics were generated by using one-way ANOVA followed by Tukey’s multiple comparisons test, Mann-Whitney test, and 2-tailed Student’s t test. ∗ p
    Figure Legend Snippet: Peripheral T Cells Display Functional Loss during Acute SARS-CoV-2 Infection (A) Frequencies of Ki67 + cells on CD4 and CD8 T cells were determined by flow cytometry. Fresh PBMCs from 13 APs and 9 CPs were collected at a median of 9 (range, 1–20 days) and 31 days (range, 23–54 days) after symptoms onset, respectively. Frequencies of CD38 + HLA-DR + and PD-1 + cells on CD4 T cells (left) and CD8 T cells (right) were also determined by flow cytometry. Samples of 17 APs and 20 CPs were collected at a median of 13 (range, 1–42 days) and 29.5 days (range, 21–54 days) after symptoms onset, respectively. Samples of 17 HDs were included as controls. Severe patients in the AP and CP groups were presented as black symbols. (B) Proliferation ability of T cells from COVID-19 patients was determined by flow cytometry. Fresh PBMCs from 6 APs (1 severe and 5 mild patients) and 6 mild CPs were obtained at a median of 12 (range, 2–25 days) and 32 days (range, 23–39 days) after symptoms onset, respectively. PBMCs were labeled with CFSE and then were cultured in the presence or absences of anti-CD3 and anti-CD28 mAbs for 3 days before the flow cytometry. PBMCs of 6 HDs were included as controls. Representative histograms (top left) and quantified results (top right) depict the CFSE profiles of CD4 and CD8 T cells, respectively. The presence of IFN-γ, TNF-α, and IL-2 in culture supernatants after anti-CD3/CD28 stimulation was also quantified by using the bead-based cytokine assays (bottom). (C) T cell responses to non-specific stimulation. Fresh PBMCs (same samples from Figure 3 B) were stimulated with PMA/Ionomycin activation cocktail in the presence of brefeldin A (BFA) for 6 h. Expression of IFN-γ and TNF-α in T cells were determined by intracellular cytokine staining analysis. Representative plots showing IFN-γ and TNF-α expression in CD4 and CD8 T cells (top). Frequencies of IFN-γ + and TNF-α + cells were gated on CD45RA − CCR7 + CM and CD45RA − CCR7 − EM CD4 T cells (middle), as well as on EM and CD45RA + CCR7 − (CD45RA + effector memory, EMRA) CD8 T cells (bottom). (D) Expression of granzyme B and perforin in unstimulated EM and EMRA CD8 T cells (same samples from Figure 3 B) was determined by intracellular staining. Representative plots (top) and quantified results (bottom) are shown. Each symbol represents an individual donor. Error bars indicate standard deviation. Statistics were generated by using one-way ANOVA followed by Tukey’s multiple comparisons test, Mann-Whitney test, and 2-tailed Student’s t test. ∗ p

    Techniques Used: Functional Assay, Infection, Flow Cytometry, Labeling, Cell Culture, Activation Assay, Expressing, Staining, Standard Deviation, Generated, MANN-WHITNEY

    11) Product Images from "Functional disruption of the Golgi apparatus protein ARF1 sensitizes MDA-MB-231 breast cancer cells to the antitumor drugs Actinomycin D and Vinblastine through ERK and AKT signaling"

    Article Title: Functional disruption of the Golgi apparatus protein ARF1 sensitizes MDA-MB-231 breast cancer cells to the antitumor drugs Actinomycin D and Vinblastine through ERK and AKT signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0195401

    Effect of the combined treatment with Golgi disrupting agents and Actinomycin D or Vinblastine on the proliferation of MDA-MB-231 cells. (A) Cells were left in normal culture medium containing 10% FBS, or transfected to transiently express the HA-epitope-tagged ARF1 constitutively-activated mutant ( ARF1-Q71L ) for 16 h. Untransfected cells were either maintained in normal culture medium containing 10% FBS for 24 h ( Serum ), or serum-starved and either left untreated for additional 24 h ( Control ) or treated 24 h either with 0.4 ng/ml Actinomycin D ( ActD ) or 1 nM Vinblastine ( VLB ). Transfected cells were serum-starved, and either left without further treatment for additional 24 h ( ARF1-Q71L ), or treated either with ActD ( ARF1-Q71L + ActD ) or VLB ( ARF1-Q71L + VLB ), as in untransfected cells. (B) Untransfected cells were treated as in A , or serum-starved and treated for 24 h either with 0.2 μg/ml Brefeldin A alone ( BFA ), or in conjunction with ActD ( BFA + ActD ) or VLB ( BFA + VLB ). (C) Untransfected cells were treated as in A , or serum-starved and treated for 24 h either with 2 μM Golgicide A alone ( GCA ), or in conjunction with ActD ( GCA + ActD ) or VLB ( GCA + VLB ). In all conditions, cells were cultured during the last 24 h in the presence of [ 3 H]-thymidine. Cells were harvested, and [ 3 H]-thymidine incorporation was quantified with a scintillation counter. Bar represents the mean ± standard deviation (n = 3). * P
    Figure Legend Snippet: Effect of the combined treatment with Golgi disrupting agents and Actinomycin D or Vinblastine on the proliferation of MDA-MB-231 cells. (A) Cells were left in normal culture medium containing 10% FBS, or transfected to transiently express the HA-epitope-tagged ARF1 constitutively-activated mutant ( ARF1-Q71L ) for 16 h. Untransfected cells were either maintained in normal culture medium containing 10% FBS for 24 h ( Serum ), or serum-starved and either left untreated for additional 24 h ( Control ) or treated 24 h either with 0.4 ng/ml Actinomycin D ( ActD ) or 1 nM Vinblastine ( VLB ). Transfected cells were serum-starved, and either left without further treatment for additional 24 h ( ARF1-Q71L ), or treated either with ActD ( ARF1-Q71L + ActD ) or VLB ( ARF1-Q71L + VLB ), as in untransfected cells. (B) Untransfected cells were treated as in A , or serum-starved and treated for 24 h either with 0.2 μg/ml Brefeldin A alone ( BFA ), or in conjunction with ActD ( BFA + ActD ) or VLB ( BFA + VLB ). (C) Untransfected cells were treated as in A , or serum-starved and treated for 24 h either with 2 μM Golgicide A alone ( GCA ), or in conjunction with ActD ( GCA + ActD ) or VLB ( GCA + VLB ). In all conditions, cells were cultured during the last 24 h in the presence of [ 3 H]-thymidine. Cells were harvested, and [ 3 H]-thymidine incorporation was quantified with a scintillation counter. Bar represents the mean ± standard deviation (n = 3). * P

    Techniques Used: Multiple Displacement Amplification, Transfection, Mutagenesis, Cell Culture, Standard Deviation

    Effect of the combined treatment with Golgi disrupting agents and Actinomycin D or Vinblastine on the apoptosis of MDA-MB-231 cells. (A) Cells were left untreated, or transfected to transiently express the HA-epitope-tagged ARF1 constitutively-activated mutant ( ARF1 ) for 16 h. Untransfected cells were left untreated for further 12 h ( Control ), or treated 12 h either with 10 ng/ml Actinomycin D ( ActD ) or 25 nM Vinblastine ( VLB ). Transfected cells were left untreated for further 12 h ( ARF1 ), or treated 12 h either with 10 ng/ml Actinomycin D ( ARF1 + ActD ) or 25 nM Vinblastine ( ARF1 + VLB ). (B) Cells were left untreated for 12 h ( Control ), or treated 12 h either with 5 μg/ml Brefeldin A ( BFA ), 10 ng/ml Actinomycin D ( ActD ) or 25 nM Vinblastine ( VLB ), or with BFA in conjunction either with ActD ( BFA + ActD ) or VLB ( BFA + VLB ). (C) Cells were left untreated for 12 h ( Control ), or treated 12 h either with 10 μM Golgicide A ( GCA ), 10 ng/ml Actinomycin D ( ActD ) or 25 nM Vinblastine ( VLB ), or with GCA in conjunction either with ActD ( GCA + ActD ) or VLB ( GCA + VLB ). (A-C) Graphs depict the quantification of the number of cells decorated with Alexa-488-conjugated Annexin V. Bar represents the mean ± standard deviation (n = 3). * P
    Figure Legend Snippet: Effect of the combined treatment with Golgi disrupting agents and Actinomycin D or Vinblastine on the apoptosis of MDA-MB-231 cells. (A) Cells were left untreated, or transfected to transiently express the HA-epitope-tagged ARF1 constitutively-activated mutant ( ARF1 ) for 16 h. Untransfected cells were left untreated for further 12 h ( Control ), or treated 12 h either with 10 ng/ml Actinomycin D ( ActD ) or 25 nM Vinblastine ( VLB ). Transfected cells were left untreated for further 12 h ( ARF1 ), or treated 12 h either with 10 ng/ml Actinomycin D ( ARF1 + ActD ) or 25 nM Vinblastine ( ARF1 + VLB ). (B) Cells were left untreated for 12 h ( Control ), or treated 12 h either with 5 μg/ml Brefeldin A ( BFA ), 10 ng/ml Actinomycin D ( ActD ) or 25 nM Vinblastine ( VLB ), or with BFA in conjunction either with ActD ( BFA + ActD ) or VLB ( BFA + VLB ). (C) Cells were left untreated for 12 h ( Control ), or treated 12 h either with 10 μM Golgicide A ( GCA ), 10 ng/ml Actinomycin D ( ActD ) or 25 nM Vinblastine ( VLB ), or with GCA in conjunction either with ActD ( GCA + ActD ) or VLB ( GCA + VLB ). (A-C) Graphs depict the quantification of the number of cells decorated with Alexa-488-conjugated Annexin V. Bar represents the mean ± standard deviation (n = 3). * P

    Techniques Used: Multiple Displacement Amplification, Transfection, Mutagenesis, Standard Deviation

    Effect of Golgi disrupting treatments on the Golgi apparatus of MDA-MB-231 cells. Cells were left untreated (A; Control ), or transfected to transiently express the HA-epitope-tagged ARF1 constitutively-activated mutant for 16 h (B; HA-ARF1-Q71L ), or treated for 60 min either with 5 μg/ml Brefeldin A (C; BFA ) or 10 μM Golgicide A (D; GCA ). Cells were fixed, permeabilized, and immunolabeled with mouse monoclonal antibody to GM130, rabbit polyclonal antibody to Giantin, and sheep antibody to TGN46. Secondary antibodies were Alexa-594-conjugated donkey anti-mouse IgG (red channel), Alexa-488-conjugated donkey anti-rabbit IgG (green channel), and Alexa-647-conjugated donkey anti-sheep IgG (blue channel). Nuclei were stained with DAPI (gray channel). Stained cells were examined by fluorescence microscopy. Merging red, green, blue, and grey channels generated the fourth image on each row; yellow indicates overlapping localization of the red and green channels, cyan indicates overlapping localization of the green and blue channels, magenta indicates overlapping localization of the red and blue channels, and white indicates overlapping localization of all three channels. Bar, 10 μm.
    Figure Legend Snippet: Effect of Golgi disrupting treatments on the Golgi apparatus of MDA-MB-231 cells. Cells were left untreated (A; Control ), or transfected to transiently express the HA-epitope-tagged ARF1 constitutively-activated mutant for 16 h (B; HA-ARF1-Q71L ), or treated for 60 min either with 5 μg/ml Brefeldin A (C; BFA ) or 10 μM Golgicide A (D; GCA ). Cells were fixed, permeabilized, and immunolabeled with mouse monoclonal antibody to GM130, rabbit polyclonal antibody to Giantin, and sheep antibody to TGN46. Secondary antibodies were Alexa-594-conjugated donkey anti-mouse IgG (red channel), Alexa-488-conjugated donkey anti-rabbit IgG (green channel), and Alexa-647-conjugated donkey anti-sheep IgG (blue channel). Nuclei were stained with DAPI (gray channel). Stained cells were examined by fluorescence microscopy. Merging red, green, blue, and grey channels generated the fourth image on each row; yellow indicates overlapping localization of the red and green channels, cyan indicates overlapping localization of the green and blue channels, magenta indicates overlapping localization of the red and blue channels, and white indicates overlapping localization of all three channels. Bar, 10 μm.

    Techniques Used: Multiple Displacement Amplification, Transfection, Mutagenesis, Immunolabeling, Staining, Fluorescence, Microscopy, Generated

    Effect of the combined treatment of Brefeldin A with Actinomycin D or Vinblastine on the levels of phospho-ERK1/2 and phospho-AKT in MDA-MB-231 cells. (A-B) Cells were left untreated for 5 h ( Control ), or treated 5 h either with 5 μg/ml Brefeldin A ( BFA ), 10 ng/ml Actinomycin D ( ActD ; A) or 25 nM Vinblastine ( VLB ; B), or with BFA in conjunction either with ActD ( BFA + ActD ; A) or VLB ( BFA + VLB ; B). After solubilizing in detergent, proteins were analyzed by SDS-PAGE followed by immunoblotting using antibodies to the proteins indicated on the right. The position of molecular mass markers is indicated on the left. (C-D) Densitometry quantification of the immunoblot signal of the levels of phospho-ERK1/2 shown as in A and B (C), and of the levels of phospho-AKT shown as in A and B (D). Bar represents the mean ± standard deviation (n = 3). ** P
    Figure Legend Snippet: Effect of the combined treatment of Brefeldin A with Actinomycin D or Vinblastine on the levels of phospho-ERK1/2 and phospho-AKT in MDA-MB-231 cells. (A-B) Cells were left untreated for 5 h ( Control ), or treated 5 h either with 5 μg/ml Brefeldin A ( BFA ), 10 ng/ml Actinomycin D ( ActD ; A) or 25 nM Vinblastine ( VLB ; B), or with BFA in conjunction either with ActD ( BFA + ActD ; A) or VLB ( BFA + VLB ; B). After solubilizing in detergent, proteins were analyzed by SDS-PAGE followed by immunoblotting using antibodies to the proteins indicated on the right. The position of molecular mass markers is indicated on the left. (C-D) Densitometry quantification of the immunoblot signal of the levels of phospho-ERK1/2 shown as in A and B (C), and of the levels of phospho-AKT shown as in A and B (D). Bar represents the mean ± standard deviation (n = 3). ** P

    Techniques Used: Multiple Displacement Amplification, SDS Page, Standard Deviation

    Effect of the combined treatment with Golgi disrupting agents and Actinomycin D or Vinblastine on the migration of MDA-MB-231 cells. (A) Cells were left untreated, or transfected to transiently express the HA-epitope-tagged ARF1 constitutively-activated mutant ( ARF1 ) for 16 h. Cultures of confluent cells were wounded with a sterile tip, cells were serum-starved, and either left untreated for additional 20 h ( Control and ARF1 ), or treated 20 h either with 10 ng/ml Actinomycin D ( ActD and ARF1 + ActD ) or 25 nM Vinblastine ( VLB and ARF1 + VLB ). (B) Cultures of confluent cells were wounded as in A , cells were serum-starved, and either left untreated for additional 20 h ( Control ), or treated 20 h either with 5 μg/ml Brefeldin A ( BFA ), 10 ng/ml Actinomycin D ( ActD ) or 25 nM Vinblastine ( VLB ), or with BFA in conjunction either with ActD ( BFA + ActD ) or VLB ( BFA + VLB ). (C) Cultures of confluent cells were wounded as in A , cells were serum-starved, and either left untreated for additional 20 h ( Control ), or treated 20 h either with 10 μM Golgicide A ( GCA ), 10 ng/ml Actinomycin D ( ActD ) or 25 nM Vinblastine ( VLB ), or with GCA in conjunction either with ActD ( GCA + ActD ) or VLB ( GCA + VLB ). Images of the same regions were taken immediately after the wounding ( 0 h ), and after 20-h of treatment ( 20 h ). (B, D and F) Cell migration, under the treatments shown in A , C and E , was estimated as the area re-occupied by cells after the 20-h treatment. Bar represents the mean ± standard deviation (n = 3). * P
    Figure Legend Snippet: Effect of the combined treatment with Golgi disrupting agents and Actinomycin D or Vinblastine on the migration of MDA-MB-231 cells. (A) Cells were left untreated, or transfected to transiently express the HA-epitope-tagged ARF1 constitutively-activated mutant ( ARF1 ) for 16 h. Cultures of confluent cells were wounded with a sterile tip, cells were serum-starved, and either left untreated for additional 20 h ( Control and ARF1 ), or treated 20 h either with 10 ng/ml Actinomycin D ( ActD and ARF1 + ActD ) or 25 nM Vinblastine ( VLB and ARF1 + VLB ). (B) Cultures of confluent cells were wounded as in A , cells were serum-starved, and either left untreated for additional 20 h ( Control ), or treated 20 h either with 5 μg/ml Brefeldin A ( BFA ), 10 ng/ml Actinomycin D ( ActD ) or 25 nM Vinblastine ( VLB ), or with BFA in conjunction either with ActD ( BFA + ActD ) or VLB ( BFA + VLB ). (C) Cultures of confluent cells were wounded as in A , cells were serum-starved, and either left untreated for additional 20 h ( Control ), or treated 20 h either with 10 μM Golgicide A ( GCA ), 10 ng/ml Actinomycin D ( ActD ) or 25 nM Vinblastine ( VLB ), or with GCA in conjunction either with ActD ( GCA + ActD ) or VLB ( GCA + VLB ). Images of the same regions were taken immediately after the wounding ( 0 h ), and after 20-h of treatment ( 20 h ). (B, D and F) Cell migration, under the treatments shown in A , C and E , was estimated as the area re-occupied by cells after the 20-h treatment. Bar represents the mean ± standard deviation (n = 3). * P

    Techniques Used: Migration, Multiple Displacement Amplification, Transfection, Mutagenesis, Standard Deviation

    12) Product Images from "T-Cell Responses to the Mycobacterium tuberculosis-Specific Antigen ESAT-6 in Brazilian Tuberculosis Patients "

    Article Title: T-Cell Responses to the Mycobacterium tuberculosis-Specific Antigen ESAT-6 in Brazilian Tuberculosis Patients

    Journal: Infection and Immunity

    doi: 10.1128/IAI.70.12.6707-6714.2002

    Intracellular flow cytometric detection of IFN-γ production in CD4 + and CD8 + T cells. PBMCs obtained from TB patients were stimulated with ESAT-6 (5 μg/ml) and anti-CD28 antibody as described in Materials and Methods. After the culture period, cells were harvested and evaluated by flow cytometry. Cells were acquired in the gated lymphocyte population, and the quadrants were set up with an isotype-matched control. The numbers in the figure represent the percentage of positive cells in each quadrant. The results are presented as already subtracted from the values obtained in nonstimulated control cultures with anti-CD28 and brefeldin A treatments. Data from one representative experiment are shown.
    Figure Legend Snippet: Intracellular flow cytometric detection of IFN-γ production in CD4 + and CD8 + T cells. PBMCs obtained from TB patients were stimulated with ESAT-6 (5 μg/ml) and anti-CD28 antibody as described in Materials and Methods. After the culture period, cells were harvested and evaluated by flow cytometry. Cells were acquired in the gated lymphocyte population, and the quadrants were set up with an isotype-matched control. The numbers in the figure represent the percentage of positive cells in each quadrant. The results are presented as already subtracted from the values obtained in nonstimulated control cultures with anti-CD28 and brefeldin A treatments. Data from one representative experiment are shown.

    Techniques Used: Flow Cytometry, Cytometry

    13) Product Images from "Poke Weed Mitogen Requires Toll-Like Receptor Ligands for Proliferative Activity in Human and Murine B Lymphocytes"

    Article Title: Poke Weed Mitogen Requires Toll-Like Receptor Ligands for Proliferative Activity in Human and Murine B Lymphocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029806

    TLR-dependency of Poke weed mitogen (PWM)-induced cell stimulation. A: B cell proliferation. Human CD19+ peripheral blood B cells were stained with CFSE and stimulated with 1 or 10 µg/ml PWM in the presence or absence of 5 µg/ml Staphylococcus aureus protein A (SpA) or 5 µg/ml anti-human Ig F(ab′) 2 fragments (aIg) as B cell receptor stimuli, with SpA or aIg alone, or left unstimulated. After 5 days B cells were harvested, stained with anti-CD20 and live gated cells were analyzed for CFSE dilution. The percentages of live gated cells (upper right corner) and proliferating cells (left) are indicated in each diagram. The diagrams depict the results from one representative experiment of n = 3. B: TNF-induction. Human PBMC were stimulated for four hours with or without PWM, TLR2 ligands FSL-1R (FSL) and Pam 3 CSK 4 (P3), phosphorothioate-modified DNA ODN CpG 2006 (CpG) and 2006 GC (GC) and LPS in the presence of Brefeldin A (BfA) or with LPS in the absence of BfA. Subsequently, intracellular staining was performed with an anti-human TNF mAb and TNF expression was analyzed by flow cytometry. The results show a summary of the data obtained in n = 4 experiments. Mean values of anti-TNF mean fluorescence intensities are provided ± SEM. C: Antagonization with Polymyxin B. CFSE-stained human CD19+ B cells were stimulated with PWM in the presence and absence of polymyxin B. Proliferation was assessed by flow cytometric analysis of CFSE dilution on day 5. The results obtained in two representative donors of n = 3 are shown. D: MyD88-dependency of B cell stimulation. B220+ B cells were isolated from the spleens of MyD88 −/− mice and their wild type counterparts. B cells were stimulated with CpG, P3, PWM and PMA/Ionomycin (P/I) and harvested after 72 hours and a 18 hour pulse with 3 H-thymidine. The diagram shows the average values in counts per minute (cpm) of n = 4 experiments ± SEM. E: TLR-dependency. Wild type, TLR2−/− and TLR9−/− B cells were isolated from murine spleen with anti-CD19 microbeads, labelled with CFSE and stimulated with TLR2 ligands Pam 3 CSK 4 (P3) and FSL-1R (FSL), PWM (10 µg/ml), TLR9 ligand CpG ODN 1668 or 1668 GC control ODN. After 4 days B cell proliferation (CFSE dilution) was quantified by flow cytometry. The diagram depicts the mean fluorescence intensity (MFI) for CFSE in live gated cells as mean value ± SEM from n = 4 experiments. Note that low MFI corresponds to strong proliferation while high MFI values reveal absence of proliferation.
    Figure Legend Snippet: TLR-dependency of Poke weed mitogen (PWM)-induced cell stimulation. A: B cell proliferation. Human CD19+ peripheral blood B cells were stained with CFSE and stimulated with 1 or 10 µg/ml PWM in the presence or absence of 5 µg/ml Staphylococcus aureus protein A (SpA) or 5 µg/ml anti-human Ig F(ab′) 2 fragments (aIg) as B cell receptor stimuli, with SpA or aIg alone, or left unstimulated. After 5 days B cells were harvested, stained with anti-CD20 and live gated cells were analyzed for CFSE dilution. The percentages of live gated cells (upper right corner) and proliferating cells (left) are indicated in each diagram. The diagrams depict the results from one representative experiment of n = 3. B: TNF-induction. Human PBMC were stimulated for four hours with or without PWM, TLR2 ligands FSL-1R (FSL) and Pam 3 CSK 4 (P3), phosphorothioate-modified DNA ODN CpG 2006 (CpG) and 2006 GC (GC) and LPS in the presence of Brefeldin A (BfA) or with LPS in the absence of BfA. Subsequently, intracellular staining was performed with an anti-human TNF mAb and TNF expression was analyzed by flow cytometry. The results show a summary of the data obtained in n = 4 experiments. Mean values of anti-TNF mean fluorescence intensities are provided ± SEM. C: Antagonization with Polymyxin B. CFSE-stained human CD19+ B cells were stimulated with PWM in the presence and absence of polymyxin B. Proliferation was assessed by flow cytometric analysis of CFSE dilution on day 5. The results obtained in two representative donors of n = 3 are shown. D: MyD88-dependency of B cell stimulation. B220+ B cells were isolated from the spleens of MyD88 −/− mice and their wild type counterparts. B cells were stimulated with CpG, P3, PWM and PMA/Ionomycin (P/I) and harvested after 72 hours and a 18 hour pulse with 3 H-thymidine. The diagram shows the average values in counts per minute (cpm) of n = 4 experiments ± SEM. E: TLR-dependency. Wild type, TLR2−/− and TLR9−/− B cells were isolated from murine spleen with anti-CD19 microbeads, labelled with CFSE and stimulated with TLR2 ligands Pam 3 CSK 4 (P3) and FSL-1R (FSL), PWM (10 µg/ml), TLR9 ligand CpG ODN 1668 or 1668 GC control ODN. After 4 days B cell proliferation (CFSE dilution) was quantified by flow cytometry. The diagram depicts the mean fluorescence intensity (MFI) for CFSE in live gated cells as mean value ± SEM from n = 4 experiments. Note that low MFI corresponds to strong proliferation while high MFI values reveal absence of proliferation.

    Techniques Used: Cell Stimulation, Staining, Modification, Expressing, Flow Cytometry, Cytometry, Fluorescence, Isolation, Mouse Assay

    14) 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

    15) Product Images from "Characterization of the Adaptor-related Protein Complex, AP-3"

    Article Title: Characterization of the Adaptor-related Protein Complex, AP-3

    Journal: The Journal of Cell Biology

    doi:

    Immunofluorescence localization of the δ subunit of the AP-3 complex. ( a , b , d , and e ) NRK cells were fixed with methanol/acetone and double labeled with anti-δ ( a and b ) and anti-transferrin receptor ( d ) or anti-lgp120 ( e ). Although all three antibodies show perinuclear labeling, many organelles in the cell are concentrated in this region, and there is little colocalization of the more peripheral elements. ( c and f ) Cells were allowed to endocytose rhodamine-conjugated wheat germ agglutinin ( f ) for 5 min before fixation in methanol/ acetone and were then labeled with anti-δ ( c ). Again, there is little colocalization of the more peripheral elements, although some structures do coincide ( arrowheads ). ( g and j ) NRK cells were fixed with paraformaldehyde and permeabilized with NP-40 and then double labeled with anti-δ ( g ) and antip200 ( j ), a putative TGN coat. Both antigens have a perinuclear distribution, but the fine details are different, indicating that they are not components of the same coat. ( h , i , k , and l ) MDBK cells were fixed with methanol/acetone, either with ( i and l ) or without ( h and k ) prior incubation for 2 min in 5 μg/ ml brefeldin A and then double labeled with anti-δ ( h and i ) and anti-γ ( k and l ). Both antigens are brefeldin A sensitive, and both have a similar perinuclear distribution in control cells, but little if any colocalization of more peripheral structures. Bar, 20 μm.
    Figure Legend Snippet: Immunofluorescence localization of the δ subunit of the AP-3 complex. ( a , b , d , and e ) NRK cells were fixed with methanol/acetone and double labeled with anti-δ ( a and b ) and anti-transferrin receptor ( d ) or anti-lgp120 ( e ). Although all three antibodies show perinuclear labeling, many organelles in the cell are concentrated in this region, and there is little colocalization of the more peripheral elements. ( c and f ) Cells were allowed to endocytose rhodamine-conjugated wheat germ agglutinin ( f ) for 5 min before fixation in methanol/ acetone and were then labeled with anti-δ ( c ). Again, there is little colocalization of the more peripheral elements, although some structures do coincide ( arrowheads ). ( g and j ) NRK cells were fixed with paraformaldehyde and permeabilized with NP-40 and then double labeled with anti-δ ( g ) and antip200 ( j ), a putative TGN coat. Both antigens have a perinuclear distribution, but the fine details are different, indicating that they are not components of the same coat. ( h , i , k , and l ) MDBK cells were fixed with methanol/acetone, either with ( i and l ) or without ( h and k ) prior incubation for 2 min in 5 μg/ ml brefeldin A and then double labeled with anti-δ ( h and i ) and anti-γ ( k and l ). Both antigens are brefeldin A sensitive, and both have a similar perinuclear distribution in control cells, but little if any colocalization of more peripheral structures. Bar, 20 μm.

    Techniques Used: Immunofluorescence, Labeling, Incubation

    16) Product Images from "Human Gingiva-Derived Mesenchymal Stem Cells Ameliorate Streptozoticin-induced T1DM in mice via Suppression of T effector cells and Up-regulating Treg Subsets"

    Article Title: Human Gingiva-Derived Mesenchymal Stem Cells Ameliorate Streptozoticin-induced T1DM in mice via Suppression of T effector cells and Up-regulating Treg Subsets

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14979-5

    GMSCs down-regulated IL-17 and IFN-γ expression on CD4 + and CD8 + T cells in STZ-induced T1DM model. T1DM was induced using multiple low dose STZ injection and 1 × 10 6 GMSCs or fibroblast cells were injected into mice via intraperitoneal route on days 0, 7, 14, 21, 28. pLN was harvested on day 10. Lymphocytes were isolated and then stimulated in vitro with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 5 hours, with brefeldin A (10 μg/ml) added in the last 4 hours, and intracellular expression of IFN-γ and IL-17 on CD4 + and CD8 + T cells was analyzed by flow cytometry. ( a , b ) Representative flow data of IFN-γ and IL-17 expression gated on CD4 + T cells and CD8 + T cells in draining LN (pLN). ( c , d ) Expression of cytokines, including IFN-γ, IL-17 on CD4 + T and CD8 + T cells in the draining LNs of T1DM mice. Data are presented as the mean ± SEM from two independent experiments (n = 5). *P
    Figure Legend Snippet: GMSCs down-regulated IL-17 and IFN-γ expression on CD4 + and CD8 + T cells in STZ-induced T1DM model. T1DM was induced using multiple low dose STZ injection and 1 × 10 6 GMSCs or fibroblast cells were injected into mice via intraperitoneal route on days 0, 7, 14, 21, 28. pLN was harvested on day 10. Lymphocytes were isolated and then stimulated in vitro with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 5 hours, with brefeldin A (10 μg/ml) added in the last 4 hours, and intracellular expression of IFN-γ and IL-17 on CD4 + and CD8 + T cells was analyzed by flow cytometry. ( a , b ) Representative flow data of IFN-γ and IL-17 expression gated on CD4 + T cells and CD8 + T cells in draining LN (pLN). ( c , d ) Expression of cytokines, including IFN-γ, IL-17 on CD4 + T and CD8 + T cells in the draining LNs of T1DM mice. Data are presented as the mean ± SEM from two independent experiments (n = 5). *P

    Techniques Used: Expressing, Injection, Mouse Assay, Isolation, In Vitro, Flow Cytometry, Cytometry

    17) Product Images from "Podocytes Produce and Secrete Functional Complement C3 and Complement Factor H"

    Article Title: Podocytes Produce and Secrete Functional Complement C3 and Complement Factor H

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.01833

    CFH and C3 were localized in the Golgi apparatus and secretion was dependent on normal Golgi function. (A) Podocytes were stained for C3 (left, green) and CFH (right, green) and with an antibody against giantin, a Golgi apparatus protein (red). Co-localization is shown in yellow for both proteins (40x, scale bar 25 μm). (B,C) Podocytes were treated with Brefeldin A (BFA), a general exocytosis inhibitor. The secretion of C3 into the cell culture supernatant was tested in Western blot, after treatment with BFA 5 μg/ml compared to SFM. (B) Western blot against C3 and quantification (C) after 0 (white bars), 2 (light gray bars), 4 (dark gray bars), and 8 h (black bars). Data are shown in mean and SD, Mann Whitney U -test, *** p
    Figure Legend Snippet: CFH and C3 were localized in the Golgi apparatus and secretion was dependent on normal Golgi function. (A) Podocytes were stained for C3 (left, green) and CFH (right, green) and with an antibody against giantin, a Golgi apparatus protein (red). Co-localization is shown in yellow for both proteins (40x, scale bar 25 μm). (B,C) Podocytes were treated with Brefeldin A (BFA), a general exocytosis inhibitor. The secretion of C3 into the cell culture supernatant was tested in Western blot, after treatment with BFA 5 μg/ml compared to SFM. (B) Western blot against C3 and quantification (C) after 0 (white bars), 2 (light gray bars), 4 (dark gray bars), and 8 h (black bars). Data are shown in mean and SD, Mann Whitney U -test, *** p

    Techniques Used: Staining, Cell Culture, Western Blot, MANN-WHITNEY

    18) Product Images from "Epstein-Barr virus BARF1-induced NFκB/miR-146a/SMAD4 alterations in stomach cancer cells"

    Article Title: Epstein-Barr virus BARF1-induced NFκB/miR-146a/SMAD4 alterations in stomach cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10511

    EBV BARF1 protein was mainly secreted and promoted cell proliferation ( A ) BARF1 mRNA was detected in SNU601 BARF1 and SNU216 BARF1 cells (stable transfection with BARF1) and in naturally Epstein-Barr virus (EBV)-infected stomach cancer cells (SNU719 and YCCEL1), whereas it was undetectable in SNU601 mock cells and SNU216 mock cells. ( B ) As seen by fluorescence microscopy, BARF1 protein was hardly observed in SNU601 BARF1 cells and was weakly detected in SNU216 BARF1, SNU719 and YCCEL1 cells, whereas BARF1 protein accumulated in cells that were treated with Brefeldin A. BARF1 antibody (MAb 6F4) was labeled in red, and nuclei were counterstained with DAPI (blue). ( C ) Cell proliferation was higher in SNU601 BARF1 and SNU216 BARF1 cells than in SNU601 mock cells and SNU216 mock cells, respectively, and lower in YCCEL1 cells transfected with BARF1-specific siRNA (siBARF1) than in YCCEL1 cells transfected with scrambled siRNA (siSCR) (* P
    Figure Legend Snippet: EBV BARF1 protein was mainly secreted and promoted cell proliferation ( A ) BARF1 mRNA was detected in SNU601 BARF1 and SNU216 BARF1 cells (stable transfection with BARF1) and in naturally Epstein-Barr virus (EBV)-infected stomach cancer cells (SNU719 and YCCEL1), whereas it was undetectable in SNU601 mock cells and SNU216 mock cells. ( B ) As seen by fluorescence microscopy, BARF1 protein was hardly observed in SNU601 BARF1 cells and was weakly detected in SNU216 BARF1, SNU719 and YCCEL1 cells, whereas BARF1 protein accumulated in cells that were treated with Brefeldin A. BARF1 antibody (MAb 6F4) was labeled in red, and nuclei were counterstained with DAPI (blue). ( C ) Cell proliferation was higher in SNU601 BARF1 and SNU216 BARF1 cells than in SNU601 mock cells and SNU216 mock cells, respectively, and lower in YCCEL1 cells transfected with BARF1-specific siRNA (siBARF1) than in YCCEL1 cells transfected with scrambled siRNA (siSCR) (* P

    Techniques Used: Stable Transfection, Infection, Fluorescence, Microscopy, Labeling, Transfection

    19) Product Images from "A major secretory defect of tumour-infiltrating T lymphocytes due to galectin impairing LFA-1-mediated synapse completion"

    Article Title: A major secretory defect of tumour-infiltrating T lymphocytes due to galectin impairing LFA-1-mediated synapse completion

    Journal: Nature Communications

    doi: 10.1038/ncomms12242

    More LFA-1/ICAM-1 interactions are required for IFN-γ secretion than for IFN-γ intracellular production. Blood CD8 T cells from non-cancerous donor LB3442 were plated in wells coated with an anti-CD3 mAb (0.5 μg ml −1 ) and increasing doses of human ICAM-1-Fc. Taking in account this strong stimulus, IFN-γ secretion and production were estimated 3 h after stimulation. ( a ) After stimulation in the presence of brefeldin A, cells were stained for intracellular IFN-γ. ( b ) IFN-γ secretion was measured by enzyme-linked immunosorbent assay (ELISA). Columns are mean±s.d. of duplicates. Results are from one representative experiment out of four.
    Figure Legend Snippet: More LFA-1/ICAM-1 interactions are required for IFN-γ secretion than for IFN-γ intracellular production. Blood CD8 T cells from non-cancerous donor LB3442 were plated in wells coated with an anti-CD3 mAb (0.5 μg ml −1 ) and increasing doses of human ICAM-1-Fc. Taking in account this strong stimulus, IFN-γ secretion and production were estimated 3 h after stimulation. ( a ) After stimulation in the presence of brefeldin A, cells were stained for intracellular IFN-γ. ( b ) IFN-γ secretion was measured by enzyme-linked immunosorbent assay (ELISA). Columns are mean±s.d. of duplicates. Results are from one representative experiment out of four.

    Techniques Used: Staining, Enzyme-linked Immunosorbent Assay

    LacNAc treatment is dispensable for TCR internalization and IFN-γ production by TILs. CD8 TILs were isolated from ascites and treated with LacNAc. TILs were cultured with sAg-pulsed EBV-B cells. Each symbol represents TILs from one patient ( Supplementary Table 1 ). ( a – e ) Data were obtained in the same experiment for each patient. Columns represent means for several patients. Paired t -test. ( a ) Cells were stained with anti-CD3ɛ mAb. Data represent the percentage of CD3ɛ surface expression on TILs, relative to unstimulated conditions. ( b , c ) Intracellular staining of IFN-γ in TILs cocultured with sAg-pulsed targets in the presence of brefeldin A. ( b ), Representative histograms obtained from patient Gdc44 (●), % of IFN-γ + TILs and the fluorescence intensity (FI) median of the positive subset are indicated ( c ), % of IFN-γ + TILs obtained from each patient. ( d ) IFN-γ secretion was measured by Bioplex. ( e ) IFN-γ messenger RNA expression was measured by quantitative PCR with reverse transcription. ( f ) After 5 h of coculture in the presence or in the absence of brefeldin A (BFA), TILs from sample Gdc42 were stained for intracellular IFN-γ and analysed by flow cytometry. Data at the right of the red line are considered as positive for IFN-γ staining. ( g ) In the same experiment, IFN-γ release was measured in the supernatants by enzyme-linked immunosorbent assay (ELISA). Columns represent mean±s.d. of triplicates. Unpaired t -test. ( f , g ) Results are from one representative experiment out of three.
    Figure Legend Snippet: LacNAc treatment is dispensable for TCR internalization and IFN-γ production by TILs. CD8 TILs were isolated from ascites and treated with LacNAc. TILs were cultured with sAg-pulsed EBV-B cells. Each symbol represents TILs from one patient ( Supplementary Table 1 ). ( a – e ) Data were obtained in the same experiment for each patient. Columns represent means for several patients. Paired t -test. ( a ) Cells were stained with anti-CD3ɛ mAb. Data represent the percentage of CD3ɛ surface expression on TILs, relative to unstimulated conditions. ( b , c ) Intracellular staining of IFN-γ in TILs cocultured with sAg-pulsed targets in the presence of brefeldin A. ( b ), Representative histograms obtained from patient Gdc44 (●), % of IFN-γ + TILs and the fluorescence intensity (FI) median of the positive subset are indicated ( c ), % of IFN-γ + TILs obtained from each patient. ( d ) IFN-γ secretion was measured by Bioplex. ( e ) IFN-γ messenger RNA expression was measured by quantitative PCR with reverse transcription. ( f ) After 5 h of coculture in the presence or in the absence of brefeldin A (BFA), TILs from sample Gdc42 were stained for intracellular IFN-γ and analysed by flow cytometry. Data at the right of the red line are considered as positive for IFN-γ staining. ( g ) In the same experiment, IFN-γ release was measured in the supernatants by enzyme-linked immunosorbent assay (ELISA). Columns represent mean±s.d. of triplicates. Unpaired t -test. ( f , g ) Results are from one representative experiment out of three.

    Techniques Used: Isolation, Cell Culture, Staining, Expressing, Fluorescence, RNA Expression, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    IFN-γ secretion and cytotoxicity depend on LFA-1 function. Blood CD8 T cells from non-cancerous donors LB2050 ( a – c ) and LB554 ( d – f ), LB554 were treated with an anti-CD11a blocking antibody at the indicated doses. ( a ) T cells were conjugated with sAg-pulsed EBV-B cells, previously loaded with CMTMR (red). After 25 min, cells were stained for F-actin (green). Scale bars, 5 μm. ( b ) Actin clearing at the synapse centre was evaluated by comparing fluorescence intensities (FI) at the synapse periphery and the synapse centre. ( c ) F-actin accumulation was evaluated by measuring FI at the synapse centre. ( b , c ) Each dot corresponds to a T-cell-target conjugate;  n > 60; Mann–Whitney  U  test. ( d ) Target cells were  51 Cr-labelled and incubated with treated T cells at the indicated ratio. Chromium release was measured after 4 h of coculture. Data represent the mean±s.d. of triplicates. ( e ) After 20 h of coculture, IFN-γ secretion was analysed by enzyme-linked immunosorbent assay (ELISA). Data represent the mean±s.d. of triplicates. ( f ) After 20 h of coculture in the presence of brefeldin A, cells were stained for intracellular IFN-γ. Per cent of IFN-γ +  T cells and FI medians of the positive subsets are indicated. Results are from one ( a – c ), two ( d ) and four ( e , f ) independent experiments. DIC, differential interference contrast.
    Figure Legend Snippet: IFN-γ secretion and cytotoxicity depend on LFA-1 function. Blood CD8 T cells from non-cancerous donors LB2050 ( a – c ) and LB554 ( d – f ), LB554 were treated with an anti-CD11a blocking antibody at the indicated doses. ( a ) T cells were conjugated with sAg-pulsed EBV-B cells, previously loaded with CMTMR (red). After 25 min, cells were stained for F-actin (green). Scale bars, 5 μm. ( b ) Actin clearing at the synapse centre was evaluated by comparing fluorescence intensities (FI) at the synapse periphery and the synapse centre. ( c ) F-actin accumulation was evaluated by measuring FI at the synapse centre. ( b , c ) Each dot corresponds to a T-cell-target conjugate; n > 60; Mann–Whitney U test. ( d ) Target cells were 51 Cr-labelled and incubated with treated T cells at the indicated ratio. Chromium release was measured after 4 h of coculture. Data represent the mean±s.d. of triplicates. ( e ) After 20 h of coculture, IFN-γ secretion was analysed by enzyme-linked immunosorbent assay (ELISA). Data represent the mean±s.d. of triplicates. ( f ) After 20 h of coculture in the presence of brefeldin A, cells were stained for intracellular IFN-γ. Per cent of IFN-γ + T cells and FI medians of the positive subsets are indicated. Results are from one ( a – c ), two ( d ) and four ( e , f ) independent experiments. DIC, differential interference contrast.

    Techniques Used: Blocking Assay, Staining, Fluorescence, MANN-WHITNEY, Incubation, Enzyme-linked Immunosorbent Assay

    20) Product Images from "TonEBP Promotes β-Cell Survival under ER Stress by Enhancing Autophagy"

    Article Title: TonEBP Promotes β-Cell Survival under ER Stress by Enhancing Autophagy

    Journal: Cells

    doi: 10.3390/cells9091928

    Tonicity-responsive enhancer-binding protein (TonEBP) prevents the accumulation of unfolded proteins. ( A , B ) MIN6-M9 cells were transfected with scrambled siRNA or TonEBP-targeted siRNA, and then treated with vehicle (VH), brefeldin A (BFA; 20 μM), or tunicamycin (TM; 1 μg/mL). Cell viability was assessed by the LDH release ( A ) and MTT reduction ( B ) after 24 h. ( C ) Cells were transfected with scrambled siRNA (Scr) or TonEBP-targeting siRNA (Ton) and then treated as above. Ubiquitin was visualized with an anti-ubiquitin antibody by immunostaining. ( D ) Percent of ubiquitin puncta positive cells were counted from 100 cells in each group. ( E ) Cells were transfected and treated as in ( C ). BiP was detected with an anti-BiP antibody by immunostaining. ( F ) Percent of BiP positive cells were counted from 100 cells in each group. VH, vehicle. Data (mean + SD) were from three independent experiments ( n = 3) each with more than three replicates. # p
    Figure Legend Snippet: Tonicity-responsive enhancer-binding protein (TonEBP) prevents the accumulation of unfolded proteins. ( A , B ) MIN6-M9 cells were transfected with scrambled siRNA or TonEBP-targeted siRNA, and then treated with vehicle (VH), brefeldin A (BFA; 20 μM), or tunicamycin (TM; 1 μg/mL). Cell viability was assessed by the LDH release ( A ) and MTT reduction ( B ) after 24 h. ( C ) Cells were transfected with scrambled siRNA (Scr) or TonEBP-targeting siRNA (Ton) and then treated as above. Ubiquitin was visualized with an anti-ubiquitin antibody by immunostaining. ( D ) Percent of ubiquitin puncta positive cells were counted from 100 cells in each group. ( E ) Cells were transfected and treated as in ( C ). BiP was detected with an anti-BiP antibody by immunostaining. ( F ) Percent of BiP positive cells were counted from 100 cells in each group. VH, vehicle. Data (mean + SD) were from three independent experiments ( n = 3) each with more than three replicates. # p

    Techniques Used: Binding Assay, Transfection, MTT Assay, Immunostaining

    TonEBP promotes autophagy in pancreatic β cells. ( A ) MIN6-M9 cells transfected with scrambled siRNA (scr) or TonEBP-targeted siRNA (Ton) were treated for 6 h with vehicle (VH), brefeldin A (BFA; 20 μM), or tunicamycin (TM; 1 μg/mL). TonEBP, LC3-II, and Hsc70 were immunoblotted. ( B ) Cells transfected and treated as above were immunostained for LC3. ( C ) Percent of LC3 positive cells and LC3 signal intensity was measured in 150 cells from each group from ( B ). ( D , E ) Cells transfected with siRNA as above were pre-treated for 1 h with chloroquine (CQ; 10 μM) or LY294002 (LY; 10 μM) followed by a 4 h treatment with TM (1 μg/mL). ( D ) Cells were immunostained for LC3. ( E ) Percent of LC3 positive cells and LC3 signal intensity was measured in 50 cells from each group. Mean + SD. # p
    Figure Legend Snippet: TonEBP promotes autophagy in pancreatic β cells. ( A ) MIN6-M9 cells transfected with scrambled siRNA (scr) or TonEBP-targeted siRNA (Ton) were treated for 6 h with vehicle (VH), brefeldin A (BFA; 20 μM), or tunicamycin (TM; 1 μg/mL). TonEBP, LC3-II, and Hsc70 were immunoblotted. ( B ) Cells transfected and treated as above were immunostained for LC3. ( C ) Percent of LC3 positive cells and LC3 signal intensity was measured in 150 cells from each group from ( B ). ( D , E ) Cells transfected with siRNA as above were pre-treated for 1 h with chloroquine (CQ; 10 μM) or LY294002 (LY; 10 μM) followed by a 4 h treatment with TM (1 μg/mL). ( D ) Cells were immunostained for LC3. ( E ) Percent of LC3 positive cells and LC3 signal intensity was measured in 50 cells from each group. Mean + SD. # p

    Techniques Used: Transfection

    RHD of TonEBP is required for endoplasmic reticulum (ER) stress-induced autophagy. ( A – D ) MIN6-M9 cells were transfected with scrambled or TonEBP-targeted siRNA followed by a second transfection with a plasmid expressing TonEBP or ΔRHD as indicated. ( A ) Cell viability was assessed by the LDH release ( A ) and MTT reduction ( B ) after a 24 h treatment of brefeldin A (BFA; 20 μM) or tunicamycin (TM; 1 μg/mL). ( B ) LC3 was detected with immunostaining. ( C ) Percent of LC3 positive cells was measured in 150 cells from each group. ( D ) LC3 signal intensity was measured in 150 cells from each group. Data (mean + SD) were from three independent experiments ( n = 3) each with more than three replicates. * p
    Figure Legend Snippet: RHD of TonEBP is required for endoplasmic reticulum (ER) stress-induced autophagy. ( A – D ) MIN6-M9 cells were transfected with scrambled or TonEBP-targeted siRNA followed by a second transfection with a plasmid expressing TonEBP or ΔRHD as indicated. ( A ) Cell viability was assessed by the LDH release ( A ) and MTT reduction ( B ) after a 24 h treatment of brefeldin A (BFA; 20 μM) or tunicamycin (TM; 1 μg/mL). ( B ) LC3 was detected with immunostaining. ( C ) Percent of LC3 positive cells was measured in 150 cells from each group. ( D ) LC3 signal intensity was measured in 150 cells from each group. Data (mean + SD) were from three independent experiments ( n = 3) each with more than three replicates. * p

    Techniques Used: Transfection, Plasmid Preparation, Expressing, MTT Assay, Immunostaining

    ER stress dramatically increases TonEBP protein stability. ( A ) MIN6-M9 cells transfected with scrambled siRNA (scr) or TonEBP-targeted siRNA (Ton) were treated for 4 h with vehicle (VH), brefeldin A (BFA; 20 μM), or tunicamycin (TM; 1 μg/mL) as indicated. TonEBP and Hsc70 were immunoblotted. Data (mean + SD) were from three independent experiments ( n = 3) each with more than three replicates. # p
    Figure Legend Snippet: ER stress dramatically increases TonEBP protein stability. ( A ) MIN6-M9 cells transfected with scrambled siRNA (scr) or TonEBP-targeted siRNA (Ton) were treated for 4 h with vehicle (VH), brefeldin A (BFA; 20 μM), or tunicamycin (TM; 1 μg/mL) as indicated. TonEBP and Hsc70 were immunoblotted. Data (mean + SD) were from three independent experiments ( n = 3) each with more than three replicates. # p

    Techniques Used: Transfection

    21) Product Images from "1-Methyl-tryptophan can interfere with TLR signaling in dendritic cells independently of IDO activity"

    Article Title: 1-Methyl-tryptophan can interfere with TLR signaling in dendritic cells independently of IDO activity

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

    doi:

    1-MT induces a Th2 function of DC stimulated with LPS. (A) Kinetic of secretion of IFNγ, IL-5 and IL-13 in MLR supernatants. MLR were conducted with control iDC (□), LPS-stimulated DC (○) and LPS-stimulated DC pre-treated with 1-MT (▲). Cytokines were measured in MLR supernatants at the indicated times. Mean ± SD of triplicates of one representative experiment out of three. (B) MLR were conducted for 5 days. After IL-2 expansion, T cells were stimulated with PMA and ionomycin in the presence of Brefeldin A and IL-5, IL-13 and IFNγ expression was analyzed by intracellular staining. Data are shown for 1/20 DC/T cell ratio and were similar for other ratios. Data of one representative experiment out of three.
    Figure Legend Snippet: 1-MT induces a Th2 function of DC stimulated with LPS. (A) Kinetic of secretion of IFNγ, IL-5 and IL-13 in MLR supernatants. MLR were conducted with control iDC (□), LPS-stimulated DC (○) and LPS-stimulated DC pre-treated with 1-MT (▲). Cytokines were measured in MLR supernatants at the indicated times. Mean ± SD of triplicates of one representative experiment out of three. (B) MLR were conducted for 5 days. After IL-2 expansion, T cells were stimulated with PMA and ionomycin in the presence of Brefeldin A and IL-5, IL-13 and IFNγ expression was analyzed by intracellular staining. Data are shown for 1/20 DC/T cell ratio and were similar for other ratios. Data of one representative experiment out of three.

    Techniques Used: Expressing, Staining

    22) Product Images from "K11- and K48-Linked Ubiquitin Chains Interact with p97 during Endoplasmic Reticulum-Associated Degradation"

    Article Title: K11- and K48-Linked Ubiquitin Chains Interact with p97 during Endoplasmic Reticulum-Associated Degradation

    Journal: The Biochemical journal

    doi: 10.1042/BJ20120662

    ER stressors upstream of p97 do not promote K11 and K48 polyubiquitin accumulation (A, B) 293 cells were treated with tunicamycin (Tn, 1 μM), Brefeldin A (Bref A, 3.6 μM), or EerI (10 μM) for 8 hours and whole cell extracts prepared (A) and fractionated into cytosol and microsomes (B) prior to analysis by immunoblotting with the indicated antibodies. (C) p97 was immunoprecipitated from the fractions prepared in (B) with p97 monoclonal antibodies and elutates were analyzed by immunoblotting.
    Figure Legend Snippet: ER stressors upstream of p97 do not promote K11 and K48 polyubiquitin accumulation (A, B) 293 cells were treated with tunicamycin (Tn, 1 μM), Brefeldin A (Bref A, 3.6 μM), or EerI (10 μM) for 8 hours and whole cell extracts prepared (A) and fractionated into cytosol and microsomes (B) prior to analysis by immunoblotting with the indicated antibodies. (C) p97 was immunoprecipitated from the fractions prepared in (B) with p97 monoclonal antibodies and elutates were analyzed by immunoblotting.

    Techniques Used: Immunoprecipitation

    23) Product Images from "Maternal immunization with ovalbumin prevents neonatal allergy development and up-regulates inhibitory receptor Fc?RIIB expression on B cells"

    Article Title: Maternal immunization with ovalbumin prevents neonatal allergy development and up-regulates inhibitory receptor Fc?RIIB expression on B cells

    Journal: BMC Immunology

    doi: 10.1186/1471-2172-11-11

    Effect of passive IgG transference to neonates on B and T cell responses . Neonate pups (3 d-o) from nonimmunized mothers injected with IgG from nonimmunized or immunized mothers and simultaneously immunized with OVA were evaluated (20 d-o) for: (A) anti-OVA IgE Ab levels by PCA reaction; (B) B cell FcγRIIb expression (B220+IgM+) and histogram of FcγRIIb expression on B cells of offspring from immunized (shaded histogram, MFI in bold numbers) or nonimmunized mothers (white histogram, MFI in light numbers); (C) intracellular cytokines of splenic B cells (B220+) or (D) CD4+ T cells after 24 h incubation with 10 μg/mL brefeldin A; data shown in B-D were obtained flow cytometry. The results represent the mean ± SEM of 9 mice per group. *  P  ≤ 0.05 compared to offspring from nonimmunized mothers.
    Figure Legend Snippet: Effect of passive IgG transference to neonates on B and T cell responses . Neonate pups (3 d-o) from nonimmunized mothers injected with IgG from nonimmunized or immunized mothers and simultaneously immunized with OVA were evaluated (20 d-o) for: (A) anti-OVA IgE Ab levels by PCA reaction; (B) B cell FcγRIIb expression (B220+IgM+) and histogram of FcγRIIb expression on B cells of offspring from immunized (shaded histogram, MFI in bold numbers) or nonimmunized mothers (white histogram, MFI in light numbers); (C) intracellular cytokines of splenic B cells (B220+) or (D) CD4+ T cells after 24 h incubation with 10 μg/mL brefeldin A; data shown in B-D were obtained flow cytometry. The results represent the mean ± SEM of 9 mice per group. * P ≤ 0.05 compared to offspring from nonimmunized mothers.

    Techniques Used: Injection, Expressing, Incubation, Flow Cytometry, Cytometry, Mouse Assay

    Effect of passive IgG transference to pregnant mice on offspring's B and T cell responses . Nonimmunized pregnant mice were injected with IgG from nonimmunized or immunized mothers. Offspring immunized with OVA were evaluated (20 d-o) for: (a) anti-OVA IgE Ab levels by PCA reaction; (b) CD80, CD86, CD40 CD23 molecule expression on splenic B cells (B220+); (c) B cell FcγRIIb expression (B220+IgM+) by flow cytometry. Histogram of FcγRIIb expression on B cells of offspring from immunized (shaded histogram, MFI in bold numbers) or nonimmunized mothers (white histogram, MFI in light numbers); (d) intracellular cytokines of splenic B cells (B220+) and (e) CD4+ T cells after 424 h incubation with 10 μg/mL brefeldin A, all by flow cytometry. The results represent the mean ± SEM of 6 mice per group. * P  ≤ 0.05 compared to offspring from nonimmunized mothers.
    Figure Legend Snippet: Effect of passive IgG transference to pregnant mice on offspring's B and T cell responses . Nonimmunized pregnant mice were injected with IgG from nonimmunized or immunized mothers. Offspring immunized with OVA were evaluated (20 d-o) for: (a) anti-OVA IgE Ab levels by PCA reaction; (b) CD80, CD86, CD40 CD23 molecule expression on splenic B cells (B220+); (c) B cell FcγRIIb expression (B220+IgM+) by flow cytometry. Histogram of FcγRIIb expression on B cells of offspring from immunized (shaded histogram, MFI in bold numbers) or nonimmunized mothers (white histogram, MFI in light numbers); (d) intracellular cytokines of splenic B cells (B220+) and (e) CD4+ T cells after 424 h incubation with 10 μg/mL brefeldin A, all by flow cytometry. The results represent the mean ± SEM of 6 mice per group. * P ≤ 0.05 compared to offspring from nonimmunized mothers.

    Techniques Used: Mouse Assay, Injection, Expressing, Flow Cytometry, Cytometry, Incubation

    Effect of maternal immunization with OVA on the immune response of nonimmunized or immunized neonates . Neonate pups (3 d-o) from control or immune mothers were immunized or not with OVA and evaluated (20 d-o) for: (A) IgG1, IgG2a and IgM by ELISA; (B) anti-OVA IgE Ab levels by PCA reaction; (C) intracellular cytokines of splenic B cells (B220+) or (d) CD4+ T cells after 24 h incubation with 10 μg/mL brefeldin A by flow cytometry. The results represent the mean ± SEM of 12 mice per group. *P ≤ 0.05 compared to offspring from nonimmunized mothers, # P ≤ 0.05 compared to nonimmunized offspring from control mothers, • P ≤ 0.05 compared to control offspring from immune mothers.
    Figure Legend Snippet: Effect of maternal immunization with OVA on the immune response of nonimmunized or immunized neonates . Neonate pups (3 d-o) from control or immune mothers were immunized or not with OVA and evaluated (20 d-o) for: (A) IgG1, IgG2a and IgM by ELISA; (B) anti-OVA IgE Ab levels by PCA reaction; (C) intracellular cytokines of splenic B cells (B220+) or (d) CD4+ T cells after 24 h incubation with 10 μg/mL brefeldin A by flow cytometry. The results represent the mean ± SEM of 12 mice per group. *P ≤ 0.05 compared to offspring from nonimmunized mothers, # P ≤ 0.05 compared to nonimmunized offspring from control mothers, • P ≤ 0.05 compared to control offspring from immune mothers.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry, Cytometry, Mouse Assay

    24) Product Images from "Mesenchymal Stromal Cells Derived From Crohn's Patients Deploy Indoleamine 2,3-dioxygenase-mediated Immune Suppression, Independent of Autophagy"

    Article Title: Mesenchymal Stromal Cells Derived From Crohn's Patients Deploy Indoleamine 2,3-dioxygenase-mediated Immune Suppression, Independent of Autophagy

    Journal: Molecular Therapy

    doi: 10.1038/mt.2015.67

    Autophagy pathway is dispensable for mesenchymal stromal cells (MSC's) suppressive effect on T-cell proliferation and cytokine secretion. ( a ) 1 mmol/l 3-Methyl Adenine (3-MA) was added to MSCs in the presence and absence of 20 ng/ml IFNγ for a period of 48 hours and surface expression of HLADR, PDL1, PDL2, and ICAM-1 was measured through flow cytometry. ( b ) Representative flow cytometry Plot, ( c ) dose-dependent effect of 3-MA on T-cell proliferation is shown. 3-MA was added at the indicated concentrations to PBMCs cocultured in the presence and absence of MSCs. T-cell proliferation was measured through intracellular Ki67 staining after 4 days. Similar results were obtained in a repeat experiment. ( d ) Cumulative % reduction of CD3+Ki67+ T-cell proliferation from two independent experiments with two independent PBMC donors was plotted against ( n = 6) Crohn's and ( n = 6) Healthy MSC donor groups with ± 1 mmol/l 3-MA. ( e ) Representative and ( f ) Cumulative percentage reduction of CD3+IFNγ+ is shown in 12-14hrs intracellular cytokine staining assay. 1 mmol/l 3-MA was added to SEB activated PBMCs cocultured in the presence and absence of ±IFNγ licensed MSCs. Brefeldin A was added to detect CD3+IFNγ+T cells. Cumulative is plotted with two independent MSC donors from healthy and Crohn's in two independent experiments performed with two independent PBMC donors. Results are plotted as mean ± SD. Two-tailed T -test was performed to obtain the P values in Prism software.
    Figure Legend Snippet: Autophagy pathway is dispensable for mesenchymal stromal cells (MSC's) suppressive effect on T-cell proliferation and cytokine secretion. ( a ) 1 mmol/l 3-Methyl Adenine (3-MA) was added to MSCs in the presence and absence of 20 ng/ml IFNγ for a period of 48 hours and surface expression of HLADR, PDL1, PDL2, and ICAM-1 was measured through flow cytometry. ( b ) Representative flow cytometry Plot, ( c ) dose-dependent effect of 3-MA on T-cell proliferation is shown. 3-MA was added at the indicated concentrations to PBMCs cocultured in the presence and absence of MSCs. T-cell proliferation was measured through intracellular Ki67 staining after 4 days. Similar results were obtained in a repeat experiment. ( d ) Cumulative % reduction of CD3+Ki67+ T-cell proliferation from two independent experiments with two independent PBMC donors was plotted against ( n = 6) Crohn's and ( n = 6) Healthy MSC donor groups with ± 1 mmol/l 3-MA. ( e ) Representative and ( f ) Cumulative percentage reduction of CD3+IFNγ+ is shown in 12-14hrs intracellular cytokine staining assay. 1 mmol/l 3-MA was added to SEB activated PBMCs cocultured in the presence and absence of ±IFNγ licensed MSCs. Brefeldin A was added to detect CD3+IFNγ+T cells. Cumulative is plotted with two independent MSC donors from healthy and Crohn's in two independent experiments performed with two independent PBMC donors. Results are plotted as mean ± SD. Two-tailed T -test was performed to obtain the P values in Prism software.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Staining, Two Tailed Test, Software

    25) Product Images from "Cytolytic CD4+-T-Cell Clones Reactive to EBNA1 Inhibit Epstein-Barr Virus-Induced B-Cell Proliferation †"

    Article Title: Cytolytic CD4+-T-Cell Clones Reactive to EBNA1 Inhibit Epstein-Barr Virus-Induced B-Cell Proliferation †

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.22.12088-12104.2003

    EBNA1-reactive CD4 + -T-cell clones do not utilize perforin to induce cytolysis of LCLs. (A) Cytotoxicity against autologous (solid bars) or MHC II-mismatched (stippled bars) LCL targets after 18 h in culture with BC.E112 or BC.E160 cells at a 1:1 ratio. Prior to exposure to LCLs, clones were incubated with concanamycin A (ConcanA), brefeldin A (BrfdA), or culture medium alone (none). (B) Loss of calcein dye by MHC I-matched LCL targets with (solid bars) or without (stippled bars) addition of EBNA3A antigenic peptide after 3 h in culture with MS.B11 CD8 + -T-cell clones at a 1:1 ratio. (C) Intracellular staining with antibody to perforin. BC EBNA1-reactive CD4 + clones were stimulated with autologous LCLs; the EBNA3A-specific CD8 + -T-cell clone MS.B11 was stimulated with peptide-loaded, MHC I-matched LCL (solid lines). Staining with a PE-conjugated isotype control is shown by the dotted lines.
    Figure Legend Snippet: EBNA1-reactive CD4 + -T-cell clones do not utilize perforin to induce cytolysis of LCLs. (A) Cytotoxicity against autologous (solid bars) or MHC II-mismatched (stippled bars) LCL targets after 18 h in culture with BC.E112 or BC.E160 cells at a 1:1 ratio. Prior to exposure to LCLs, clones were incubated with concanamycin A (ConcanA), brefeldin A (BrfdA), or culture medium alone (none). (B) Loss of calcein dye by MHC I-matched LCL targets with (solid bars) or without (stippled bars) addition of EBNA3A antigenic peptide after 3 h in culture with MS.B11 CD8 + -T-cell clones at a 1:1 ratio. (C) Intracellular staining with antibody to perforin. BC EBNA1-reactive CD4 + clones were stimulated with autologous LCLs; the EBNA3A-specific CD8 + -T-cell clone MS.B11 was stimulated with peptide-loaded, MHC I-matched LCL (solid lines). Staining with a PE-conjugated isotype control is shown by the dotted lines.

    Techniques Used: Clone Assay, Incubation, Mass Spectrometry, Staining

    26) Product Images from "Actin- and microtubule-dependent regulation of Golgi morphology by FHDC1"

    Article Title: Actin- and microtubule-dependent regulation of Golgi morphology by FHDC1

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E15-02-0070

    Brefeldin A treatment decreases FHDC1 microtubule localization. (A) In untreated cells, FHDC1 is found primarily on the microtubule network and in close association with the Golgi. (B) After 45 min of treatment with 5.0 μg/ml brefeldin A, FHDC1 subcellular localization in many cells is less filamentous and has a more punctuate and diffuse cytoplasmic distribution, as typified by the indicated cell (arrow). (C) At15 min after removal of brefeldin A, the Golgi ribbon has begun to reassemble; FHDC1 is regaining a more filamentous distribution and is concentrated at the Golgi. (D) In fully recovered cells, FHDC1 is primarily filamentous and preferentially associated with the Golgi. Scale bar, 10 μm. (E) Quantification of data shown in A–D. In untreated cells, FHDC1 is primarily filamentous (blue bars). In contrast, after 45 min of treatment with brefeldin A, FHDC1 exhibits a more punctuate distribution. N = 3, > 100 cells counted per experiment. Error bars indicate SEM.
    Figure Legend Snippet: Brefeldin A treatment decreases FHDC1 microtubule localization. (A) In untreated cells, FHDC1 is found primarily on the microtubule network and in close association with the Golgi. (B) After 45 min of treatment with 5.0 μg/ml brefeldin A, FHDC1 subcellular localization in many cells is less filamentous and has a more punctuate and diffuse cytoplasmic distribution, as typified by the indicated cell (arrow). (C) At15 min after removal of brefeldin A, the Golgi ribbon has begun to reassemble; FHDC1 is regaining a more filamentous distribution and is concentrated at the Golgi. (D) In fully recovered cells, FHDC1 is primarily filamentous and preferentially associated with the Golgi. Scale bar, 10 μm. (E) Quantification of data shown in A–D. In untreated cells, FHDC1 is primarily filamentous (blue bars). In contrast, after 45 min of treatment with brefeldin A, FHDC1 exhibits a more punctuate distribution. N = 3, > 100 cells counted per experiment. Error bars indicate SEM.

    Techniques Used:

    Brefeldin A blocks FHDC1 recruitment to microtubules during recovery from nocodazole treatment. (A) In untreated cells, FHDC1 (red) is concentrated on microtubules associated with the Golgi (GM130; green). (B) Cells were treated for 2 h with 2.5 μg/ml nocodazole and 5.0 μg/ml brefeldin A. The Golgi is dispersed throughout the cytoplasm; FHDC1 has a punctate cytoplasmic distribution. (C) Cells were allowed to recover after nocodazole washout for 120 min in the presence of brefeldin A. FHDC1 does not recover its filamentous staining pattern and maintains its punctate cytoplasmic distribution. (D) Cells were allowed to recover for 120 min in the absence of BFA, with FHDC1 regaining its association with the microtubule network. Scale bar, 10 μm.
    Figure Legend Snippet: Brefeldin A blocks FHDC1 recruitment to microtubules during recovery from nocodazole treatment. (A) In untreated cells, FHDC1 (red) is concentrated on microtubules associated with the Golgi (GM130; green). (B) Cells were treated for 2 h with 2.5 μg/ml nocodazole and 5.0 μg/ml brefeldin A. The Golgi is dispersed throughout the cytoplasm; FHDC1 has a punctate cytoplasmic distribution. (C) Cells were allowed to recover after nocodazole washout for 120 min in the presence of brefeldin A. FHDC1 does not recover its filamentous staining pattern and maintains its punctate cytoplasmic distribution. (D) Cells were allowed to recover for 120 min in the absence of BFA, with FHDC1 regaining its association with the microtubule network. Scale bar, 10 μm.

    Techniques Used: Staining

    27) Product Images from "Co-Expression of miRNA Targeting the Expression of PERK, but Not PKR, Enhances Cellular Immunity from an HIV-1 Env DNA Vaccine"

    Article Title: Co-Expression of miRNA Targeting the Expression of PERK, but Not PKR, Enhances Cellular Immunity from an HIV-1 Env DNA Vaccine

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018225

    miRNA knockdown of PERK increases the immunogenicity of DNA vaccination. ( A ) Immunisation schedule. BALB/c mice were vaccinated three times at one week intervals with the indicated plasmids. Two weeks after the final vaccination, mice were sacrificed and spleens and blood samples were harvested. Splenocytes were isolated and restimulated with ( B ) 1 µM/ml p18 peptide or ( C ) recombinant NL4.3 gp140 protein. Cytokine secretion was prevented by the addition of 2 µg/ml brefeldin-A and samples were stained with monoclonal antibodies for muCD4, muCD8β, and muIFN-γ and analysed by flow cytometry. Data is shown as the proportion of CD4+ or CD8+ T-cells positive for IFN-γ secretion, minus the background stimulation observed with media alone. Pooled results are shown from two independent vaccination experiments and the mean indicated by the solid bars (n = 16 except Group 2 (pNL-140) and Group 5 (pNL-140 miR-muPERK) where n = 15). Results were compared using a Mann-Whitney U test.
    Figure Legend Snippet: miRNA knockdown of PERK increases the immunogenicity of DNA vaccination. ( A ) Immunisation schedule. BALB/c mice were vaccinated three times at one week intervals with the indicated plasmids. Two weeks after the final vaccination, mice were sacrificed and spleens and blood samples were harvested. Splenocytes were isolated and restimulated with ( B ) 1 µM/ml p18 peptide or ( C ) recombinant NL4.3 gp140 protein. Cytokine secretion was prevented by the addition of 2 µg/ml brefeldin-A and samples were stained with monoclonal antibodies for muCD4, muCD8β, and muIFN-γ and analysed by flow cytometry. Data is shown as the proportion of CD4+ or CD8+ T-cells positive for IFN-γ secretion, minus the background stimulation observed with media alone. Pooled results are shown from two independent vaccination experiments and the mean indicated by the solid bars (n = 16 except Group 2 (pNL-140) and Group 5 (pNL-140 miR-muPERK) where n = 15). Results were compared using a Mann-Whitney U test.

    Techniques Used: Mouse Assay, Isolation, Recombinant, Staining, Flow Cytometry, Cytometry, MANN-WHITNEY

    28) Product Images from "Cytokine Production by V?+-T-Cell Subsets Is an Important Factor Determining CD4+-Th-Cell Phenotype and Susceptibility of BALB/c Mice to Coxsackievirus B3-Induced Myocarditis"

    Article Title: Cytokine Production by V?+-T-Cell Subsets Is an Important Factor Determining CD4+-Th-Cell Phenotype and Susceptibility of BALB/c Mice to Coxsackievirus B3-Induced Myocarditis

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.13.5860-5869.2001

    Cytokine staining of Vγ1 and Vγ4 subsets in uninfected H3- and H310A1-infected BALB/c mice. Spleen cells were stimulated for 4 h with PMA, ionomycin, and brefeldin A; surface stained for either Vγ1 or Vγ4; and then intracellularly stained for IFN-γ and IL-4. Cells were gated on the Vγ1 or Vγ4 stained population and then analyzed for cytokine-positive subpopulations. Numbers in the upper right-hand corner represent the percentages of gated cells in the quadrants.
    Figure Legend Snippet: Cytokine staining of Vγ1 and Vγ4 subsets in uninfected H3- and H310A1-infected BALB/c mice. Spleen cells were stimulated for 4 h with PMA, ionomycin, and brefeldin A; surface stained for either Vγ1 or Vγ4; and then intracellularly stained for IFN-γ and IL-4. Cells were gated on the Vγ1 or Vγ4 stained population and then analyzed for cytokine-positive subpopulations. Numbers in the upper right-hand corner represent the percentages of gated cells in the quadrants.

    Techniques Used: Staining, Infection, Mouse Assay

    29) Product Images from "The Antimicrobial Peptide hLF1–11 Drives Monocyte-Dendritic Cell Differentiation toward Dendritic Cells That Promote Antifungal Responses and Enhance Th17 Polarization"

    Article Title: The Antimicrobial Peptide hLF1–11 Drives Monocyte-Dendritic Cell Differentiation toward Dendritic Cells That Promote Antifungal Responses and Enhance Th17 Polarization

    Journal: Journal of Innate Immunity

    doi: 10.1159/000332941

    Cytokine profile by CD4+ T cells after coculture with hLF1–11-differentiated and control (peptide-incubated) DCs. Monocytes were cultured with rhGM-CSF and IL-4 in the presence of hLF1–11 (100 µg/ml; dark gray bars), control peptide (CP, 100 µg/ml; light gray bars), or saline (open bars) for 6 days. Thereafter, cells were stimulated with heat-killed C. albicans (5 × 10 5 /ml), purified protein derivative of M. tuberculosis (PPD; 5 µg/ml), TT; 150 lf/ml) and LPS (100 ng/ml) for 24 h. Next, cells were washed and CD4+ T cells from the same donor and TT were added to the culture. Seventy-two hours later, supernatants were harvested and assessed for IL-17, IL-10 and IFN-γ ( a ) and IL-4, IL-2 levels (data not shown). Next, intracellular cytokine production by the T-lymphocytes in the coculture was assessed by addition of brefeldin A for the last 16 h of the coculture (3 µg/ml). T cells were stained for intracellular IL-17, IL-10 and IFN-γ ( b ). Data are expressed as boxes and whiskers; boxes represent medians and second and third interquartiles, whiskers represent ranges in experiments with 6–8 different donors. * p
    Figure Legend Snippet: Cytokine profile by CD4+ T cells after coculture with hLF1–11-differentiated and control (peptide-incubated) DCs. Monocytes were cultured with rhGM-CSF and IL-4 in the presence of hLF1–11 (100 µg/ml; dark gray bars), control peptide (CP, 100 µg/ml; light gray bars), or saline (open bars) for 6 days. Thereafter, cells were stimulated with heat-killed C. albicans (5 × 10 5 /ml), purified protein derivative of M. tuberculosis (PPD; 5 µg/ml), TT; 150 lf/ml) and LPS (100 ng/ml) for 24 h. Next, cells were washed and CD4+ T cells from the same donor and TT were added to the culture. Seventy-two hours later, supernatants were harvested and assessed for IL-17, IL-10 and IFN-γ ( a ) and IL-4, IL-2 levels (data not shown). Next, intracellular cytokine production by the T-lymphocytes in the coculture was assessed by addition of brefeldin A for the last 16 h of the coculture (3 µg/ml). T cells were stained for intracellular IL-17, IL-10 and IFN-γ ( b ). Data are expressed as boxes and whiskers; boxes represent medians and second and third interquartiles, whiskers represent ranges in experiments with 6–8 different donors. * p

    Techniques Used: Incubation, Cell Culture, Purification, Staining

    30) Product Images from "Efficacy of a Virus-Like Nanoparticle As Treatment for a Chronic Viral Infection Is Hindered by IRAK1 Regulation and Antibody Interference"

    Article Title: Efficacy of a Virus-Like Nanoparticle As Treatment for a Chronic Viral Infection Is Hindered by IRAK1 Regulation and Antibody Interference

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01885

    PapMV induces Sca-1 on splenic and bone marrow-derived plasmacytoid dendritic cell (pDCs), despite its expression not being associated with interferon (IFN)-α production. (A) Percentages of pDCs from spleen (left) and bone marrow (right) positive for Sca-1 expression 1, 5, and 25 days following an immunization with control (black squares), R837 (open inverted triangles), and PapMV (open circles) ( n = 2–4, 1–3 mice per group). (B) Representative flow cytometry plots of IFN-α production by pDCs according to their Sca-1 expression profile 4 h postimmunization followed by a 4-h incubation with Brefeldin A (BFA). Spleen (top) and bone marrow (bottom) samples are represented. (C) Percentages of intracellular IFN-α + pDCs found in the spleen (left) and the bone marrow (right) 4 h following an immunization with control or PapMV followed by 4-h incubation with BFA before intracellular staining (* p
    Figure Legend Snippet: PapMV induces Sca-1 on splenic and bone marrow-derived plasmacytoid dendritic cell (pDCs), despite its expression not being associated with interferon (IFN)-α production. (A) Percentages of pDCs from spleen (left) and bone marrow (right) positive for Sca-1 expression 1, 5, and 25 days following an immunization with control (black squares), R837 (open inverted triangles), and PapMV (open circles) ( n = 2–4, 1–3 mice per group). (B) Representative flow cytometry plots of IFN-α production by pDCs according to their Sca-1 expression profile 4 h postimmunization followed by a 4-h incubation with Brefeldin A (BFA). Spleen (top) and bone marrow (bottom) samples are represented. (C) Percentages of intracellular IFN-α + pDCs found in the spleen (left) and the bone marrow (right) 4 h following an immunization with control or PapMV followed by 4-h incubation with BFA before intracellular staining (* p

    Techniques Used: Derivative Assay, Expressing, Mouse Assay, Flow Cytometry, Cytometry, Incubation, Staining

    31) Product Images from "APP dimer formation is initiated in the endoplasmic reticulum and differs between APP isoforms"

    Article Title: APP dimer formation is initiated in the endoplasmic reticulum and differs between APP isoforms

    Journal: Cellular and Molecular Life Sciences

    doi: 10.1007/s00018-011-0882-4

    Homodimerization of APP wt can be induced by inhibiting protein traffic out of the ER with brefeldin A. a CHO-K1 cells stably overexpressing either APP695 wt or APP695 fused to the ER retention motif KKAA (APP695 ER) were treated with 5 μg/ml brefeldin A (BFA) for the indicated time points (0–8 h). Aliquots of cell lysates were mixed with SDS sample buffer, containing BME without heat denaturing and separated on a 4–12% SDS-PAGE. APP was detected with CT15 antibody, shown on one representative Western blot. The bottom panel shows a longer exposure time, indicating dimer formation of APP695 wt when export of the ER is blocked by BFA. b Diagram showing the ratio of APP wt dimer formation during BFA treatment, plotted against time, with ± SEM of three independent experiments, statistical significance: *** p
    Figure Legend Snippet: Homodimerization of APP wt can be induced by inhibiting protein traffic out of the ER with brefeldin A. a CHO-K1 cells stably overexpressing either APP695 wt or APP695 fused to the ER retention motif KKAA (APP695 ER) were treated with 5 μg/ml brefeldin A (BFA) for the indicated time points (0–8 h). Aliquots of cell lysates were mixed with SDS sample buffer, containing BME without heat denaturing and separated on a 4–12% SDS-PAGE. APP was detected with CT15 antibody, shown on one representative Western blot. The bottom panel shows a longer exposure time, indicating dimer formation of APP695 wt when export of the ER is blocked by BFA. b Diagram showing the ratio of APP wt dimer formation during BFA treatment, plotted against time, with ± SEM of three independent experiments, statistical significance: *** p

    Techniques Used: Stable Transfection, SDS Page, Western Blot

    APP wt homodimers are formed in the ER but also occur at the cell surface. a N2a cells were transiently co-transfected with NT HA APP695 CT Split GFP 1–10 and NT HA APP695 CT Split GFP 11. The cells were fixed with paraformaldehyde and immunostained with ER marker Grp78, followed by incubation with AlexaFlour-594 conjugated secondary antibody. The green fluorescence represents the APP dimer, induced by bimolecular fluorescence complementation (BiFC). Co-staining with Grp78 revealed colocalization of the APP dimer ( inset ) with the ER. b Single transfections of N2a cells with either NT HA APP695 CT Split GFP 1–10 or NT HA APP695 CT Split GFP 11. APP expression was verified with HA antibody, confirming a non-perturbed distribution ( right panel , red ). The green channel does not show any specific fluorescence. Scale bar is 10 μm. c N2a cells were transiently transfected with NT HA APP695 CT Split GFP 1–10, NT HA APP695 CT Split GFP 11 or GFP only. Cell lysates were analyzed via Western blotting for APP full length ( top part ), APP CTFs ( middle part ) and GFP, or GFP-containing CTFs ( bottom part ). Note that GFP antibody does not recognize GFP 11 alone. d CHO-K1 cells stably overexpressing APP 695 wt were pretreated for 1 h at 37°C in the presence of 10 μg/ml brefeldin A (BFA), or DMSO (vehicle) for 1 h to block protein secretion from the ER. Subsequently, cells were either directly lysed (BFA), or medium containing BFA was removed, and cells were incubated for additional 30 min at 37°C with 10 μM chlorpromazine (BFA/CPZ), to allow transport to the cell surface, but inhibit further endocytosis. Lysates were analyzed either under non-reducing (+β mercaptoethanol; −95°C), or reducing conditions (+β-mercaptoethanol; +95°C). Note that cells pretreated with BFA, followed by chlorpromazine exposure (BFA/CPZ), display two upper SDS-resistant bands migrating with a slight size shift, indicating different glycosylation patterns (dimer i/m). Note that strong denaturing conditions (+95°C) result in the disappearance of the upper migrating bands, suggesting disulfide bond formation. The separating line indicates different exposure times to visualize dimer formation in treated cells, whereas APP ER overexpressing cells served as size reference for dimers. e Cells were treated as described under d , and subjected to cell surface biotinylation to confirm drug treatments. APP ER is completely absent from the surface, whereas only minor quantities of mature APP reach the surface upon BFA treatment. Removal of BFA, and subsequent incubation with chlorpromazine (BFA/CPZ), strongly increases surface exposed mature APP, compared to vehicle control. Note that no dimers are recovered in biotinylation, because NeutrAvidin beads were boiled in BME containing sample buffer, to release biotin conjugated proteins
    Figure Legend Snippet: APP wt homodimers are formed in the ER but also occur at the cell surface. a N2a cells were transiently co-transfected with NT HA APP695 CT Split GFP 1–10 and NT HA APP695 CT Split GFP 11. The cells were fixed with paraformaldehyde and immunostained with ER marker Grp78, followed by incubation with AlexaFlour-594 conjugated secondary antibody. The green fluorescence represents the APP dimer, induced by bimolecular fluorescence complementation (BiFC). Co-staining with Grp78 revealed colocalization of the APP dimer ( inset ) with the ER. b Single transfections of N2a cells with either NT HA APP695 CT Split GFP 1–10 or NT HA APP695 CT Split GFP 11. APP expression was verified with HA antibody, confirming a non-perturbed distribution ( right panel , red ). The green channel does not show any specific fluorescence. Scale bar is 10 μm. c N2a cells were transiently transfected with NT HA APP695 CT Split GFP 1–10, NT HA APP695 CT Split GFP 11 or GFP only. Cell lysates were analyzed via Western blotting for APP full length ( top part ), APP CTFs ( middle part ) and GFP, or GFP-containing CTFs ( bottom part ). Note that GFP antibody does not recognize GFP 11 alone. d CHO-K1 cells stably overexpressing APP 695 wt were pretreated for 1 h at 37°C in the presence of 10 μg/ml brefeldin A (BFA), or DMSO (vehicle) for 1 h to block protein secretion from the ER. Subsequently, cells were either directly lysed (BFA), or medium containing BFA was removed, and cells were incubated for additional 30 min at 37°C with 10 μM chlorpromazine (BFA/CPZ), to allow transport to the cell surface, but inhibit further endocytosis. Lysates were analyzed either under non-reducing (+β mercaptoethanol; −95°C), or reducing conditions (+β-mercaptoethanol; +95°C). Note that cells pretreated with BFA, followed by chlorpromazine exposure (BFA/CPZ), display two upper SDS-resistant bands migrating with a slight size shift, indicating different glycosylation patterns (dimer i/m). Note that strong denaturing conditions (+95°C) result in the disappearance of the upper migrating bands, suggesting disulfide bond formation. The separating line indicates different exposure times to visualize dimer formation in treated cells, whereas APP ER overexpressing cells served as size reference for dimers. e Cells were treated as described under d , and subjected to cell surface biotinylation to confirm drug treatments. APP ER is completely absent from the surface, whereas only minor quantities of mature APP reach the surface upon BFA treatment. Removal of BFA, and subsequent incubation with chlorpromazine (BFA/CPZ), strongly increases surface exposed mature APP, compared to vehicle control. Note that no dimers are recovered in biotinylation, because NeutrAvidin beads were boiled in BME containing sample buffer, to release biotin conjugated proteins

    Techniques Used: Transfection, Marker, Incubation, Fluorescence, Bimolecular Fluorescence Complementation Assay, Staining, Expressing, Western Blot, Stable Transfection, Blocking Assay

    32) Product Images from "Immunomodulation of human intestinal T cells by the synthetic CD80 antagonist RhuDex®"

    Article Title: Immunomodulation of human intestinal T cells by the synthetic CD80 antagonist RhuDex®

    Journal: Immunity, Inflammation and Disease

    doi: 10.1002/iid3.34

    RhuDex® impairs cytokine release of CD4 + T cells. WO-LPL and PBL were stimulated with anti-CD3 or anti-CD2 for 6 h and Brefeldin A was added for the last 4 h. The fraction of T cells expressing intracellular cytokines (IL-17, IL-2, IFN-γ, and TNF-α) as gated on CD3 + CD4 + T cells were determined. Shown is the normalized intracellular cytokine expression of (A) CD4 + WO-LP T cells (2 tissue donors) and (B) CD4 + PB T cells (2 allogeneic donors) in the absence of inhibitors (medium set to 100%) and in the presence of inhibitors (Aba, Abatacept, Rhu, RhuDex®). Data points for each donor are shown in grey circles, and the mean of all data points in each condition is shown as columns.
    Figure Legend Snippet: RhuDex® impairs cytokine release of CD4 + T cells. WO-LPL and PBL were stimulated with anti-CD3 or anti-CD2 for 6 h and Brefeldin A was added for the last 4 h. The fraction of T cells expressing intracellular cytokines (IL-17, IL-2, IFN-γ, and TNF-α) as gated on CD3 + CD4 + T cells were determined. Shown is the normalized intracellular cytokine expression of (A) CD4 + WO-LP T cells (2 tissue donors) and (B) CD4 + PB T cells (2 allogeneic donors) in the absence of inhibitors (medium set to 100%) and in the presence of inhibitors (Aba, Abatacept, Rhu, RhuDex®). Data points for each donor are shown in grey circles, and the mean of all data points in each condition is shown as columns.

    Techniques Used: Expressing

    33) 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

    34) 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

    35) Product Images from "Endophilin A2 deficiency protects rodents from autoimmune arthritis by modulating T cell activation"

    Article Title: Endophilin A2 deficiency protects rodents from autoimmune arthritis by modulating T cell activation

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20586-2

    Endophilin A2 co-localizes with the TCR upon activation and regulates TCR internalization and signaling. a EA2 co-localization with the TCR in Jurkat cells after anti-CD3/CD28 stimulation determined by proximity-ligase assay and visualized as TexasRed + spots using confocal imaging. b Percentage of internalized TCR in T cells from 5 DA and 5 DA Mut rats after anti-CD3/CD28 stimulation. Data has been reproduced two times. c Percentage of internalized TCR in CD4 + CD3 + T cells from three SH3gl1 −/− and SH3gl1 +/+ mice stimulated with anti-CD3/CD28 for 30 min in the presence or absence of Brefeldin A at 1.25 μg/ml. Experiment has been repeated twice with the same results. d Representative Western blot analysis from two experiments of phosphorylated and unphosphorylated ZAP70 and ERK1/2 in T cells from two SH3gl1 −/− and SH3gl1 +/+ mice stimulated with anti-CD3/CD28 for 0, 3, and 7 min. Histone 2B was used as loading control. e Normalized OD values of phosphorylated Zap70 compared to unphosphorylated Zap70 from two SH3gl1 −/− and SH3gl1 +/+ mice. f Normalized OD values of phosphorylated ERK1/2 compared to unphosphorylated ERK1/2 from two SH3gl1 −/− and SH3gl1 +/+ mice. g Flow cytometry blot showing in vitro cell proliferation of DA Mut and DA T cells 72 h after anti-CD3/CD28 stimulation. h Division index of sorted T cells from 7 DA and 5 DA Mut rats after 72 h of anti-CD3/CD28 stimulation. Non-parametrical Mann–Whitney U test was used for statistical evaluation of data. Data are presented as mean with error bars indicating ± SEM with each dot representing an individual value.
    Figure Legend Snippet: Endophilin A2 co-localizes with the TCR upon activation and regulates TCR internalization and signaling. a EA2 co-localization with the TCR in Jurkat cells after anti-CD3/CD28 stimulation determined by proximity-ligase assay and visualized as TexasRed + spots using confocal imaging. b Percentage of internalized TCR in T cells from 5 DA and 5 DA Mut rats after anti-CD3/CD28 stimulation. Data has been reproduced two times. c Percentage of internalized TCR in CD4 + CD3 + T cells from three SH3gl1 −/− and SH3gl1 +/+ mice stimulated with anti-CD3/CD28 for 30 min in the presence or absence of Brefeldin A at 1.25 μg/ml. Experiment has been repeated twice with the same results. d Representative Western blot analysis from two experiments of phosphorylated and unphosphorylated ZAP70 and ERK1/2 in T cells from two SH3gl1 −/− and SH3gl1 +/+ mice stimulated with anti-CD3/CD28 for 0, 3, and 7 min. Histone 2B was used as loading control. e Normalized OD values of phosphorylated Zap70 compared to unphosphorylated Zap70 from two SH3gl1 −/− and SH3gl1 +/+ mice. f Normalized OD values of phosphorylated ERK1/2 compared to unphosphorylated ERK1/2 from two SH3gl1 −/− and SH3gl1 +/+ mice. g Flow cytometry blot showing in vitro cell proliferation of DA Mut and DA T cells 72 h after anti-CD3/CD28 stimulation. h Division index of sorted T cells from 7 DA and 5 DA Mut rats after 72 h of anti-CD3/CD28 stimulation. Non-parametrical Mann–Whitney U test was used for statistical evaluation of data. Data are presented as mean with error bars indicating ± SEM with each dot representing an individual value.

    Techniques Used: Activation Assay, Imaging, Mouse Assay, Western Blot, Flow Cytometry, In Vitro, MANN-WHITNEY

    36) Product Images from "Bacillus Calmette-Guérin Induces PD-L1 Expression on Antigen-Presenting Cells via Autocrine and Paracrine Interleukin-STAT3 Circuits"

    Article Title: Bacillus Calmette-Guérin Induces PD-L1 Expression on Antigen-Presenting Cells via Autocrine and Paracrine Interleukin-STAT3 Circuits

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-40145-0

    In vivo PD-L1 blockade during BCG immunisation boosts CD4 T-cell cytokine production. Splenocytes from immunised mice were cultured in duplicate with 2 μg/mL α-CD28 and either Ag85B/Acr (5 μg/mL) or PPD (5 μg/mL) for 6 hours, in the presence of brefeldin A (10 μg/mL). (A) Cells were then stained for Th1-Th17 cytokines and gated by size/viability → CD3 + → CD4 + /CD8 + . (B) CD4 T-cell cytokine responses. (C) CD8 T-cell cytokine responses. Significance was tested by two-way ANOVA with Dunnett’s post-test. Data are derived from n = 3 mice per group. Bars depict means ± SEM. **** p
    Figure Legend Snippet: In vivo PD-L1 blockade during BCG immunisation boosts CD4 T-cell cytokine production. Splenocytes from immunised mice were cultured in duplicate with 2 μg/mL α-CD28 and either Ag85B/Acr (5 μg/mL) or PPD (5 μg/mL) for 6 hours, in the presence of brefeldin A (10 μg/mL). (A) Cells were then stained for Th1-Th17 cytokines and gated by size/viability → CD3 + → CD4 + /CD8 + . (B) CD4 T-cell cytokine responses. (C) CD8 T-cell cytokine responses. Significance was tested by two-way ANOVA with Dunnett’s post-test. Data are derived from n = 3 mice per group. Bars depict means ± SEM. **** p

    Techniques Used: In Vivo, Mouse Assay, Cell Culture, Staining, Derivative Assay

    37) Product Images from "The Role of CD38 on the Function of Regulatory B Cells in a Murine Model of Lupus"

    Article Title: The Role of CD38 on the Function of Regulatory B Cells in a Murine Model of Lupus

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19102906

    Increased frequencies of B10 + B10pro cells in spleen from Cd38 −/− mice. ( a ) Spleen cells from naïve WT and Cd38 −/− mice were cultured with medium alone (control cells, left panels), with LPS + PMA + Ionomycin + Brefeldin A (LPIB) for 5 h (B10 cells, middle panels), or with anti-CD40 agonistic mAb for 48 h, with LPIB added during the last 5 h of culture (B10 + B10pro cells, right panels). Then cells were stained with CD19 mAb, permeabilized, and stained with IL-10 mAb for flow cytometry analysis. Gates show the percentages of IL-10 producing cells among total CD19 + B cells. Pseudocolor/smoothing/outliers plots are represented. Blue and green colors correspond to areas of lower cell density, red and orange are areas of high cell density, and yellow is mid-range. Outliers represent the lowest density of cells and are represented as black dots. ( b ) Bars represent the mean ± SEM of the percentages of IL-10 + CD19 + B cells in controls, B10 cells, and B10 + B10pro cells from WT splenocytes (closed bars) or Cd38 −/− splenocytes (open bars). The frequency of CD19 + B cells that express IL-10 + increases significantly after 48 h of anti-CD40 stimulation, reflecting maturation of B10pro cells into IL-10 competent B10 cells. ** p = 0.0051; *** p = 0.0003; **** p
    Figure Legend Snippet: Increased frequencies of B10 + B10pro cells in spleen from Cd38 −/− mice. ( a ) Spleen cells from naïve WT and Cd38 −/− mice were cultured with medium alone (control cells, left panels), with LPS + PMA + Ionomycin + Brefeldin A (LPIB) for 5 h (B10 cells, middle panels), or with anti-CD40 agonistic mAb for 48 h, with LPIB added during the last 5 h of culture (B10 + B10pro cells, right panels). Then cells were stained with CD19 mAb, permeabilized, and stained with IL-10 mAb for flow cytometry analysis. Gates show the percentages of IL-10 producing cells among total CD19 + B cells. Pseudocolor/smoothing/outliers plots are represented. Blue and green colors correspond to areas of lower cell density, red and orange are areas of high cell density, and yellow is mid-range. Outliers represent the lowest density of cells and are represented as black dots. ( b ) Bars represent the mean ± SEM of the percentages of IL-10 + CD19 + B cells in controls, B10 cells, and B10 + B10pro cells from WT splenocytes (closed bars) or Cd38 −/− splenocytes (open bars). The frequency of CD19 + B cells that express IL-10 + increases significantly after 48 h of anti-CD40 stimulation, reflecting maturation of B10pro cells into IL-10 competent B10 cells. ** p = 0.0051; *** p = 0.0003; **** p

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

    38) Product Images from "Pollen-associated phytoprostanes inhibit dendritic cell interleukin-12 production and augment T helper type 2 cell polarization"

    Article Title: Pollen-associated phytoprostanes inhibit dendritic cell interleukin-12 production and augment T helper type 2 cell polarization

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20041065

    DCs matured in the presence of Bet.- APE display reduced Th1- and increased Th2-polarizing capacity. (A) DCs were left untreated or stimulated with Bet .-APE (3 mg/ml) in the presence or absence of LPS (100 ng/ml). After 24 h DCs were washed and cocultured with CD4 + CD45RA + allogenic T cells (DC/T cell ratio 1:4) that were expanded for 12 d in the presence of IL-2. T cell polarization was determined by analyzing intracellular IFN-γ and IL-4 accumulation via flow cytometry after restimulation with PMA and ionomycin in the presence of brefeldin A during the last 2 h of stimulation. To address, if the Bet.- APE–dependent Th2 polarization could be reverted by exogenous IL-12, hrIL-12 (10 ng/ml) was added at the beginning of the coculture of Bet .-APE/LPS-treated DCs and T cells. Representative experiment of n = 3–6 (compare Table I ). To explore the role of IL-4 in the Th2 polarization induced by Bet .-APE–treated DCs, IL-4–neutralizing antibodies (10 μg/ml) were added at the beginning of the DC/T cell coculture. Representative experiment of n = 3.
    Figure Legend Snippet: DCs matured in the presence of Bet.- APE display reduced Th1- and increased Th2-polarizing capacity. (A) DCs were left untreated or stimulated with Bet .-APE (3 mg/ml) in the presence or absence of LPS (100 ng/ml). After 24 h DCs were washed and cocultured with CD4 + CD45RA + allogenic T cells (DC/T cell ratio 1:4) that were expanded for 12 d in the presence of IL-2. T cell polarization was determined by analyzing intracellular IFN-γ and IL-4 accumulation via flow cytometry after restimulation with PMA and ionomycin in the presence of brefeldin A during the last 2 h of stimulation. To address, if the Bet.- APE–dependent Th2 polarization could be reverted by exogenous IL-12, hrIL-12 (10 ng/ml) was added at the beginning of the coculture of Bet .-APE/LPS-treated DCs and T cells. Representative experiment of n = 3–6 (compare Table I ). To explore the role of IL-4 in the Th2 polarization induced by Bet .-APE–treated DCs, IL-4–neutralizing antibodies (10 μg/ml) were added at the beginning of the DC/T cell coculture. Representative experiment of n = 3.

    Techniques Used: Flow Cytometry, Cytometry

    39) Product Images from "Regulated Degradation of the HIV-1 Vpu Protein through a ?TrCP-Independent Pathway Limits the Release of Viral Particles"

    Article Title: Regulated Degradation of the HIV-1 Vpu Protein through a ?TrCP-Independent Pathway Limits the Release of Viral Particles

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0030104

    Vpu Degradation in Nocodazole-Treated Cells Is Independent of the SCF βTrCP Activity (A) Vpu degradation occurs via the proteasome pathway. HeLa cells either mock-transfected (lanes 1, 3, and 5) or expressing Vpu-HA-GFP (lanes 2, 4, and 6) were left untreated or treated for 18 h with nocodazole (lanes 3–6). Where indicated, MG132 was added 6 h prior to harvest of cells. Cell lysates were analyzed by western blot using the indicated antibodies. (B) Vpu expression is not sensitive to brefeldin A or to short nocodazole treatments. HeLa cells expressing Vpu-HA-GFP were treated with nocodazole during either 18 h (lane 2) or 2.5 h (lane 3) and with BFA during 1.5 h (lane 4) or DMSO alone (lane 1). Cell lysates were separated by SDS-PAGE and analyzed by western blot using the indicated antibodies. (C) Degradadion of Vpu in nocodazole-treated cells does not require a functional βTrCP-binding site. HeLa cells were transfected with the expression vectors encoding either wild-type Vpu-HA-GFP (lanes 1 and 3) or the Vpu 2/6-HA-GFP mutant defective in βTrCP binding (lanes 2 and 4), together with a plasmid expressing Myc-βgalactosidase used as an internal reporter. Cells were left untreated or treated 18 h with nocodazole as indicated. Vpu-HA-GFP and Myc-βgalactosidase were revealed by western blot using anti-Vpu and anti-myc antibodies, respectively. The histogram represents the Vpu/βgalactosidase ratios, calculated from the respective quantified signals. (D) Vpu expression from the HIV-1 provirus is reduced in nocodazole-treated cells. HeLa cells were transfected with pNL4–3ΔVpr provirus together with a plasmid expressing GFP used as an internal control. Cells were treated with nocodazole where indicated. Vpu was detected by western blot using anti-Vpu antibodies. (E) Vpu is not detectable in infected mitotic cells. HeLa cells were transfected by the pNL4–3 provirus. 48 h after transfection, cells were fixed and analyzed by immunofluorescence using anti-Gag and anti-Vpu antibodies. DNA was revealed by DAPI staining. Arrows 1 and 2 indicate mitotic cells and arrows 3 and 4 indicate interphasic cells. wt, wild-type.
    Figure Legend Snippet: Vpu Degradation in Nocodazole-Treated Cells Is Independent of the SCF βTrCP Activity (A) Vpu degradation occurs via the proteasome pathway. HeLa cells either mock-transfected (lanes 1, 3, and 5) or expressing Vpu-HA-GFP (lanes 2, 4, and 6) were left untreated or treated for 18 h with nocodazole (lanes 3–6). Where indicated, MG132 was added 6 h prior to harvest of cells. Cell lysates were analyzed by western blot using the indicated antibodies. (B) Vpu expression is not sensitive to brefeldin A or to short nocodazole treatments. HeLa cells expressing Vpu-HA-GFP were treated with nocodazole during either 18 h (lane 2) or 2.5 h (lane 3) and with BFA during 1.5 h (lane 4) or DMSO alone (lane 1). Cell lysates were separated by SDS-PAGE and analyzed by western blot using the indicated antibodies. (C) Degradadion of Vpu in nocodazole-treated cells does not require a functional βTrCP-binding site. HeLa cells were transfected with the expression vectors encoding either wild-type Vpu-HA-GFP (lanes 1 and 3) or the Vpu 2/6-HA-GFP mutant defective in βTrCP binding (lanes 2 and 4), together with a plasmid expressing Myc-βgalactosidase used as an internal reporter. Cells were left untreated or treated 18 h with nocodazole as indicated. Vpu-HA-GFP and Myc-βgalactosidase were revealed by western blot using anti-Vpu and anti-myc antibodies, respectively. The histogram represents the Vpu/βgalactosidase ratios, calculated from the respective quantified signals. (D) Vpu expression from the HIV-1 provirus is reduced in nocodazole-treated cells. HeLa cells were transfected with pNL4–3ΔVpr provirus together with a plasmid expressing GFP used as an internal control. Cells were treated with nocodazole where indicated. Vpu was detected by western blot using anti-Vpu antibodies. (E) Vpu is not detectable in infected mitotic cells. HeLa cells were transfected by the pNL4–3 provirus. 48 h after transfection, cells were fixed and analyzed by immunofluorescence using anti-Gag and anti-Vpu antibodies. DNA was revealed by DAPI staining. Arrows 1 and 2 indicate mitotic cells and arrows 3 and 4 indicate interphasic cells. wt, wild-type.

    Techniques Used: Activity Assay, Transfection, Expressing, Western Blot, SDS Page, Functional Assay, Binding Assay, Mutagenesis, Plasmid Preparation, Infection, Immunofluorescence, Staining

    40) Product Images from "Type I Interferon Plays Opposing Roles in Cytotoxicity and Interferon-γ Production by Natural Killer and CD8+ T Cells after Influenza A Virus Infection in Mice"

    Article Title: Type I Interferon Plays Opposing Roles in Cytotoxicity and Interferon-γ Production by Natural Killer and CD8+ T Cells after Influenza A Virus Infection in Mice

    Journal: Journal of Innate Immunity

    doi: 10.1159/000356824

    IFN-γ-producing cells in the lungs of Ifnar1 −/− mice after infection with influenza virus A/FM/1/47. Lung cells were harvested on days 3, 6 and 9 after infection with 25 pfu of influenza virus A/FM/1/47. The samples were incubated with 10 μg/ml brefeldin A for 4 h at 37°C. Expression of IFN-γ was detected by intracellular staining and analyzed by flow cytometry. Each group consists of 3 mice. The data are representative of at least 3 independent experiments. Error bars represent the mean ± SEM. * p
    Figure Legend Snippet: IFN-γ-producing cells in the lungs of Ifnar1 −/− mice after infection with influenza virus A/FM/1/47. Lung cells were harvested on days 3, 6 and 9 after infection with 25 pfu of influenza virus A/FM/1/47. The samples were incubated with 10 μg/ml brefeldin A for 4 h at 37°C. Expression of IFN-γ was detected by intracellular staining and analyzed by flow cytometry. Each group consists of 3 mice. The data are representative of at least 3 independent experiments. Error bars represent the mean ± SEM. * p

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

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    Article Snippet: Superantigen toxic shock syndrome toxin 1 (TSST1), kindly given by the laboratory of Andres Alcover (Institut Pasteur), were used at a final concentration of 0·2 μg/ml. .. Brefeldin A (BFA; Sigma Aldrich) was used at a final concentration of 5 μg/ml. .. Statistical analyses were performed using GraphPad Prism version 6.0 software.

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    Article Snippet: Tunicamycin and brefeldin A were obtained from Sigma.

    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: M1- and M2-Mφ were seeded into 96 well microplates (Greiner Bio-One GmbH) and were infected with TB40E, ΔUS2-11 (MOI of 5) or left untreated. .. 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.

    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: M1- and M2-Mφ were seeded into 96 well microplates (Greiner Bio-One GmbH) and were infected with TB40E, ΔUS2-11 (MOI of 5) or left untreated. .. 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.

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    Article Title: Identification of novel HIV-1 dependency factors in primary CCR4+CCR6+Th17 cells via a genome-wide transcriptional approach
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    Millipore brefeldin a
    Effect of passive IgG transference to neonates on B and T cell responses . Neonate pups (3 d-o) from nonimmunized mothers injected with IgG from nonimmunized or immunized mothers and simultaneously immunized with OVA were evaluated (20 d-o) for: (A) anti-OVA IgE Ab levels by PCA reaction; (B) B cell FcγRIIb expression (B220+IgM+) and histogram of FcγRIIb expression on B cells of offspring from immunized (shaded histogram, MFI in bold numbers) or nonimmunized mothers (white histogram, MFI in light numbers); (C) intracellular cytokines of splenic B cells (B220+) or (D) CD4+ T cells after 24 h incubation with 10 μg/mL brefeldin A; data shown in B-D were obtained flow cytometry. The results represent the mean ± SEM of 9 mice per group. *  P  ≤ 0.05 compared to offspring from nonimmunized mothers.
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    Effect of passive IgG transference to neonates on B and T cell responses . Neonate pups (3 d-o) from nonimmunized mothers injected with IgG from nonimmunized or immunized mothers and simultaneously immunized with OVA were evaluated (20 d-o) for: (A) anti-OVA IgE Ab levels by PCA reaction; (B) B cell FcγRIIb expression (B220+IgM+) and histogram of FcγRIIb expression on B cells of offspring from immunized (shaded histogram, MFI in bold numbers) or nonimmunized mothers (white histogram, MFI in light numbers); (C) intracellular cytokines of splenic B cells (B220+) or (D) CD4+ T cells after 24 h incubation with 10 μg/mL brefeldin A; data shown in B-D were obtained flow cytometry. The results represent the mean ± SEM of 9 mice per group. *  P  ≤ 0.05 compared to offspring from nonimmunized mothers.

    Journal: BMC Immunology

    Article Title: Maternal immunization with ovalbumin prevents neonatal allergy development and up-regulates inhibitory receptor Fc?RIIB expression on B cells

    doi: 10.1186/1471-2172-11-11

    Figure Lengend Snippet: Effect of passive IgG transference to neonates on B and T cell responses . Neonate pups (3 d-o) from nonimmunized mothers injected with IgG from nonimmunized or immunized mothers and simultaneously immunized with OVA were evaluated (20 d-o) for: (A) anti-OVA IgE Ab levels by PCA reaction; (B) B cell FcγRIIb expression (B220+IgM+) and histogram of FcγRIIb expression on B cells of offspring from immunized (shaded histogram, MFI in bold numbers) or nonimmunized mothers (white histogram, MFI in light numbers); (C) intracellular cytokines of splenic B cells (B220+) or (D) CD4+ T cells after 24 h incubation with 10 μg/mL brefeldin A; data shown in B-D were obtained flow cytometry. The results represent the mean ± SEM of 9 mice per group. * P ≤ 0.05 compared to offspring from nonimmunized mothers.

    Article Snippet: To determine intracellular cytokines, SMCs were cultivated in 24-well plates (Costar) with Brefeldin A (10 μg/mL, Sigma) for 24 h. Next, cells were washed with PBS-BSA solution, labeled with fluorochrome-conjugated CD4 or B220.

    Techniques: Injection, Expressing, Incubation, Flow Cytometry, Cytometry, Mouse Assay

    Effect of passive IgG transference to pregnant mice on offspring's B and T cell responses . Nonimmunized pregnant mice were injected with IgG from nonimmunized or immunized mothers. Offspring immunized with OVA were evaluated (20 d-o) for: (a) anti-OVA IgE Ab levels by PCA reaction; (b) CD80, CD86, CD40 CD23 molecule expression on splenic B cells (B220+); (c) B cell FcγRIIb expression (B220+IgM+) by flow cytometry. Histogram of FcγRIIb expression on B cells of offspring from immunized (shaded histogram, MFI in bold numbers) or nonimmunized mothers (white histogram, MFI in light numbers); (d) intracellular cytokines of splenic B cells (B220+) and (e) CD4+ T cells after 424 h incubation with 10 μg/mL brefeldin A, all by flow cytometry. The results represent the mean ± SEM of 6 mice per group. * P  ≤ 0.05 compared to offspring from nonimmunized mothers.

    Journal: BMC Immunology

    Article Title: Maternal immunization with ovalbumin prevents neonatal allergy development and up-regulates inhibitory receptor Fc?RIIB expression on B cells

    doi: 10.1186/1471-2172-11-11

    Figure Lengend Snippet: Effect of passive IgG transference to pregnant mice on offspring's B and T cell responses . Nonimmunized pregnant mice were injected with IgG from nonimmunized or immunized mothers. Offspring immunized with OVA were evaluated (20 d-o) for: (a) anti-OVA IgE Ab levels by PCA reaction; (b) CD80, CD86, CD40 CD23 molecule expression on splenic B cells (B220+); (c) B cell FcγRIIb expression (B220+IgM+) by flow cytometry. Histogram of FcγRIIb expression on B cells of offspring from immunized (shaded histogram, MFI in bold numbers) or nonimmunized mothers (white histogram, MFI in light numbers); (d) intracellular cytokines of splenic B cells (B220+) and (e) CD4+ T cells after 424 h incubation with 10 μg/mL brefeldin A, all by flow cytometry. The results represent the mean ± SEM of 6 mice per group. * P ≤ 0.05 compared to offspring from nonimmunized mothers.

    Article Snippet: To determine intracellular cytokines, SMCs were cultivated in 24-well plates (Costar) with Brefeldin A (10 μg/mL, Sigma) for 24 h. Next, cells were washed with PBS-BSA solution, labeled with fluorochrome-conjugated CD4 or B220.

    Techniques: Mouse Assay, Injection, Expressing, Flow Cytometry, Cytometry, Incubation

    Effect of maternal immunization with OVA on the immune response of nonimmunized or immunized neonates . Neonate pups (3 d-o) from control or immune mothers were immunized or not with OVA and evaluated (20 d-o) for: (A) IgG1, IgG2a and IgM by ELISA; (B) anti-OVA IgE Ab levels by PCA reaction; (C) intracellular cytokines of splenic B cells (B220+) or (d) CD4+ T cells after 24 h incubation with 10 μg/mL brefeldin A by flow cytometry. The results represent the mean ± SEM of 12 mice per group. *P ≤ 0.05 compared to offspring from nonimmunized mothers, # P ≤ 0.05 compared to nonimmunized offspring from control mothers, • P ≤ 0.05 compared to control offspring from immune mothers.

    Journal: BMC Immunology

    Article Title: Maternal immunization with ovalbumin prevents neonatal allergy development and up-regulates inhibitory receptor Fc?RIIB expression on B cells

    doi: 10.1186/1471-2172-11-11

    Figure Lengend Snippet: Effect of maternal immunization with OVA on the immune response of nonimmunized or immunized neonates . Neonate pups (3 d-o) from control or immune mothers were immunized or not with OVA and evaluated (20 d-o) for: (A) IgG1, IgG2a and IgM by ELISA; (B) anti-OVA IgE Ab levels by PCA reaction; (C) intracellular cytokines of splenic B cells (B220+) or (d) CD4+ T cells after 24 h incubation with 10 μg/mL brefeldin A by flow cytometry. The results represent the mean ± SEM of 12 mice per group. *P ≤ 0.05 compared to offspring from nonimmunized mothers, # P ≤ 0.05 compared to nonimmunized offspring from control mothers, • P ≤ 0.05 compared to control offspring from immune mothers.

    Article Snippet: To determine intracellular cytokines, SMCs were cultivated in 24-well plates (Costar) with Brefeldin A (10 μg/mL, Sigma) for 24 h. Next, cells were washed with PBS-BSA solution, labeled with fluorochrome-conjugated CD4 or B220.

    Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry, Cytometry, Mouse Assay

    TLR-dependency of Poke weed mitogen (PWM)-induced cell stimulation. A: B cell proliferation. Human CD19+ peripheral blood B cells were stained with CFSE and stimulated with 1 or 10 µg/ml PWM in the presence or absence of 5 µg/ml Staphylococcus aureus protein A (SpA) or 5 µg/ml anti-human Ig F(ab′) 2 fragments (aIg) as B cell receptor stimuli, with SpA or aIg alone, or left unstimulated. After 5 days B cells were harvested, stained with anti-CD20 and live gated cells were analyzed for CFSE dilution. The percentages of live gated cells (upper right corner) and proliferating cells (left) are indicated in each diagram. The diagrams depict the results from one representative experiment of n = 3. B: TNF-induction. Human PBMC were stimulated for four hours with or without PWM, TLR2 ligands FSL-1R (FSL) and Pam 3 CSK 4 (P3), phosphorothioate-modified DNA ODN CpG 2006 (CpG) and 2006 GC (GC) and LPS in the presence of Brefeldin A (BfA) or with LPS in the absence of BfA. Subsequently, intracellular staining was performed with an anti-human TNF mAb and TNF expression was analyzed by flow cytometry. The results show a summary of the data obtained in n = 4 experiments. Mean values of anti-TNF mean fluorescence intensities are provided ± SEM. C: Antagonization with Polymyxin B. CFSE-stained human CD19+ B cells were stimulated with PWM in the presence and absence of polymyxin B. Proliferation was assessed by flow cytometric analysis of CFSE dilution on day 5. The results obtained in two representative donors of n = 3 are shown. D: MyD88-dependency of B cell stimulation. B220+ B cells were isolated from the spleens of MyD88 −/− mice and their wild type counterparts. B cells were stimulated with CpG, P3, PWM and PMA/Ionomycin (P/I) and harvested after 72 hours and a 18 hour pulse with 3 H-thymidine. The diagram shows the average values in counts per minute (cpm) of n = 4 experiments ± SEM. E: TLR-dependency. Wild type, TLR2−/− and TLR9−/− B cells were isolated from murine spleen with anti-CD19 microbeads, labelled with CFSE and stimulated with TLR2 ligands Pam 3 CSK 4 (P3) and FSL-1R (FSL), PWM (10 µg/ml), TLR9 ligand CpG ODN 1668 or 1668 GC control ODN. After 4 days B cell proliferation (CFSE dilution) was quantified by flow cytometry. The diagram depicts the mean fluorescence intensity (MFI) for CFSE in live gated cells as mean value ± SEM from n = 4 experiments. Note that low MFI corresponds to strong proliferation while high MFI values reveal absence of proliferation.

    Journal: PLoS ONE

    Article Title: Poke Weed Mitogen Requires Toll-Like Receptor Ligands for Proliferative Activity in Human and Murine B Lymphocytes

    doi: 10.1371/journal.pone.0029806

    Figure Lengend Snippet: TLR-dependency of Poke weed mitogen (PWM)-induced cell stimulation. A: B cell proliferation. Human CD19+ peripheral blood B cells were stained with CFSE and stimulated with 1 or 10 µg/ml PWM in the presence or absence of 5 µg/ml Staphylococcus aureus protein A (SpA) or 5 µg/ml anti-human Ig F(ab′) 2 fragments (aIg) as B cell receptor stimuli, with SpA or aIg alone, or left unstimulated. After 5 days B cells were harvested, stained with anti-CD20 and live gated cells were analyzed for CFSE dilution. The percentages of live gated cells (upper right corner) and proliferating cells (left) are indicated in each diagram. The diagrams depict the results from one representative experiment of n = 3. B: TNF-induction. Human PBMC were stimulated for four hours with or without PWM, TLR2 ligands FSL-1R (FSL) and Pam 3 CSK 4 (P3), phosphorothioate-modified DNA ODN CpG 2006 (CpG) and 2006 GC (GC) and LPS in the presence of Brefeldin A (BfA) or with LPS in the absence of BfA. Subsequently, intracellular staining was performed with an anti-human TNF mAb and TNF expression was analyzed by flow cytometry. The results show a summary of the data obtained in n = 4 experiments. Mean values of anti-TNF mean fluorescence intensities are provided ± SEM. C: Antagonization with Polymyxin B. CFSE-stained human CD19+ B cells were stimulated with PWM in the presence and absence of polymyxin B. Proliferation was assessed by flow cytometric analysis of CFSE dilution on day 5. The results obtained in two representative donors of n = 3 are shown. D: MyD88-dependency of B cell stimulation. B220+ B cells were isolated from the spleens of MyD88 −/− mice and their wild type counterparts. B cells were stimulated with CpG, P3, PWM and PMA/Ionomycin (P/I) and harvested after 72 hours and a 18 hour pulse with 3 H-thymidine. The diagram shows the average values in counts per minute (cpm) of n = 4 experiments ± SEM. E: TLR-dependency. Wild type, TLR2−/− and TLR9−/− B cells were isolated from murine spleen with anti-CD19 microbeads, labelled with CFSE and stimulated with TLR2 ligands Pam 3 CSK 4 (P3) and FSL-1R (FSL), PWM (10 µg/ml), TLR9 ligand CpG ODN 1668 or 1668 GC control ODN. After 4 days B cell proliferation (CFSE dilution) was quantified by flow cytometry. The diagram depicts the mean fluorescence intensity (MFI) for CFSE in live gated cells as mean value ± SEM from n = 4 experiments. Note that low MFI corresponds to strong proliferation while high MFI values reveal absence of proliferation.

    Article Snippet: Analysis of intracellular TNF expression in human monocytes was performed as previously described ; briefly, PBMC (2×10*6/ml) were incubated with stimulatory reagents with/without Brefeldin A (1 µg/ml, Sigma) for 4 hours, fixed, permeabilized, stained with PE-conjugated anti-human TNF (Becton Dickinson) and analyzed by flow cytometry.

    Techniques: Cell Stimulation, Staining, Modification, Expressing, Flow Cytometry, Cytometry, Fluorescence, Isolation, Mouse Assay

    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.

    Journal: Current Alzheimer research

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

    doi:

    Figure Lengend 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.

    Article Snippet: Brefeldin A and monensin were from Sigma.

    Techniques: Protein Extraction, Western Blot

    RhuDex® impairs cytokine release of CD4 + T cells. WO-LPL and PBL were stimulated with anti-CD3 or anti-CD2 for 6 h and Brefeldin A was added for the last 4 h. The fraction of T cells expressing intracellular cytokines (IL-17, IL-2, IFN-γ, and TNF-α) as gated on CD3 + CD4 + T cells were determined. Shown is the normalized intracellular cytokine expression of (A) CD4 + WO-LP T cells (2 tissue donors) and (B) CD4 + PB T cells (2 allogeneic donors) in the absence of inhibitors (medium set to 100%) and in the presence of inhibitors (Aba, Abatacept, Rhu, RhuDex®). Data points for each donor are shown in grey circles, and the mean of all data points in each condition is shown as columns.

    Journal: Immunity, Inflammation and Disease

    Article Title: Immunomodulation of human intestinal T cells by the synthetic CD80 antagonist RhuDex®

    doi: 10.1002/iid3.34

    Figure Lengend Snippet: RhuDex® impairs cytokine release of CD4 + T cells. WO-LPL and PBL were stimulated with anti-CD3 or anti-CD2 for 6 h and Brefeldin A was added for the last 4 h. The fraction of T cells expressing intracellular cytokines (IL-17, IL-2, IFN-γ, and TNF-α) as gated on CD3 + CD4 + T cells were determined. Shown is the normalized intracellular cytokine expression of (A) CD4 + WO-LP T cells (2 tissue donors) and (B) CD4 + PB T cells (2 allogeneic donors) in the absence of inhibitors (medium set to 100%) and in the presence of inhibitors (Aba, Abatacept, Rhu, RhuDex®). Data points for each donor are shown in grey circles, and the mean of all data points in each condition is shown as columns.

    Article Snippet: After 2 h of incubation, 10 µg/mL Brefeldin A (Sigma–Aldrich) was added for an additional 4 h. Subsequently, cells were harvested, pooling two wells per condition, and the intracellular staining procedure was performed using BD Cytofix/Cytoperm™ (BD Biosciences) solutions according to manufacturer's instructions.

    Techniques: Expressing