monensin  (Thermo Fisher)


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    eBioscience Monensin Solution 1000X
    Description:
    Monensin is an inhibitor of intracellular protein transport Incubation of cells in culture with Monensin leads to blockade of protein transport to the Golgi complex GC and accumulation of proteins in the endoplasmic reticulum ER Addition of Monensin during the last hours of in vitro activation of cells results in enhanced detection of intracellular cytokines Monensin is effective for enhanced detection of a majority of mouse and human intracellular cytokines however it is advised that the investigators evaluate the use and efficacy of this reagent as well as other protein transport inhibitors such as Brefeldin A in their specific assay system Reported ApplicationIntracellular Staining Followed by Flow Cytometric Analysis
    Catalog Number:
    00-4505-51
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    Lab Reagents and Chemicals
    Applications:
    Cell Analysis|Cellular Imaging|Flow Cytometry
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    Structured Review

    Thermo Fisher monensin
    AREG Induces EGFR Phosphorylation at Tyr-992, which Allows for the Interaction between T1/ST2 and EGFR WT (gray) and Areg −/− (purple) mice were infected with H. polygyrus , and on day 14 post infection, mLN were harvested. (A and B) mLN cells were stimulated with rIL-33, anti-CD3, or media, and the IL-13 (A) and p-ERK (B) expression was determined by intra-cellular staining and flow cytometry analysis. (C) MHCII-deficient mice were infected with H. polygyrus and 7 days post-infection received flow cytometry-sorted CD4 + T cells derived from mLN of naive or H. polygyrus -infected WT, Egfr fl/fl xCd4-cre , or Areg −/− mice. Worm burden and egg counts were determined 2 weeks post infection. (D–F) mLN cells were stimulated with rIL-33, rAREG, both, or media only, and EGFR p-Y1068 (D), p-ERK (E), and IL-13 (F) expression was determined by flow cytometry analysis. (G) EGFR phosphorylation at position Y992 on CD4 + T cells derived from mLN of WT or Areg −/− H. polygyrus -infected mice in the presence or absence of rAREG. (H) HEK293T cells were transfected as indicated with T1/ST2 and the IL-1RacP in combination with WT EGFR or EGFR Y992F mutant. Subsequently, the cell lysates were analyzed for the expression of the transfected proteins (input, left panel). The same lysates were also subjected to an EGFR-specific immunoprecipitation (EGFR-IP, right panel) or were treated with the isotype control (iso, right panel). Precipitates were analyzed by immunoblot. (I) mLN cells were stimulated with rIL-33 in the presence of <t>monensin,</t> and EGFR expression on CD4 + T cells was analyzed by flow cytometry. All data are representative of at least two independent experiments (mean ± SEM); results for individual mice are shown as dots. See also Figure S7 .
    Monensin is an inhibitor of intracellular protein transport Incubation of cells in culture with Monensin leads to blockade of protein transport to the Golgi complex GC and accumulation of proteins in the endoplasmic reticulum ER Addition of Monensin during the last hours of in vitro activation of cells results in enhanced detection of intracellular cytokines Monensin is effective for enhanced detection of a majority of mouse and human intracellular cytokines however it is advised that the investigators evaluate the use and efficacy of this reagent as well as other protein transport inhibitors such as Brefeldin A in their specific assay system Reported ApplicationIntracellular Staining Followed by Flow Cytometric Analysis
    https://www.bioz.com/result/monensin/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monensin - by Bioz Stars, 2021-03
    86/100 stars

    Images

    1) Product Images from "Epidermal Growth Factor Receptor Expression Licenses Type-2 Helper T Cells to Function in a T Cell Receptor-Independent Fashion"

    Article Title: Epidermal Growth Factor Receptor Expression Licenses Type-2 Helper T Cells to Function in a T Cell Receptor-Independent Fashion

    Journal: Immunity

    doi: 10.1016/j.immuni.2017.09.013

    AREG Induces EGFR Phosphorylation at Tyr-992, which Allows for the Interaction between T1/ST2 and EGFR WT (gray) and Areg −/− (purple) mice were infected with H. polygyrus , and on day 14 post infection, mLN were harvested. (A and B) mLN cells were stimulated with rIL-33, anti-CD3, or media, and the IL-13 (A) and p-ERK (B) expression was determined by intra-cellular staining and flow cytometry analysis. (C) MHCII-deficient mice were infected with H. polygyrus and 7 days post-infection received flow cytometry-sorted CD4 + T cells derived from mLN of naive or H. polygyrus -infected WT, Egfr fl/fl xCd4-cre , or Areg −/− mice. Worm burden and egg counts were determined 2 weeks post infection. (D–F) mLN cells were stimulated with rIL-33, rAREG, both, or media only, and EGFR p-Y1068 (D), p-ERK (E), and IL-13 (F) expression was determined by flow cytometry analysis. (G) EGFR phosphorylation at position Y992 on CD4 + T cells derived from mLN of WT or Areg −/− H. polygyrus -infected mice in the presence or absence of rAREG. (H) HEK293T cells were transfected as indicated with T1/ST2 and the IL-1RacP in combination with WT EGFR or EGFR Y992F mutant. Subsequently, the cell lysates were analyzed for the expression of the transfected proteins (input, left panel). The same lysates were also subjected to an EGFR-specific immunoprecipitation (EGFR-IP, right panel) or were treated with the isotype control (iso, right panel). Precipitates were analyzed by immunoblot. (I) mLN cells were stimulated with rIL-33 in the presence of monensin, and EGFR expression on CD4 + T cells was analyzed by flow cytometry. All data are representative of at least two independent experiments (mean ± SEM); results for individual mice are shown as dots. See also Figure S7 .
    Figure Legend Snippet: AREG Induces EGFR Phosphorylation at Tyr-992, which Allows for the Interaction between T1/ST2 and EGFR WT (gray) and Areg −/− (purple) mice were infected with H. polygyrus , and on day 14 post infection, mLN were harvested. (A and B) mLN cells were stimulated with rIL-33, anti-CD3, or media, and the IL-13 (A) and p-ERK (B) expression was determined by intra-cellular staining and flow cytometry analysis. (C) MHCII-deficient mice were infected with H. polygyrus and 7 days post-infection received flow cytometry-sorted CD4 + T cells derived from mLN of naive or H. polygyrus -infected WT, Egfr fl/fl xCd4-cre , or Areg −/− mice. Worm burden and egg counts were determined 2 weeks post infection. (D–F) mLN cells were stimulated with rIL-33, rAREG, both, or media only, and EGFR p-Y1068 (D), p-ERK (E), and IL-13 (F) expression was determined by flow cytometry analysis. (G) EGFR phosphorylation at position Y992 on CD4 + T cells derived from mLN of WT or Areg −/− H. polygyrus -infected mice in the presence or absence of rAREG. (H) HEK293T cells were transfected as indicated with T1/ST2 and the IL-1RacP in combination with WT EGFR or EGFR Y992F mutant. Subsequently, the cell lysates were analyzed for the expression of the transfected proteins (input, left panel). The same lysates were also subjected to an EGFR-specific immunoprecipitation (EGFR-IP, right panel) or were treated with the isotype control (iso, right panel). Precipitates were analyzed by immunoblot. (I) mLN cells were stimulated with rIL-33 in the presence of monensin, and EGFR expression on CD4 + T cells was analyzed by flow cytometry. All data are representative of at least two independent experiments (mean ± SEM); results for individual mice are shown as dots. See also Figure S7 .

    Techniques Used: Mouse Assay, Infection, Expressing, Staining, Flow Cytometry, Cytometry, Derivative Assay, Transfection, Mutagenesis, Immunoprecipitation

    IL-33-Induced IL-13 Production by Th2 Cells Is Dependent on a Signaling Complex between T1/ST2 and EGFR WT and Egfr fl/fl xCd4-cre (EGFR ΔCD4 ) mice were infected with H. polygyrus larvae, and on day 14 post infection, mLN were harvested. (A–C) Cells were stimulated with rIL-33, anti-CD3, or media in the presence of gefitinib, marimastat, or vehicle, and p-ERK (A and C) or IL-13 (B) expression was determined by intra-cellular staining and flow cytometry analysis. (D and E) Cells were stimulated with rHB-EGF, anti-CD3, or media, and IL-13 (D) and p-ERK (E) expression was determined by intra-cellular staining and flow cytometry analysis. (F) EGFR expression on stimulated mLN WT CD4 + T cells in the presence of monensin was analyzed by flow cytometry. (G) HEK293T cells were transfected as indicated with T1/ST2, the IL-1RacP, or the EGFR alone or in combination. Subsequently, the cells lysates were analyzed for the expression of the transfected proteins (input, left panel). The same lysates were also subjected to an EGFR-specific immunoprecipitation (EGFR-IP, right panel) or were treated with the isotype control (iso, right panel). Precipitates were analyzed by immunoblot. (H) mLN (upper) or HaCaT cells (lower) were stimulated with rIL-33, rHB-EGF, or media, and the EGFR phosphorylation at position Y1068 was determined by intra-cellular staining and flow cytometry analysis (upper) or immunoblot (lower). All data are representative of at least two independent experiments (mean ± SEM); results for individual mice are shown as dots. See also Figure S6 .
    Figure Legend Snippet: IL-33-Induced IL-13 Production by Th2 Cells Is Dependent on a Signaling Complex between T1/ST2 and EGFR WT and Egfr fl/fl xCd4-cre (EGFR ΔCD4 ) mice were infected with H. polygyrus larvae, and on day 14 post infection, mLN were harvested. (A–C) Cells were stimulated with rIL-33, anti-CD3, or media in the presence of gefitinib, marimastat, or vehicle, and p-ERK (A and C) or IL-13 (B) expression was determined by intra-cellular staining and flow cytometry analysis. (D and E) Cells were stimulated with rHB-EGF, anti-CD3, or media, and IL-13 (D) and p-ERK (E) expression was determined by intra-cellular staining and flow cytometry analysis. (F) EGFR expression on stimulated mLN WT CD4 + T cells in the presence of monensin was analyzed by flow cytometry. (G) HEK293T cells were transfected as indicated with T1/ST2, the IL-1RacP, or the EGFR alone or in combination. Subsequently, the cells lysates were analyzed for the expression of the transfected proteins (input, left panel). The same lysates were also subjected to an EGFR-specific immunoprecipitation (EGFR-IP, right panel) or were treated with the isotype control (iso, right panel). Precipitates were analyzed by immunoblot. (H) mLN (upper) or HaCaT cells (lower) were stimulated with rIL-33, rHB-EGF, or media, and the EGFR phosphorylation at position Y1068 was determined by intra-cellular staining and flow cytometry analysis (upper) or immunoblot (lower). All data are representative of at least two independent experiments (mean ± SEM); results for individual mice are shown as dots. See also Figure S6 .

    Techniques Used: Mouse Assay, Infection, Expressing, Staining, Flow Cytometry, Cytometry, Transfection, Immunoprecipitation

    2) Product Images from "Protective and Pathogenic Roles for B Cells During Systemic Autoimmunity in NZB/W F1 Mice"

    Article Title: Protective and Pathogenic Roles for B Cells During Systemic Autoimmunity in NZB/W F1 Mice

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

    doi: 10.4049/jimmunol.0902391

    The expanded B10 cell subset in NZB/W F 1 mice is depleted by CD20 mAb. A, Representative analysis of IL-10-producing spleen B cell frequencies in NZB/W F 1 and C57BL/6 mice. Splenocytes were cultured with LPS, PMA, ionomycin, and monensin for 5 h, stained for cell-surface CD19, CD1d, and CD5, permeabilized before staining for intracellular IL-10, with subsequent multi-color flow cytometry analysis. B , Mean (±SEM) spleen cytoplasmic IL-10 + B (CD19 + ) cell frequencies and numbers in 10 wk-old NZB/W F 1 (control mAb-treated) and C57BL/6 mice (n≥3). C , Relative cell surface CD19 and CD5 expression levels by cytoplasmic IL-10 + and IL-10 − spleen B cells from NZB/W F 1 mice as assessed in ( A ). D , Representative B220 expression by CD1d hi CD5 + , CD1d hi CD5 − , and CD1d int CD5 + spleen B220 + B cell subsets in 10 wk-old NZB/W F 1 mice. Bar graphs indicate mean (±SEM) frequencies and numbers of CD1d hi CD5 + spleen B cells in NZB/W F 1 and C57BL/6 mice. E , Representative frequencies of spleen CD5 + B220 lo B cells in 10 wk-old NZB/W F 1 and C57BL/6 mice. Bar graphs indicate mean (±SEM) frequencies and numbers of CD5 + B220 lo B cells. F , Depletion of IL-10-producing spleen B cells in NZB/W F 1 mice by CD20 mAb. CD20 or control mAbs (100 μg) were given i.p. with IL-10-producing B cells quantified 7 days later as in ( A ). Bar graphs indicate mean (±SEM) frequencies and numbers of IL-10-producing CD19 + cells. A–F , All data represent results obtained from ≥3 mice (age 10 weeks) per group, with significant differences between mean (±SEM) values indicated; *p
    Figure Legend Snippet: The expanded B10 cell subset in NZB/W F 1 mice is depleted by CD20 mAb. A, Representative analysis of IL-10-producing spleen B cell frequencies in NZB/W F 1 and C57BL/6 mice. Splenocytes were cultured with LPS, PMA, ionomycin, and monensin for 5 h, stained for cell-surface CD19, CD1d, and CD5, permeabilized before staining for intracellular IL-10, with subsequent multi-color flow cytometry analysis. B , Mean (±SEM) spleen cytoplasmic IL-10 + B (CD19 + ) cell frequencies and numbers in 10 wk-old NZB/W F 1 (control mAb-treated) and C57BL/6 mice (n≥3). C , Relative cell surface CD19 and CD5 expression levels by cytoplasmic IL-10 + and IL-10 − spleen B cells from NZB/W F 1 mice as assessed in ( A ). D , Representative B220 expression by CD1d hi CD5 + , CD1d hi CD5 − , and CD1d int CD5 + spleen B220 + B cell subsets in 10 wk-old NZB/W F 1 mice. Bar graphs indicate mean (±SEM) frequencies and numbers of CD1d hi CD5 + spleen B cells in NZB/W F 1 and C57BL/6 mice. E , Representative frequencies of spleen CD5 + B220 lo B cells in 10 wk-old NZB/W F 1 and C57BL/6 mice. Bar graphs indicate mean (±SEM) frequencies and numbers of CD5 + B220 lo B cells. F , Depletion of IL-10-producing spleen B cells in NZB/W F 1 mice by CD20 mAb. CD20 or control mAbs (100 μg) were given i.p. with IL-10-producing B cells quantified 7 days later as in ( A ). Bar graphs indicate mean (±SEM) frequencies and numbers of IL-10-producing CD19 + cells. A–F , All data represent results obtained from ≥3 mice (age 10 weeks) per group, with significant differences between mean (±SEM) values indicated; *p

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

    3) Product Images from "Cd1d regulates B cell development but not B cell accumulation and IL10 production in mice with pathologic CD5+ B cell expansion"

    Article Title: Cd1d regulates B cell development but not B cell accumulation and IL10 production in mice with pathologic CD5+ B cell expansion

    Journal: BMC Immunology

    doi: 10.1186/s12865-015-0130-z

    CD5 + B cells in 12 week-old dnRAG1 and DTG mice support inducible IL10 expression in vitro, but IL10 competence is CD1d-independent. a - b Splenocytes from 12 week-old wild-type, dnRAG1, Eμ-TCL1, and DTG mice on a Cd1d +/+ ( a ) or Cd1d del/del ( b ) background were incubated with monensin alone or LPS + PIM for 4 h. After permeabilization, the cells were stained with IL-10-specific or isotype-control monoclonal antibody (Ab) as indicated. Gated CD19 + B cells were analyzed for CD5 and IL10 expression. The percentage of cells in each quadrant is shown for representative animals. c The mean percentage of total CD5 + B cells staining positive for IL10 for n = 4 mice/genotype is plotted in bar graph format. Error bars represent the SEM. Statistically significant ( p
    Figure Legend Snippet: CD5 + B cells in 12 week-old dnRAG1 and DTG mice support inducible IL10 expression in vitro, but IL10 competence is CD1d-independent. a - b Splenocytes from 12 week-old wild-type, dnRAG1, Eμ-TCL1, and DTG mice on a Cd1d +/+ ( a ) or Cd1d del/del ( b ) background were incubated with monensin alone or LPS + PIM for 4 h. After permeabilization, the cells were stained with IL-10-specific or isotype-control monoclonal antibody (Ab) as indicated. Gated CD19 + B cells were analyzed for CD5 and IL10 expression. The percentage of cells in each quadrant is shown for representative animals. c The mean percentage of total CD5 + B cells staining positive for IL10 for n = 4 mice/genotype is plotted in bar graph format. Error bars represent the SEM. Statistically significant ( p

    Techniques Used: Mouse Assay, Expressing, In Vitro, Incubation, Staining

    Related Articles

    Staining:

    Article Title: Intestinal Microbiota Promotes Psoriasis-Like Skin Inflammation by Enhancing Th17 Response
    Article Snippet: To analyze intracellular RORγt, the cells were first stained with FITC conjugated anti-CD3 (clone 145-2C11, dilution 1:100) and then fixed, permeabilized by Intracellular fixation & permeabilization buffer set (eBioscience, San Diego, CA, USA) and stained intracellularly for PE-conjugated anti-RORγt (clone AFJKS-9, dilution 1:50). .. For intracellular staining of produced cytokines, cells were first stimulated with 1 μg/ml PMA and 2.8 μM Ionomycin (both from Sigma-Aldrich) for 8 hours and then treated with a mixture of Brefeldin A and Monensin (both from eBioscience) for 4 hours, according to the manufacturer's instructions. .. The cells were stained with extracellular FITC conjugated anti-CD3 (clone 145-2C11, dilution 1:100), fixed, permeabilized and stained for intracellular PE conjugated anti-IL-17 (clone eBio17B7, dilution 1:50) and APC conjugated anti-IFN-γ (clone XMG1.2, dilution 1:50) both from eBioscience.

    Article Title: ILC2s regulate adaptive Th2 cell functions via PD-L1 checkpoint control
    Article Snippet: For staining of transcription factors, cells were fixed and permeabilized using the FoxP3 staining buffer kit (eBioscience) and stained with the following antibodies: PE-, PE-Cy7–, or e660-conjugated GATA3 (TWAJ), FITC- or APC-conjugated anti-Tbet (O4-46; BD), Brilliant Violet 421–conjugated anti-RORγT (Q31-378), and APC- or PE-CF594–conjugated anti-FoxP3 (MF23; BD). .. For intracellular detection of cytokines, cells were stimulated with 50 ng/ml PMA (Sigma) and 1 µg/ml ionomycin (R & D Systems) for 5 h in the presence of monensin (eBioscience) for the last 3 h. After staining of surface antigens, cells were fixed and permeabilized using the BD Cytofix/Cytoperm kit (BD), and the following antibodies were used for staining: FITC- or APC-conjugated anti–IL-4 (BVD5-24G2), PE-conjugated anti–IL-5 (TRFK5; BD), APC- or PE-Cy7–conjugated anti–IL-13, PerCP-Cy5.5– or APC-conjugated anti-IFNγ (XMG1.2 and eBio13A), PE-conjugated anti-IL17A (TC11-18H10; BD), and PerCP-Cy5.5–conjugated anti–IL-9 (D9302C12; BD). .. Cell sorting ILC2s were sorted by FACS from the lungs of day 5 N. brasiliensis –infected mice.

    Article Title: IL-4R?-Independent Expression of Mannose Receptor and Ym1 by Macrophages Depends on their IL-10 Responsiveness
    Article Snippet: Dead cells were excluded from analysis by using 7-amino-actinomycin D (7-AAD, Sigma). .. For detection of intracellular IL-13, cells were further incubated 4h with monensin, fixed for 20 min on ice in 2% (wt/ol) paraformaldehyde and permeabilized during 30 min with 1× permeabilization buffer (eBioscience) before being stained with anti-IL-13-PE (eBioscience, eBio13a ). ..

    Article Title: Regulatory T Cells Limit Induction of Protective Immunity and Promote Immune Pathology Following Intestinal Helminth Infection
    Article Snippet: .. For intracellular cytokine staining, cells were stimulated at 5×106 cells/ml in DMEM media with PMA/Ionomycin (100ng/ml and 500ng/ml, respectively; Sigma-Aldrich) or 5µg/ml of Trichuris E/S antigen for 12–16 hours in the presence of Monensin (1:1000; eBioscience). ..

    Article Title: B Cell Presentation of Chlamydia Antigen Selects Out Protective CD4γ13 T Cells: Implications for Genital Tract Tissue-Resident Memory Lymphocyte Clusters
    Article Snippet: T cells were stained for 20 min at 4°C with 1 μg per 1 million T cells in RPMI complete medium with 10% fetal bovine serum (FBS), fixed with 1% paraformaldehyde, and analyzed by flow cytometry (BD Facscalibur or LSRII). .. For intracellular staining for IL-13 (ebio13A coupled to PE) and IFN-γ (XMG1.2 coupled to allophycocyanin), T cells were activated for 5 h in a cocktail of phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A, and monensin (cell stimulation cocktail; Ebioscience), stained for CD8a, and then fixed and permeabilized (Fix/Perm buffer; Ebioscience), stained for IL-13–PE/IFN-γ–allophycocyanin or control antibody (eBRG1-PE/IFN-γ–allophycocyanin) in the presence of 2 mg/ml donkey IgG (Jackson ImmunoResearch) for 30 min at room temperature, washed, suspended in 1% paraformaldehyde, and analyzed. .. All of the T cell populations are > 90% CD4 T cells; negative staining based on CD8a was chosen because PMA-ionomycin activation resulted in shedding of cell surface CD4 and diminished CD4 staining; CD8a staining was not affected (data not shown).

    Produced:

    Article Title: Intestinal Microbiota Promotes Psoriasis-Like Skin Inflammation by Enhancing Th17 Response
    Article Snippet: To analyze intracellular RORγt, the cells were first stained with FITC conjugated anti-CD3 (clone 145-2C11, dilution 1:100) and then fixed, permeabilized by Intracellular fixation & permeabilization buffer set (eBioscience, San Diego, CA, USA) and stained intracellularly for PE-conjugated anti-RORγt (clone AFJKS-9, dilution 1:50). .. For intracellular staining of produced cytokines, cells were first stimulated with 1 μg/ml PMA and 2.8 μM Ionomycin (both from Sigma-Aldrich) for 8 hours and then treated with a mixture of Brefeldin A and Monensin (both from eBioscience) for 4 hours, according to the manufacturer's instructions. .. The cells were stained with extracellular FITC conjugated anti-CD3 (clone 145-2C11, dilution 1:100), fixed, permeabilized and stained for intracellular PE conjugated anti-IL-17 (clone eBio17B7, dilution 1:50) and APC conjugated anti-IFN-γ (clone XMG1.2, dilution 1:50) both from eBioscience.

    Expressing:

    Article Title: Lymphocytic choriomeningitis virus expands a population of NKG2D+CD8+ T cells that exacerbate disease in mice co-infected with Leishmania major
    Article Snippet: For intracellular staining, cells were previously permeabilized with 0.2% of saponin buffer and stained for IFN-γ, gzmB, and/or IL-17A (eBioscience or Invitrogen). .. To assess CD107a expression, cells were incubated with BFA, monensin, and anti-CD107a (eBioscience) for 6 hours. .. Fixable Aqua dye (Invitrogen) was added to assess cell viability.

    Incubation:

    Article Title: Lymphocytic choriomeningitis virus expands a population of NKG2D+CD8+ T cells that exacerbate disease in mice co-infected with Leishmania major
    Article Snippet: For intracellular staining, cells were previously permeabilized with 0.2% of saponin buffer and stained for IFN-γ, gzmB, and/or IL-17A (eBioscience or Invitrogen). .. To assess CD107a expression, cells were incubated with BFA, monensin, and anti-CD107a (eBioscience) for 6 hours. .. Fixable Aqua dye (Invitrogen) was added to assess cell viability.

    Article Title: IL-4R?-Independent Expression of Mannose Receptor and Ym1 by Macrophages Depends on their IL-10 Responsiveness
    Article Snippet: Dead cells were excluded from analysis by using 7-amino-actinomycin D (7-AAD, Sigma). .. For detection of intracellular IL-13, cells were further incubated 4h with monensin, fixed for 20 min on ice in 2% (wt/ol) paraformaldehyde and permeabilized during 30 min with 1× permeabilization buffer (eBioscience) before being stained with anti-IL-13-PE (eBioscience, eBio13a ). ..

    Cell Stimulation:

    Article Title: B Cell Presentation of Chlamydia Antigen Selects Out Protective CD4γ13 T Cells: Implications for Genital Tract Tissue-Resident Memory Lymphocyte Clusters
    Article Snippet: T cells were stained for 20 min at 4°C with 1 μg per 1 million T cells in RPMI complete medium with 10% fetal bovine serum (FBS), fixed with 1% paraformaldehyde, and analyzed by flow cytometry (BD Facscalibur or LSRII). .. For intracellular staining for IL-13 (ebio13A coupled to PE) and IFN-γ (XMG1.2 coupled to allophycocyanin), T cells were activated for 5 h in a cocktail of phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A, and monensin (cell stimulation cocktail; Ebioscience), stained for CD8a, and then fixed and permeabilized (Fix/Perm buffer; Ebioscience), stained for IL-13–PE/IFN-γ–allophycocyanin or control antibody (eBRG1-PE/IFN-γ–allophycocyanin) in the presence of 2 mg/ml donkey IgG (Jackson ImmunoResearch) for 30 min at room temperature, washed, suspended in 1% paraformaldehyde, and analyzed. .. All of the T cell populations are > 90% CD4 T cells; negative staining based on CD8a was chosen because PMA-ionomycin activation resulted in shedding of cell surface CD4 and diminished CD4 staining; CD8a staining was not affected (data not shown).

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    • monensin  (Thermo Fisher)
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    Thermo Fisher monensin
    Circulating frequencies and mRNA expression of T helper type 1 (Th1), Th2 and Th17 in human peripheral blood mononuclear cells (PBMCs). PBMCs from patients with dilated cardiomyopathy (DCM; n = 18) and control groups ( n = 20) were stimulated with PMA, ionomycin and <t>monensin</t> for 4 hr, and then stained with labelled antibodies as described in the Materials and methods. (a) Representative dot plot shows the gated CD4 T cells on the FSC/SSC. (b–d) Representative FACS pictures from a single patient in each group and the percentages of interferon‐ γ (IFN‐ γ ) (b), IL‐17 (c) and interleukin‐4 (IL‐4) (d) were comparable between the patients with DCM and control groups. (e) Relative mRNA expression of IFN‐ γ , IL‐17 and IL‐4 in stimulated PBMCs of patients with DCM and controls. * P
    Monensin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monensin/product/Thermo Fisher
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    Price from $9.99 to $1999.99
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    Circulating frequencies and mRNA expression of T helper type 1 (Th1), Th2 and Th17 in human peripheral blood mononuclear cells (PBMCs). PBMCs from patients with dilated cardiomyopathy (DCM; n = 18) and control groups ( n = 20) were stimulated with PMA, ionomycin and monensin for 4 hr, and then stained with labelled antibodies as described in the Materials and methods. (a) Representative dot plot shows the gated CD4 T cells on the FSC/SSC. (b–d) Representative FACS pictures from a single patient in each group and the percentages of interferon‐ γ (IFN‐ γ ) (b), IL‐17 (c) and interleukin‐4 (IL‐4) (d) were comparable between the patients with DCM and control groups. (e) Relative mRNA expression of IFN‐ γ , IL‐17 and IL‐4 in stimulated PBMCs of patients with DCM and controls. * P

    Journal: Immunology

    Article Title: CD4+ CD25+ GARP+ regulatory T cells display a compromised suppressive function in patients with dilated cardiomyopathy

    doi: 10.1111/imm.12728

    Figure Lengend Snippet: Circulating frequencies and mRNA expression of T helper type 1 (Th1), Th2 and Th17 in human peripheral blood mononuclear cells (PBMCs). PBMCs from patients with dilated cardiomyopathy (DCM; n = 18) and control groups ( n = 20) were stimulated with PMA, ionomycin and monensin for 4 hr, and then stained with labelled antibodies as described in the Materials and methods. (a) Representative dot plot shows the gated CD4 T cells on the FSC/SSC. (b–d) Representative FACS pictures from a single patient in each group and the percentages of interferon‐ γ (IFN‐ γ ) (b), IL‐17 (c) and interleukin‐4 (IL‐4) (d) were comparable between the patients with DCM and control groups. (e) Relative mRNA expression of IFN‐ γ , IL‐17 and IL‐4 in stimulated PBMCs of patients with DCM and controls. * P

    Article Snippet: For Th cell analysis, PBMCs were seeded at a density of 2 × 106 cells/well in RPMI‐1640 medium with 10% heat‐inactivated fetal calf serum (Gibco BRL) and stimulated with PMA (50 ng/ml; Sigma, St Louis, MO) plus ionomycin (1 μ m , Sigma) and monensin (500 ng/ml, eBioscience) for 4 hr at 37° under 5% CO2 .

    Techniques: Expressing, Staining, FACS

    Cytokine profile of B cell-derived polyclonal B2 versus immune splenocyte-derived polyclonal CD4 T cell lines, shown by the presence of CD4γ13 T cells in the genital tract and spleen. (A) Polyclonal B cell-derived line 2 (B2) and splenocyte-derived line 2 (spl2) were activated for 5 h with PMA-ionomycin-brefeldin A-monensin and then stained for CD8 (negative stain for CD4 T cells) versus IL-13 and IFN-γ versus IL-13. (B) Specificity and cytokine profiles for B2 and five splenocyte-derived T cell lines (spl1 to -5). 2.5 × 10 4 T cells were coincubated with 5 × 10 4 purified immune B cells unpulsed (APCs) or pulsed with uvMoPn (antigen [Ag]) in triplicate. Cell culture supernatants harvested at 48 h, and levels of IFN-γ, IL-13, and IL-4 were determined by ELISA. Shown are comparisons between control (Con) and antigen-pulsed (Ag) B cell activation for each cytokine and T cell line. *, P

    Journal: Infection and Immunity

    Article Title: B Cell Presentation of Chlamydia Antigen Selects Out Protective CD4γ13 T Cells: Implications for Genital Tract Tissue-Resident Memory Lymphocyte Clusters

    doi: 10.1128/IAI.00614-17

    Figure Lengend Snippet: Cytokine profile of B cell-derived polyclonal B2 versus immune splenocyte-derived polyclonal CD4 T cell lines, shown by the presence of CD4γ13 T cells in the genital tract and spleen. (A) Polyclonal B cell-derived line 2 (B2) and splenocyte-derived line 2 (spl2) were activated for 5 h with PMA-ionomycin-brefeldin A-monensin and then stained for CD8 (negative stain for CD4 T cells) versus IL-13 and IFN-γ versus IL-13. (B) Specificity and cytokine profiles for B2 and five splenocyte-derived T cell lines (spl1 to -5). 2.5 × 10 4 T cells were coincubated with 5 × 10 4 purified immune B cells unpulsed (APCs) or pulsed with uvMoPn (antigen [Ag]) in triplicate. Cell culture supernatants harvested at 48 h, and levels of IFN-γ, IL-13, and IL-4 were determined by ELISA. Shown are comparisons between control (Con) and antigen-pulsed (Ag) B cell activation for each cytokine and T cell line. *, P

    Article Snippet: For intracellular staining for IL-13 (ebio13A coupled to PE) and IFN-γ (XMG1.2 coupled to allophycocyanin), T cells were activated for 5 h in a cocktail of phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A, and monensin (cell stimulation cocktail; Ebioscience), stained for CD8a, and then fixed and permeabilized (Fix/Perm buffer; Ebioscience), stained for IL-13–PE/IFN-γ–allophycocyanin or control antibody (eBRG1-PE/IFN-γ–allophycocyanin) in the presence of 2 mg/ml donkey IgG (Jackson ImmunoResearch) for 30 min at room temperature, washed, suspended in 1% paraformaldehyde, and analyzed.

    Techniques: Derivative Assay, Staining, Purification, Cell Culture, Enzyme-linked Immunosorbent Assay, Activation Assay

    B cell dynamics in the genital tract during C. muridarum infection and differential expansion of memory T cell subsets. (A) Single-cell suspensions of genital tracts from the following conditions were gated on CD79a (B cells) and analyzed for the level of B220: high expression of B220 indicates B lymphocytes and low expression indicates plasma B cells. Uninfected mice (Naive), day 7 primary infection (D7_pri_inf), day 35 primary infection (D35_pri_Cm inf), and day 5 secondary infection (D5_sec_inf) were investigated. Mice were pretreated with medroxyprogesterone and infected 1 week later with 1,500 IFU of C. muridarum ; representative data are shown from a minimum of 2 experiments for each experimental group. (B) Analysis of B cell APC-derived and immune splenocyte-derived Chlamydia -specific polyclonal T cell lines for production of IL-13. T cell lines were activated for 5 h with PMA-ionomycin-brefeldin A-monensin and stained for CD8 versus IL-13. CD4 T cells are identified as CD8 neg in this assay (see Materials and Methods).

    Journal: Infection and Immunity

    Article Title: B Cell Presentation of Chlamydia Antigen Selects Out Protective CD4γ13 T Cells: Implications for Genital Tract Tissue-Resident Memory Lymphocyte Clusters

    doi: 10.1128/IAI.00614-17

    Figure Lengend Snippet: B cell dynamics in the genital tract during C. muridarum infection and differential expansion of memory T cell subsets. (A) Single-cell suspensions of genital tracts from the following conditions were gated on CD79a (B cells) and analyzed for the level of B220: high expression of B220 indicates B lymphocytes and low expression indicates plasma B cells. Uninfected mice (Naive), day 7 primary infection (D7_pri_inf), day 35 primary infection (D35_pri_Cm inf), and day 5 secondary infection (D5_sec_inf) were investigated. Mice were pretreated with medroxyprogesterone and infected 1 week later with 1,500 IFU of C. muridarum ; representative data are shown from a minimum of 2 experiments for each experimental group. (B) Analysis of B cell APC-derived and immune splenocyte-derived Chlamydia -specific polyclonal T cell lines for production of IL-13. T cell lines were activated for 5 h with PMA-ionomycin-brefeldin A-monensin and stained for CD8 versus IL-13. CD4 T cells are identified as CD8 neg in this assay (see Materials and Methods).

    Article Snippet: For intracellular staining for IL-13 (ebio13A coupled to PE) and IFN-γ (XMG1.2 coupled to allophycocyanin), T cells were activated for 5 h in a cocktail of phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A, and monensin (cell stimulation cocktail; Ebioscience), stained for CD8a, and then fixed and permeabilized (Fix/Perm buffer; Ebioscience), stained for IL-13–PE/IFN-γ–allophycocyanin or control antibody (eBRG1-PE/IFN-γ–allophycocyanin) in the presence of 2 mg/ml donkey IgG (Jackson ImmunoResearch) for 30 min at room temperature, washed, suspended in 1% paraformaldehyde, and analyzed.

    Techniques: Infection, Expressing, Mouse Assay, Size-exclusion Chromatography, Derivative Assay, Staining

    IL-4Rα-expressing macrophages do not affect cytokine responses in granulomas. Il4ra −/lox , LysM cre Il4ra −/lox , iLck cre Il4ra −/lox and Il4ra −/− mice were infected with 100 S. mansoni cercariae and killed 8 weeks later. Mesenteric lymph node (mLN) cells or liver granuloma-associated leukocytes (Liver) were then isolated and restimulated overnight with 20 µg/ml SEA before analyzed for ex vivo cytokine production capability. Staining of surface CD4 was followed by detection of IL-4, IL-10 and IFN-γ secretion in a cytokine catch assay or detection of intracellular levels of IL-13 after 4h of monensin treatment, as described in Methods . Live gate was placed on lymphocytes according to their forward- and side-scatter by flow cytometry. (A) Representative contour plots of IFN-γ, IL-4, IL-10 and IL-13-producing lymphocytes. Quadrant bars were set up on unstimulated cells and numbers represent percent cells in each quadrant. Data represent one out of three independent experiments with similar results. (B) Percentages of IFN-γ, IL-4, IL-10 or IL-13-producing cells were determined in gated CD4 + or non-CD4 + cell populations from mLN or liver granuloma-associated lymphocytes (Liver) (mean±SEM, n = 4). Data are representative of three independent experiments. * p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: IL-4R?-Independent Expression of Mannose Receptor and Ym1 by Macrophages Depends on their IL-10 Responsiveness

    doi: 10.1371/journal.pntd.0000689

    Figure Lengend Snippet: IL-4Rα-expressing macrophages do not affect cytokine responses in granulomas. Il4ra −/lox , LysM cre Il4ra −/lox , iLck cre Il4ra −/lox and Il4ra −/− mice were infected with 100 S. mansoni cercariae and killed 8 weeks later. Mesenteric lymph node (mLN) cells or liver granuloma-associated leukocytes (Liver) were then isolated and restimulated overnight with 20 µg/ml SEA before analyzed for ex vivo cytokine production capability. Staining of surface CD4 was followed by detection of IL-4, IL-10 and IFN-γ secretion in a cytokine catch assay or detection of intracellular levels of IL-13 after 4h of monensin treatment, as described in Methods . Live gate was placed on lymphocytes according to their forward- and side-scatter by flow cytometry. (A) Representative contour plots of IFN-γ, IL-4, IL-10 and IL-13-producing lymphocytes. Quadrant bars were set up on unstimulated cells and numbers represent percent cells in each quadrant. Data represent one out of three independent experiments with similar results. (B) Percentages of IFN-γ, IL-4, IL-10 or IL-13-producing cells were determined in gated CD4 + or non-CD4 + cell populations from mLN or liver granuloma-associated lymphocytes (Liver) (mean±SEM, n = 4). Data are representative of three independent experiments. * p

    Article Snippet: For detection of intracellular IL-13, cells were further incubated 4h with monensin, fixed for 20 min on ice in 2% (wt/ol) paraformaldehyde and permeabilized during 30 min with 1× permeabilization buffer (eBioscience) before being stained with anti-IL-13-PE (eBioscience, eBio13a ).

    Techniques: Expressing, Mouse Assay, Infection, Isolation, Ex Vivo, Staining, Flow Cytometry, Cytometry

    Preferential Th2 expansion upon T reg depletion (A) Representative flow plots gated on CD4 + T cells stained for the Th1 cytokine, IFNγ. DLN and NDLN cells were isolated from T. muris infected mice treated with Early DT (left) or Late DT (right) approaches and analyzed at day 35. Cells were stimulated with PMA and Ionomycin for 12–16 hours in the presence of Monensin for cytokine secretion analysis. (B) Percentages of IFN + CD4 T cells in the DLN and NDLNs following T reg depletion in T.muris infected mice as discussed in (A). Fold induction of IFNγ + CD4 T cells (ratio of +DT/−DT) indicated. (C) Representative flow plots gated on CD4 + T cells stained for the Th2 cytokine, IL-4. DLN and NDLN cells were isolated from T. muris infected mice as discussed in (A). (D) Percentages of IL-4 + CD4 T cells in the DLN and NDLN following T reg depletion in T. muris infected mice as discussed in (A). Fold induction of IL-4 + CD4 T cells (ratio of +DT/−DT) indicated. Each data point on the scatter plots represents an individual mouse with mean indicated. Data represents two independent experiments for both Early and Late DT treatments (n=8–10 mice per group). *** p

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

    Article Title: Regulatory T Cells Limit Induction of Protective Immunity and Promote Immune Pathology Following Intestinal Helminth Infection

    doi: 10.4049/jimmunol.1202502

    Figure Lengend Snippet: Preferential Th2 expansion upon T reg depletion (A) Representative flow plots gated on CD4 + T cells stained for the Th1 cytokine, IFNγ. DLN and NDLN cells were isolated from T. muris infected mice treated with Early DT (left) or Late DT (right) approaches and analyzed at day 35. Cells were stimulated with PMA and Ionomycin for 12–16 hours in the presence of Monensin for cytokine secretion analysis. (B) Percentages of IFN + CD4 T cells in the DLN and NDLNs following T reg depletion in T.muris infected mice as discussed in (A). Fold induction of IFNγ + CD4 T cells (ratio of +DT/−DT) indicated. (C) Representative flow plots gated on CD4 + T cells stained for the Th2 cytokine, IL-4. DLN and NDLN cells were isolated from T. muris infected mice as discussed in (A). (D) Percentages of IL-4 + CD4 T cells in the DLN and NDLN following T reg depletion in T. muris infected mice as discussed in (A). Fold induction of IL-4 + CD4 T cells (ratio of +DT/−DT) indicated. Each data point on the scatter plots represents an individual mouse with mean indicated. Data represents two independent experiments for both Early and Late DT treatments (n=8–10 mice per group). *** p

    Article Snippet: For intracellular cytokine staining, cells were stimulated at 5×106 cells/ml in DMEM media with PMA/Ionomycin (100ng/ml and 500ng/ml, respectively; Sigma-Aldrich) or 5µg/ml of Trichuris E/S antigen for 12–16 hours in the presence of Monensin (1:1000; eBioscience).

    Techniques: Flow Cytometry, Staining, Isolation, Infection, Mouse Assay

    T reg depletion alters T. muris -specific Th1/Th2 cell polarization (A) Percentages of Ag-specific IFNγ + CD4 T cells in the DLN and NDLNs following T reg depletion by Early DT (left) and Late DT (right) approaches in T.muris infected mice and analyzed at day 35. Cells were stimulated with T. muris antigen for 12–16 hours in the presence of Monensin for cytokine secretion analysis. (B) Percentages of Ag-specific IL-4 + CD4 T cells in the DLN and NDLNs following T reg depletion as discussed in (A). Each data point on the scatter plots represents an individual mouse with mean indicated. Data represents two independent experiments for both Early and Late DT treatments (n=8–10 mice per group). * p

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

    Article Title: Regulatory T Cells Limit Induction of Protective Immunity and Promote Immune Pathology Following Intestinal Helminth Infection

    doi: 10.4049/jimmunol.1202502

    Figure Lengend Snippet: T reg depletion alters T. muris -specific Th1/Th2 cell polarization (A) Percentages of Ag-specific IFNγ + CD4 T cells in the DLN and NDLNs following T reg depletion by Early DT (left) and Late DT (right) approaches in T.muris infected mice and analyzed at day 35. Cells were stimulated with T. muris antigen for 12–16 hours in the presence of Monensin for cytokine secretion analysis. (B) Percentages of Ag-specific IL-4 + CD4 T cells in the DLN and NDLNs following T reg depletion as discussed in (A). Each data point on the scatter plots represents an individual mouse with mean indicated. Data represents two independent experiments for both Early and Late DT treatments (n=8–10 mice per group). * p

    Article Snippet: For intracellular cytokine staining, cells were stimulated at 5×106 cells/ml in DMEM media with PMA/Ionomycin (100ng/ml and 500ng/ml, respectively; Sigma-Aldrich) or 5µg/ml of Trichuris E/S antigen for 12–16 hours in the presence of Monensin (1:1000; eBioscience).

    Techniques: Infection, Mouse Assay

    T reg -depletion mediated Th2 induction limits T. muris driven intestinal pathology (A) Schematic of IL-4 neutralization in low dose T. muris infected mice following T reg depletion by the Early DT approach. Infected mice received α-IL-4 (11B11) neutralizing antibody or isotype (IgG1) control on days 2, 5 and 8, alongside the early DT treatments (d0–d8) and were analyzed at day 35. Thus, the four treatment groups included: (−DT+IgG1, −DT+α-IL-4, +DT+IgG1 and +DT+α-IL-4). (B) Total worm burden from ceca of T. muris infected mice as treated in (A). Worms counted at day 35 for the four treatment groups. (C) Representative flow plots gated on CD4 + T cells stained for the Th1 cytokine, IFNγ. DLN and NDLN cells were isolated from T. muris infected mice treated with IL-4 neutralizing antibody or IgG1 isotype alongside early T reg depletion and analyzed at day 35. Cells stimulated with PMA and Ionomycin for 12–16 hours in the presence of Monensin. Data represented for only the two DT treatment groups (+DT+IgG1 and +DT+ α-IL-4). (D) Representative flow plots gated on CD4 + T cells stained for the Th2 cytokine, IL-4. DLN and NDLN cells were isolated and treated as in (C). (E) Inflammation and edema scores as assessed by H E staining of day 35 cecal sections for the four treatment groups as treated in (A). (F) Representative H E stained cecal sections for the four treatment groups as treated in (A). Each data point on scatter plots represents an individual mouse with mean indicated. Data represents two independent experiments (n=5–8 mice per group) * p

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

    Article Title: Regulatory T Cells Limit Induction of Protective Immunity and Promote Immune Pathology Following Intestinal Helminth Infection

    doi: 10.4049/jimmunol.1202502

    Figure Lengend Snippet: T reg -depletion mediated Th2 induction limits T. muris driven intestinal pathology (A) Schematic of IL-4 neutralization in low dose T. muris infected mice following T reg depletion by the Early DT approach. Infected mice received α-IL-4 (11B11) neutralizing antibody or isotype (IgG1) control on days 2, 5 and 8, alongside the early DT treatments (d0–d8) and were analyzed at day 35. Thus, the four treatment groups included: (−DT+IgG1, −DT+α-IL-4, +DT+IgG1 and +DT+α-IL-4). (B) Total worm burden from ceca of T. muris infected mice as treated in (A). Worms counted at day 35 for the four treatment groups. (C) Representative flow plots gated on CD4 + T cells stained for the Th1 cytokine, IFNγ. DLN and NDLN cells were isolated from T. muris infected mice treated with IL-4 neutralizing antibody or IgG1 isotype alongside early T reg depletion and analyzed at day 35. Cells stimulated with PMA and Ionomycin for 12–16 hours in the presence of Monensin. Data represented for only the two DT treatment groups (+DT+IgG1 and +DT+ α-IL-4). (D) Representative flow plots gated on CD4 + T cells stained for the Th2 cytokine, IL-4. DLN and NDLN cells were isolated and treated as in (C). (E) Inflammation and edema scores as assessed by H E staining of day 35 cecal sections for the four treatment groups as treated in (A). (F) Representative H E stained cecal sections for the four treatment groups as treated in (A). Each data point on scatter plots represents an individual mouse with mean indicated. Data represents two independent experiments (n=5–8 mice per group) * p

    Article Snippet: For intracellular cytokine staining, cells were stimulated at 5×106 cells/ml in DMEM media with PMA/Ionomycin (100ng/ml and 500ng/ml, respectively; Sigma-Aldrich) or 5µg/ml of Trichuris E/S antigen for 12–16 hours in the presence of Monensin (1:1000; eBioscience).

    Techniques: Neutralization, Infection, Mouse Assay, Flow Cytometry, Staining, Isolation