brefeldin a solution  (Thermo Fisher)


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    eBioscience Brefeldin A Solution 1000X
    Description:
    Brefeldin A is an inhibitor of intracellular protein transport Incubation of cells in culture with Brefeldin A leads to blockade of protein transport to the Golgi complex GC and accumulation of proteins in the endoplasmic reticulum ER Addition of Brefeldin A during the last hours of in vitro activation of cells results in enhanced detection of intracellular cytokines Brefeldin A 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 Monensin in their specific assay system Reported ApplicationIntracellular Staining Followed by Flow Cytometric Analysis
    Catalog Number:
    00-4506-51
    Price:
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    Applications:
    Cell Analysis|Cellular Imaging|Flow Cytometry
    Category:
    Lab Reagents and Chemicals
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    Structured Review

    Thermo Fisher brefeldin a solution
    Infected burn mice have a higher percentage of IL-10 + neutrophils and a lower percentage of IL-12 + neutrophils, dendritic cells, and macrophages than infected sham mice. A) Splenocytes were harvested at 48 hours post infection and underwent intracellular staining for cytokine analysis without further stimulation in vitro . Shown is a representative histogram from an infected burn mouse, which indicates that IL-10 is being produced by Gr1 + CD11b + cells within the spleen. B–E) Splenocytes were collected at 48 following infection and underwent CD11b enrichment by magnetic selection. CD11b + cells were cultured in the presence of LPS and <t>brefeldin-A</t> then were subjected to cell surface and intracellular staining. Percentage of B) IL-10 + neutrophils, as well as IL-12 + C) neutrophils, D) dendritic cells, and E) macrophages were measured for infected sham (open) and burn (solid) mice. Data expressed as mean ± SEM. (n = 6, 7) **, p≤0.005. ***, p≤0.0005. ****, p
    Brefeldin A is an inhibitor of intracellular protein transport Incubation of cells in culture with Brefeldin A leads to blockade of protein transport to the Golgi complex GC and accumulation of proteins in the endoplasmic reticulum ER Addition of Brefeldin A during the last hours of in vitro activation of cells results in enhanced detection of intracellular cytokines Brefeldin A 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 Monensin in their specific assay system Reported ApplicationIntracellular Staining Followed by Flow Cytometric Analysis
    https://www.bioz.com/result/brefeldin a solution/product/Thermo Fisher
    Average 99 stars, based on 40 article reviews
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    Images

    1) Product Images from "Flagellin Treatment Prevents Increased Susceptibility to Systemic Bacterial Infection after Injury by Inhibiting Anti-Inflammatory IL-10+ IL-12- Neutrophil Polarization"

    Article Title: Flagellin Treatment Prevents Increased Susceptibility to Systemic Bacterial Infection after Injury by Inhibiting Anti-Inflammatory IL-10+ IL-12- Neutrophil Polarization

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085623

    Infected burn mice have a higher percentage of IL-10 + neutrophils and a lower percentage of IL-12 + neutrophils, dendritic cells, and macrophages than infected sham mice. A) Splenocytes were harvested at 48 hours post infection and underwent intracellular staining for cytokine analysis without further stimulation in vitro . Shown is a representative histogram from an infected burn mouse, which indicates that IL-10 is being produced by Gr1 + CD11b + cells within the spleen. B–E) Splenocytes were collected at 48 following infection and underwent CD11b enrichment by magnetic selection. CD11b + cells were cultured in the presence of LPS and brefeldin-A then were subjected to cell surface and intracellular staining. Percentage of B) IL-10 + neutrophils, as well as IL-12 + C) neutrophils, D) dendritic cells, and E) macrophages were measured for infected sham (open) and burn (solid) mice. Data expressed as mean ± SEM. (n = 6, 7) **, p≤0.005. ***, p≤0.0005. ****, p
    Figure Legend Snippet: Infected burn mice have a higher percentage of IL-10 + neutrophils and a lower percentage of IL-12 + neutrophils, dendritic cells, and macrophages than infected sham mice. A) Splenocytes were harvested at 48 hours post infection and underwent intracellular staining for cytokine analysis without further stimulation in vitro . Shown is a representative histogram from an infected burn mouse, which indicates that IL-10 is being produced by Gr1 + CD11b + cells within the spleen. B–E) Splenocytes were collected at 48 following infection and underwent CD11b enrichment by magnetic selection. CD11b + cells were cultured in the presence of LPS and brefeldin-A then were subjected to cell surface and intracellular staining. Percentage of B) IL-10 + neutrophils, as well as IL-12 + C) neutrophils, D) dendritic cells, and E) macrophages were measured for infected sham (open) and burn (solid) mice. Data expressed as mean ± SEM. (n = 6, 7) **, p≤0.005. ***, p≤0.0005. ****, p

    Techniques Used: Infection, Mouse Assay, Staining, In Vitro, Produced, Selection, Cell Culture

    2) Product Images from "IL-22 hinders antiviral T cell responses and exacerbates ZIKV encephalitis in immunocompetent neonatal mice"

    Article Title: IL-22 hinders antiviral T cell responses and exacerbates ZIKV encephalitis in immunocompetent neonatal mice

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-020-01928-9

    IL-22 dampens anti-ZIKV CD8 +  T cell responses. Neonatal WT and  IL-22 -/-  mice were  s.c.  infected with ZIKV.  a ,  b  Viral loads of the spleen and brain were measured at 2, 7, 13, and 20 dpi.  c ,  d  Lymphocytes were harvested from the spleen and brain at 13 dpi and stimulated with ZIKV peptide for 5 hrs in the presence of Brefeldin A. ZIKV-specific CD8 +  T cells were quantified by intracellular flow cytometry staining.  e  Neonatal WT mice were  s.c.  infected with ZIKV, followed by rIL-22 treatment as indicated in Fig.   2 e . Viral loads of the brains were measured at 13 dpi, and  f  ZIKV-specific CD8 +  T cells were quantified in the spleen and brains. All experiments were repeated three times independently. Data are shown as means ± SEM and a two-tailed Student’s  t  test was used for statistical analysis. * p
    Figure Legend Snippet: IL-22 dampens anti-ZIKV CD8 + T cell responses. Neonatal WT and IL-22 -/- mice were s.c. infected with ZIKV. a , b Viral loads of the spleen and brain were measured at 2, 7, 13, and 20 dpi. c , d Lymphocytes were harvested from the spleen and brain at 13 dpi and stimulated with ZIKV peptide for 5 hrs in the presence of Brefeldin A. ZIKV-specific CD8 + T cells were quantified by intracellular flow cytometry staining. e Neonatal WT mice were s.c. infected with ZIKV, followed by rIL-22 treatment as indicated in Fig. 2 e . Viral loads of the brains were measured at 13 dpi, and f ZIKV-specific CD8 + T cells were quantified in the spleen and brains. All experiments were repeated three times independently. Data are shown as means ± SEM and a two-tailed Student’s t test was used for statistical analysis. * p

    Techniques Used: Mouse Assay, Infection, Flow Cytometry, Staining, Two Tailed Test

    3) Product Images from "Peripheral PDGFRα+gp38+ mesenchymal cells support the differentiation of fetal liver–derived ILC2 "

    Article Title: Peripheral PDGFRα+gp38+ mesenchymal cells support the differentiation of fetal liver–derived ILC2

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20172310

    ILCP, CCR9 + ILCP, and immature ILCs exist in the fetal mesentery. (A) Flow-cytometric analyses of CD45 + Lin − cells from the E17 fetal or adult mesentery. Numbers indicate the percentage of cells in the boxed areas. (B and C) Expression levels of the indicated markers on Lin − IL-7Rα + T1/ST2 − cells from the E17 fetal mesentery. (D) Percentages of T-bet + cells (pink frame), RORγt + cells (blue frame), and T-bet − RORγt − T1/ST2 + ILC2 (red frame) in the CD45 + Lin − fraction of the mesentery from fetal (E17) or adult mice were determined by flow cytometry. (E) Flow-cytometric analyses of CD45 + Lin − cells from the E17 fetal mesentery of RORγ GFP/+ mice. (F) Intracellular cytokine staining of several lymphocytes and progenitors. Whole splenic cells, adult mesenteric cells, and fetal E18 mesenteric cells were stimulated with 50 ng/ml PMA and 1 µg/ml ionomycin and 3 µg/ml brefeldin A solution for 3 h, and the expression levels of IFNγ, IL-13, and IL-17A were analyzed by flow cytometry. NK/ILC1 (Lin − NK1.1 + ) and ILC3 (Lin − IL-7Rα + RORγt + ) in the spleen, mature ILC2 (Lin − NK1.1 − IL-7Rα + T1/ST2 + ) in the adult mesentery, immature ILC2 (Lin − NK1.1 − IL-7Rα + T1/ST2 + ) and NK1.1 + cells (Lin − NK1.1 + ) in the fetal mesentery, and RORγt + CCR9 − cells, RORγt + CCR9 + cells, RORγt − CCR9 + cells, and RORγt − CCR9 − cells in the Lin − NK1.1 − IL-7Rα + T1/ST2 − gated cells in the fetal mesentery were assessed. (G) CCR9 expression on Lin − IL-7Rα + T1/ST2 − cells and Lin − IL-7Rα + T1/ST2 + fetal ILC2 from the E17 fetal mesentery. (H) ILC differentiation from Lin − IL-7Rα + T1/ST2 − CCR9 − or Lin − IL-7Rα + T1/ST2 − CCR9 + cells cultured with TSt4-DLL1 and 10 ng/ml IL-7 for 10 d. Blue, red, and pink boxes indicate T-bet + ILC1, GATA3 hi ILC2, and RORγt + ILC3, respectively. (I) Numbers of ILC1, ILC2, and ILC3 in H. n = 4–8. Error bars show means ± SD. (J) Lin − NK1.1 − IL-7Rα + T1/ST2 − RORγt − CCR9 − cells from the E18 fetal mesentery from RORγt GFP/+ mice were cultured with TSt4-DLL1 cells in the presence of 10 ng/ml IL-7 for 10 d. After 10 d, differentiated cells were assessed for T-bet, GATA3, and RORγt expression by flow cytometry. Blue, red, and pink boxes indicate T-bet + ILC1, GATA3 hi ILC2, and RORγt + ILC3, respectively. Results are representative of two independent experiments. *, P
    Figure Legend Snippet: ILCP, CCR9 + ILCP, and immature ILCs exist in the fetal mesentery. (A) Flow-cytometric analyses of CD45 + Lin − cells from the E17 fetal or adult mesentery. Numbers indicate the percentage of cells in the boxed areas. (B and C) Expression levels of the indicated markers on Lin − IL-7Rα + T1/ST2 − cells from the E17 fetal mesentery. (D) Percentages of T-bet + cells (pink frame), RORγt + cells (blue frame), and T-bet − RORγt − T1/ST2 + ILC2 (red frame) in the CD45 + Lin − fraction of the mesentery from fetal (E17) or adult mice were determined by flow cytometry. (E) Flow-cytometric analyses of CD45 + Lin − cells from the E17 fetal mesentery of RORγ GFP/+ mice. (F) Intracellular cytokine staining of several lymphocytes and progenitors. Whole splenic cells, adult mesenteric cells, and fetal E18 mesenteric cells were stimulated with 50 ng/ml PMA and 1 µg/ml ionomycin and 3 µg/ml brefeldin A solution for 3 h, and the expression levels of IFNγ, IL-13, and IL-17A were analyzed by flow cytometry. NK/ILC1 (Lin − NK1.1 + ) and ILC3 (Lin − IL-7Rα + RORγt + ) in the spleen, mature ILC2 (Lin − NK1.1 − IL-7Rα + T1/ST2 + ) in the adult mesentery, immature ILC2 (Lin − NK1.1 − IL-7Rα + T1/ST2 + ) and NK1.1 + cells (Lin − NK1.1 + ) in the fetal mesentery, and RORγt + CCR9 − cells, RORγt + CCR9 + cells, RORγt − CCR9 + cells, and RORγt − CCR9 − cells in the Lin − NK1.1 − IL-7Rα + T1/ST2 − gated cells in the fetal mesentery were assessed. (G) CCR9 expression on Lin − IL-7Rα + T1/ST2 − cells and Lin − IL-7Rα + T1/ST2 + fetal ILC2 from the E17 fetal mesentery. (H) ILC differentiation from Lin − IL-7Rα + T1/ST2 − CCR9 − or Lin − IL-7Rα + T1/ST2 − CCR9 + cells cultured with TSt4-DLL1 and 10 ng/ml IL-7 for 10 d. Blue, red, and pink boxes indicate T-bet + ILC1, GATA3 hi ILC2, and RORγt + ILC3, respectively. (I) Numbers of ILC1, ILC2, and ILC3 in H. n = 4–8. Error bars show means ± SD. (J) Lin − NK1.1 − IL-7Rα + T1/ST2 − RORγt − CCR9 − cells from the E18 fetal mesentery from RORγt GFP/+ mice were cultured with TSt4-DLL1 cells in the presence of 10 ng/ml IL-7 for 10 d. After 10 d, differentiated cells were assessed for T-bet, GATA3, and RORγt expression by flow cytometry. Blue, red, and pink boxes indicate T-bet + ILC1, GATA3 hi ILC2, and RORγt + ILC3, respectively. Results are representative of two independent experiments. *, P

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

    4) Product Images from "Basophil-associated OX40 Ligand Participates in the Initiation of Th2 Responses during Airway Inflammation *"

    Article Title: Basophil-associated OX40 Ligand Participates in the Initiation of Th2 Responses during Airway Inflammation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.642637

    Blocking OX40-OX40L interaction reduced Th2 cell differentiation induced by basophils in vitro . A , flow cytometric analysis of Th1/Th2 subsets in co-culture of splenic naïve T cells (5 × 10 5 /well) from B6 WT mice or OX40 −/− mice (with C57BL/6 genetic background) and BM-Bas (2.5 × 10 5 /well) from B6 WT mice following 5 days of treatment with DNP-OVA (100 μg/ml) plus anti-DNP IgE (10 μg/ml), rIL-2 (20 units/ml), and rIL-3 (30 ng/ml) in the absence or presence of αOX40L (20 μg/ml). Cells were gated on CD4 + T cells. The percentage of Th2 cell (CD4 + IL-4 + cells) in the co-culture cells ( right ); B , flow cytometric analysis of Th1/Th2 subsets in co-culture of splenic naïve T cells (5 × 10 5 /well) from DO11.10 mice and BM-Bas (2.5 × 10 5 /well) from WT BALB/c mice or CD40 −/− mice (with BALB/c genetic background) following 5 days of treatment with DNP-OVA (100 μg/ml) plus anti-DNP IgE (10 μg/ml), rIL-2 (20 units/ml), and rIL-3 (30 ng/ml). Cells were gated on CD4 + T cells. The percentage of Th2 cell (CD4 + IL-4 + cells) in the co-culture cells ( right ); C , ELISA analysis of IL-4, IL-5 and IL13 levels in the supernatants of co-cultured cells stimulated with PMA (50 ng/ml) plus ionomycin (1 μg/ml) for 6 h and Brefeldin A (1:1000) for the last 2 h. Results are presented as mean ± S.D. and are representative of three independent experiments. *, p
    Figure Legend Snippet: Blocking OX40-OX40L interaction reduced Th2 cell differentiation induced by basophils in vitro . A , flow cytometric analysis of Th1/Th2 subsets in co-culture of splenic naïve T cells (5 × 10 5 /well) from B6 WT mice or OX40 −/− mice (with C57BL/6 genetic background) and BM-Bas (2.5 × 10 5 /well) from B6 WT mice following 5 days of treatment with DNP-OVA (100 μg/ml) plus anti-DNP IgE (10 μg/ml), rIL-2 (20 units/ml), and rIL-3 (30 ng/ml) in the absence or presence of αOX40L (20 μg/ml). Cells were gated on CD4 + T cells. The percentage of Th2 cell (CD4 + IL-4 + cells) in the co-culture cells ( right ); B , flow cytometric analysis of Th1/Th2 subsets in co-culture of splenic naïve T cells (5 × 10 5 /well) from DO11.10 mice and BM-Bas (2.5 × 10 5 /well) from WT BALB/c mice or CD40 −/− mice (with BALB/c genetic background) following 5 days of treatment with DNP-OVA (100 μg/ml) plus anti-DNP IgE (10 μg/ml), rIL-2 (20 units/ml), and rIL-3 (30 ng/ml). Cells were gated on CD4 + T cells. The percentage of Th2 cell (CD4 + IL-4 + cells) in the co-culture cells ( right ); C , ELISA analysis of IL-4, IL-5 and IL13 levels in the supernatants of co-cultured cells stimulated with PMA (50 ng/ml) plus ionomycin (1 μg/ml) for 6 h and Brefeldin A (1:1000) for the last 2 h. Results are presented as mean ± S.D. and are representative of three independent experiments. *, p

    Techniques Used: Blocking Assay, Cell Differentiation, In Vitro, Flow Cytometry, Co-Culture Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Cell Culture

    5) Product Images from "Peripheral PDGFRα+gp38+ mesenchymal cells support the differentiation of fetal liver–derived ILC2"

    Article Title: Peripheral PDGFRα+gp38+ mesenchymal cells support the differentiation of fetal liver–derived ILC2

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20172310

    ILCP, CCR9 + ILCP, and immature ILCs exist in the fetal mesentery. (A) Flow-cytometric analyses of CD45 + Lin − cells from the E17 fetal or adult mesentery. Numbers indicate the percentage of cells in the boxed areas. (B and C) Expression levels of the indicated markers on Lin − IL-7Rα + T1/ST2 − cells from the E17 fetal mesentery. (D) Percentages of T-bet + cells (pink frame), RORγt + cells (blue frame), and T-bet − RORγt − T1/ST2 + ILC2 (red frame) in the CD45 + Lin − fraction of the mesentery from fetal (E17) or adult mice were determined by flow cytometry. (E) Flow-cytometric analyses of CD45 + Lin − cells from the E17 fetal mesentery of RORγ GFP/+ mice. (F) Intracellular cytokine staining of several lymphocytes and progenitors. Whole splenic cells, adult mesenteric cells, and fetal E18 mesenteric cells were stimulated with 50 ng/ml PMA and 1 µg/ml ionomycin and 3 µg/ml brefeldin A solution for 3 h, and the expression levels of IFNγ, IL-13, and IL-17A were analyzed by flow cytometry. NK/ILC1 (Lin − NK1.1 + ) and ILC3 (Lin − IL-7Rα + RORγt + ) in the spleen, mature ILC2 (Lin − NK1.1 − IL-7Rα + T1/ST2 + ) in the adult mesentery, immature ILC2 (Lin − NK1.1 − IL-7Rα + T1/ST2 + ) and NK1.1 + cells (Lin − NK1.1 + ) in the fetal mesentery, and RORγt + CCR9 − cells, RORγt + CCR9 + cells, RORγt − CCR9 + cells, and RORγt − CCR9 − cells in the Lin − NK1.1 − IL-7Rα + T1/ST2 − gated cells in the fetal mesentery were assessed. (G) CCR9 expression on Lin − IL-7Rα + T1/ST2 − cells and Lin − IL-7Rα + T1/ST2 + fetal ILC2 from the E17 fetal mesentery. (H) ILC differentiation from Lin − IL-7Rα + T1/ST2 − CCR9 − or Lin − IL-7Rα + T1/ST2 − CCR9 + cells cultured with TSt4-DLL1 and 10 ng/ml IL-7 for 10 d. Blue, red, and pink boxes indicate T-bet + ILC1, GATA3 hi ILC2, and RORγt + ILC3, respectively. (I) Numbers of ILC1, ILC2, and ILC3 in H. n = 4–8. Error bars show means ± SD. (J) Lin − NK1.1 − IL-7Rα + T1/ST2 − RORγt − CCR9 − cells from the E18 fetal mesentery from RORγt GFP/+ mice were cultured with TSt4-DLL1 cells in the presence of 10 ng/ml IL-7 for 10 d. After 10 d, differentiated cells were assessed for T-bet, GATA3, and RORγt expression by flow cytometry. Blue, red, and pink boxes indicate T-bet + ILC1, GATA3 hi ILC2, and RORγt + ILC3, respectively. Results are representative of two independent experiments. *, P
    Figure Legend Snippet: ILCP, CCR9 + ILCP, and immature ILCs exist in the fetal mesentery. (A) Flow-cytometric analyses of CD45 + Lin − cells from the E17 fetal or adult mesentery. Numbers indicate the percentage of cells in the boxed areas. (B and C) Expression levels of the indicated markers on Lin − IL-7Rα + T1/ST2 − cells from the E17 fetal mesentery. (D) Percentages of T-bet + cells (pink frame), RORγt + cells (blue frame), and T-bet − RORγt − T1/ST2 + ILC2 (red frame) in the CD45 + Lin − fraction of the mesentery from fetal (E17) or adult mice were determined by flow cytometry. (E) Flow-cytometric analyses of CD45 + Lin − cells from the E17 fetal mesentery of RORγ GFP/+ mice. (F) Intracellular cytokine staining of several lymphocytes and progenitors. Whole splenic cells, adult mesenteric cells, and fetal E18 mesenteric cells were stimulated with 50 ng/ml PMA and 1 µg/ml ionomycin and 3 µg/ml brefeldin A solution for 3 h, and the expression levels of IFNγ, IL-13, and IL-17A were analyzed by flow cytometry. NK/ILC1 (Lin − NK1.1 + ) and ILC3 (Lin − IL-7Rα + RORγt + ) in the spleen, mature ILC2 (Lin − NK1.1 − IL-7Rα + T1/ST2 + ) in the adult mesentery, immature ILC2 (Lin − NK1.1 − IL-7Rα + T1/ST2 + ) and NK1.1 + cells (Lin − NK1.1 + ) in the fetal mesentery, and RORγt + CCR9 − cells, RORγt + CCR9 + cells, RORγt − CCR9 + cells, and RORγt − CCR9 − cells in the Lin − NK1.1 − IL-7Rα + T1/ST2 − gated cells in the fetal mesentery were assessed. (G) CCR9 expression on Lin − IL-7Rα + T1/ST2 − cells and Lin − IL-7Rα + T1/ST2 + fetal ILC2 from the E17 fetal mesentery. (H) ILC differentiation from Lin − IL-7Rα + T1/ST2 − CCR9 − or Lin − IL-7Rα + T1/ST2 − CCR9 + cells cultured with TSt4-DLL1 and 10 ng/ml IL-7 for 10 d. Blue, red, and pink boxes indicate T-bet + ILC1, GATA3 hi ILC2, and RORγt + ILC3, respectively. (I) Numbers of ILC1, ILC2, and ILC3 in H. n = 4–8. Error bars show means ± SD. (J) Lin − NK1.1 − IL-7Rα + T1/ST2 − RORγt − CCR9 − cells from the E18 fetal mesentery from RORγt GFP/+ mice were cultured with TSt4-DLL1 cells in the presence of 10 ng/ml IL-7 for 10 d. After 10 d, differentiated cells were assessed for T-bet, GATA3, and RORγt expression by flow cytometry. Blue, red, and pink boxes indicate T-bet + ILC1, GATA3 hi ILC2, and RORγt + ILC3, respectively. Results are representative of two independent experiments. *, P

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

    Related Articles

    Enzyme-linked Immunosorbent Assay:

    Article Title: Exacerbated Leishmaniasis Caused by a Viral Endosymbiont can be Prevented by Immunization with Its Viral Capsid
    Article Snippet: .. Spleen cell supernatants cultured without BrefeldinA were then analyzed 72 hours post incubation by ELISA to detect IL-10, IL-4 and IFN-γ (eBioscience) as described previously. .. T cell purification and transfer One week after the third recall, lymphocytes were collected from spleen of immunized or control group mice.

    Staining:

    Article Title: Thioesterase PPT1 balances viral resistance and efficient T cell crosspriming in dendritic cells
    Article Snippet: .. In the case of intracellular cytokine staining, brefeldin A (eBioscience) was added with peptide (10 nM SIINFEKL for OT-I cells) or TLR ligands (for BMDCs) for 4–8 h before staining with the intracellular staining kit (eBioscience). .. The following antibodies were all purchased from eBioscience unless otherwise indicated: anti-CD8α (53-6.7), IL-2 (JES6-5H4), CD69 (H1.2F3), TNF-α (MP6-XT22), CD45.1 (A20), CD44 (IM7), CD80 (16-10A1), IFN-α (RMMA-1), CD11C (N418), Clec9A (42D2), IFN-γ (XMG1.2), MHC II (M5-114.15.2), CD24 (M1-69), CD86 (GL1), PD-L1 (NIH5), CD127 (A7R34), XCR1 (ZET; BioLegend), CD103 (2E7), KLRG1 (2F1), IL-12p40 (C17.8), PD-1(J43), CD40 (IC40), SIINFEKL-peptide bound to H2-Kb (25.D1.16), LCMV gp33-H-2Db (gp33-41; MBL), and H-2Kb (AF6-88.5.5.3).

    Article Title: Tissue-specific control of latent CMV reactivation by regulatory T cells
    Article Snippet: .. For CD4 T cells, cells were stimulated with 50ng/ml PMA and 1μg/ml ionomycin for 5 hours, in the presence of brefeldinA for the final 4 h and fixed with 2%MF FA for 1 hour followed by intracellular staining for IL-10, IFN-γ, TNF-α (all from eBioscience) and Foxp3 (eBioscience) staining was done according to eBioscience Foxp3 staining kit and protocol. .. Data were acquired on an LSRII flow cytometer (BD Biosciences) and analyzed using FACSDiva software (BD Biosciences).

    Article Title: SP-R210 (Myo18A) Isoforms as Intrinsic Modulators of Macrophage Priming and Activation
    Article Snippet: .. Brefeldin A, and fix/permealizing solution for intracellular cytokine staining were from eBioscience. .. Antibodies to NFκB subunit RelA(p65), RelA(p65)S536 , IRAK-1, and IκB were purchased from Cell Signaling.

    Incubation:

    Article Title: Exacerbated Leishmaniasis Caused by a Viral Endosymbiont can be Prevented by Immunization with Its Viral Capsid
    Article Snippet: .. Spleen cell supernatants cultured without BrefeldinA were then analyzed 72 hours post incubation by ELISA to detect IL-10, IL-4 and IFN-γ (eBioscience) as described previously. .. T cell purification and transfer One week after the third recall, lymphocytes were collected from spleen of immunized or control group mice.

    Cell Culture:

    Article Title: Peripheral PDGFRα+gp38+ mesenchymal cells support the differentiation of fetal liver–derived ILC2
    Article Snippet: .. To determine the potential for cytokine production, cells were cultured at 5 × 105 cells per well in 24-well flat-bottom plates with 50 ng/ml PMA (Sigma-Aldrich), 1 µg/ml ionomycin (Sigma-Aldrich), and 3 µg/ml brefeldin A solution (eBioscience). ..

    Article Title: Exacerbated Leishmaniasis Caused by a Viral Endosymbiont can be Prevented by Immunization with Its Viral Capsid
    Article Snippet: .. Spleen cell supernatants cultured without BrefeldinA were then analyzed 72 hours post incubation by ELISA to detect IL-10, IL-4 and IFN-γ (eBioscience) as described previously. .. T cell purification and transfer One week after the third recall, lymphocytes were collected from spleen of immunized or control group mice.

    Blocking Assay:

    Article Title: Flagellin Treatment Prevents Increased Susceptibility to Systemic Bacterial Infection after Injury by Inhibiting Anti-Inflammatory IL-10+ IL-12- Neutrophil Polarization
    Article Snippet: .. During the last 4 hours of the stimulation, 3.0 ul/mL of brefeldin-A solution (eBioscience; San Diego, CA) was added to block protein secretion. .. Flow cytometric analysis Splenocytes were incubated with anti-mouse CD16/32 (BD Biosciences; San Jose, CA) at a concentration of 1 ug per million cells for 5 min at 4°C to block Fc receptors.

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    Rvs161/Rvs167 and Rvs162/Rvs167-3 heterodimers bind membranes in vitro . <t>Coomassie-stained</t> SDS-PAA gels used in lipid cosedimentation experiments are shown. Purified Rvs162(+H 0 )/Rvs167-3(−H 0 ) (A) and Rvs161(−H 0 )/Rvs167(−H 0 ) (B)
    Coomassie Brilliant Blue, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 191 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher sds running buffer coomassie brilliant blue solution purification dialysis buffer
    Representative human La antigen purification The 6xHis-tagged human La antigen (approximately 32 kDa, arrowhead) elutes from pre-charged Ni 2+ -beads using an imidazole gradient. Fractions can be analyzed by <t>SDS-PAGE</t> (shown here 15% SDS, <t>Coomassie</t> Brilliant Blue stain). The purest fractions containing the desired La antigen product are pooled for use in ALARM NMR. IPTG, bacterial culture aliquots before (–) and after (+) IPTG induction; LMW, low molecular weight protein ladder.
    Sds Running Buffer Coomassie Brilliant Blue Solution Purification Dialysis Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher coomassie blue solution
    Rif1-associated complexes are enriched for chromatin-related proteins and PP1 substrates/regulators. ( A ) Immunoprecipitation of Rif1-associated proteins from extracts obtained from ESCs homozygous for Rif1 FH or Rif1 + alleles, visualized by <t>Coomassie</t> blue-stained SDS-PAGE. ( B ) Determination of Rif1 interactome using label-free quantitation pipeline MaxQuant 49 . For volcano plot, t -test was performed on data from 3 Rif1 FH/+ and 2 Rif1 +/+ ESC independent lines analysed in 2 independent experiments. t -test difference ratios were plotted against the negative logarithmic P -value of the t -test. Rif1, Lamin B1 and PP1 LFQs are indicated in red. ( C ) Top gene ontology (GO) terms enriched within nuclear proteins associated to Rif1 FH , listed in supplemental table C (cut-off of enrichment over negative control 1.5). The analysis is a Panther overrepresentation test against the complete GO biological process annotation dataset. Bonferroni correction was applied. ( D ) PP1 interaction with Rif1 was confirmed by immunoprecipitation of Rif1-associated proteins from Rif1 FH/+ ESCs and immunoblotting with anti-PP1 antibody. IN = input; FT = flow through; IP = immunoprecipitated. ( E ) List of the known PP1 substrates/regulators identified in the Rif1-associated complexes. The Rif1 interactome enrichment for PP1 substrates/regulators is statistically significant as evaluated by Fisher’s exact test ( P - value : 0.00111).
    Coomassie Blue Solution, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Rvs161/Rvs167 and Rvs162/Rvs167-3 heterodimers bind membranes in vitro . Coomassie-stained SDS-PAA gels used in lipid cosedimentation experiments are shown. Purified Rvs162(+H 0 )/Rvs167-3(−H 0 ) (A) and Rvs161(−H 0 )/Rvs167(−H 0 ) (B)

    Journal: Eukaryotic Cell

    Article Title: Identification and Characterization of Rvs162/Rvs167-3, a Novel N-BAR Heterodimer in the Human Fungal Pathogen Candida albicans

    doi: 10.1128/EC.00282-14

    Figure Lengend Snippet: Rvs161/Rvs167 and Rvs162/Rvs167-3 heterodimers bind membranes in vitro . Coomassie-stained SDS-PAA gels used in lipid cosedimentation experiments are shown. Purified Rvs162(+H 0 )/Rvs167-3(−H 0 ) (A) and Rvs161(−H 0 )/Rvs167(−H 0 ) (B)

    Article Snippet: After separation, protein bands were visualized with Coomassie brilliant blue (Thermo Scientific PageBlue protein staining solution).

    Techniques: In Vitro, Staining, Purification

    Representative human La antigen purification The 6xHis-tagged human La antigen (approximately 32 kDa, arrowhead) elutes from pre-charged Ni 2+ -beads using an imidazole gradient. Fractions can be analyzed by SDS-PAGE (shown here 15% SDS, Coomassie Brilliant Blue stain). The purest fractions containing the desired La antigen product are pooled for use in ALARM NMR. IPTG, bacterial culture aliquots before (–) and after (+) IPTG induction; LMW, low molecular weight protein ladder.

    Journal: Current protocols in chemical biology

    Article Title: ALARM NMR for HTS triage and chemical probe validation

    doi: 10.1002/cpch.35

    Figure Lengend Snippet: Representative human La antigen purification The 6xHis-tagged human La antigen (approximately 32 kDa, arrowhead) elutes from pre-charged Ni 2+ -beads using an imidazole gradient. Fractions can be analyzed by SDS-PAGE (shown here 15% SDS, Coomassie Brilliant Blue stain). The purest fractions containing the desired La antigen product are pooled for use in ALARM NMR. IPTG, bacterial culture aliquots before (–) and after (+) IPTG induction; LMW, low molecular weight protein ladder.

    Article Snippet: Rosetta (DE3)pLysS cells (Novagen, Cat # 70956) (plasmid ID 90209) ; see also ) LB agar petri plates LB liquid media (see recipe) M9 media (see recipe) Kanamycyin stock solution (see recipe) Chloramphenicol stock solution (see recipe) [3-13 C]-α-Ketobutyrate (Cambridge Isotope Laboratories, Cat # CLM-6820) [3,3-13 C]-α-Ketoisovalerate (Cambridge Isotope Laboratories, Cat # CLM-6821) IPTG solution (see recipe) Phosphate-buffered saline (PBS; see recipe) Lysis buffer (see recipe) Pre-charged Ni-NTA agarose beads (Qiagen) Wash buffer (see recipe) Elution buffer (see recipe) Bradford reagent solution or microvolume spectrophotometer (e.g., ThermoFisher Nanodrop™) SDS-PAGE gels (e.g., 10-15%) 5× SDS loading buffer 1× SDS running buffer Coomassie brilliant blue solution Purification dialysis buffer (see recipe) SnakeSkin™ dialysis membrane 10 kDa MWCO (ThermoFisher, Cat # 68305) Incubator oven Temperature-controlled bacterial incubated shaker Tabletop centrifuge (e.g., Beckman-Coulter Allegra X-12) High-performance centrifuge (e.g., Beckman-Coulter Avanti J-26XP) Compatible high-speed rotor (e.g., Beckman-Coulter JA-30.50 fixed-angle titanium rotor) with compatible 50 mL bottles (e.g., Beckman-Coulter Cat # 361694) Compatible large-volume capacity rotor (e.g., Beckman-Coulter JLA-8.1000 fixed-angle rotor) with compatible 1 L bottles (e.g., Beckman-Coulter Cat # 366751) French press or probe sonicator (e.g., Qsonica Q125) Gravity drip columns (e.g., Bio-Rad Econo-Pac® or Econo-Column®) Spectrophotometer Gel electrophoresis apparatus with power supply Rocker Gel imaging system Note: additional required reagents are listed under recipes.

    Techniques: Purification, SDS Page, Staining, Nuclear Magnetic Resonance, Molecular Weight

    Rif1-associated complexes are enriched for chromatin-related proteins and PP1 substrates/regulators. ( A ) Immunoprecipitation of Rif1-associated proteins from extracts obtained from ESCs homozygous for Rif1 FH or Rif1 + alleles, visualized by Coomassie blue-stained SDS-PAGE. ( B ) Determination of Rif1 interactome using label-free quantitation pipeline MaxQuant 49 . For volcano plot, t -test was performed on data from 3 Rif1 FH/+ and 2 Rif1 +/+ ESC independent lines analysed in 2 independent experiments. t -test difference ratios were plotted against the negative logarithmic P -value of the t -test. Rif1, Lamin B1 and PP1 LFQs are indicated in red. ( C ) Top gene ontology (GO) terms enriched within nuclear proteins associated to Rif1 FH , listed in supplemental table C (cut-off of enrichment over negative control 1.5). The analysis is a Panther overrepresentation test against the complete GO biological process annotation dataset. Bonferroni correction was applied. ( D ) PP1 interaction with Rif1 was confirmed by immunoprecipitation of Rif1-associated proteins from Rif1 FH/+ ESCs and immunoblotting with anti-PP1 antibody. IN = input; FT = flow through; IP = immunoprecipitated. ( E ) List of the known PP1 substrates/regulators identified in the Rif1-associated complexes. The Rif1 interactome enrichment for PP1 substrates/regulators is statistically significant as evaluated by Fisher’s exact test ( P - value : 0.00111).

    Journal: Scientific Reports

    Article Title: Mouse Rif1 is a regulatory subunit of protein phosphatase 1 (PP1)

    doi: 10.1038/s41598-017-01910-1

    Figure Lengend Snippet: Rif1-associated complexes are enriched for chromatin-related proteins and PP1 substrates/regulators. ( A ) Immunoprecipitation of Rif1-associated proteins from extracts obtained from ESCs homozygous for Rif1 FH or Rif1 + alleles, visualized by Coomassie blue-stained SDS-PAGE. ( B ) Determination of Rif1 interactome using label-free quantitation pipeline MaxQuant 49 . For volcano plot, t -test was performed on data from 3 Rif1 FH/+ and 2 Rif1 +/+ ESC independent lines analysed in 2 independent experiments. t -test difference ratios were plotted against the negative logarithmic P -value of the t -test. Rif1, Lamin B1 and PP1 LFQs are indicated in red. ( C ) Top gene ontology (GO) terms enriched within nuclear proteins associated to Rif1 FH , listed in supplemental table C (cut-off of enrichment over negative control 1.5). The analysis is a Panther overrepresentation test against the complete GO biological process annotation dataset. Bonferroni correction was applied. ( D ) PP1 interaction with Rif1 was confirmed by immunoprecipitation of Rif1-associated proteins from Rif1 FH/+ ESCs and immunoblotting with anti-PP1 antibody. IN = input; FT = flow through; IP = immunoprecipitated. ( E ) List of the known PP1 substrates/regulators identified in the Rif1-associated complexes. The Rif1 interactome enrichment for PP1 substrates/regulators is statistically significant as evaluated by Fisher’s exact test ( P - value : 0.00111).

    Article Snippet: Staining was performed with Coomassie blue solution (3 g/l Coomassie Brilliant blue G-250, Thermo 20279, in 45% methanol, 10% acetic acid) for up to 3 h at room temperature.

    Techniques: Immunoprecipitation, Staining, SDS Page, Quantitation Assay, Negative Control, Flow Cytometry

    The Rif1 CRI region contains two canonical RVSF and SILK motifs interacting with PP1. ( A ) Beads loaded with GST-Rif1 fragments were incubated with cell lysates containing hexahistidine-PP1. PP1 retained by means of interaction with Rif1 was eluted and analysed by imunoblotting using antibodies against the hexahistidine tag. ( B ) The affinity of Rif1 CRI mutants and peptide for PP1 was determined by the SPR and expressed as percentage of wild-type. SAAA indicates the mutation of the residues within the SILK motif. ( C ) Analysis of Rif1 CRI interaction with PP1 by size exclusion chromatography. PP1 (light blue), Rif1 CRI (red), PP1/CRI at molar ratio 1:1 (magenta), PP1/CRI at molar ratio 1:3 (green) and PP1/CRI at molar ratio 2:1 (blue) were subjected to analytical gel-filtration. The Coomassie-stained gel shows recombinant PP1 and Rif1 CRI co-eluting from the column. mAU = milli Absorbance Unit. ( D ) Superposition of the 1 H- 15 N HSQC spectra of 15 N-labeled CRI (red) and 15 N-labeled CRI in the presence of PP1 (blue). In the inset, assignments are shown for CRI for a selected region of the HSQC spectrum. ( E ) PP1-interacting region in mouse Rif1. Interacting residues identified by NMR analysis are highlighted in yellow, residues present in the synthetic peptide (Pept) are shown in bold, and residues subjected to mutagenesis are shown in red.

    Journal: Scientific Reports

    Article Title: Mouse Rif1 is a regulatory subunit of protein phosphatase 1 (PP1)

    doi: 10.1038/s41598-017-01910-1

    Figure Lengend Snippet: The Rif1 CRI region contains two canonical RVSF and SILK motifs interacting with PP1. ( A ) Beads loaded with GST-Rif1 fragments were incubated with cell lysates containing hexahistidine-PP1. PP1 retained by means of interaction with Rif1 was eluted and analysed by imunoblotting using antibodies against the hexahistidine tag. ( B ) The affinity of Rif1 CRI mutants and peptide for PP1 was determined by the SPR and expressed as percentage of wild-type. SAAA indicates the mutation of the residues within the SILK motif. ( C ) Analysis of Rif1 CRI interaction with PP1 by size exclusion chromatography. PP1 (light blue), Rif1 CRI (red), PP1/CRI at molar ratio 1:1 (magenta), PP1/CRI at molar ratio 1:3 (green) and PP1/CRI at molar ratio 2:1 (blue) were subjected to analytical gel-filtration. The Coomassie-stained gel shows recombinant PP1 and Rif1 CRI co-eluting from the column. mAU = milli Absorbance Unit. ( D ) Superposition of the 1 H- 15 N HSQC spectra of 15 N-labeled CRI (red) and 15 N-labeled CRI in the presence of PP1 (blue). In the inset, assignments are shown for CRI for a selected region of the HSQC spectrum. ( E ) PP1-interacting region in mouse Rif1. Interacting residues identified by NMR analysis are highlighted in yellow, residues present in the synthetic peptide (Pept) are shown in bold, and residues subjected to mutagenesis are shown in red.

    Article Snippet: Staining was performed with Coomassie blue solution (3 g/l Coomassie Brilliant blue G-250, Thermo 20279, in 45% methanol, 10% acetic acid) for up to 3 h at room temperature.

    Techniques: Incubation, SPR Assay, Mutagenesis, Size-exclusion Chromatography, Filtration, Staining, Recombinant, Labeling, Nuclear Magnetic Resonance