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Pharmingen brefeldin a
The Fas-mediated pathway is favored. Two hours prior to the 51 Cr release assay, TMEV-induced autoreactive cells were incubated with <t>brefeldin</t> A (BFA) (○), concanamycin A (CMA) (▵), or vehicle (□). Brefeldin A and concanamycin A are known to inhibit Fas- and perforin-mediated cytotoxicities, respectively. Effector cells together with 51 Cr-labeled PSJL target cells were cultured for 5 h. Only brefeldin A showed dose-dependent inhibition of autoreactive killing. Results are representative of those from two independent experiments. Error bars indicate standard errors.
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Images

1) Product Images from "Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity"

Article Title: Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity

Journal: Journal of Virology

doi: 10.1128/JVI.76.24.12834-12844.2002

The Fas-mediated pathway is favored. Two hours prior to the 51 Cr release assay, TMEV-induced autoreactive cells were incubated with brefeldin A (BFA) (○), concanamycin A (CMA) (▵), or vehicle (□). Brefeldin A and concanamycin A are known to inhibit Fas- and perforin-mediated cytotoxicities, respectively. Effector cells together with 51 Cr-labeled PSJL target cells were cultured for 5 h. Only brefeldin A showed dose-dependent inhibition of autoreactive killing. Results are representative of those from two independent experiments. Error bars indicate standard errors.
Figure Legend Snippet: The Fas-mediated pathway is favored. Two hours prior to the 51 Cr release assay, TMEV-induced autoreactive cells were incubated with brefeldin A (BFA) (○), concanamycin A (CMA) (▵), or vehicle (□). Brefeldin A and concanamycin A are known to inhibit Fas- and perforin-mediated cytotoxicities, respectively. Effector cells together with 51 Cr-labeled PSJL target cells were cultured for 5 h. Only brefeldin A showed dose-dependent inhibition of autoreactive killing. Results are representative of those from two independent experiments. Error bars indicate standard errors.

Techniques Used: Release Assay, Incubation, Labeling, Cell Culture, Inhibition

2) Product Images from "Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity"

Article Title: Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity

Journal: Journal of Virology

doi: 10.1128/JVI.76.24.12834-12844.2002

The Fas-mediated pathway is favored. Two hours prior to the 51 Cr release assay, TMEV-induced autoreactive cells were incubated with brefeldin A (BFA) (○), concanamycin A (CMA) (▵), or vehicle (□). Brefeldin A and concanamycin A are known to inhibit Fas- and perforin-mediated cytotoxicities, respectively. Effector cells together with 51 Cr-labeled PSJL target cells were cultured for 5 h. Only brefeldin A showed dose-dependent inhibition of autoreactive killing. Results are representative of those from two independent experiments. Error bars indicate standard errors.
Figure Legend Snippet: The Fas-mediated pathway is favored. Two hours prior to the 51 Cr release assay, TMEV-induced autoreactive cells were incubated with brefeldin A (BFA) (○), concanamycin A (CMA) (▵), or vehicle (□). Brefeldin A and concanamycin A are known to inhibit Fas- and perforin-mediated cytotoxicities, respectively. Effector cells together with 51 Cr-labeled PSJL target cells were cultured for 5 h. Only brefeldin A showed dose-dependent inhibition of autoreactive killing. Results are representative of those from two independent experiments. Error bars indicate standard errors.

Techniques Used: Release Assay, Incubation, Labeling, Cell Culture, Inhibition

3) Product Images from "The Class A Macrophage Scavenger Receptor Is a Major Pattern Recognition Receptor for Neisseria meningitidis Which Is Independent of Lipopolysaccharide and Not Required for Secretory Responses "

Article Title: The Class A Macrophage Scavenger Receptor Is a Major Pattern Recognition Receptor for Neisseria meningitidis Which Is Independent of Lipopolysaccharide and Not Required for Secretory Responses

Journal: Infection and Immunity

doi: 10.1128/IAI.70.10.5346-5354.2002

Mφ proinflammatory cytokine secretion following challenge with  N. meningitidis . (a) WT and SR-A −/−  BMMφ were incubated with ethanol-fixed MC58 bacteria at 37°C. At different times, the culture supernatant was harvested and analyzed by cytokine ELISA for production of TNF-α, IL-6, IL-10, and IL-12. The results are representative of at least two similar experiments, and the average of triplicate conditions is plotted. (b) BMMφ were incubated with ethanol-fixed RdGnX-MC58 (40 bacteria per cell) in the presence of brefeldin A. After incubation, the Mφ were fixed with 4% paraformaldehyde, stained with a phycoerythrin (PE)-labeled anti-TNF-α antibody, and analyzed by flow cytometry. The results are representative of at least two similar experiments. Note that preincubation of the anti-TNF-α antibody with recombinant mouse TNF-α or of the cells with an unlabeled anti-TNF-α antibody blocked the binding of the PE-labeled anti-TNF-α (not shown), confirming specificity. The error bars indicate standard deviations.
Figure Legend Snippet: Mφ proinflammatory cytokine secretion following challenge with N. meningitidis . (a) WT and SR-A −/− BMMφ were incubated with ethanol-fixed MC58 bacteria at 37°C. At different times, the culture supernatant was harvested and analyzed by cytokine ELISA for production of TNF-α, IL-6, IL-10, and IL-12. The results are representative of at least two similar experiments, and the average of triplicate conditions is plotted. (b) BMMφ were incubated with ethanol-fixed RdGnX-MC58 (40 bacteria per cell) in the presence of brefeldin A. After incubation, the Mφ were fixed with 4% paraformaldehyde, stained with a phycoerythrin (PE)-labeled anti-TNF-α antibody, and analyzed by flow cytometry. The results are representative of at least two similar experiments. Note that preincubation of the anti-TNF-α antibody with recombinant mouse TNF-α or of the cells with an unlabeled anti-TNF-α antibody blocked the binding of the PE-labeled anti-TNF-α (not shown), confirming specificity. The error bars indicate standard deviations.

Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay, Staining, Labeling, Flow Cytometry, Cytometry, Recombinant, Binding Assay

4) Product Images from "Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity"

Article Title: Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity

Journal: Journal of Virology

doi: 10.1128/JVI.76.24.12834-12844.2002

The Fas-mediated pathway is favored. Two hours prior to the 51 Cr release assay, TMEV-induced autoreactive cells were incubated with brefeldin A (BFA) (○), concanamycin A (CMA) (▵), or vehicle (□). Brefeldin A and concanamycin A are known to inhibit Fas- and perforin-mediated cytotoxicities, respectively. Effector cells together with 51 Cr-labeled PSJL target cells were cultured for 5 h. Only brefeldin A showed dose-dependent inhibition of autoreactive killing. Results are representative of those from two independent experiments. Error bars indicate standard errors.
Figure Legend Snippet: The Fas-mediated pathway is favored. Two hours prior to the 51 Cr release assay, TMEV-induced autoreactive cells were incubated with brefeldin A (BFA) (○), concanamycin A (CMA) (▵), or vehicle (□). Brefeldin A and concanamycin A are known to inhibit Fas- and perforin-mediated cytotoxicities, respectively. Effector cells together with 51 Cr-labeled PSJL target cells were cultured for 5 h. Only brefeldin A showed dose-dependent inhibition of autoreactive killing. Results are representative of those from two independent experiments. Error bars indicate standard errors.

Techniques Used: Release Assay, Incubation, Labeling, Cell Culture, Inhibition

5) Product Images from "Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity"

Article Title: Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity

Journal: Journal of Virology

doi: 10.1128/JVI.76.24.12834-12844.2002

The Fas-mediated pathway is favored. Two hours prior to the 51 Cr release assay, TMEV-induced autoreactive cells were incubated with brefeldin A (BFA) (○), concanamycin A (CMA) (▵), or vehicle (□). Brefeldin A and concanamycin A are known to inhibit Fas- and perforin-mediated cytotoxicities, respectively. Effector cells together with 51 Cr-labeled PSJL target cells were cultured for 5 h. Only brefeldin A showed dose-dependent inhibition of autoreactive killing. Results are representative of those from two independent experiments. Error bars indicate standard errors.
Figure Legend Snippet: The Fas-mediated pathway is favored. Two hours prior to the 51 Cr release assay, TMEV-induced autoreactive cells were incubated with brefeldin A (BFA) (○), concanamycin A (CMA) (▵), or vehicle (□). Brefeldin A and concanamycin A are known to inhibit Fas- and perforin-mediated cytotoxicities, respectively. Effector cells together with 51 Cr-labeled PSJL target cells were cultured for 5 h. Only brefeldin A showed dose-dependent inhibition of autoreactive killing. Results are representative of those from two independent experiments. Error bars indicate standard errors.

Techniques Used: Release Assay, Incubation, Labeling, Cell Culture, Inhibition

6) Product Images from "Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity"

Article Title: Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity

Journal: Journal of Virology

doi: 10.1128/JVI.76.24.12834-12844.2002

The Fas-mediated pathway is favored. Two hours prior to the 51 Cr release assay, TMEV-induced autoreactive cells were incubated with brefeldin A (BFA) (○), concanamycin A (CMA) (▵), or vehicle (□). Brefeldin A and concanamycin A are known to inhibit Fas- and perforin-mediated cytotoxicities, respectively. Effector cells together with 51 Cr-labeled PSJL target cells were cultured for 5 h. Only brefeldin A showed dose-dependent inhibition of autoreactive killing. Results are representative of those from two independent experiments. Error bars indicate standard errors.
Figure Legend Snippet: The Fas-mediated pathway is favored. Two hours prior to the 51 Cr release assay, TMEV-induced autoreactive cells were incubated with brefeldin A (BFA) (○), concanamycin A (CMA) (▵), or vehicle (□). Brefeldin A and concanamycin A are known to inhibit Fas- and perforin-mediated cytotoxicities, respectively. Effector cells together with 51 Cr-labeled PSJL target cells were cultured for 5 h. Only brefeldin A showed dose-dependent inhibition of autoreactive killing. Results are representative of those from two independent experiments. Error bars indicate standard errors.

Techniques Used: Release Assay, Incubation, Labeling, Cell Culture, Inhibition

7) Product Images from "Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity"

Article Title: Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity

Journal: Journal of Virology

doi: 10.1128/JVI.76.24.12834-12844.2002

The Fas-mediated pathway is favored. Two hours prior to the 51 Cr release assay, TMEV-induced autoreactive cells were incubated with brefeldin A (BFA) (○), concanamycin A (CMA) (▵), or vehicle (□). Brefeldin A and concanamycin A are known to inhibit Fas- and perforin-mediated cytotoxicities, respectively. Effector cells together with 51 Cr-labeled PSJL target cells were cultured for 5 h. Only brefeldin A showed dose-dependent inhibition of autoreactive killing. Results are representative of those from two independent experiments. Error bars indicate standard errors.
Figure Legend Snippet: The Fas-mediated pathway is favored. Two hours prior to the 51 Cr release assay, TMEV-induced autoreactive cells were incubated with brefeldin A (BFA) (○), concanamycin A (CMA) (▵), or vehicle (□). Brefeldin A and concanamycin A are known to inhibit Fas- and perforin-mediated cytotoxicities, respectively. Effector cells together with 51 Cr-labeled PSJL target cells were cultured for 5 h. Only brefeldin A showed dose-dependent inhibition of autoreactive killing. Results are representative of those from two independent experiments. Error bars indicate standard errors.

Techniques Used: Release Assay, Incubation, Labeling, Cell Culture, Inhibition

8) Product Images from "Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity"

Article Title: Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity

Journal: Journal of Virology

doi: 10.1128/JVI.76.24.12834-12844.2002

The Fas-mediated pathway is favored. Two hours prior to the 51 Cr release assay, TMEV-induced autoreactive cells were incubated with brefeldin A (BFA) (○), concanamycin A (CMA) (▵), or vehicle (□). Brefeldin A and concanamycin A are known to inhibit Fas- and perforin-mediated cytotoxicities, respectively. Effector cells together with 51 Cr-labeled PSJL target cells were cultured for 5 h. Only brefeldin A showed dose-dependent inhibition of autoreactive killing. Results are representative of those from two independent experiments. Error bars indicate standard errors.
Figure Legend Snippet: The Fas-mediated pathway is favored. Two hours prior to the 51 Cr release assay, TMEV-induced autoreactive cells were incubated with brefeldin A (BFA) (○), concanamycin A (CMA) (▵), or vehicle (□). Brefeldin A and concanamycin A are known to inhibit Fas- and perforin-mediated cytotoxicities, respectively. Effector cells together with 51 Cr-labeled PSJL target cells were cultured for 5 h. Only brefeldin A showed dose-dependent inhibition of autoreactive killing. Results are representative of those from two independent experiments. Error bars indicate standard errors.

Techniques Used: Release Assay, Incubation, Labeling, Cell Culture, Inhibition

9) Product Images from "A subset of natural killer cells achieves self-tolerance without expressing inhibitory receptors specific for self-MHC molecules"

Article Title: A subset of natural killer cells achieves self-tolerance without expressing inhibitory receptors specific for self-MHC molecules

Journal:

doi: 10.1182/blood-2004-08-3156

CI/NKG2 - NK cells in B6 mice exhibit reduced responsiveness to NK-sensitive target cells. Splenocytes from poly(I:C)-treated β2m -/- , B6, or B10.M mice were stimulated for 5 hours in the presence of brefeldin A with Con A–activated lymphoblasts
Figure Legend Snippet: CI/NKG2 - NK cells in B6 mice exhibit reduced responsiveness to NK-sensitive target cells. Splenocytes from poly(I:C)-treated β2m -/- , B6, or B10.M mice were stimulated for 5 hours in the presence of brefeldin A with Con A–activated lymphoblasts

Techniques Used: Mouse Assay

10) Product Images from "Unanticipated Antigens: Translation Initiation at CUG with LeucineWhere to Start? Alternate Protein Translation Mechanism Creates Unanticipated Antigens"

Article Title: Unanticipated Antigens: Translation Initiation at CUG with LeucineWhere to Start? Alternate Protein Translation Mechanism Creates Unanticipated Antigens

Journal: PLoS Biology

doi: 10.1371/journal.pbio.0020366

The Leucine Start Is Enhanced in the Presence of Phosphorylated eIF2α HeLa cells transfected with cDNA encoding K b together with cDNA encoding either the ATG- or CTG- initiated peptides were treated for 4 h with 50 μM NaAs, with brefeldin A (BfA), or left untreated (UT). (A) Transfected cells treated with NaAs or without (UT) were lysed and tested for phosphorylation of eIF2α by Western blot and for tubulin as a loading control. (B) The transfected cells were titrated and tested for their ability to stimulate BCZ103 T cells.
Figure Legend Snippet: The Leucine Start Is Enhanced in the Presence of Phosphorylated eIF2α HeLa cells transfected with cDNA encoding K b together with cDNA encoding either the ATG- or CTG- initiated peptides were treated for 4 h with 50 μM NaAs, with brefeldin A (BfA), or left untreated (UT). (A) Transfected cells treated with NaAs or without (UT) were lysed and tested for phosphorylation of eIF2α by Western blot and for tubulin as a loading control. (B) The transfected cells were titrated and tested for their ability to stimulate BCZ103 T cells.

Techniques Used: Transfection, CTG Assay, Western Blot

11) Product Images from "Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity"

Article Title: Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity

Journal: Journal of Virology

doi: 10.1128/JVI.76.24.12834-12844.2002

The Fas-mediated pathway is favored. Two hours prior to the 51 Cr release assay, TMEV-induced autoreactive cells were incubated with brefeldin A (BFA) (○), concanamycin A (CMA) (▵), or vehicle (□). Brefeldin A and concanamycin A are known to inhibit Fas- and perforin-mediated cytotoxicities, respectively. Effector cells together with 51 Cr-labeled PSJL target cells were cultured for 5 h. Only brefeldin A showed dose-dependent inhibition of autoreactive killing. Results are representative of those from two independent experiments. Error bars indicate standard errors.
Figure Legend Snippet: The Fas-mediated pathway is favored. Two hours prior to the 51 Cr release assay, TMEV-induced autoreactive cells were incubated with brefeldin A (BFA) (○), concanamycin A (CMA) (▵), or vehicle (□). Brefeldin A and concanamycin A are known to inhibit Fas- and perforin-mediated cytotoxicities, respectively. Effector cells together with 51 Cr-labeled PSJL target cells were cultured for 5 h. Only brefeldin A showed dose-dependent inhibition of autoreactive killing. Results are representative of those from two independent experiments. Error bars indicate standard errors.

Techniques Used: Release Assay, Incubation, Labeling, Cell Culture, Inhibition

12) 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

13) Product Images from "Characteristics of virus-specific CD8+ T cells in the liver during the control and resolution phases of influenza pneumonia"

Article Title: Characteristics of virus-specific CD8+ T cells in the liver during the control and resolution phases of influenza pneumonia

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

doi:

Comparison of influenza virus-specific DbNP366 tetramer staining of CD8 +  T cells and the NP peptide-specific response (IFN-γ) of liver ( Upper ) and BAL ( Lower ) and lymphocytes isolated 10 days after i.n. infection of PR8-immune mice with HKx31. Lymphocytes were pooled from six mice, then stained with anti-CD8 and DbNP366 tetramer and analyzed by flow cytometry ( Left ) or cultured for 6 h in complete medium/brefeldin A in the presence or absence of the NP peptide ( Center ) or anti-CD3ɛ ( Right ), then stained for surface CD8 and intracellular IFN-γ. For samples incubated without peptide, IFN-γ staining was
Figure Legend Snippet: Comparison of influenza virus-specific DbNP366 tetramer staining of CD8 + T cells and the NP peptide-specific response (IFN-γ) of liver ( Upper ) and BAL ( Lower ) and lymphocytes isolated 10 days after i.n. infection of PR8-immune mice with HKx31. Lymphocytes were pooled from six mice, then stained with anti-CD8 and DbNP366 tetramer and analyzed by flow cytometry ( Left ) or cultured for 6 h in complete medium/brefeldin A in the presence or absence of the NP peptide ( Center ) or anti-CD3ɛ ( Right ), then stained for surface CD8 and intracellular IFN-γ. For samples incubated without peptide, IFN-γ staining was

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

14) Product Images from "Two Roads Diverged: Interferon ?/?- and Interleukin 12-mediated Pathways in Promoting T Cell Interferon ? Responses during Viral Infection "

Article Title: Two Roads Diverged: Interferon ?/?- and Interleukin 12-mediated Pathways in Promoting T Cell Interferon ? Responses during Viral Infection

Journal: The Journal of Experimental Medicine

doi:

T cell responses in mice lacking endogenous sources of either IL-12 or IFN-α/β. Mice, WT and IL-12p35 KO 129/B6 (A–C) or WT and IFN-α/βR KO 129 (D–F), were either uninfected (open bars or symbols) or infected i.p. (closed bars or symbols) on day 0 with 2 × 10 4 PFU LCMV Armstrong strain. On day 8 after infection, blood and spleens were harvested and processed. Total numbers of CD8 T cell splenic leukocytes were calculated by multiplying the percentages of CD8 cells, determined by flow cytometry, with the total cell yields (A and D). IFN-γ levels were measured, in media conditioned with splenic leukocytes and in serum samples, by ELISA (B and E). For flow cytometric analysis of cytoplasmic IFN-γ expression, splenic leukocytes were stimulated for 6 h with anti–CD3, with Brefeldin A during the last 2 h. Cells were collected and stained for cell surface expression of CD8, CD4, and cytoplasmic expression of IFN-γ (C and F). Histogram plots were formed by gating on the CD8 population, and displaying CD8 T cell number versus IFN-γ fluorescence intensity. Numbers of CD8 T cells expressing IFN-γ were calculated by multiplying the percentage of CD8 T cells expressing IFN-γ by the number of CD8 T cells per spleen (× 10 6 ), and given as means ± SEM. Results are representative of two or more repetitive experiments with two to four mice per uninfected group and three to four mice per infected group. Data are shown as means ± SEM.
Figure Legend Snippet: T cell responses in mice lacking endogenous sources of either IL-12 or IFN-α/β. Mice, WT and IL-12p35 KO 129/B6 (A–C) or WT and IFN-α/βR KO 129 (D–F), were either uninfected (open bars or symbols) or infected i.p. (closed bars or symbols) on day 0 with 2 × 10 4 PFU LCMV Armstrong strain. On day 8 after infection, blood and spleens were harvested and processed. Total numbers of CD8 T cell splenic leukocytes were calculated by multiplying the percentages of CD8 cells, determined by flow cytometry, with the total cell yields (A and D). IFN-γ levels were measured, in media conditioned with splenic leukocytes and in serum samples, by ELISA (B and E). For flow cytometric analysis of cytoplasmic IFN-γ expression, splenic leukocytes were stimulated for 6 h with anti–CD3, with Brefeldin A during the last 2 h. Cells were collected and stained for cell surface expression of CD8, CD4, and cytoplasmic expression of IFN-γ (C and F). Histogram plots were formed by gating on the CD8 population, and displaying CD8 T cell number versus IFN-γ fluorescence intensity. Numbers of CD8 T cells expressing IFN-γ were calculated by multiplying the percentage of CD8 T cells expressing IFN-γ by the number of CD8 T cells per spleen (× 10 6 ), and given as means ± SEM. Results are representative of two or more repetitive experiments with two to four mice per uninfected group and three to four mice per infected group. Data are shown as means ± SEM.

Techniques Used: Mouse Assay, Infection, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Staining, Fluorescence

Related Articles

Flow Cytometry:

Article Title: Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity
Article Snippet: .. Cells were treated with 0.016 to 10 μg of brefeldin A per ml, and MHC class I expression was measured by flow cytometry with the anti-MHC class I antibody M1/42. .. No decrease in MHC class I expression was observed over this concentration range of brefeldin A.

Article Title: The Class A Macrophage Scavenger Receptor Is a Major Pattern Recognition Receptor for Neisseria meningitidis Which Is Independent of Lipopolysaccharide and Not Required for Secretory Responses
Article Snippet: .. Cytokine detection using flow cytometry kits, brefeldin A, and enzyme-linked immunosorbent assay (ELISA) kits were obtained from PharMingen (San Diego, Calif.), and brain heart infusion broth was from Merck (Poole, United Kingdom). .. Purified 2F8, rat anti-mouse SR-A monoclonal antibody, has been described previously ( ) and was prepared using affinity chromatography.

Cytometry:

Article Title: Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity
Article Snippet: .. Cells were treated with 0.016 to 10 μg of brefeldin A per ml, and MHC class I expression was measured by flow cytometry with the anti-MHC class I antibody M1/42. .. No decrease in MHC class I expression was observed over this concentration range of brefeldin A.

Article Title: The Class A Macrophage Scavenger Receptor Is a Major Pattern Recognition Receptor for Neisseria meningitidis Which Is Independent of Lipopolysaccharide and Not Required for Secretory Responses
Article Snippet: .. Cytokine detection using flow cytometry kits, brefeldin A, and enzyme-linked immunosorbent assay (ELISA) kits were obtained from PharMingen (San Diego, Calif.), and brain heart infusion broth was from Merck (Poole, United Kingdom). .. Purified 2F8, rat anti-mouse SR-A monoclonal antibody, has been described previously ( ) and was prepared using affinity chromatography.

Blocking Assay:

Article Title: Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity
Article Snippet: .. Kataoka et al. ( ) clearly demonstrated that brefeldin A and concanamycin A are selective inhibitors that block Fas-based cytotoxicity and perforin-based killing, respectively. .. Since then, brefeldin A and concanamycin A have been used to clarify the contributions of the two distinct cytolytic pathways ( , ).

Enzyme-linked Immunosorbent Assay:

Article Title: The Class A Macrophage Scavenger Receptor Is a Major Pattern Recognition Receptor for Neisseria meningitidis Which Is Independent of Lipopolysaccharide and Not Required for Secretory Responses
Article Snippet: .. Cytokine detection using flow cytometry kits, brefeldin A, and enzyme-linked immunosorbent assay (ELISA) kits were obtained from PharMingen (San Diego, Calif.), and brain heart infusion broth was from Merck (Poole, United Kingdom). .. Purified 2F8, rat anti-mouse SR-A monoclonal antibody, has been described previously ( ) and was prepared using affinity chromatography.

Incubation:

Article Title: Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity
Article Snippet: .. We incubated effector cells with 51 Cr-labeled PSJL target cells for 5 h. Only brefeldin A showed a dose-dependent inhibition of the killing (Fig. ). .. The killing was also inhibited by incubation with anti-Fas antibody (data not shown).

Article Title: Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity
Article Snippet: .. A negative control group was incubated with dimethyl sulfoxide, since both brefeldin A and concanamycin A were dissolved in dimethyl sulfoxide as stock solutions. .. After a 2-h incubation, effector cells were added to the 51 Cr-labeled PSJL target cells.

Inhibition:

Article Title: Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity
Article Snippet: .. We incubated effector cells with 51 Cr-labeled PSJL target cells for 5 h. Only brefeldin A showed a dose-dependent inhibition of the killing (Fig. ). .. The killing was also inhibited by incubation with anti-Fas antibody (data not shown).

Negative Control:

Article Title: Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity
Article Snippet: .. A negative control group was incubated with dimethyl sulfoxide, since both brefeldin A and concanamycin A were dissolved in dimethyl sulfoxide as stock solutions. .. After a 2-h incubation, effector cells were added to the 51 Cr-labeled PSJL target cells.

Expressing:

Article Title: Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity
Article Snippet: .. Cells were treated with 0.016 to 10 μg of brefeldin A per ml, and MHC class I expression was measured by flow cytometry with the anti-MHC class I antibody M1/42. .. No decrease in MHC class I expression was observed over this concentration range of brefeldin A.

Lysis:

Article Title: Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity
Article Snippet: .. To analyze a pathway for cell lysis, effector cells were preincubated with either brefeldin A (PharMingen, San Diego, Calif.) or concanamycin A (Sigma, St. Louis, Mo.) as an inhibitor for a Fas-FasL or a perforin pathway, respectively ( , , ). .. A negative control group was incubated with dimethyl sulfoxide, since both brefeldin A and concanamycin A were dissolved in dimethyl sulfoxide as stock solutions.

other:

Article Title: Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity
Article Snippet: Since then, brefeldin A and concanamycin A have been used to clarify the contributions of the two distinct cytolytic pathways ( , ).

Article Title: Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity
Article Snippet: TMEV-induced autoreactive cells were preincubated with brefeldin A, concanamycin A, or vehicle alone.

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    Pharmingen brefeldin a
    The Fas-mediated pathway is favored. Two hours prior to the 51 Cr release assay, TMEV-induced autoreactive cells were incubated with <t>brefeldin</t> A (BFA) (○), concanamycin A (CMA) (▵), or vehicle (□). Brefeldin A and concanamycin A are known to inhibit Fas- and perforin-mediated cytotoxicities, respectively. Effector cells together with 51 Cr-labeled PSJL target cells were cultured for 5 h. Only brefeldin A showed dose-dependent inhibition of autoreactive killing. Results are representative of those from two independent experiments. Error bars indicate standard errors.
    Brefeldin A, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The Fas-mediated pathway is favored. Two hours prior to the 51 Cr release assay, TMEV-induced autoreactive cells were incubated with brefeldin A (BFA) (○), concanamycin A (CMA) (▵), or vehicle (□). Brefeldin A and concanamycin A are known to inhibit Fas- and perforin-mediated cytotoxicities, respectively. Effector cells together with 51 Cr-labeled PSJL target cells were cultured for 5 h. Only brefeldin A showed dose-dependent inhibition of autoreactive killing. Results are representative of those from two independent experiments. Error bars indicate standard errors.

    Journal: Journal of Virology

    Article Title: Induction of Autoreactive CD8+ Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity

    doi: 10.1128/JVI.76.24.12834-12844.2002

    Figure Lengend Snippet: The Fas-mediated pathway is favored. Two hours prior to the 51 Cr release assay, TMEV-induced autoreactive cells were incubated with brefeldin A (BFA) (○), concanamycin A (CMA) (▵), or vehicle (□). Brefeldin A and concanamycin A are known to inhibit Fas- and perforin-mediated cytotoxicities, respectively. Effector cells together with 51 Cr-labeled PSJL target cells were cultured for 5 h. Only brefeldin A showed dose-dependent inhibition of autoreactive killing. Results are representative of those from two independent experiments. Error bars indicate standard errors.

    Article Snippet: Since then, brefeldin A and concanamycin A have been used to clarify the contributions of the two distinct cytolytic pathways ( , ).

    Techniques: Release Assay, Incubation, Labeling, Cell Culture, Inhibition

    Mφ proinflammatory cytokine secretion following challenge with  N. meningitidis . (a) WT and SR-A −/−  BMMφ were incubated with ethanol-fixed MC58 bacteria at 37°C. At different times, the culture supernatant was harvested and analyzed by cytokine ELISA for production of TNF-α, IL-6, IL-10, and IL-12. The results are representative of at least two similar experiments, and the average of triplicate conditions is plotted. (b) BMMφ were incubated with ethanol-fixed RdGnX-MC58 (40 bacteria per cell) in the presence of brefeldin A. After incubation, the Mφ were fixed with 4% paraformaldehyde, stained with a phycoerythrin (PE)-labeled anti-TNF-α antibody, and analyzed by flow cytometry. The results are representative of at least two similar experiments. Note that preincubation of the anti-TNF-α antibody with recombinant mouse TNF-α or of the cells with an unlabeled anti-TNF-α antibody blocked the binding of the PE-labeled anti-TNF-α (not shown), confirming specificity. The error bars indicate standard deviations.

    Journal: Infection and Immunity

    Article Title: The Class A Macrophage Scavenger Receptor Is a Major Pattern Recognition Receptor for Neisseria meningitidis Which Is Independent of Lipopolysaccharide and Not Required for Secretory Responses

    doi: 10.1128/IAI.70.10.5346-5354.2002

    Figure Lengend Snippet: Mφ proinflammatory cytokine secretion following challenge with N. meningitidis . (a) WT and SR-A −/− BMMφ were incubated with ethanol-fixed MC58 bacteria at 37°C. At different times, the culture supernatant was harvested and analyzed by cytokine ELISA for production of TNF-α, IL-6, IL-10, and IL-12. The results are representative of at least two similar experiments, and the average of triplicate conditions is plotted. (b) BMMφ were incubated with ethanol-fixed RdGnX-MC58 (40 bacteria per cell) in the presence of brefeldin A. After incubation, the Mφ were fixed with 4% paraformaldehyde, stained with a phycoerythrin (PE)-labeled anti-TNF-α antibody, and analyzed by flow cytometry. The results are representative of at least two similar experiments. Note that preincubation of the anti-TNF-α antibody with recombinant mouse TNF-α or of the cells with an unlabeled anti-TNF-α antibody blocked the binding of the PE-labeled anti-TNF-α (not shown), confirming specificity. The error bars indicate standard deviations.

    Article Snippet: Cytokine detection using flow cytometry kits, brefeldin A, and enzyme-linked immunosorbent assay (ELISA) kits were obtained from PharMingen (San Diego, Calif.), and brain heart infusion broth was from Merck (Poole, United Kingdom).

    Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Staining, Labeling, Flow Cytometry, Cytometry, Recombinant, Binding Assay