brefeldin a Search Results


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  • 99
    Thermo Fisher brefeldin a
    Brefeldin A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8963 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore brefeldin a
    Brefeldin A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 33791 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson brefeldin a
    Unprotected mice have higher regulatory immune responses compared to protected mice. Hepatic CD4 T cells (gated Foxp3 − ) and Foxp3 + CD25 + Treg cells of protected and unprotected ID immunized mice were analyzed for regulatory marker expression at day 7 after challenge under artesunate treatment. (A) Representative FACS plots of Foxp3 − gated CD4 T cells for intracellular IL-10 expression after 36 h culture of hepatic leukocytes with CSP and sporozoites and addition of PMA/ionomycin plus <t>brefeldin</t> A in the last 4 h, and for surface expression of CTLA-4 or GITR ex vivo . Graphs show a summary of (B) intracellular IL-10 expression, (C) surface CTLA-4 and (D) GITR expression of Foxp3 − CD4 T cells and Treg cells from 2 experiments with 8–14 mice per group. The dotted line indicates the mean frequency for protected IV immunized mice (N = 9–16). Significant difference by Mann-Whitney test is indicated by *p
    Brefeldin A, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 15653 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    BioLegend brefeldin a
    Unprotected mice have higher regulatory immune responses compared to protected mice. Hepatic CD4 T cells (gated Foxp3 − ) and Foxp3 + CD25 + Treg cells of protected and unprotected ID immunized mice were analyzed for regulatory marker expression at day 7 after challenge under artesunate treatment. (A) Representative FACS plots of Foxp3 − gated CD4 T cells for intracellular IL-10 expression after 36 h culture of hepatic leukocytes with CSP and sporozoites and addition of PMA/ionomycin plus <t>brefeldin</t> A in the last 4 h, and for surface expression of CTLA-4 or GITR ex vivo . Graphs show a summary of (B) intracellular IL-10 expression, (C) surface CTLA-4 and (D) GITR expression of Foxp3 − CD4 T cells and Treg cells from 2 experiments with 8–14 mice per group. The dotted line indicates the mean frequency for protected IV immunized mice (N = 9–16). Significant difference by Mann-Whitney test is indicated by *p
    Brefeldin A, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 3716 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher permeabilization
    Unprotected mice have higher regulatory immune responses compared to protected mice. Hepatic CD4 T cells (gated Foxp3 − ) and Foxp3 + CD25 + Treg cells of protected and unprotected ID immunized mice were analyzed for regulatory marker expression at day 7 after challenge under artesunate treatment. (A) Representative FACS plots of Foxp3 − gated CD4 T cells for intracellular IL-10 expression after 36 h culture of hepatic leukocytes with CSP and sporozoites and addition of PMA/ionomycin plus <t>brefeldin</t> A in the last 4 h, and for surface expression of CTLA-4 or GITR ex vivo . Graphs show a summary of (B) intracellular IL-10 expression, (C) surface CTLA-4 and (D) GITR expression of Foxp3 − CD4 T cells and Treg cells from 2 experiments with 8–14 mice per group. The dotted line indicates the mean frequency for protected IV immunized mice (N = 9–16). Significant difference by Mann-Whitney test is indicated by *p
    Permeabilization, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11609 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pharmingen brefeldin a
    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.
    Brefeldin A, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 462 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc brefeldin a
    Similar APP expression pattern by brefeldin A, a protein ER-to-Golgi traffic blocker. (A) Chemical structures of isoginkgetin and brefeldin A. (B) Western blots of time-coursed APP expression in HEK293T cells treated with 30 μM IGK, 5 μM brefeldin A, and DMSO.
    Brefeldin A, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 217 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    BioLegend cell activation cocktail
    Similar APP expression pattern by brefeldin A, a protein ER-to-Golgi traffic blocker. (A) Chemical structures of isoginkgetin and brefeldin A. (B) Western blots of time-coursed APP expression in HEK293T cells treated with 30 μM IGK, 5 μM brefeldin A, and DMSO.
    Cell Activation Cocktail, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 370 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Enzo Biochem brefeldin a
    The magnitude of IL-27 produced by cDC1 cells predicts the magnitude of the vaccine adjuvant-elicited CD8 +  T cell response Mice were immunized i.v. with OVA plus the indicated adjuvants. Seven days later, the spleens were harvested and stained with SIINFEKL–MHC class I K b  tetramers, as previously described ( 20 ), to identify OVA-specific T cells. ( A ) Representative dot plots showing tetramer staining on all live singlet B220 − CD8 + CD3 +  spleen cells isolated from mice immunized with the indicated adjuvants. ( B ) Quantification of T cell data shown in (A) after immunization with the indicated adjuvants. Error bars indicate SD derived from three mice each. ( C  and  D ) The number of tetramer +  T cells generated by each adjuvant, as shown in (B), was plotted against eGFP gMFI of cDC1 cells (C) or the percentage of eGFP +  cDC1 cells (D) 6 h postimmunization with each adjuvant, as shown in  Fig. 3 .  r 2  and  p  values were determined by linear regression analysis (Prism). ( E ) Mice were challenged with the indicated innate stimuli, and the splenocytes were isolated at 6 h, as in  Fig. 3 . Spleens were incubated in vitro for 6 h in the presence of brefeldin A, after which they were stained to identify XCR1 +/−  DCs, as in  Fig. 2 . The cells were then fixed, permeabilized, and stained for intracellular IL-12. The percentage of IL-12 +  XCR1 +  DCs is shown. ( F ) Tetramer +  T cell numbers in the spleen 7 d after immunization with the indicated adjuvants were plotted against the percentage of IL-12 +  cells shown in (E).  r 2  and  p  values were determined by linear regression analysis (Prism). Data in (E) and (F) are from one of two experiments performed. ( G ) Mice were immunized with OVA + LTA, as in (A), with or without the addition of 10 μg of rIL-27. T cell responses were measured as in (A) and (B). * p
    Brefeldin A, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 282 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher brefeldin a solution
    The magnitude of IL-27 produced by cDC1 cells predicts the magnitude of the vaccine adjuvant-elicited CD8 +  T cell response Mice were immunized i.v. with OVA plus the indicated adjuvants. Seven days later, the spleens were harvested and stained with SIINFEKL–MHC class I K b  tetramers, as previously described ( 20 ), to identify OVA-specific T cells. ( A ) Representative dot plots showing tetramer staining on all live singlet B220 − CD8 + CD3 +  spleen cells isolated from mice immunized with the indicated adjuvants. ( B ) Quantification of T cell data shown in (A) after immunization with the indicated adjuvants. Error bars indicate SD derived from three mice each. ( C  and  D ) The number of tetramer +  T cells generated by each adjuvant, as shown in (B), was plotted against eGFP gMFI of cDC1 cells (C) or the percentage of eGFP +  cDC1 cells (D) 6 h postimmunization with each adjuvant, as shown in  Fig. 3 .  r 2  and  p  values were determined by linear regression analysis (Prism). ( E ) Mice were challenged with the indicated innate stimuli, and the splenocytes were isolated at 6 h, as in  Fig. 3 . Spleens were incubated in vitro for 6 h in the presence of brefeldin A, after which they were stained to identify XCR1 +/−  DCs, as in  Fig. 2 . The cells were then fixed, permeabilized, and stained for intracellular IL-12. The percentage of IL-12 +  XCR1 +  DCs is shown. ( F ) Tetramer +  T cell numbers in the spleen 7 d after immunization with the indicated adjuvants were plotted against the percentage of IL-12 +  cells shown in (E).  r 2  and  p  values were determined by linear regression analysis (Prism). Data in (E) and (F) are from one of two experiments performed. ( G ) Mice were immunized with OVA + LTA, as in (A), with or without the addition of 10 μg of rIL-27. T cell responses were measured as in (A) and (B). * p
    Brefeldin A Solution, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 365 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cayman Chemical brefeldin a
    The magnitude of IL-27 produced by cDC1 cells predicts the magnitude of the vaccine adjuvant-elicited CD8 +  T cell response Mice were immunized i.v. with OVA plus the indicated adjuvants. Seven days later, the spleens were harvested and stained with SIINFEKL–MHC class I K b  tetramers, as previously described ( 20 ), to identify OVA-specific T cells. ( A ) Representative dot plots showing tetramer staining on all live singlet B220 − CD8 + CD3 +  spleen cells isolated from mice immunized with the indicated adjuvants. ( B ) Quantification of T cell data shown in (A) after immunization with the indicated adjuvants. Error bars indicate SD derived from three mice each. ( C  and  D ) The number of tetramer +  T cells generated by each adjuvant, as shown in (B), was plotted against eGFP gMFI of cDC1 cells (C) or the percentage of eGFP +  cDC1 cells (D) 6 h postimmunization with each adjuvant, as shown in  Fig. 3 .  r 2  and  p  values were determined by linear regression analysis (Prism). ( E ) Mice were challenged with the indicated innate stimuli, and the splenocytes were isolated at 6 h, as in  Fig. 3 . Spleens were incubated in vitro for 6 h in the presence of brefeldin A, after which they were stained to identify XCR1 +/−  DCs, as in  Fig. 2 . The cells were then fixed, permeabilized, and stained for intracellular IL-12. The percentage of IL-12 +  XCR1 +  DCs is shown. ( F ) Tetramer +  T cell numbers in the spleen 7 d after immunization with the indicated adjuvants were plotted against the percentage of IL-12 +  cells shown in (E).  r 2  and  p  values were determined by linear regression analysis (Prism). Data in (E) and (F) are from one of two experiments performed. ( G ) Mice were immunized with OVA + LTA, as in (A), with or without the addition of 10 μg of rIL-27. T cell responses were measured as in (A) and (B). * p
    Brefeldin A, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 99/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    brefeldin a - by Bioz Stars, 2020-10
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    95
    Tocris brefeldin a
    The magnitude of IL-27 produced by cDC1 cells predicts the magnitude of the vaccine adjuvant-elicited CD8 +  T cell response Mice were immunized i.v. with OVA plus the indicated adjuvants. Seven days later, the spleens were harvested and stained with SIINFEKL–MHC class I K b  tetramers, as previously described ( 20 ), to identify OVA-specific T cells. ( A ) Representative dot plots showing tetramer staining on all live singlet B220 − CD8 + CD3 +  spleen cells isolated from mice immunized with the indicated adjuvants. ( B ) Quantification of T cell data shown in (A) after immunization with the indicated adjuvants. Error bars indicate SD derived from three mice each. ( C  and  D ) The number of tetramer +  T cells generated by each adjuvant, as shown in (B), was plotted against eGFP gMFI of cDC1 cells (C) or the percentage of eGFP +  cDC1 cells (D) 6 h postimmunization with each adjuvant, as shown in  Fig. 3 .  r 2  and  p  values were determined by linear regression analysis (Prism). ( E ) Mice were challenged with the indicated innate stimuli, and the splenocytes were isolated at 6 h, as in  Fig. 3 . Spleens were incubated in vitro for 6 h in the presence of brefeldin A, after which they were stained to identify XCR1 +/−  DCs, as in  Fig. 2 . The cells were then fixed, permeabilized, and stained for intracellular IL-12. The percentage of IL-12 +  XCR1 +  DCs is shown. ( F ) Tetramer +  T cell numbers in the spleen 7 d after immunization with the indicated adjuvants were plotted against the percentage of IL-12 +  cells shown in (E).  r 2  and  p  values were determined by linear regression analysis (Prism). Data in (E) and (F) are from one of two experiments performed. ( G ) Mice were immunized with OVA + LTA, as in (A), with or without the addition of 10 μg of rIL-27. T cell responses were measured as in (A) and (B). * p
    Brefeldin A, supplied by Tocris, used in various techniques. Bioz Stars score: 95/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson brefeldin a bfa
    The magnitude of IL-27 produced by cDC1 cells predicts the magnitude of the vaccine adjuvant-elicited CD8 +  T cell response Mice were immunized i.v. with OVA plus the indicated adjuvants. Seven days later, the spleens were harvested and stained with SIINFEKL–MHC class I K b  tetramers, as previously described ( 20 ), to identify OVA-specific T cells. ( A ) Representative dot plots showing tetramer staining on all live singlet B220 − CD8 + CD3 +  spleen cells isolated from mice immunized with the indicated adjuvants. ( B ) Quantification of T cell data shown in (A) after immunization with the indicated adjuvants. Error bars indicate SD derived from three mice each. ( C  and  D ) The number of tetramer +  T cells generated by each adjuvant, as shown in (B), was plotted against eGFP gMFI of cDC1 cells (C) or the percentage of eGFP +  cDC1 cells (D) 6 h postimmunization with each adjuvant, as shown in  Fig. 3 .  r 2  and  p  values were determined by linear regression analysis (Prism). ( E ) Mice were challenged with the indicated innate stimuli, and the splenocytes were isolated at 6 h, as in  Fig. 3 . Spleens were incubated in vitro for 6 h in the presence of brefeldin A, after which they were stained to identify XCR1 +/−  DCs, as in  Fig. 2 . The cells were then fixed, permeabilized, and stained for intracellular IL-12. The percentage of IL-12 +  XCR1 +  DCs is shown. ( F ) Tetramer +  T cell numbers in the spleen 7 d after immunization with the indicated adjuvants were plotted against the percentage of IL-12 +  cells shown in (E).  r 2  and  p  values were determined by linear regression analysis (Prism). Data in (E) and (F) are from one of two experiments performed. ( G ) Mice were immunized with OVA + LTA, as in (A), with or without the addition of 10 μg of rIL-27. T cell responses were measured as in (A) and (B). * p
    Brefeldin A Bfa, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Applichem brefeldin a
    The absence of Tregs or IL-10 does not compromise persistence of strain 101 in the oral epithelium.  (A–C)  DEREG mice and control littermates were sublingually infected with  C. albicans  strain 101 and treated with diphtheria toxin on day 11 and 13 after the antifungal response was fully established. One day after the last treatment, the mice were sacrificed for analysis.  (A)  Treg depletion efficiency was analyzed in the cervical lymph nodes by flow cytometry. Data are the % of Foxp3 +  cells within the population of CD4 +  viable cells.  (B)  Lymph node cells were re-stimulated with MutuDC1940 cells that were pulsed with heat-killed  C. albicans  or left unpulsed for 5 h in the presence of Brefeldin A. IL-17A (left) and IFN-γ (right) production by CD3 + CD4 +  cells was analyzed by intracellular cytokine staining and flow cytometry.  (C)  The fungal burden was determined by plating tongue homogenates on YPD agar. Each bar represents the mean + SD of 3 to 4 mice per group. Data are from one out of two independent experiments.  (D,E)  IL-10-deficient mice and WT controls were sublingually infected with  C. albicans  strain 101 and analyzed on day 9 post-infection.  (D)  Lymph node cells were re-stimulated and analyzed for IL-17 (left) and IFN-γ (right) production as in B.  (E)  Tongue fungal burdens were analyzed as in C. Each bar represents the mean + SD of 8–9 mice per group pooled from two independent experiments. Statistics were calculated using unpaired t-Test. *** p
    Brefeldin A, supplied by Applichem, used in various techniques. Bioz Stars score: 92/100, based on 147 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    brefeldin a - by Bioz Stars, 2020-10
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    Image Search Results


    Unprotected mice have higher regulatory immune responses compared to protected mice. Hepatic CD4 T cells (gated Foxp3 − ) and Foxp3 + CD25 + Treg cells of protected and unprotected ID immunized mice were analyzed for regulatory marker expression at day 7 after challenge under artesunate treatment. (A) Representative FACS plots of Foxp3 − gated CD4 T cells for intracellular IL-10 expression after 36 h culture of hepatic leukocytes with CSP and sporozoites and addition of PMA/ionomycin plus brefeldin A in the last 4 h, and for surface expression of CTLA-4 or GITR ex vivo . Graphs show a summary of (B) intracellular IL-10 expression, (C) surface CTLA-4 and (D) GITR expression of Foxp3 − CD4 T cells and Treg cells from 2 experiments with 8–14 mice per group. The dotted line indicates the mean frequency for protected IV immunized mice (N = 9–16). Significant difference by Mann-Whitney test is indicated by *p

    Journal: Scientific Reports

    Article Title: Protective immunity differs between routes of administration of attenuated malaria parasites independent of parasite liver load

    doi: 10.1038/s41598-017-10480-1

    Figure Lengend Snippet: Unprotected mice have higher regulatory immune responses compared to protected mice. Hepatic CD4 T cells (gated Foxp3 − ) and Foxp3 + CD25 + Treg cells of protected and unprotected ID immunized mice were analyzed for regulatory marker expression at day 7 after challenge under artesunate treatment. (A) Representative FACS plots of Foxp3 − gated CD4 T cells for intracellular IL-10 expression after 36 h culture of hepatic leukocytes with CSP and sporozoites and addition of PMA/ionomycin plus brefeldin A in the last 4 h, and for surface expression of CTLA-4 or GITR ex vivo . Graphs show a summary of (B) intracellular IL-10 expression, (C) surface CTLA-4 and (D) GITR expression of Foxp3 − CD4 T cells and Treg cells from 2 experiments with 8–14 mice per group. The dotted line indicates the mean frequency for protected IV immunized mice (N = 9–16). Significant difference by Mann-Whitney test is indicated by *p

    Article Snippet: For the detection of intracellular IL-10 it was required to culture cells with CSP peptide (10 µg/ml) plus P. yoelii sporozoites (Py wt-GFP-Luccon , 25,000/ml) for 36 h including PMA, ionomycin and brefeldin A in the last 4 h. For flow cytometric detection of surface-exposed CD107a, cells were cultured for 4 hours with 0.25% GolgiStop (BD Biosciences), 10 µg/ml brefeldin A and anti-mouse CD107a-AlexaFluor488 (eBioscience, Vienna, Austria).

    Techniques: Mouse Assay, Marker, Expressing, FACS, Ex Vivo, MANN-WHITNEY

    ID immunization induces stronger regulatory immune responses in liver compared to IV. Hepatic leukocytes were analyzed 7 days after IV or ID primary immunization or boost for regulatory marker expression by flow cytometry directly ex vivo ( A , B ) or after 36 h culture with CSP and sporozoites ( C – G ). (A) Number of Foxp3 + CD25 + Treg cells per liver. (B) Frequency of Treg cells within the CD4 + T cell population. (C) Intracellular IL-10 expression of Treg cells after addition of PMA, ionomycin and brefeldin A for 4 h to the culture. (D) Representative FACS plots of CD4 + Foxp3-negative T cells (CD4 T) in one IV or ID immunized and a naïve control mouse. (E,F) Summary of intracellular IL-10 expression of CD4 + Foxp3 − T cells after addition of PMA, ionomycin and brefeldin A expressed as frequencies ( E ) and cell number per liver ( F ). Representative FACS plots ( G ) and summary ( H ) of intracellular IL-10 expression in CD19 + gated B cells after addition of PMA, ionomycin and brefeldin A. Graphs show 1 representative out of 2 similar experiments ( C ) or a summary of 2 experiments with 8–10 mice per group. Significant difference by Mann-Whitney test is indicated by *p

    Journal: Scientific Reports

    Article Title: Protective immunity differs between routes of administration of attenuated malaria parasites independent of parasite liver load

    doi: 10.1038/s41598-017-10480-1

    Figure Lengend Snippet: ID immunization induces stronger regulatory immune responses in liver compared to IV. Hepatic leukocytes were analyzed 7 days after IV or ID primary immunization or boost for regulatory marker expression by flow cytometry directly ex vivo ( A , B ) or after 36 h culture with CSP and sporozoites ( C – G ). (A) Number of Foxp3 + CD25 + Treg cells per liver. (B) Frequency of Treg cells within the CD4 + T cell population. (C) Intracellular IL-10 expression of Treg cells after addition of PMA, ionomycin and brefeldin A for 4 h to the culture. (D) Representative FACS plots of CD4 + Foxp3-negative T cells (CD4 T) in one IV or ID immunized and a naïve control mouse. (E,F) Summary of intracellular IL-10 expression of CD4 + Foxp3 − T cells after addition of PMA, ionomycin and brefeldin A expressed as frequencies ( E ) and cell number per liver ( F ). Representative FACS plots ( G ) and summary ( H ) of intracellular IL-10 expression in CD19 + gated B cells after addition of PMA, ionomycin and brefeldin A. Graphs show 1 representative out of 2 similar experiments ( C ) or a summary of 2 experiments with 8–10 mice per group. Significant difference by Mann-Whitney test is indicated by *p

    Article Snippet: For the detection of intracellular IL-10 it was required to culture cells with CSP peptide (10 µg/ml) plus P. yoelii sporozoites (Py wt-GFP-Luccon , 25,000/ml) for 36 h including PMA, ionomycin and brefeldin A in the last 4 h. For flow cytometric detection of surface-exposed CD107a, cells were cultured for 4 hours with 0.25% GolgiStop (BD Biosciences), 10 µg/ml brefeldin A and anti-mouse CD107a-AlexaFluor488 (eBioscience, Vienna, Austria).

    Techniques: Marker, Expressing, Flow Cytometry, Cytometry, Ex Vivo, FACS, Mouse Assay, MANN-WHITNEY

    ID but not IV immunization increases regulatory immune responses in the skin-draining lymph node. Inguinal lymph node cells were analyzed 7 days after IV or ID primary immunization for regulatory marker and cytokine expression. Flow cytometry was performed after 36 h culture with CSP and sporozoites and addition of PMA, ionomycin and brefeldin A in the last 4 h ( A , B ), or directly ex vivo ( C ). Frequencies calculated relative to naïve control are given for (A) IFN-γ + CD4 T cells and CD8 T cells, (B) IL-10 + Foxp3 − CD4 T cells, IL-10 + Foxp3 + CD25 + Treg cells, IL-10 + CD19 + B cells, and (C) CTLA-4 + Foxp3 − CD4 T cells and CTLA-4 + Foxp3 + CD25 + Treg cells. Summary of 2 experiments with 10 mice per group. Significant difference by unpaired t-test is indicated by *p

    Journal: Scientific Reports

    Article Title: Protective immunity differs between routes of administration of attenuated malaria parasites independent of parasite liver load

    doi: 10.1038/s41598-017-10480-1

    Figure Lengend Snippet: ID but not IV immunization increases regulatory immune responses in the skin-draining lymph node. Inguinal lymph node cells were analyzed 7 days after IV or ID primary immunization for regulatory marker and cytokine expression. Flow cytometry was performed after 36 h culture with CSP and sporozoites and addition of PMA, ionomycin and brefeldin A in the last 4 h ( A , B ), or directly ex vivo ( C ). Frequencies calculated relative to naïve control are given for (A) IFN-γ + CD4 T cells and CD8 T cells, (B) IL-10 + Foxp3 − CD4 T cells, IL-10 + Foxp3 + CD25 + Treg cells, IL-10 + CD19 + B cells, and (C) CTLA-4 + Foxp3 − CD4 T cells and CTLA-4 + Foxp3 + CD25 + Treg cells. Summary of 2 experiments with 10 mice per group. Significant difference by unpaired t-test is indicated by *p

    Article Snippet: For the detection of intracellular IL-10 it was required to culture cells with CSP peptide (10 µg/ml) plus P. yoelii sporozoites (Py wt-GFP-Luccon , 25,000/ml) for 36 h including PMA, ionomycin and brefeldin A in the last 4 h. For flow cytometric detection of surface-exposed CD107a, cells were cultured for 4 hours with 0.25% GolgiStop (BD Biosciences), 10 µg/ml brefeldin A and anti-mouse CD107a-AlexaFluor488 (eBioscience, Vienna, Austria).

    Techniques: Marker, Expressing, Flow Cytometry, Cytometry, Ex Vivo, Mouse Assay

    Unprotected mice after ID immunization show reduced liver T cell effector responses compared to protected mice. ID immunized mice were challenged followed by artesunate treatment, and distinguished as protected (p, i.e. luciferase negative) or unprotected (unp, i.e. luciferase positive). Hepatic CD8 and CD4 T cells were analyzed at day 7 after challenge. (A) IFN-γ concentration in supernatant of total leukocytes after culture for 36 h with CSP and sporozoites as measured by ELISA. (B) Representative FACS plots of CD8 + gated T cells for intracellular expression of IFN-γ or TNF after culture for 4 h with CSP and brefeldin A, and for surface expression of CD107a after 4 h culture with CSP, brefeldin A and monensin. Numbers indicate the frequency of the gated cell population. Graphs show a summary of 2 experiments with 8–14 mice per group for frequency of IFN-γ ( C ), TNF ( D ), or CD107a ( E ) -expressing CD8 T cells and CD4 T cells. The dotted line indicates the mean cytokine or expression level for protected IV immunized mice (N = 9–16). Significant difference by Mann-Whitney test is indicated by *p

    Journal: Scientific Reports

    Article Title: Protective immunity differs between routes of administration of attenuated malaria parasites independent of parasite liver load

    doi: 10.1038/s41598-017-10480-1

    Figure Lengend Snippet: Unprotected mice after ID immunization show reduced liver T cell effector responses compared to protected mice. ID immunized mice were challenged followed by artesunate treatment, and distinguished as protected (p, i.e. luciferase negative) or unprotected (unp, i.e. luciferase positive). Hepatic CD8 and CD4 T cells were analyzed at day 7 after challenge. (A) IFN-γ concentration in supernatant of total leukocytes after culture for 36 h with CSP and sporozoites as measured by ELISA. (B) Representative FACS plots of CD8 + gated T cells for intracellular expression of IFN-γ or TNF after culture for 4 h with CSP and brefeldin A, and for surface expression of CD107a after 4 h culture with CSP, brefeldin A and monensin. Numbers indicate the frequency of the gated cell population. Graphs show a summary of 2 experiments with 8–14 mice per group for frequency of IFN-γ ( C ), TNF ( D ), or CD107a ( E ) -expressing CD8 T cells and CD4 T cells. The dotted line indicates the mean cytokine or expression level for protected IV immunized mice (N = 9–16). Significant difference by Mann-Whitney test is indicated by *p

    Article Snippet: For the detection of intracellular IL-10 it was required to culture cells with CSP peptide (10 µg/ml) plus P. yoelii sporozoites (Py wt-GFP-Luccon , 25,000/ml) for 36 h including PMA, ionomycin and brefeldin A in the last 4 h. For flow cytometric detection of surface-exposed CD107a, cells were cultured for 4 hours with 0.25% GolgiStop (BD Biosciences), 10 µg/ml brefeldin A and anti-mouse CD107a-AlexaFluor488 (eBioscience, Vienna, Austria).

    Techniques: Mouse Assay, Luciferase, Concentration Assay, Enzyme-linked Immunosorbent Assay, FACS, Expressing, MANN-WHITNEY

    CD8 dendritic cell subset frequencies are smaller after ID compared to IV immunization. Dendritic cell (DC) subsets in liver were analyzed by flow cytometry at 7 days after primary or boost immunization via IV or ID route. (A) MHCII + CD11c hi/int DC were gated for CD11c int CD64 hi monocyte-derived DC (mo-DC) and CD11c hi CD64 low conventional DC (cDC) after excluding F4/80-expressing macrophages. Representative FACS plots of both DC subsets for CD8 and intracellular IL-12p40 expression in mice 7 days after IV or ID boost or in naïve control mice are shown. Numbers indicate the frequency of the gated cell population. (B) Frequencies of DC subsets within total leukocytes. (C) Number of CD8-expressing DC per liver. (D) Frequencies of CD8-expressing DC within the mo-DC or cDC subset. (E) Intracellular IL-12p40 expression of DC subsets after culture of leukocytes with brefeldin A for 4 h. Summary of 2 experiments with 8–10 mice per group. Significant difference by Mann-Whitney test is indicated by *p

    Journal: Scientific Reports

    Article Title: Protective immunity differs between routes of administration of attenuated malaria parasites independent of parasite liver load

    doi: 10.1038/s41598-017-10480-1

    Figure Lengend Snippet: CD8 dendritic cell subset frequencies are smaller after ID compared to IV immunization. Dendritic cell (DC) subsets in liver were analyzed by flow cytometry at 7 days after primary or boost immunization via IV or ID route. (A) MHCII + CD11c hi/int DC were gated for CD11c int CD64 hi monocyte-derived DC (mo-DC) and CD11c hi CD64 low conventional DC (cDC) after excluding F4/80-expressing macrophages. Representative FACS plots of both DC subsets for CD8 and intracellular IL-12p40 expression in mice 7 days after IV or ID boost or in naïve control mice are shown. Numbers indicate the frequency of the gated cell population. (B) Frequencies of DC subsets within total leukocytes. (C) Number of CD8-expressing DC per liver. (D) Frequencies of CD8-expressing DC within the mo-DC or cDC subset. (E) Intracellular IL-12p40 expression of DC subsets after culture of leukocytes with brefeldin A for 4 h. Summary of 2 experiments with 8–10 mice per group. Significant difference by Mann-Whitney test is indicated by *p

    Article Snippet: For the detection of intracellular IL-10 it was required to culture cells with CSP peptide (10 µg/ml) plus P. yoelii sporozoites (Py wt-GFP-Luccon , 25,000/ml) for 36 h including PMA, ionomycin and brefeldin A in the last 4 h. For flow cytometric detection of surface-exposed CD107a, cells were cultured for 4 hours with 0.25% GolgiStop (BD Biosciences), 10 µg/ml brefeldin A and anti-mouse CD107a-AlexaFluor488 (eBioscience, Vienna, Austria).

    Techniques: Flow Cytometry, Cytometry, Derivative Assay, Expressing, FACS, Mouse Assay, MANN-WHITNEY

    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

    Similar APP expression pattern by brefeldin A, a protein ER-to-Golgi traffic blocker. (A) Chemical structures of isoginkgetin and brefeldin A. (B) Western blots of time-coursed APP expression in HEK293T cells treated with 30 μM IGK, 5 μM brefeldin A, and DMSO.

    Journal: ACS Omega

    Article Title: Effects of RNA Splicing Inhibitors on Amyloid Precursor Protein Expression

    doi: 10.1021/acsomega.7b02073

    Figure Lengend Snippet: Similar APP expression pattern by brefeldin A, a protein ER-to-Golgi traffic blocker. (A) Chemical structures of isoginkgetin and brefeldin A. (B) Western blots of time-coursed APP expression in HEK293T cells treated with 30 μM IGK, 5 μM brefeldin A, and DMSO.

    Article Snippet: Chemicals used in other experiments were CHX (239764, Calbiochem), bafilomycin A1 (B1793, Sigma-Aldrich), MG-132 (1748, Boston Biochem), and brefeldin A (9972S, Cell Signaling Technology).

    Techniques: Expressing, Western Blot

    The magnitude of IL-27 produced by cDC1 cells predicts the magnitude of the vaccine adjuvant-elicited CD8 +  T cell response Mice were immunized i.v. with OVA plus the indicated adjuvants. Seven days later, the spleens were harvested and stained with SIINFEKL–MHC class I K b  tetramers, as previously described ( 20 ), to identify OVA-specific T cells. ( A ) Representative dot plots showing tetramer staining on all live singlet B220 − CD8 + CD3 +  spleen cells isolated from mice immunized with the indicated adjuvants. ( B ) Quantification of T cell data shown in (A) after immunization with the indicated adjuvants. Error bars indicate SD derived from three mice each. ( C  and  D ) The number of tetramer +  T cells generated by each adjuvant, as shown in (B), was plotted against eGFP gMFI of cDC1 cells (C) or the percentage of eGFP +  cDC1 cells (D) 6 h postimmunization with each adjuvant, as shown in  Fig. 3 .  r 2  and  p  values were determined by linear regression analysis (Prism). ( E ) Mice were challenged with the indicated innate stimuli, and the splenocytes were isolated at 6 h, as in  Fig. 3 . Spleens were incubated in vitro for 6 h in the presence of brefeldin A, after which they were stained to identify XCR1 +/−  DCs, as in  Fig. 2 . The cells were then fixed, permeabilized, and stained for intracellular IL-12. The percentage of IL-12 +  XCR1 +  DCs is shown. ( F ) Tetramer +  T cell numbers in the spleen 7 d after immunization with the indicated adjuvants were plotted against the percentage of IL-12 +  cells shown in (E).  r 2  and  p  values were determined by linear regression analysis (Prism). Data in (E) and (F) are from one of two experiments performed. ( G ) Mice were immunized with OVA + LTA, as in (A), with or without the addition of 10 μg of rIL-27. T cell responses were measured as in (A) and (B). * p

    Journal: ImmunoHorizons

    Article Title: IL-27p28 Production by XCR1+ Dendritic Cells and Monocytes Effectively Predicts Adjuvant-Elicited CD8+ T Cell Responses

    doi: 10.4049/immunohorizons.1700054

    Figure Lengend Snippet: The magnitude of IL-27 produced by cDC1 cells predicts the magnitude of the vaccine adjuvant-elicited CD8 + T cell response Mice were immunized i.v. with OVA plus the indicated adjuvants. Seven days later, the spleens were harvested and stained with SIINFEKL–MHC class I K b tetramers, as previously described ( 20 ), to identify OVA-specific T cells. ( A ) Representative dot plots showing tetramer staining on all live singlet B220 − CD8 + CD3 + spleen cells isolated from mice immunized with the indicated adjuvants. ( B ) Quantification of T cell data shown in (A) after immunization with the indicated adjuvants. Error bars indicate SD derived from three mice each. ( C and D ) The number of tetramer + T cells generated by each adjuvant, as shown in (B), was plotted against eGFP gMFI of cDC1 cells (C) or the percentage of eGFP + cDC1 cells (D) 6 h postimmunization with each adjuvant, as shown in Fig. 3 . r 2 and p values were determined by linear regression analysis (Prism). ( E ) Mice were challenged with the indicated innate stimuli, and the splenocytes were isolated at 6 h, as in Fig. 3 . Spleens were incubated in vitro for 6 h in the presence of brefeldin A, after which they were stained to identify XCR1 +/− DCs, as in Fig. 2 . The cells were then fixed, permeabilized, and stained for intracellular IL-12. The percentage of IL-12 + XCR1 + DCs is shown. ( F ) Tetramer + T cell numbers in the spleen 7 d after immunization with the indicated adjuvants were plotted against the percentage of IL-12 + cells shown in (E). r 2 and p values were determined by linear regression analysis (Prism). Data in (E) and (F) are from one of two experiments performed. ( G ) Mice were immunized with OVA + LTA, as in (A), with or without the addition of 10 μg of rIL-27. T cell responses were measured as in (A) and (B). * p

    Article Snippet: Intracellular cytokine staining was performed by incubating splenocytes for 6 h with 5 μg/ml Brefeldin A (Enzo Life Sciences), surface staining, fixing with 1% paraformaldehyde, and cytokine staining in 1× Perm Buffer (Invitrogen).

    Techniques: Produced, Mouse Assay, Staining, Isolation, Derivative Assay, Generated, Incubation, In Vitro

    The absence of Tregs or IL-10 does not compromise persistence of strain 101 in the oral epithelium.  (A–C)  DEREG mice and control littermates were sublingually infected with  C. albicans  strain 101 and treated with diphtheria toxin on day 11 and 13 after the antifungal response was fully established. One day after the last treatment, the mice were sacrificed for analysis.  (A)  Treg depletion efficiency was analyzed in the cervical lymph nodes by flow cytometry. Data are the % of Foxp3 +  cells within the population of CD4 +  viable cells.  (B)  Lymph node cells were re-stimulated with MutuDC1940 cells that were pulsed with heat-killed  C. albicans  or left unpulsed for 5 h in the presence of Brefeldin A. IL-17A (left) and IFN-γ (right) production by CD3 + CD4 +  cells was analyzed by intracellular cytokine staining and flow cytometry.  (C)  The fungal burden was determined by plating tongue homogenates on YPD agar. Each bar represents the mean + SD of 3 to 4 mice per group. Data are from one out of two independent experiments.  (D,E)  IL-10-deficient mice and WT controls were sublingually infected with  C. albicans  strain 101 and analyzed on day 9 post-infection.  (D)  Lymph node cells were re-stimulated and analyzed for IL-17 (left) and IFN-γ (right) production as in B.  (E)  Tongue fungal burdens were analyzed as in C. Each bar represents the mean + SD of 8–9 mice per group pooled from two independent experiments. Statistics were calculated using unpaired t-Test. *** p

    Journal: Frontiers in Immunology

    Article Title: Persistence of Candida albicans in the Oral Mucosa Induces a Curbed Inflammatory Host Response That Is Independent of Immunosuppression

    doi: 10.3389/fimmu.2019.00330

    Figure Lengend Snippet: The absence of Tregs or IL-10 does not compromise persistence of strain 101 in the oral epithelium. (A–C) DEREG mice and control littermates were sublingually infected with C. albicans strain 101 and treated with diphtheria toxin on day 11 and 13 after the antifungal response was fully established. One day after the last treatment, the mice were sacrificed for analysis. (A) Treg depletion efficiency was analyzed in the cervical lymph nodes by flow cytometry. Data are the % of Foxp3 + cells within the population of CD4 + viable cells. (B) Lymph node cells were re-stimulated with MutuDC1940 cells that were pulsed with heat-killed C. albicans or left unpulsed for 5 h in the presence of Brefeldin A. IL-17A (left) and IFN-γ (right) production by CD3 + CD4 + cells was analyzed by intracellular cytokine staining and flow cytometry. (C) The fungal burden was determined by plating tongue homogenates on YPD agar. Each bar represents the mean + SD of 3 to 4 mice per group. Data are from one out of two independent experiments. (D,E) IL-10-deficient mice and WT controls were sublingually infected with C. albicans strain 101 and analyzed on day 9 post-infection. (D) Lymph node cells were re-stimulated and analyzed for IL-17 (left) and IFN-γ (right) production as in B. (E) Tongue fungal burdens were analyzed as in C. Each bar represents the mean + SD of 8–9 mice per group pooled from two independent experiments. Statistics were calculated using unpaired t-Test. *** p

    Article Snippet: Brefeldin A (10 μg/ml, AppliChem) was added for the last 5 h to inhibit the secretory pathway.

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

    The Treg response during persistent colonization of the oral mucosa with  C. albicans .  (A–F)  WT mice were sublingually infected with strain 101 or SC5314 and cervical lymph node cells were analyzed on day 7 or day 18–21 as indicated.  (A,B)  Lymph node cells were re-stimulated with MutuDC1940 cells that were pulsed with heat-killed  C. albicans  or left unpulsed for 5 h in the presence of Brefeldin A. IL-17A production by CD3 + CD4 +  cells was analyzed by intracellular cytokine staining and flow cytometry.  (C,D)  The frequency of Foxp3-expressing cells within the CD4 +  lymphocyte compartment was assessed by flow cytometry.  (E,F)  PD-1, TIGIT, and Tim-3 expression by CD4 + Foxp3 +  Treg cells was analyzed by flow cytometry. ( G–J )  Il10 -Thy1.1 reporter mice were sublingually infected with strain 101 or SC5314 or left naive. IL-10 expression by Foxp3 +  Tregs and Foxp3 −  effector T cells was assessed in the cervical lymph nodes  (G,H)  and in the tongue  (I,J)  on day 21 post-infection by flow cytometry. Cells were pregated on CD90 + CD4 + (G,H)  or on CD45.2 + CD3 + (I,J) , respectively. Representative FACS plots are shown in (A, C, G, I); summary plots with data pooled from at least 2 experiments with 6–9 animals per infected group and 3–4 animals per naive group are shown in (B, D–F, H, J), with the exception of the right plot in  (D) , where data are from a single experiment with 3 animals per group. In B, Statistics were calculated using  t -test. In  (D–F, H, J) , statistics were calculated using one-way ANOVA. * p

    Journal: Frontiers in Immunology

    Article Title: Persistence of Candida albicans in the Oral Mucosa Induces a Curbed Inflammatory Host Response That Is Independent of Immunosuppression

    doi: 10.3389/fimmu.2019.00330

    Figure Lengend Snippet: The Treg response during persistent colonization of the oral mucosa with C. albicans . (A–F) WT mice were sublingually infected with strain 101 or SC5314 and cervical lymph node cells were analyzed on day 7 or day 18–21 as indicated. (A,B) Lymph node cells were re-stimulated with MutuDC1940 cells that were pulsed with heat-killed C. albicans or left unpulsed for 5 h in the presence of Brefeldin A. IL-17A production by CD3 + CD4 + cells was analyzed by intracellular cytokine staining and flow cytometry. (C,D) The frequency of Foxp3-expressing cells within the CD4 + lymphocyte compartment was assessed by flow cytometry. (E,F) PD-1, TIGIT, and Tim-3 expression by CD4 + Foxp3 + Treg cells was analyzed by flow cytometry. ( G–J ) Il10 -Thy1.1 reporter mice were sublingually infected with strain 101 or SC5314 or left naive. IL-10 expression by Foxp3 + Tregs and Foxp3 − effector T cells was assessed in the cervical lymph nodes (G,H) and in the tongue (I,J) on day 21 post-infection by flow cytometry. Cells were pregated on CD90 + CD4 + (G,H) or on CD45.2 + CD3 + (I,J) , respectively. Representative FACS plots are shown in (A, C, G, I); summary plots with data pooled from at least 2 experiments with 6–9 animals per infected group and 3–4 animals per naive group are shown in (B, D–F, H, J), with the exception of the right plot in (D) , where data are from a single experiment with 3 animals per group. In B, Statistics were calculated using t -test. In (D–F, H, J) , statistics were calculated using one-way ANOVA. * p

    Article Snippet: Brefeldin A (10 μg/ml, AppliChem) was added for the last 5 h to inhibit the secretory pathway.

    Techniques: Mouse Assay, Infection, Staining, Flow Cytometry, Cytometry, Expressing, FACS