brefeldin a  (BioLegend)

 
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    Name:
    Brefeldin A Solution 1 000X
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    Brefeldin A Solution 1 000X Apps ICFC Size 1 ml
    Catalog Number:
    420601
    Price:
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    Structured Review

    BioLegend brefeldin a
    SQLLNAKYL-specific CD8 + T cells are functional and cytotoxic in vivo A,B. Combined tetramer and intracellular cytokine staining were performed on splenocytes and brain-sequestered leukocytes from PbA-infected mice 7 days p.i. Cells were incubated with <t>Brefeldin</t> A for 2 h without restimulation. (A) Representative IFN-γ and GrB profiles of live CD8 + T cells (CD8a + CD16/32 − ). (B) The indicated IFN-γ + GrB + gate was used to analyze tetramer-labelled CD8 + T cells. Bars represent medians. C. Equal numbers of CFSE hi unpulsed naïve splenocytes and CFSE lo SQLLNAKYL-pulsed splenocytes were transferred into naïve or PbA-infected mice 6 days p.i. The mice were sacrificed 20 h later to analyze the CFSE-labelled cells in the spleens. The infected mouse is representative of n = 4, all with 96–97% specific lysis.
    Brefeldin A Solution 1 000X Apps ICFC Size 1 ml
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    Images

    1) Product Images from "Brain microvessel cross-presentation is a hallmark of experimental cerebral malaria"

    Article Title: Brain microvessel cross-presentation is a hallmark of experimental cerebral malaria

    Journal: EMBO Molecular Medicine

    doi: 10.1002/emmm.201202273

    SQLLNAKYL-specific CD8 + T cells are functional and cytotoxic in vivo A,B. Combined tetramer and intracellular cytokine staining were performed on splenocytes and brain-sequestered leukocytes from PbA-infected mice 7 days p.i. Cells were incubated with Brefeldin A for 2 h without restimulation. (A) Representative IFN-γ and GrB profiles of live CD8 + T cells (CD8a + CD16/32 − ). (B) The indicated IFN-γ + GrB + gate was used to analyze tetramer-labelled CD8 + T cells. Bars represent medians. C. Equal numbers of CFSE hi unpulsed naïve splenocytes and CFSE lo SQLLNAKYL-pulsed splenocytes were transferred into naïve or PbA-infected mice 6 days p.i. The mice were sacrificed 20 h later to analyze the CFSE-labelled cells in the spleens. The infected mouse is representative of n = 4, all with 96–97% specific lysis.
    Figure Legend Snippet: SQLLNAKYL-specific CD8 + T cells are functional and cytotoxic in vivo A,B. Combined tetramer and intracellular cytokine staining were performed on splenocytes and brain-sequestered leukocytes from PbA-infected mice 7 days p.i. Cells were incubated with Brefeldin A for 2 h without restimulation. (A) Representative IFN-γ and GrB profiles of live CD8 + T cells (CD8a + CD16/32 − ). (B) The indicated IFN-γ + GrB + gate was used to analyze tetramer-labelled CD8 + T cells. Bars represent medians. C. Equal numbers of CFSE hi unpulsed naïve splenocytes and CFSE lo SQLLNAKYL-pulsed splenocytes were transferred into naïve or PbA-infected mice 6 days p.i. The mice were sacrificed 20 h later to analyze the CFSE-labelled cells in the spleens. The infected mouse is representative of n = 4, all with 96–97% specific lysis.

    Techniques Used: Functional Assay, In Vivo, Staining, Infection, Mouse Assay, Incubation, Lysis

    2) Product Images from "Human Cytomegalovirus (HCMV)-Specific CD4+ T Cells Are Polyfunctional and Can Respond to HCMV-Infected Dendritic Cells In Vitro"

    Article Title: Human Cytomegalovirus (HCMV)-Specific CD4+ T Cells Are Polyfunctional and Can Respond to HCMV-Infected Dendritic Cells In Vitro

    Journal: Journal of Virology

    doi: 10.1128/JVI.02128-16

    A portion of the HCMV-specific CD4 +  T cells has a cytotoxic capacity and can secrete MIP-1β. PBMCs were stimulated overnight with HCMV peptide pools in the presence of an anti-CD107a antibody, brefeldin A, and monensin to measure the degranulation
    Figure Legend Snippet: A portion of the HCMV-specific CD4 + T cells has a cytotoxic capacity and can secrete MIP-1β. PBMCs were stimulated overnight with HCMV peptide pools in the presence of an anti-CD107a antibody, brefeldin A, and monensin to measure the degranulation

    Techniques Used:

    HCMV-specific CD4 +  T cells have a predominantly effector memory cell phenotype and are not highly differentiated. PBMCs were stimulated overnight with HCMV protein peptide pools in the presence of brefeldin A. HCMV-specific CD4 +  T cell responses were
    Figure Legend Snippet: HCMV-specific CD4 + T cells have a predominantly effector memory cell phenotype and are not highly differentiated. PBMCs were stimulated overnight with HCMV protein peptide pools in the presence of brefeldin A. HCMV-specific CD4 + T cell responses were

    Techniques Used:

    3) Product Images from "Adipocyte-specific CD1d-deficiency mitigates diet-induced obesity and insulin resistance in mice"

    Article Title: Adipocyte-specific CD1d-deficiency mitigates diet-induced obesity and insulin resistance in mice

    Journal: Scientific Reports

    doi: 10.1038/srep28473

    IFN-γ enhances CD1d expression in 3T3-L1 adipocytes. ( a–c ) IFN-γ expression by NKT cells and NK cells in adipose tissue from WT and Jα18 −/− mice fed on SFD or HFD (n = 3 in each group). Representative flow cytometric data are shown for NKT cells (gated on TCRβ + NK1.1 + ) in WT mice ( a ), the proportion and cell number (/g adipose tissue) of IFN-γ + NKT cells or NK cells (gated on TCRβ - NK1.1 + ) in WT mice ( b ), and in Jα18 −/− mice ( c ). Intracellular staining was performed after stromal vascular fraction was incubated with PMA/ionomycin in the presence of brefeldin A for 4 h. ( d,e ) The gene expression in 3T3-L1 adipocytes stimulated with IFN-γ (0, 30, 150 ng/ml) for 3 days was quantified by qPCR. ( f ) The expression of Cd1d1 in 3T3-L1 adipocytes stimulated with the supernatant from 3T3-L1 adipocytes + iNKT cells from WT mice or NKT cells from Jα18 −/− mice. ( g,h ) The expression of CD1d, CD80 and CD86 ( g ) and α-GalCer:CD1d complex ( h ), in 3T3-L1 adipocytes stimulated with IFN-γ (gray: isotype, black line: no treatment, red line: IFN−γ treatment) was analyzed by flow cytometry. Numbers are shown as MFI. Representative data from at least 2 independent experiments are shown. Data are shown as mean ± s.d. Statistical analysis was performed according to the Student’s t -test and the Tukey-Kramer test. * P
    Figure Legend Snippet: IFN-γ enhances CD1d expression in 3T3-L1 adipocytes. ( a–c ) IFN-γ expression by NKT cells and NK cells in adipose tissue from WT and Jα18 −/− mice fed on SFD or HFD (n = 3 in each group). Representative flow cytometric data are shown for NKT cells (gated on TCRβ + NK1.1 + ) in WT mice ( a ), the proportion and cell number (/g adipose tissue) of IFN-γ + NKT cells or NK cells (gated on TCRβ - NK1.1 + ) in WT mice ( b ), and in Jα18 −/− mice ( c ). Intracellular staining was performed after stromal vascular fraction was incubated with PMA/ionomycin in the presence of brefeldin A for 4 h. ( d,e ) The gene expression in 3T3-L1 adipocytes stimulated with IFN-γ (0, 30, 150 ng/ml) for 3 days was quantified by qPCR. ( f ) The expression of Cd1d1 in 3T3-L1 adipocytes stimulated with the supernatant from 3T3-L1 adipocytes + iNKT cells from WT mice or NKT cells from Jα18 −/− mice. ( g,h ) The expression of CD1d, CD80 and CD86 ( g ) and α-GalCer:CD1d complex ( h ), in 3T3-L1 adipocytes stimulated with IFN-γ (gray: isotype, black line: no treatment, red line: IFN−γ treatment) was analyzed by flow cytometry. Numbers are shown as MFI. Representative data from at least 2 independent experiments are shown. Data are shown as mean ± s.d. Statistical analysis was performed according to the Student’s t -test and the Tukey-Kramer test. * P

    Techniques Used: Expressing, Mouse Assay, Flow Cytometry, Staining, Incubation, Real-time Polymerase Chain Reaction, Cytometry

    4) Product Images from "CD36 Ecto-domain Phosphorylation Blocks Thrombospondin-1 Binding: Structure - Function Relationships and Regulation by Protein Kinase C"

    Article Title: CD36 Ecto-domain Phosphorylation Blocks Thrombospondin-1 Binding: Structure - Function Relationships and Regulation by Protein Kinase C

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    doi: 10.1161/ATVBAHA.111.242511

    PMA-induced CD36 phosphorylation inhibits TSR-mediated Src recruitment to CD36.  A.  CD36 transfected Bowes melanoma cells were incubated for 16h in low serum media and then treated for 4h with 0.1μg/ml PMA or vehicle control. Some cells were then treated for 1h with 40u/ml alkaline phosphatase (AP) or vehicle control. Cells were then exposed to 100nM recombinant TSR2/3 or thioredoxin control for 15min. CD36 was then immunoprecipitated and the precipitates were analyzed by western blot with an antibody to Src family proteins. Blots were re-probed with anti-CD36 antibodies. Blots were scanned and the amount of co-precipitated Src was normalized to total CD36.  B.  CD36 transfected Bowes melanoma cells were incubated for 16h in low serum media and then treated for 4h with 0.1μg/ml PMA along with either 100μg/ml cycloheximide or 5ng/ml Brefeldin A. Cells were then exposed to 100nM recombinant TSR2/3 or thioredoxin control for 15min. Src family association was then assessed by western blot and analyzed as in panel A. C) Cells prepared as in Panel B were analyzed by western blot using antibodies to phospho-PKCα (upper blot). Blots were then stripped and re-probed with an antibody to total PKC (lower blot). All blots are representative of n=3.
    Figure Legend Snippet: PMA-induced CD36 phosphorylation inhibits TSR-mediated Src recruitment to CD36. A. CD36 transfected Bowes melanoma cells were incubated for 16h in low serum media and then treated for 4h with 0.1μg/ml PMA or vehicle control. Some cells were then treated for 1h with 40u/ml alkaline phosphatase (AP) or vehicle control. Cells were then exposed to 100nM recombinant TSR2/3 or thioredoxin control for 15min. CD36 was then immunoprecipitated and the precipitates were analyzed by western blot with an antibody to Src family proteins. Blots were re-probed with anti-CD36 antibodies. Blots were scanned and the amount of co-precipitated Src was normalized to total CD36. B. CD36 transfected Bowes melanoma cells were incubated for 16h in low serum media and then treated for 4h with 0.1μg/ml PMA along with either 100μg/ml cycloheximide or 5ng/ml Brefeldin A. Cells were then exposed to 100nM recombinant TSR2/3 or thioredoxin control for 15min. Src family association was then assessed by western blot and analyzed as in panel A. C) Cells prepared as in Panel B were analyzed by western blot using antibodies to phospho-PKCα (upper blot). Blots were then stripped and re-probed with an antibody to total PKC (lower blot). All blots are representative of n=3.

    Techniques Used: Transfection, Incubation, Recombinant, Immunoprecipitation, Western Blot

    5) Product Images from "CD36 Ecto-domain Phosphorylation Blocks Thrombospondin-1 Binding: Structure - Function Relationships and Regulation by Protein Kinase C"

    Article Title: CD36 Ecto-domain Phosphorylation Blocks Thrombospondin-1 Binding: Structure - Function Relationships and Regulation by Protein Kinase C

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    doi: 10.1161/ATVBAHA.111.242511

    PMA-induced CD36 phosphorylation inhibits TSR-mediated Src recruitment to CD36.  A.  CD36 transfected Bowes melanoma cells were incubated for 16h in low serum media and then treated for 4h with 0.1μg/ml PMA or vehicle control. Some cells were then treated for 1h with 40u/ml alkaline phosphatase (AP) or vehicle control. Cells were then exposed to 100nM recombinant TSR2/3 or thioredoxin control for 15min. CD36 was then immunoprecipitated and the precipitates were analyzed by western blot with an antibody to Src family proteins. Blots were re-probed with anti-CD36 antibodies. Blots were scanned and the amount of co-precipitated Src was normalized to total CD36.  B.  CD36 transfected Bowes melanoma cells were incubated for 16h in low serum media and then treated for 4h with 0.1μg/ml PMA along with either 100μg/ml cycloheximide or 5ng/ml Brefeldin A. Cells were then exposed to 100nM recombinant TSR2/3 or thioredoxin control for 15min. Src family association was then assessed by western blot and analyzed as in panel A. C) Cells prepared as in Panel B were analyzed by western blot using antibodies to phospho-PKCα (upper blot). Blots were then stripped and re-probed with an antibody to total PKC (lower blot). All blots are representative of n=3.
    Figure Legend Snippet: PMA-induced CD36 phosphorylation inhibits TSR-mediated Src recruitment to CD36. A. CD36 transfected Bowes melanoma cells were incubated for 16h in low serum media and then treated for 4h with 0.1μg/ml PMA or vehicle control. Some cells were then treated for 1h with 40u/ml alkaline phosphatase (AP) or vehicle control. Cells were then exposed to 100nM recombinant TSR2/3 or thioredoxin control for 15min. CD36 was then immunoprecipitated and the precipitates were analyzed by western blot with an antibody to Src family proteins. Blots were re-probed with anti-CD36 antibodies. Blots were scanned and the amount of co-precipitated Src was normalized to total CD36. B. CD36 transfected Bowes melanoma cells were incubated for 16h in low serum media and then treated for 4h with 0.1μg/ml PMA along with either 100μg/ml cycloheximide or 5ng/ml Brefeldin A. Cells were then exposed to 100nM recombinant TSR2/3 or thioredoxin control for 15min. Src family association was then assessed by western blot and analyzed as in panel A. C) Cells prepared as in Panel B were analyzed by western blot using antibodies to phospho-PKCα (upper blot). Blots were then stripped and re-probed with an antibody to total PKC (lower blot). All blots are representative of n=3.

    Techniques Used: Transfection, Incubation, Recombinant, Immunoprecipitation, Western Blot

    6) Product Images from "CCR6 Deficiency Impairs IgA Production and Dysregulates Antimicrobial Peptide Production, Altering the Intestinal Flora"

    Article Title: CCR6 Deficiency Impairs IgA Production and Dysregulates Antimicrobial Peptide Production, Altering the Intestinal Flora

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00805

    ILC3s show significantly decreased IL-17 expression but significantly increased MHCII expression in Peyer’s patches (PPs) of CCR6 −/− mice. PP lymphocytes were subjected to immunofluorescence staining of surface markers (Lin, CD45, CD117, CD127, and MHCII) followed by intracellular staining of RORγt. (A) Representative contour plots for the identification of ILC3–LTi (Lin − RORγt + CD117 + CD127 + ) in PPs are shown. (B) The frequency of ILC3–LTi in PPs of WT and CCR6 −/− mice is shown. (C) PP lymphocytes were stimulated with 20 µg/ml PMA, 1 µM ionomycin, and 5 µg/ml brefeldin A for 4 h followed by surface staining of ILC3–LTi and intracellular staining of IL-17 and RORγt. The frequency of IL-17-producing ILC3–LTi in PPs of WT and CCR6 −/− mice is shown. (D) The frequency of MHCII-expressi ng ILC3–LTi in PPs of WT and CCR6 −/− mice is shown. Each symbol represents one mouse. Data are a compilation of six (B) , three (C) , or four (D) independent experiments (* p
    Figure Legend Snippet: ILC3s show significantly decreased IL-17 expression but significantly increased MHCII expression in Peyer’s patches (PPs) of CCR6 −/− mice. PP lymphocytes were subjected to immunofluorescence staining of surface markers (Lin, CD45, CD117, CD127, and MHCII) followed by intracellular staining of RORγt. (A) Representative contour plots for the identification of ILC3–LTi (Lin − RORγt + CD117 + CD127 + ) in PPs are shown. (B) The frequency of ILC3–LTi in PPs of WT and CCR6 −/− mice is shown. (C) PP lymphocytes were stimulated with 20 µg/ml PMA, 1 µM ionomycin, and 5 µg/ml brefeldin A for 4 h followed by surface staining of ILC3–LTi and intracellular staining of IL-17 and RORγt. The frequency of IL-17-producing ILC3–LTi in PPs of WT and CCR6 −/− mice is shown. (D) The frequency of MHCII-expressi ng ILC3–LTi in PPs of WT and CCR6 −/− mice is shown. Each symbol represents one mouse. Data are a compilation of six (B) , three (C) , or four (D) independent experiments (* p

    Techniques Used: Expressing, Mouse Assay, Immunofluorescence, Staining

    7) Product Images from "CD4+ T cells promote humoral immunity and viral control during Zika virus infection"

    Article Title: CD4+ T cells promote humoral immunity and viral control during Zika virus infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007474

    Mapping of the CD4 +  T cell response in the  LysMCre + Ifnar1 fl/fl  mouse model of primary ZIKV infection. Five-week-old  LysMCre + Ifnar1 fl/fl  C57BL/6 mice were infected retro-orbitally with 10 4  FFU of ZIKV strain MR766 or FSS13025 in 10% FBS/PBS or were mock-infected (10% FBS/PBS alone). Data are the mean ± SEM of  n  = 4–6 mice/group. ( A–C ) Splenocytes were removed on day 7 post-infection and analyzed for the percentage of ( A ) CD44 + CD4 +  T cells, ( B ) CD49d + CD11a +  T cells, and ( C ) granzyme B + CD4 +  T cells. ( D–F ) Splenocytes were removed on day 7 post-infection and stimulated with the indicated ZIKV-derived peptides and brefeldin A. The percentage CD44 + CD4 +  T cells producing ( D ) IFNγ, ( E ) IFNγ and TNF, and ( F ) IL-2 was measured by ICS. Cells stimulated with DMSO or PMA/ionomycin served as negative and positive controls, respectively. ( G ) Summary of the data shown in ( D–F ). ( H )  In vivo  killing of ZIKV peptide-pulsed target cells.  LysMCre + Ifnar1 fl/fl  mice were retro-orbitally mock-infected ( n  = 4) or infected with 10 4  FFU ZIKV MR766 ( n  = 5) and FSS13025 ( n  = 4) for 7 days, and then injected retro-orbitally with naïve C57BL/6 splenocytes ( n  = 4) pulsed with a pool of ZIKV peptides (E 346-360,  E 644-658 , NS3 1740-1754 , NS4B 2480-2494 , NS5 2604-2618 , NS5 2738-2752 ) or treated with DMSO. After 12 h, the splenocytes were harvested from recipient mice, analyzed by flow cytometry, and the percentage ZIKV-specific cytotoxicity was calculated. * P
    Figure Legend Snippet: Mapping of the CD4 + T cell response in the LysMCre + Ifnar1 fl/fl mouse model of primary ZIKV infection. Five-week-old LysMCre + Ifnar1 fl/fl C57BL/6 mice were infected retro-orbitally with 10 4 FFU of ZIKV strain MR766 or FSS13025 in 10% FBS/PBS or were mock-infected (10% FBS/PBS alone). Data are the mean ± SEM of n = 4–6 mice/group. ( A–C ) Splenocytes were removed on day 7 post-infection and analyzed for the percentage of ( A ) CD44 + CD4 + T cells, ( B ) CD49d + CD11a + T cells, and ( C ) granzyme B + CD4 + T cells. ( D–F ) Splenocytes were removed on day 7 post-infection and stimulated with the indicated ZIKV-derived peptides and brefeldin A. The percentage CD44 + CD4 + T cells producing ( D ) IFNγ, ( E ) IFNγ and TNF, and ( F ) IL-2 was measured by ICS. Cells stimulated with DMSO or PMA/ionomycin served as negative and positive controls, respectively. ( G ) Summary of the data shown in ( D–F ). ( H ) In vivo killing of ZIKV peptide-pulsed target cells. LysMCre + Ifnar1 fl/fl mice were retro-orbitally mock-infected ( n = 4) or infected with 10 4 FFU ZIKV MR766 ( n = 5) and FSS13025 ( n = 4) for 7 days, and then injected retro-orbitally with naïve C57BL/6 splenocytes ( n = 4) pulsed with a pool of ZIKV peptides (E 346-360, E 644-658 , NS3 1740-1754 , NS4B 2480-2494 , NS5 2604-2618 , NS5 2738-2752 ) or treated with DMSO. After 12 h, the splenocytes were harvested from recipient mice, analyzed by flow cytometry, and the percentage ZIKV-specific cytotoxicity was calculated. * P

    Techniques Used: Infection, Mouse Assay, Derivative Assay, In Vivo, Injection, Flow Cytometry, Cytometry

    8) Product Images from "Deletion of cannabinoid receptors 1 and 2 exacerbates APC function to increase inflammation and cellular immunity during influenza infection"

    Article Title: Deletion of cannabinoid receptors 1 and 2 exacerbates APC function to increase inflammation and cellular immunity during influenza infection

    Journal: Journal of Leukocyte Biology

    doi: 10.1189/jlb.0511219

    Greater cytokine production by leukocytes from CB 1 −/− CB 2 −/−  mice compared with WT mice. Lungs from mice were mechanically disrupted and passed through a sieve ( n =5). After removal of connective tissue, cells were counted and restimulated with PMA/Io for 5 h in the presence of Brefeldin A. (A) Cells were blocked with FcRs and stained with CD4, CD8, and NK1.1, in addition to cytokine staining with IFN-γ and IL-17 (B). Flow cytometry samples were gated as depicted in A. Cytokine secretors were identified as percent of positive cells within surface delineation, and unstimulated samples were used as controls. (C) Immune cell populations were enumerated for percent cytokine expression in FlowJo (type of cytokine indicated on the left) within effector cell populations (surface marker indicated on the right) in FlowJo, and bar graphs were generated using GraphPad Prism. Friedman's test for percentages was performed, and significance is indicated in figures as  # P
    Figure Legend Snippet: Greater cytokine production by leukocytes from CB 1 −/− CB 2 −/− mice compared with WT mice. Lungs from mice were mechanically disrupted and passed through a sieve ( n =5). After removal of connective tissue, cells were counted and restimulated with PMA/Io for 5 h in the presence of Brefeldin A. (A) Cells were blocked with FcRs and stained with CD4, CD8, and NK1.1, in addition to cytokine staining with IFN-γ and IL-17 (B). Flow cytometry samples were gated as depicted in A. Cytokine secretors were identified as percent of positive cells within surface delineation, and unstimulated samples were used as controls. (C) Immune cell populations were enumerated for percent cytokine expression in FlowJo (type of cytokine indicated on the left) within effector cell populations (surface marker indicated on the right) in FlowJo, and bar graphs were generated using GraphPad Prism. Friedman's test for percentages was performed, and significance is indicated in figures as # P

    Techniques Used: Mouse Assay, Staining, Flow Cytometry, Cytometry, Expressing, Marker, Generated

    9) Product Images from "Programmed Death 1 Regulates Development of Central Memory CD8 T Cells after Acute Viral Infection"

    Article Title: Programmed Death 1 Regulates Development of Central Memory CD8 T Cells after Acute Viral Infection

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

    doi: 10.4049/jimmunol.1003870

    Increased cytokine production by Ag-specific memory CD8 T cells in the absence of PD-1 signaling in the BMC model. Spleen cells from chimeric mice were incubated for 5 h with or without B8R peptide in the presence of brefeldin A. IFN-γ ( A ) or
    Figure Legend Snippet: Increased cytokine production by Ag-specific memory CD8 T cells in the absence of PD-1 signaling in the BMC model. Spleen cells from chimeric mice were incubated for 5 h with or without B8R peptide in the presence of brefeldin A. IFN-γ ( A ) or

    Techniques Used: Mouse Assay, Incubation

    10) Product Images from "Activation of Eosinophils Interacting with Bronchial Epithelial Cells by Antimicrobial Peptide LL-37: Implications in Allergic Asthma"

    Article Title: Activation of Eosinophils Interacting with Bronchial Epithelial Cells by Antimicrobial Peptide LL-37: Implications in Allergic Asthma

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-02085-5

    Sources of cytokine/chemokine expressions in co-culture of eosinophils and BEAS-2B cells under the stimulation of LL-37. Human eosinophils (3 × 10 5 ) and BEAS-2B cells (1 × 10 5 ) were treated with or without paraformaldehyde before being cultured together with or without 10 µg/ml LL-37 for 20 h. Release of IL-6 ( A ), CXCL8 ( B ) and CCL4 ( C ) in culture supernatant was determined. ( D ) 3 × 10 5 eosinophils and 1 × 10 5 BEAS-2B cells were co-cultured with or without 10 µg/ml LL-37 for 16 h, followed by the incubation with 10 µg/ml brefeldin A for further 4 h. Intracellular staining of IL-6, CXCL8 and CCL4 were then performed. Representative histograms of intracellular staining of IL-6, CXCL8 and CCL4 are shown from triplicate independent experiments showing essentially similar results. E: unfixed eosinophils; E^: fixed eosinophils; B: unfixed BEAS-2B cells; B^: fixed BEAS-2B cells. Results are shown as arithmetic mean plus SD of triplicate independent experiments. *P
    Figure Legend Snippet: Sources of cytokine/chemokine expressions in co-culture of eosinophils and BEAS-2B cells under the stimulation of LL-37. Human eosinophils (3 × 10 5 ) and BEAS-2B cells (1 × 10 5 ) were treated with or without paraformaldehyde before being cultured together with or without 10 µg/ml LL-37 for 20 h. Release of IL-6 ( A ), CXCL8 ( B ) and CCL4 ( C ) in culture supernatant was determined. ( D ) 3 × 10 5 eosinophils and 1 × 10 5 BEAS-2B cells were co-cultured with or without 10 µg/ml LL-37 for 16 h, followed by the incubation with 10 µg/ml brefeldin A for further 4 h. Intracellular staining of IL-6, CXCL8 and CCL4 were then performed. Representative histograms of intracellular staining of IL-6, CXCL8 and CCL4 are shown from triplicate independent experiments showing essentially similar results. E: unfixed eosinophils; E^: fixed eosinophils; B: unfixed BEAS-2B cells; B^: fixed BEAS-2B cells. Results are shown as arithmetic mean plus SD of triplicate independent experiments. *P

    Techniques Used: Co-Culture Assay, Cell Culture, Incubation, Staining

    11) Product Images from "PD-1 and Tim-3 pathways are associated with regulatory CD8+ T-cell function in decidua and maintenance of normal pregnancy"

    Article Title: PD-1 and Tim-3 pathways are associated with regulatory CD8+ T-cell function in decidua and maintenance of normal pregnancy

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2015.112

    In vivo roles of Tim-3 and PD-1 during early pregnancy. ( a ) The weight of pregnant CBA/J females treated with PBS, anti-Tim-3 antibody, anti-PD-1 antibody, or both antibodies i.p. at doses of 500, 250, and 250 mg at days 4.5, 6.5, and 8.5, respectively. ( b ) The number of live fetuses per uterus from pregnant CBA/J females following treatment with the indicated blocking antibodies. Pregnant mice were kiled at day 10.5 of pregnancy, the uteri were removed, and the implantation sites and resorbed/live embryos were counted to assess the effect of anti-Tim-3 and anti-PD-1 antibody treatment on pregnancy. ( c – f ) Quantification of flow-cytometric analysis of cytokine production and transcription factor expression by dCD8 + T cells of mice following treatment with the indicated blocking antibodies. Freshly isolated DICs were treated with brefeldin A (10 μ g/ml), PMA (50 ng/ml), and ionomycin (1 μ g/ml) for 4 h, then the cells were harvested and analyzed by flow cytometry. Data represent mean±S.E.M. of n =6–8 mice per group and are representative of four independent analyses. * P
    Figure Legend Snippet: In vivo roles of Tim-3 and PD-1 during early pregnancy. ( a ) The weight of pregnant CBA/J females treated with PBS, anti-Tim-3 antibody, anti-PD-1 antibody, or both antibodies i.p. at doses of 500, 250, and 250 mg at days 4.5, 6.5, and 8.5, respectively. ( b ) The number of live fetuses per uterus from pregnant CBA/J females following treatment with the indicated blocking antibodies. Pregnant mice were kiled at day 10.5 of pregnancy, the uteri were removed, and the implantation sites and resorbed/live embryos were counted to assess the effect of anti-Tim-3 and anti-PD-1 antibody treatment on pregnancy. ( c – f ) Quantification of flow-cytometric analysis of cytokine production and transcription factor expression by dCD8 + T cells of mice following treatment with the indicated blocking antibodies. Freshly isolated DICs were treated with brefeldin A (10 μ g/ml), PMA (50 ng/ml), and ionomycin (1 μ g/ml) for 4 h, then the cells were harvested and analyzed by flow cytometry. Data represent mean±S.E.M. of n =6–8 mice per group and are representative of four independent analyses. * P

    Techniques Used: In Vivo, Crocin Bleaching Assay, Blocking Assay, Mouse Assay, Flow Cytometry, Expressing, Isolation, Cytometry

    Proliferation and cytokine production in dCD8 + T cells during normal pregnancy. ( a ) Freshly isolated DICs were stained with antibodies against Ki-67 to detect the proliferation of dCD8 + T cells by flow cytometry. A representative flow-cytometry plot (right) and quantification (left) of Ki-67 staining in dCD8 + T cells are shown. n =9. ( b and c ) Freshly isolated DICs were treated with brefeldin A (10 μ g/ml), phorbol 12-myrstate 13-acetate (PMA) (50 ng/ml), and ionomycin (1 μ g/ml) for 4 h, then the cells were harvested and analyzed by flow cytometry. ( b ) Expression of the Th2-type cytokines IL-4 and IL-10 in Tim-3 + PD-1 + , Tim-3 − PD-1 + , Tim-3 + PD-1 − , and Tim-3 − PD-1 − dCD8 + T cells. A representative flow-cytometry plot (right) and quantitation (left) are shown. n =9. ( c ) Quantitation of flow-cytometric analysis of the Th1-type cytokines IFN- γ and TNF- α in Tim-3 + PD-1 + , Tim-3 − PD-1 + , Tim-3 + PD-1 − , and Tim-3 − PD-1 − dCD8 + T cells. n =12. Data represent mean±S.E.M. The flow-cytometry plots are representative of three independent experiments. * P
    Figure Legend Snippet: Proliferation and cytokine production in dCD8 + T cells during normal pregnancy. ( a ) Freshly isolated DICs were stained with antibodies against Ki-67 to detect the proliferation of dCD8 + T cells by flow cytometry. A representative flow-cytometry plot (right) and quantification (left) of Ki-67 staining in dCD8 + T cells are shown. n =9. ( b and c ) Freshly isolated DICs were treated with brefeldin A (10 μ g/ml), phorbol 12-myrstate 13-acetate (PMA) (50 ng/ml), and ionomycin (1 μ g/ml) for 4 h, then the cells were harvested and analyzed by flow cytometry. ( b ) Expression of the Th2-type cytokines IL-4 and IL-10 in Tim-3 + PD-1 + , Tim-3 − PD-1 + , Tim-3 + PD-1 − , and Tim-3 − PD-1 − dCD8 + T cells. A representative flow-cytometry plot (right) and quantitation (left) are shown. n =9. ( c ) Quantitation of flow-cytometric analysis of the Th1-type cytokines IFN- γ and TNF- α in Tim-3 + PD-1 + , Tim-3 − PD-1 + , Tim-3 + PD-1 − , and Tim-3 − PD-1 − dCD8 + T cells. n =12. Data represent mean±S.E.M. The flow-cytometry plots are representative of three independent experiments. * P

    Techniques Used: Isolation, Staining, Flow Cytometry, Cytometry, Expressing, Quantitation Assay

    Decreased number of CD8 + T cells co-expressing Tim-3 and PD-1 with disregulated cytokine production is observed in human early pregnancy loss. ( a ) Frequency of Tim-3 and PD-1 expression on gated CD8 + T cells from DICs and PBMCs from normal pregnancy (NP; n =30) and miscarriage (abnormal pregnancy, AP; n =36) as measured by flow cytometry. ( b ) Flow-cytometric analysis of Ki-67 staining in dCD8 + T cells from normal pregnancy ( n =30) and miscarriage ( n =36). ( c and d ) Quantification of flow-cytometric analysis of cytokine production by dCD8 + T cells from normal pregnancy ( n =30) and miscarriage ( n =36). Freshly isolated DICs were treated with brefeldin A (10 μ g/ml), PMA (50 ng/ml), and ionomycin (1 μ g/ml) for 4 h, then the cells were harvested, fixed, permeabilized, and stained with antibodies against IL-4, IL-10, IFN- γ , TNF- α , CD8, PD-1, and Tim-3. Data represent mean±S.E.M. The flow-cytometry plot is representative of six independent experiments. * P
    Figure Legend Snippet: Decreased number of CD8 + T cells co-expressing Tim-3 and PD-1 with disregulated cytokine production is observed in human early pregnancy loss. ( a ) Frequency of Tim-3 and PD-1 expression on gated CD8 + T cells from DICs and PBMCs from normal pregnancy (NP; n =30) and miscarriage (abnormal pregnancy, AP; n =36) as measured by flow cytometry. ( b ) Flow-cytometric analysis of Ki-67 staining in dCD8 + T cells from normal pregnancy ( n =30) and miscarriage ( n =36). ( c and d ) Quantification of flow-cytometric analysis of cytokine production by dCD8 + T cells from normal pregnancy ( n =30) and miscarriage ( n =36). Freshly isolated DICs were treated with brefeldin A (10 μ g/ml), PMA (50 ng/ml), and ionomycin (1 μ g/ml) for 4 h, then the cells were harvested, fixed, permeabilized, and stained with antibodies against IL-4, IL-10, IFN- γ , TNF- α , CD8, PD-1, and Tim-3. Data represent mean±S.E.M. The flow-cytometry plot is representative of six independent experiments. * P

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Staining, Isolation

    12) Product Images from "Type 1 Conventional CD103+ Dendritic Cells Control Effector CD8+ T Cell Migration, Survival, and Memory Responses During Influenza Infection"

    Article Title: Type 1 Conventional CD103+ Dendritic Cells Control Effector CD8+ T Cell Migration, Survival, and Memory Responses During Influenza Infection

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.03043

    Impaired effector CD8 + T cell responses in the absence of pulmonary CD103 + cDC1s. (A) NP 366−374 -specific CD8 + T cells in the lungs of uninfected, infected wild type, and Clec9A-DTR mice (day 6, 10, and 15 post infection). (B) Kinetics of total CD8 + T cells and NP 366−374 -specific CD8 + T cells in the lungs of uninfected, infected wild type, and Clec9A-DTR mice. Absolute numbers are shown. (C) Lung virus load was measured by relative quantification of M1 viral protein in infected wild type and Clec9A-DTR mice on day 10 post infection. (D–F) Lung cells were harvested from uninfected, infected wild type, and Clec9A-DTR mice on day 10 post infection and stimulated with PMA/Ionomycin for 3 h followed by Brefeldin A incubation for an additional 3 h. Intracellular IFN-γ and IL-10 staining profiles (d) of pulmonary CD8 + T cells, frequency (E) and total numbers (F) of IFN-γ-producing, IL-10-producing, and IFN-γ/IL-10 double-producing CD8 + T cells in the lungs. (G) IFN-γ and IL-10 BAL levels as measured by sandwich ELISA (H) Relative quantification of IFN-γ and IL-10 transcripts. Data are shown as mean ± SEM. * p
    Figure Legend Snippet: Impaired effector CD8 + T cell responses in the absence of pulmonary CD103 + cDC1s. (A) NP 366−374 -specific CD8 + T cells in the lungs of uninfected, infected wild type, and Clec9A-DTR mice (day 6, 10, and 15 post infection). (B) Kinetics of total CD8 + T cells and NP 366−374 -specific CD8 + T cells in the lungs of uninfected, infected wild type, and Clec9A-DTR mice. Absolute numbers are shown. (C) Lung virus load was measured by relative quantification of M1 viral protein in infected wild type and Clec9A-DTR mice on day 10 post infection. (D–F) Lung cells were harvested from uninfected, infected wild type, and Clec9A-DTR mice on day 10 post infection and stimulated with PMA/Ionomycin for 3 h followed by Brefeldin A incubation for an additional 3 h. Intracellular IFN-γ and IL-10 staining profiles (d) of pulmonary CD8 + T cells, frequency (E) and total numbers (F) of IFN-γ-producing, IL-10-producing, and IFN-γ/IL-10 double-producing CD8 + T cells in the lungs. (G) IFN-γ and IL-10 BAL levels as measured by sandwich ELISA (H) Relative quantification of IFN-γ and IL-10 transcripts. Data are shown as mean ± SEM. * p

    Techniques Used: Infection, Mouse Assay, Incubation, Staining, Sandwich ELISA

    13) Product Images from "ATP release drives heightened immune responses associated with hypertension"

    Article Title: ATP release drives heightened immune responses associated with hypertension

    Journal: Science immunology

    doi: 10.1126/sciimmunol.aau6426

    Blocking CD86 eliminates the overactivation of immune responses in hypertensive mice. C57BL/6 mice were sham-treated (normotensive) or Ang Il-treated (hypertensive) for 2 weeks. ( A ) Splenic DCs were purified. Some of the cells were pulsed with SIINFEKL peptide. Some cells were also treated with an anti-CD80 antibody or an anti-CD86 antibody or both. They were then coincubated with OT-I T cells, and the expression of T cell CD69 and IFN-γ was measured. ( B  and  C ) Mice were regularly treated with an anti-CD86 antibody or an isotype control antibody from 3 days before being immunized with OVA until sacrifice. Seven days after immunization, the numbers of OVA-specific CD8 +  T cells in the blood (B) and splenocyte IL-2 production after SIINFEKL restimulation were measured (C). ( D  and  E ) Acute hepatitis was induced by intravenous injection of ConA (5 mg/kg) into normotensive and hypertensive mice. Some hypertensive mice were injected intraperitoneally with either CD86 blocking antibody (α-CD86) or an isotype control antibody (IgG) every other day for three times before ConA administration. (D) Blood ALT levels were measured 6 hours after ConA injection. (E) Leukocytes in the liver were isolated with Percoll gradients. Cells were cultured in medium for 6 hours in the presence of brefeldin A, and then intra-cellular staining was performed to examine the production of IFN-γ and TNF-γ by CD4 +  T cells. Data are means ± SEM.  *P
    Figure Legend Snippet: Blocking CD86 eliminates the overactivation of immune responses in hypertensive mice. C57BL/6 mice were sham-treated (normotensive) or Ang Il-treated (hypertensive) for 2 weeks. ( A ) Splenic DCs were purified. Some of the cells were pulsed with SIINFEKL peptide. Some cells were also treated with an anti-CD80 antibody or an anti-CD86 antibody or both. They were then coincubated with OT-I T cells, and the expression of T cell CD69 and IFN-γ was measured. ( B and C ) Mice were regularly treated with an anti-CD86 antibody or an isotype control antibody from 3 days before being immunized with OVA until sacrifice. Seven days after immunization, the numbers of OVA-specific CD8 + T cells in the blood (B) and splenocyte IL-2 production after SIINFEKL restimulation were measured (C). ( D and E ) Acute hepatitis was induced by intravenous injection of ConA (5 mg/kg) into normotensive and hypertensive mice. Some hypertensive mice were injected intraperitoneally with either CD86 blocking antibody (α-CD86) or an isotype control antibody (IgG) every other day for three times before ConA administration. (D) Blood ALT levels were measured 6 hours after ConA injection. (E) Leukocytes in the liver were isolated with Percoll gradients. Cells were cultured in medium for 6 hours in the presence of brefeldin A, and then intra-cellular staining was performed to examine the production of IFN-γ and TNF-γ by CD4 + T cells. Data are means ± SEM. *P

    Techniques Used: Blocking Assay, Mouse Assay, Purification, Expressing, Injection, Isolation, Cell Culture, Staining

    Hypertensive mice are predisposed to autoimmune diseases. ( A  to  C ) RIP-mOVA mice were made hypertensive by treatment with either Ang II or L-NAME. After 2 to 3 weeks, when hypertension was established, 5 × 10 6  OT-I T cells (CD8 +  T cells from OT-I transgenic mice) were transferred intravenously into normotensive and hypertensive RIP-mOVA mice. (A) Blood glucose levels were measured for 2 weeks after OT-I T cell transfer. (B and C) Two weeks after OT-I T cell transfer, the percentages of OT-I T cells among CD8 +  T cells in the blood, spleen, and pancreatic draining lymph nodes (DLN) were quantified by tetramer analysis (B), and the numbers of inflamed islets, identified by anti-CD3 staining, were counted in a blinded fashion (C). ( D  to  F ) ConA (5 mg/kg) was injected intravenously into normotensive and hypertensive mice. Blood ALT levels were measured after 6 hours. (D) Hepatic inflammatory cells were prepared by tissue enzymatic digestion followed by Percoll centrifugation. (E) Cells were cultured in medium for 6 hours in the presence of brefeldin A, and then intracellular staining was performed to examine the production of IFN-γ and TNF-α by CD4 +  T cells. Liver necrosis area was measured after hematoxylin and eosin staining. MFI, mean fluorescence intensity. (F) For each liver, necrosis area represents the average of 10 separate fields. In (A) to (C),  n  = 17, 6, and 12 for normotensive, Ang II-treated, and L-NAME-treated mice, respectively. Data are means ± SEM.  *P
    Figure Legend Snippet: Hypertensive mice are predisposed to autoimmune diseases. ( A to C ) RIP-mOVA mice were made hypertensive by treatment with either Ang II or L-NAME. After 2 to 3 weeks, when hypertension was established, 5 × 10 6 OT-I T cells (CD8 + T cells from OT-I transgenic mice) were transferred intravenously into normotensive and hypertensive RIP-mOVA mice. (A) Blood glucose levels were measured for 2 weeks after OT-I T cell transfer. (B and C) Two weeks after OT-I T cell transfer, the percentages of OT-I T cells among CD8 + T cells in the blood, spleen, and pancreatic draining lymph nodes (DLN) were quantified by tetramer analysis (B), and the numbers of inflamed islets, identified by anti-CD3 staining, were counted in a blinded fashion (C). ( D to F ) ConA (5 mg/kg) was injected intravenously into normotensive and hypertensive mice. Blood ALT levels were measured after 6 hours. (D) Hepatic inflammatory cells were prepared by tissue enzymatic digestion followed by Percoll centrifugation. (E) Cells were cultured in medium for 6 hours in the presence of brefeldin A, and then intracellular staining was performed to examine the production of IFN-γ and TNF-α by CD4 + T cells. Liver necrosis area was measured after hematoxylin and eosin staining. MFI, mean fluorescence intensity. (F) For each liver, necrosis area represents the average of 10 separate fields. In (A) to (C), n = 17, 6, and 12 for normotensive, Ang II-treated, and L-NAME-treated mice, respectively. Data are means ± SEM. *P

    Techniques Used: Mouse Assay, Transgenic Assay, Staining, Injection, Centrifugation, Cell Culture, Fluorescence

    APCs from hypertensive mice present antigens more efficiently. ( A  and  B ) Splenic DCs and peritoneal macrophages (PM) from normotensive or Ang Il-induced hypertensive C57BL/6 mice were pulsed with 10 μM OVA (A) or 50 pM SIINFEKL peptide (SKL) (B) in vitro and were then coincubated with OT-I T cells. The percentages of CD69 +  (after 4 hours) or IFN-γ +  cells (after 6 hours in the presence of brefeldin A) among OT-I T cells were measured by flow cytometry. Representative flow cytometry dot plots of IFN-γ and CD69 expression are shown. ( C ) Splenic DCs from normotensive or L-NAME-induced hypertensive mice were pulsed with SIINFEKL and then coincubated with OT-I T cells. The percentages of CD69 +  or IFN-γ +  cells among all CD8 +  T cells were measured. ( D ) Splenic DCs from normotensive or Ang II-induced hypertensive mice were loaded with SIINFEKL for 4 hours ex vivo. Cells were then transferred intravenously into naїve C57BL/6 mice. Seven days later, splenocytes from the recipients were restimulated with SIINFEKL and the secretion of IL-2 and IFN-γ was measured. Data are means ± SEM. * P
    Figure Legend Snippet: APCs from hypertensive mice present antigens more efficiently. ( A and B ) Splenic DCs and peritoneal macrophages (PM) from normotensive or Ang Il-induced hypertensive C57BL/6 mice were pulsed with 10 μM OVA (A) or 50 pM SIINFEKL peptide (SKL) (B) in vitro and were then coincubated with OT-I T cells. The percentages of CD69 + (after 4 hours) or IFN-γ + cells (after 6 hours in the presence of brefeldin A) among OT-I T cells were measured by flow cytometry. Representative flow cytometry dot plots of IFN-γ and CD69 expression are shown. ( C ) Splenic DCs from normotensive or L-NAME-induced hypertensive mice were pulsed with SIINFEKL and then coincubated with OT-I T cells. The percentages of CD69 + or IFN-γ + cells among all CD8 + T cells were measured. ( D ) Splenic DCs from normotensive or Ang II-induced hypertensive mice were loaded with SIINFEKL for 4 hours ex vivo. Cells were then transferred intravenously into naїve C57BL/6 mice. Seven days later, splenocytes from the recipients were restimulated with SIINFEKL and the secretion of IL-2 and IFN-γ was measured. Data are means ± SEM. * P

    Techniques Used: Mouse Assay, In Vitro, Flow Cytometry, Cytometry, Expressing, Ex Vivo

    14) Product Images from "Tethering soluble meprin α in an enzyme complex to the cell surface affects IBD-associated genes"

    Article Title: Tethering soluble meprin α in an enzyme complex to the cell surface affects IBD-associated genes

    Journal: The FASEB Journal

    doi: 10.1096/fj.201802391R

    Meprin interaction on the cell surface and secretory pathway. A ) Transfection of HeLa cells with meprin α WT and meprin β WT constructs. Cell surface proteins were labeled by primary amine biotinylation, pulled down with streptavidin sepharose beads, and analyzed via immunoblotting. GAPDH and transferrin receptor served as loading controls. B ) Immunofluorescence staining of transfected HeLa cells with meprin β SF and meprin α WT constructs (left panel) or meprin α SF and meprin β WT constructs (right panel). Nuclear staining was visualized using DAPI. Images were taken by confocal fluorescence microscopy. Scale bar, 20 µm. C ) Immunofluorescence staining of transfected HeLa cells using meprin α SF, meprin α C308S SF, and meprin β WT constructs. Cells were treated overnight with brefeldin A at 5 µg/ml or DMSO control. Cell surface staining was performed by excluding saponin from buffers. Nuclear staining was visualized using DAPI. Images were taken by confocal fluorescence microscopy. Scale bars, 20 µm. D ) Transfection of HeLa cells using meprin α SF and meprin β WT constructs. Cells were treated overnight with brefeldin A at 5 µg/ml or DMSO control. Coimmunoprecipitation was performed using Flag-tag antibody against meprin α C terminus or Strep-tag antibody against meprin α N terminus as indicated. Protein fractions were analyzed by immunoblotting. E ) Diagram of meprin α and meprin β interacting in the ER and being transported to the cell surface. Treatment of cells with brefeldin A inhibits the transport from the ER to Golgi apparatus. IP, immunoprecipitation; Mep, meprin.
    Figure Legend Snippet: Meprin interaction on the cell surface and secretory pathway. A ) Transfection of HeLa cells with meprin α WT and meprin β WT constructs. Cell surface proteins were labeled by primary amine biotinylation, pulled down with streptavidin sepharose beads, and analyzed via immunoblotting. GAPDH and transferrin receptor served as loading controls. B ) Immunofluorescence staining of transfected HeLa cells with meprin β SF and meprin α WT constructs (left panel) or meprin α SF and meprin β WT constructs (right panel). Nuclear staining was visualized using DAPI. Images were taken by confocal fluorescence microscopy. Scale bar, 20 µm. C ) Immunofluorescence staining of transfected HeLa cells using meprin α SF, meprin α C308S SF, and meprin β WT constructs. Cells were treated overnight with brefeldin A at 5 µg/ml or DMSO control. Cell surface staining was performed by excluding saponin from buffers. Nuclear staining was visualized using DAPI. Images were taken by confocal fluorescence microscopy. Scale bars, 20 µm. D ) Transfection of HeLa cells using meprin α SF and meprin β WT constructs. Cells were treated overnight with brefeldin A at 5 µg/ml or DMSO control. Coimmunoprecipitation was performed using Flag-tag antibody against meprin α C terminus or Strep-tag antibody against meprin α N terminus as indicated. Protein fractions were analyzed by immunoblotting. E ) Diagram of meprin α and meprin β interacting in the ER and being transported to the cell surface. Treatment of cells with brefeldin A inhibits the transport from the ER to Golgi apparatus. IP, immunoprecipitation; Mep, meprin.

    Techniques Used: Transfection, Construct, Labeling, Immunofluorescence, Staining, Fluorescence, Microscopy, FLAG-tag, Strep-tag, Immunoprecipitation

    15) Product Images from "Role of Lymphocyte Activation Gene-3 (Lag-3) in Conventional and Regulatory T Cell Function in Allogeneic Transplantation"

    Article Title: Role of Lymphocyte Activation Gene-3 (Lag-3) in Conventional and Regulatory T Cell Function in Allogeneic Transplantation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0086551

    Lag-3 −/− Tcon show increased activation and proliferation. Lethally irradiated Balb/c recipients were transplanted with 5×10 6 TCD-BM and 7.5×10 5 CFSE-labeled WT or Lag-3 −/− Tcon. Donor T cells were re-isolated 3 days after transplant. (A) CFSE histograms of donor CD4 T cells (left panels) and CD8 T cells (right panels) re-isolated from spleen (top), pLN (middle), and MLN (bottom). Histograms are representative of 5 mice/group. Numbers represent percentage of proliferated cells. (B) Bar graphs indicating the percentage of proliferated donor CD4 T cells (left panels) and CD8 T cells (right panels) re-isolated from spleen (top), pLN (middle), and MLN (bottom). Bar depicts mean plus or minus SD, n = 5 mice/group. (C) and (D) Mice were transplanted with 5×10 6 TCD-BM and 7.5×10 5 WT or Lag-3 −/− Tcon. Donor T cells were re-isolated 4 days after transplant. To facilitate retention of intracellular cytokines in vivo , mice were injected i.p. with 250 µg brefeldin A 6 h before spleen and LN harvest. (C) Frequency of indicated activation markers gated on donor CD4 T cells (upper panels) and donor CD8 T cells (lower panels) re-isolated from spleen. (D) Mean fluorescence intensity (MFI) of IFN-γ and IL-10 gated on donor CD4 and CD8 T cells re-isolated from spleen. Bar depicts mean plus or minus SD, each data point represents two pooled mice, n = 4/group. Statistical significance indicated by *(* P
    Figure Legend Snippet: Lag-3 −/− Tcon show increased activation and proliferation. Lethally irradiated Balb/c recipients were transplanted with 5×10 6 TCD-BM and 7.5×10 5 CFSE-labeled WT or Lag-3 −/− Tcon. Donor T cells were re-isolated 3 days after transplant. (A) CFSE histograms of donor CD4 T cells (left panels) and CD8 T cells (right panels) re-isolated from spleen (top), pLN (middle), and MLN (bottom). Histograms are representative of 5 mice/group. Numbers represent percentage of proliferated cells. (B) Bar graphs indicating the percentage of proliferated donor CD4 T cells (left panels) and CD8 T cells (right panels) re-isolated from spleen (top), pLN (middle), and MLN (bottom). Bar depicts mean plus or minus SD, n = 5 mice/group. (C) and (D) Mice were transplanted with 5×10 6 TCD-BM and 7.5×10 5 WT or Lag-3 −/− Tcon. Donor T cells were re-isolated 4 days after transplant. To facilitate retention of intracellular cytokines in vivo , mice were injected i.p. with 250 µg brefeldin A 6 h before spleen and LN harvest. (C) Frequency of indicated activation markers gated on donor CD4 T cells (upper panels) and donor CD8 T cells (lower panels) re-isolated from spleen. (D) Mean fluorescence intensity (MFI) of IFN-γ and IL-10 gated on donor CD4 and CD8 T cells re-isolated from spleen. Bar depicts mean plus or minus SD, each data point represents two pooled mice, n = 4/group. Statistical significance indicated by *(* P

    Techniques Used: Activation Assay, Irradiation, Labeling, Isolation, Mouse Assay, In Vivo, Injection, Fluorescence

    16) Product Images from "CD36 Ecto-domain Phosphorylation Blocks Thrombospondin-1 Binding: Structure - Function Relationships and Regulation by Protein Kinase C"

    Article Title: CD36 Ecto-domain Phosphorylation Blocks Thrombospondin-1 Binding: Structure - Function Relationships and Regulation by Protein Kinase C

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    doi: 10.1161/ATVBAHA.111.242511

    PMA-induced CD36 phosphorylation inhibits TSR-mediated Src recruitment to CD36.  A.  CD36 transfected Bowes melanoma cells were incubated for 16h in low serum media and then treated for 4h with 0.1μg/ml PMA or vehicle control. Some cells were then treated for 1h with 40u/ml alkaline phosphatase (AP) or vehicle control. Cells were then exposed to 100nM recombinant TSR2/3 or thioredoxin control for 15min. CD36 was then immunoprecipitated and the precipitates were analyzed by western blot with an antibody to Src family proteins. Blots were re-probed with anti-CD36 antibodies. Blots were scanned and the amount of co-precipitated Src was normalized to total CD36.  B.  CD36 transfected Bowes melanoma cells were incubated for 16h in low serum media and then treated for 4h with 0.1μg/ml PMA along with either 100μg/ml cycloheximide or 5ng/ml Brefeldin A. Cells were then exposed to 100nM recombinant TSR2/3 or thioredoxin control for 15min. Src family association was then assessed by western blot and analyzed as in panel A. C) Cells prepared as in Panel B were analyzed by western blot using antibodies to phospho-PKCα (upper blot). Blots were then stripped and re-probed with an antibody to total PKC (lower blot). All blots are representative of n=3.
    Figure Legend Snippet: PMA-induced CD36 phosphorylation inhibits TSR-mediated Src recruitment to CD36. A. CD36 transfected Bowes melanoma cells were incubated for 16h in low serum media and then treated for 4h with 0.1μg/ml PMA or vehicle control. Some cells were then treated for 1h with 40u/ml alkaline phosphatase (AP) or vehicle control. Cells were then exposed to 100nM recombinant TSR2/3 or thioredoxin control for 15min. CD36 was then immunoprecipitated and the precipitates were analyzed by western blot with an antibody to Src family proteins. Blots were re-probed with anti-CD36 antibodies. Blots were scanned and the amount of co-precipitated Src was normalized to total CD36. B. CD36 transfected Bowes melanoma cells were incubated for 16h in low serum media and then treated for 4h with 0.1μg/ml PMA along with either 100μg/ml cycloheximide or 5ng/ml Brefeldin A. Cells were then exposed to 100nM recombinant TSR2/3 or thioredoxin control for 15min. Src family association was then assessed by western blot and analyzed as in panel A. C) Cells prepared as in Panel B were analyzed by western blot using antibodies to phospho-PKCα (upper blot). Blots were then stripped and re-probed with an antibody to total PKC (lower blot). All blots are representative of n=3.

    Techniques Used: Transfection, Incubation, Recombinant, Immunoprecipitation, Western Blot

    17) Product Images from "Nlrp12 mutation causes C57BL/6J strain-specific defect in neutrophil recruitment"

    Article Title: Nlrp12 mutation causes C57BL/6J strain-specific defect in neutrophil recruitment

    Journal: Nature Communications

    doi: 10.1038/ncomms13180

    BMDM from C57BL/6J and Nlrp12 −/− mice have defective CXCL1 production. ( a ) WT (B6N) or Nlrp12 −/− mice were infected i.n. with 5 × 10 3 colony-forming unit of F. tularensis LVS. Three days post infection BAL were performed and cytokine levels were determined by ELISA ( n =8, WT; n =9, Nlrp12 −/−) . ( b ) BMDM from WT (B6N) and Nlrp12 −/− mice were challenged with either F. tularensis LVS, S. aureus or P. aeruginosa . Eight hours later supernatants were collected and secretion of the indicated cytokine or chemokine quantified by ELISA. ( c ) BMDM from WT (B6N) and Nlrp12 −/− mice were stimulated with LPS (50 ng ml −1 ) for the indicated amount of time; supernatants were collected and assayed for CXCL1 production by ELISA. ( d ) BMDM from WT (B6N) and Nlrp12 −/− mice were left unstimulated or stimulated for 4 h with LPS in the presence of brefeldin A and monensin; intracellular CXCL1 was then assessed by flow cytometry. ( e ) BMDM from WT (B6N) and Nlrp12 −/− mice were stimulated with LPS for the indicated amount of time; Cxcl1 expression was quantified by real-time PCR. ( f ) BMDM from WT (B6N), WT (B6J) and Nlrp12 −/− mice were stimulated with LPS for 8 h; supernatants were collected and assayed for the indicated cytokines by ELISA. ( g ) BMDM from B6N, B6J, B6N-B6J F1 and B6N-B6J F2 mice were challenged with LPS for 8 h; supernatants were collected and CXCL1 secretion assessed by ELISA. The Nlrp12 allele was sequenced for all B6N-B6J F2 mice and cohorts stratified based on their Nlrp12 genotype ( Nlrp12 B6N/B6N , Nlrp12 B6N/B6J or Nlrp12 B6J/B6J ) ( n =14, B6N; n =13, B6J; n =14, F1; n =52, F2). Pooled data from three ( b , c ) independent experiments are depicted or are representative of two ( d ) or three ( e , f ) independent experiments. ( b , c , e , f ) Data are expressed as the mean±s.e.m. * P
    Figure Legend Snippet: BMDM from C57BL/6J and Nlrp12 −/− mice have defective CXCL1 production. ( a ) WT (B6N) or Nlrp12 −/− mice were infected i.n. with 5 × 10 3 colony-forming unit of F. tularensis LVS. Three days post infection BAL were performed and cytokine levels were determined by ELISA ( n =8, WT; n =9, Nlrp12 −/−) . ( b ) BMDM from WT (B6N) and Nlrp12 −/− mice were challenged with either F. tularensis LVS, S. aureus or P. aeruginosa . Eight hours later supernatants were collected and secretion of the indicated cytokine or chemokine quantified by ELISA. ( c ) BMDM from WT (B6N) and Nlrp12 −/− mice were stimulated with LPS (50 ng ml −1 ) for the indicated amount of time; supernatants were collected and assayed for CXCL1 production by ELISA. ( d ) BMDM from WT (B6N) and Nlrp12 −/− mice were left unstimulated or stimulated for 4 h with LPS in the presence of brefeldin A and monensin; intracellular CXCL1 was then assessed by flow cytometry. ( e ) BMDM from WT (B6N) and Nlrp12 −/− mice were stimulated with LPS for the indicated amount of time; Cxcl1 expression was quantified by real-time PCR. ( f ) BMDM from WT (B6N), WT (B6J) and Nlrp12 −/− mice were stimulated with LPS for 8 h; supernatants were collected and assayed for the indicated cytokines by ELISA. ( g ) BMDM from B6N, B6J, B6N-B6J F1 and B6N-B6J F2 mice were challenged with LPS for 8 h; supernatants were collected and CXCL1 secretion assessed by ELISA. The Nlrp12 allele was sequenced for all B6N-B6J F2 mice and cohorts stratified based on their Nlrp12 genotype ( Nlrp12 B6N/B6N , Nlrp12 B6N/B6J or Nlrp12 B6J/B6J ) ( n =14, B6N; n =13, B6J; n =14, F1; n =52, F2). Pooled data from three ( b , c ) independent experiments are depicted or are representative of two ( d ) or three ( e , f ) independent experiments. ( b , c , e , f ) Data are expressed as the mean±s.e.m. * P

    Techniques Used: Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Expressing, Real-time Polymerase Chain Reaction

    18) Product Images from "A20 Curtails Primary but Augments Secondary CD8+ T Cell Responses in Intracellular Bacterial Infection"

    Article Title: A20 Curtails Primary but Augments Secondary CD8+ T Cell Responses in Intracellular Bacterial Infection

    Journal: Scientific Reports

    doi: 10.1038/srep39796

    Improved primary but impaired secondary CD8 +  T cell response in CD4-Cre A20 fl/fl  mice. CD4-Cre A20 fl/fl  and A20 fl/fl  control mice were infected with a non-lethal dose of Lm OVA and CD8 +  T cell response in spleen was analyzed at the indicated time points. ( a ) Representative dot plots and ( b ) absolute number of H2-Kb SIINFEKL pentamer +  CD8 +  T cells after primary infection (day 0, 7 and 21 p.i.) and after reinfection (day 50 and 53 p.i.) with Lm OVA. ( c ) Representative dot plots and ( d ) absolute number of IFN-γ producing CD8 +  T cells p.i. with Lm OVA and  ex vivo  restimulation with SIINFEKL peptide for 4 h in the presence of Brefeldin A. ( e ) IFN-γ-producing CD8 +  T cells were gated and representative histograms of IFN-γ is displayed for the indicated time points after Lm OVA infection. ( f ) IFN-γ MFI from IFN-γ-producing CD8 +  T cells. ( g ) Representative histograms and ( h ) granzyme B MFI of CD8 +  T cells after Lm OVA infection and  ex vivo  restimulation with SIINFEKL peptide. ( i ) Representative histograms and ( j ) MFI of PD-1 expression on bulk CD8 +  T cells (0 d.p.i.) or Lm OVA-specific CD8 +  T cells (7, 21, 50 and 53 d.p.i.). A representative of 3 independent experiments is shown with 3 mice per group. Error bars indicate + SEM. Student’s  t -test, *p 
    Figure Legend Snippet: Improved primary but impaired secondary CD8 + T cell response in CD4-Cre A20 fl/fl mice. CD4-Cre A20 fl/fl and A20 fl/fl control mice were infected with a non-lethal dose of Lm OVA and CD8 + T cell response in spleen was analyzed at the indicated time points. ( a ) Representative dot plots and ( b ) absolute number of H2-Kb SIINFEKL pentamer + CD8 + T cells after primary infection (day 0, 7 and 21 p.i.) and after reinfection (day 50 and 53 p.i.) with Lm OVA. ( c ) Representative dot plots and ( d ) absolute number of IFN-γ producing CD8 + T cells p.i. with Lm OVA and ex vivo restimulation with SIINFEKL peptide for 4 h in the presence of Brefeldin A. ( e ) IFN-γ-producing CD8 + T cells were gated and representative histograms of IFN-γ is displayed for the indicated time points after Lm OVA infection. ( f ) IFN-γ MFI from IFN-γ-producing CD8 + T cells. ( g ) Representative histograms and ( h ) granzyme B MFI of CD8 + T cells after Lm OVA infection and ex vivo restimulation with SIINFEKL peptide. ( i ) Representative histograms and ( j ) MFI of PD-1 expression on bulk CD8 + T cells (0 d.p.i.) or Lm OVA-specific CD8 + T cells (7, 21, 50 and 53 d.p.i.). A representative of 3 independent experiments is shown with 3 mice per group. Error bars indicate + SEM. Student’s t -test, *p 

    Techniques Used: Mouse Assay, Infection, Ex Vivo, Expressing

    19) Product Images from "KLRG1 Negatively Regulates Natural Killer Cell Functions through the Akt Pathway in Individuals with Chronic Hepatitis C Virus Infection"

    Article Title: KLRG1 Negatively Regulates Natural Killer Cell Functions through the Akt Pathway in Individuals with Chronic Hepatitis C Virus Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.01515-13

    KLRG1 expression is inversely associated with the low levels of IFN-γ production by NK cells in HCV infection. (A) PBMCs from chronically HCV-infected patients and HS were stimulated with rhIL-12 (10 ng/ml) for 18 h and incubated for the last 4 h with brefeldin A (10 μg/ml) to halt cytokine secretion. The cells were immunostained with PE-CD3, PerCP-CD56, APC–IFN-γ, and Alexa Fluor 488-KLRG1, and the IFN-γ production by KLRG1 + and KLRG1 − NK cells was analyzed by flow cytometry. Representative dot plots for IFN-γ production by KLRG1 + and KLRG1 − NK cells, including total CD3 − CD56 + , CD3 − CD56 bright , and CD3 − CD56 dim subsets from an HCV-infected subject and HS. (B) The average percentage of IFN-γ production by the KLRG1 + versus KLRG1 − fraction of total CD3 − CD56 + NK cells, or in the CD3 − CD56 bright and CD3 − CD56 dim subsets. Results are expressed as means ± SD of the percentages of IFN-γ production by NK cells from 24 HCV-infected patients versus 8 HS. *, P
    Figure Legend Snippet: KLRG1 expression is inversely associated with the low levels of IFN-γ production by NK cells in HCV infection. (A) PBMCs from chronically HCV-infected patients and HS were stimulated with rhIL-12 (10 ng/ml) for 18 h and incubated for the last 4 h with brefeldin A (10 μg/ml) to halt cytokine secretion. The cells were immunostained with PE-CD3, PerCP-CD56, APC–IFN-γ, and Alexa Fluor 488-KLRG1, and the IFN-γ production by KLRG1 + and KLRG1 − NK cells was analyzed by flow cytometry. Representative dot plots for IFN-γ production by KLRG1 + and KLRG1 − NK cells, including total CD3 − CD56 + , CD3 − CD56 bright , and CD3 − CD56 dim subsets from an HCV-infected subject and HS. (B) The average percentage of IFN-γ production by the KLRG1 + versus KLRG1 − fraction of total CD3 − CD56 + NK cells, or in the CD3 − CD56 bright and CD3 − CD56 dim subsets. Results are expressed as means ± SD of the percentages of IFN-γ production by NK cells from 24 HCV-infected patients versus 8 HS. *, P

    Techniques Used: Expressing, Infection, Incubation, Flow Cytometry, Cytometry

    KLRG1 expression is positively associated with apoptosis of NK cells following HCV infection. (A) PBMCs from HCV-infected patients and HS were stimulated with 10 ng/ml rhIL-12 for 18 h, followed by 1 μg/ml brefeldin A 4 h prior to harvest of the cells (to block cytokine secretion). PBMCs were immunostained and then analyzed for Annexin V expression on total CD3 − CD56 + and CD3 − CD56 bright and CD3 − CD56 dim NK cells by flow cytometry. Representative dot plots for Annexin V expression on KLRG1 + and KLRG1 − NK cells, including CD3 − CD56 + and the CD3 − CD56 bright and CD3 − CD56 dim subsets from an HCV-infected patient and HS are shown. (B) Average percentage of Annexin V expression from the KLRG1 + versus KLRG1 − cell fraction of total CD3 − CD56 + NK cells and the CD3 − CD56 bright and CD3 − CD56 dim subsets. Results are expressed as means ± SD of the percentages of Annexin V expression by NK cells from 24 HCV-infected patients versus 8 HS. *, P
    Figure Legend Snippet: KLRG1 expression is positively associated with apoptosis of NK cells following HCV infection. (A) PBMCs from HCV-infected patients and HS were stimulated with 10 ng/ml rhIL-12 for 18 h, followed by 1 μg/ml brefeldin A 4 h prior to harvest of the cells (to block cytokine secretion). PBMCs were immunostained and then analyzed for Annexin V expression on total CD3 − CD56 + and CD3 − CD56 bright and CD3 − CD56 dim NK cells by flow cytometry. Representative dot plots for Annexin V expression on KLRG1 + and KLRG1 − NK cells, including CD3 − CD56 + and the CD3 − CD56 bright and CD3 − CD56 dim subsets from an HCV-infected patient and HS are shown. (B) Average percentage of Annexin V expression from the KLRG1 + versus KLRG1 − cell fraction of total CD3 − CD56 + NK cells and the CD3 − CD56 bright and CD3 − CD56 dim subsets. Results are expressed as means ± SD of the percentages of Annexin V expression by NK cells from 24 HCV-infected patients versus 8 HS. *, P

    Techniques Used: Expressing, Infection, Blocking Assay, Flow Cytometry, Cytometry

    20) Product Images from "TIM-3 regulates CD103+ dendritic cell function and response to chemotherapy in breast cancer"

    Article Title: TIM-3 regulates CD103+ dendritic cell function and response to chemotherapy in breast cancer

    Journal: Cancer cell

    doi: 10.1016/j.ccell.2017.11.019

    αTIM-3 increases CXCR3 ligand expression by cDCs (A) Detection of mCherry fluorescence in macrophages and cDCs from PyMTchOVA tumors. Mice were treated with isotype control or αTIM-3 for 7 days prior to analysis. n=9 per group, data pooled over 5 experiments. (B) mRNA expression levels in tumor macrophages and cDCs isolated from mice bearing orthotopically implanted PyMT tumors 2 days following second dose of PTX (day 7). Expression of Cxcl9 , Cxcl10 , Cxcl11 , and Il12b were determined by NanoString, with normalized counts displayed. n=8 per group, data pooled from 2 experiments. (C) Intracellular flow cytometric analysis of CXCL9 in macrophages and cDCs from MMTV-PyMT animals following i.v. injection of brefeldin A for 4-6 hr. Representative staining as well as a fluorescence minus one (FMO) control is shown above. n=7, data pooled over 2 experiments. (D) Tumor volume shown as a relative change from the initiation of chemotherapy (day 0) in MMTV-PyMT animals. Mice were treated with an IgG 2a isotype control, αTIM-3 and/or αCXCR3 antibodies, together with 10 mg/kg PTX as indicated. n=8 mice per group, pooled over 4 cohorts. (E) Orthotopic PyMT tumor volume in C57BL/6J animals treated with an IgG 2a .
    Figure Legend Snippet: αTIM-3 increases CXCR3 ligand expression by cDCs (A) Detection of mCherry fluorescence in macrophages and cDCs from PyMTchOVA tumors. Mice were treated with isotype control or αTIM-3 for 7 days prior to analysis. n=9 per group, data pooled over 5 experiments. (B) mRNA expression levels in tumor macrophages and cDCs isolated from mice bearing orthotopically implanted PyMT tumors 2 days following second dose of PTX (day 7). Expression of Cxcl9 , Cxcl10 , Cxcl11 , and Il12b were determined by NanoString, with normalized counts displayed. n=8 per group, data pooled from 2 experiments. (C) Intracellular flow cytometric analysis of CXCL9 in macrophages and cDCs from MMTV-PyMT animals following i.v. injection of brefeldin A for 4-6 hr. Representative staining as well as a fluorescence minus one (FMO) control is shown above. n=7, data pooled over 2 experiments. (D) Tumor volume shown as a relative change from the initiation of chemotherapy (day 0) in MMTV-PyMT animals. Mice were treated with an IgG 2a isotype control, αTIM-3 and/or αCXCR3 antibodies, together with 10 mg/kg PTX as indicated. n=8 mice per group, pooled over 4 cohorts. (E) Orthotopic PyMT tumor volume in C57BL/6J animals treated with an IgG 2a .

    Techniques Used: Expressing, Fluorescence, Mouse Assay, Isolation, Flow Cytometry, Injection, Staining

    21) Product Images from "TIM-3 regulates CD103+ dendritic cell function and response to chemotherapy in breast cancer"

    Article Title: TIM-3 regulates CD103+ dendritic cell function and response to chemotherapy in breast cancer

    Journal: Cancer cell

    doi: 10.1016/j.ccell.2017.11.019

    αTIM-3 increases CXCR3 ligand expression by cDCs (A) Detection of mCherry fluorescence in macrophages and cDCs from PyMTchOVA tumors. Mice were treated with isotype control or αTIM-3 for 7 days prior to analysis. n=9 per group, data pooled over 5 experiments. (B) mRNA expression levels in tumor macrophages and cDCs isolated from mice bearing orthotopically implanted PyMT tumors 2 days following second dose of PTX (day 7). Expression of Cxcl9 , Cxcl10 , Cxcl11 , and Il12b were determined by NanoString, with normalized counts displayed. n=8 per group, data pooled from 2 experiments. (C) Intracellular flow cytometric analysis of CXCL9 in macrophages and cDCs from MMTV-PyMT animals following i.v. injection of brefeldin A for 4-6 hr. Representative staining as well as a fluorescence minus one (FMO) control is shown above. n=7, data pooled over 2 experiments. (D) Tumor volume shown as a relative change from the initiation of chemotherapy (day 0) in MMTV-PyMT animals. Mice were treated with an IgG 2a isotype control, αTIM-3 and/or αCXCR3 antibodies, together with 10 mg/kg PTX as indicated. n=8 mice per group, pooled over 4 cohorts. (E) Orthotopic PyMT tumor volume in C57BL/6J animals treated with an IgG 2a .
    Figure Legend Snippet: αTIM-3 increases CXCR3 ligand expression by cDCs (A) Detection of mCherry fluorescence in macrophages and cDCs from PyMTchOVA tumors. Mice were treated with isotype control or αTIM-3 for 7 days prior to analysis. n=9 per group, data pooled over 5 experiments. (B) mRNA expression levels in tumor macrophages and cDCs isolated from mice bearing orthotopically implanted PyMT tumors 2 days following second dose of PTX (day 7). Expression of Cxcl9 , Cxcl10 , Cxcl11 , and Il12b were determined by NanoString, with normalized counts displayed. n=8 per group, data pooled from 2 experiments. (C) Intracellular flow cytometric analysis of CXCL9 in macrophages and cDCs from MMTV-PyMT animals following i.v. injection of brefeldin A for 4-6 hr. Representative staining as well as a fluorescence minus one (FMO) control is shown above. n=7, data pooled over 2 experiments. (D) Tumor volume shown as a relative change from the initiation of chemotherapy (day 0) in MMTV-PyMT animals. Mice were treated with an IgG 2a isotype control, αTIM-3 and/or αCXCR3 antibodies, together with 10 mg/kg PTX as indicated. n=8 mice per group, pooled over 4 cohorts. (E) Orthotopic PyMT tumor volume in C57BL/6J animals treated with an IgG 2a .

    Techniques Used: Expressing, Fluorescence, Mouse Assay, Isolation, Flow Cytometry, Injection, Staining

    22) Product Images from "TIM-3 regulates CD103+ dendritic cell function and response to chemotherapy in breast cancer"

    Article Title: TIM-3 regulates CD103+ dendritic cell function and response to chemotherapy in breast cancer

    Journal: Cancer cell

    doi: 10.1016/j.ccell.2017.11.019

    αTIM-3 increases CXCR3 ligand expression by cDCs (A) Detection of mCherry fluorescence in macrophages and cDCs from PyMTchOVA tumors. Mice were treated with isotype control or αTIM-3 for 7 days prior to analysis. n=9 per group, data pooled over 5 experiments. (B) mRNA expression levels in tumor macrophages and cDCs isolated from mice bearing orthotopically implanted PyMT tumors 2 days following second dose of PTX (day 7). Expression of Cxcl9 , Cxcl10 , Cxcl11 , and Il12b were determined by NanoString, with normalized counts displayed. n=8 per group, data pooled from 2 experiments. (C) Intracellular flow cytometric analysis of CXCL9 in macrophages and cDCs from MMTV-PyMT animals following i.v. injection of brefeldin A for 4-6 hr. Representative staining as well as a fluorescence minus one (FMO) control is shown above. n=7, data pooled over 2 experiments. (D) Tumor volume shown as a relative change from the initiation of chemotherapy (day 0) in MMTV-PyMT animals. Mice were treated with an IgG 2a isotype control, αTIM-3 and/or αCXCR3 antibodies, together with 10 mg/kg PTX as indicated. n=8 mice per group, pooled over 4 cohorts. (E) Orthotopic PyMT tumor volume in C57BL/6J animals treated with an IgG 2a .
    Figure Legend Snippet: αTIM-3 increases CXCR3 ligand expression by cDCs (A) Detection of mCherry fluorescence in macrophages and cDCs from PyMTchOVA tumors. Mice were treated with isotype control or αTIM-3 for 7 days prior to analysis. n=9 per group, data pooled over 5 experiments. (B) mRNA expression levels in tumor macrophages and cDCs isolated from mice bearing orthotopically implanted PyMT tumors 2 days following second dose of PTX (day 7). Expression of Cxcl9 , Cxcl10 , Cxcl11 , and Il12b were determined by NanoString, with normalized counts displayed. n=8 per group, data pooled from 2 experiments. (C) Intracellular flow cytometric analysis of CXCL9 in macrophages and cDCs from MMTV-PyMT animals following i.v. injection of brefeldin A for 4-6 hr. Representative staining as well as a fluorescence minus one (FMO) control is shown above. n=7, data pooled over 2 experiments. (D) Tumor volume shown as a relative change from the initiation of chemotherapy (day 0) in MMTV-PyMT animals. Mice were treated with an IgG 2a isotype control, αTIM-3 and/or αCXCR3 antibodies, together with 10 mg/kg PTX as indicated. n=8 mice per group, pooled over 4 cohorts. (E) Orthotopic PyMT tumor volume in C57BL/6J animals treated with an IgG 2a .

    Techniques Used: Expressing, Fluorescence, Mouse Assay, Isolation, Flow Cytometry, Injection, Staining

    23) Product Images from "The adaptor molecule CD2AP in CD4 T cells modulates differentiation of follicular helper T cells during chronic LCMV infection"

    Article Title: The adaptor molecule CD2AP in CD4 T cells modulates differentiation of follicular helper T cells during chronic LCMV infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007053

    Cd2ap -deficiency causes sustained TCR signaling specifically in T H 1 cells in vitro . (A) Schematic of experimental strategy for culture of CD4 T cells. (B) Concentration of IFN-γ or IL-4 in supernatant measured by ELISA 24 hours after re-stimulation of polarized Cd2ap F/F and Cd4 -cre + Cd2ap F/F T H 1 cells and T H 2 cells with plate-bound anti-CD3/anti-CD28 antibodies. (C) Immunoblotting showing phosphorylation of MEK1/2 in polarized Cd2ap F/F and Cd4 -cre + Cd2ap F/F T H 1 or T H 2 cells following re-stimulation with plate-bound anti-CD3/anti-CD28 for the indicated times at 37°C. Anti-CD2AP, -CIN85 and ERK were used as control. Data are representative of three experiments. (D) Downregulation of surface TCR of Cd2ap F/F and Cd4 -cre + Cd2ap F/F T cells that were polarized under indicated conditions. Cells were stimulated with plate-bound anti-CD3 and surface TCR levels were quantitated at the indicated time points. Data are representative of three experiments. (E, F) Intracellular staining for IFN-γ and TNF-α of polarized Cd2ap F/F and Cd4 -cre + Cd2ap F/F T H 1 cells after re-stimulation with PMA and Ionomycin or plate-bound anti-CD3 and anti-CD28 for 4 or 24 hours in the presence of Brefeldin A for the last 2 hours before harvest. Representative plots (E) and statistical analyses with means and standard deviations (F) from three experiments are shown.
    Figure Legend Snippet: Cd2ap -deficiency causes sustained TCR signaling specifically in T H 1 cells in vitro . (A) Schematic of experimental strategy for culture of CD4 T cells. (B) Concentration of IFN-γ or IL-4 in supernatant measured by ELISA 24 hours after re-stimulation of polarized Cd2ap F/F and Cd4 -cre + Cd2ap F/F T H 1 cells and T H 2 cells with plate-bound anti-CD3/anti-CD28 antibodies. (C) Immunoblotting showing phosphorylation of MEK1/2 in polarized Cd2ap F/F and Cd4 -cre + Cd2ap F/F T H 1 or T H 2 cells following re-stimulation with plate-bound anti-CD3/anti-CD28 for the indicated times at 37°C. Anti-CD2AP, -CIN85 and ERK were used as control. Data are representative of three experiments. (D) Downregulation of surface TCR of Cd2ap F/F and Cd4 -cre + Cd2ap F/F T cells that were polarized under indicated conditions. Cells were stimulated with plate-bound anti-CD3 and surface TCR levels were quantitated at the indicated time points. Data are representative of three experiments. (E, F) Intracellular staining for IFN-γ and TNF-α of polarized Cd2ap F/F and Cd4 -cre + Cd2ap F/F T H 1 cells after re-stimulation with PMA and Ionomycin or plate-bound anti-CD3 and anti-CD28 for 4 or 24 hours in the presence of Brefeldin A for the last 2 hours before harvest. Representative plots (E) and statistical analyses with means and standard deviations (F) from three experiments are shown.

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

    24) Product Images from "Selective targeting of pro-inflammatory Th1 cells by microRNA-148a-specific antagomirs in vivo"

    Article Title: Selective targeting of pro-inflammatory Th1 cells by microRNA-148a-specific antagomirs in vivo

    Journal: Journal of Autoimmunity

    doi: 10.1016/j.jaut.2017.11.005

    Systemic antagomir-148a treatment of colitic mice depletes IFN-γ-expressing Th1 cells selectively in the inflamed colon.  (A) Schematic overview depicting the experimental procedure for inducing colitis by adoptive transfer of repeatedly activated Th1 cells into  Rag1 −/−  mice and subsequent antagomir treatment. (B) Representative dot plots showing the frequencies of CD3 + CD4 +  Th cells isolated from inflamed colons of colitic mice that were treated with antagomir-Scr or antagomir-148a. Displayed frequencies are percentages of Th cells among isolated viable cells. (C) Total cell numbers of viable CD3 + CD4 +  Th cells that were isolated from the colons and spleens of colitic mice following antagomir-148a or antagomir-Scr treatment as shown in (A). (D–G) Lymphocytes from colitic mice were isolated from the inflamed colons and stimulated with PMA/ionomycin in the presence of Brefeldin A. Shown are the absolute cell numbers of CD3 + CD4 +  Th cells that expressed IFN-γ (D), IL-10 (E), IL-17A (F) or IL-22 (G). (H) Quantification of different myeloid subsets isolated from inflamed colonic mucosae of colitic mice. Depicted data are pooled from two independent experiments with n = 12 and n = 13 for antagomir-148a- and antagomir-Scr-treated mice, respectively.
    Figure Legend Snippet: Systemic antagomir-148a treatment of colitic mice depletes IFN-γ-expressing Th1 cells selectively in the inflamed colon. (A) Schematic overview depicting the experimental procedure for inducing colitis by adoptive transfer of repeatedly activated Th1 cells into Rag1 −/− mice and subsequent antagomir treatment. (B) Representative dot plots showing the frequencies of CD3 + CD4 + Th cells isolated from inflamed colons of colitic mice that were treated with antagomir-Scr or antagomir-148a. Displayed frequencies are percentages of Th cells among isolated viable cells. (C) Total cell numbers of viable CD3 + CD4 + Th cells that were isolated from the colons and spleens of colitic mice following antagomir-148a or antagomir-Scr treatment as shown in (A). (D–G) Lymphocytes from colitic mice were isolated from the inflamed colons and stimulated with PMA/ionomycin in the presence of Brefeldin A. Shown are the absolute cell numbers of CD3 + CD4 + Th cells that expressed IFN-γ (D), IL-10 (E), IL-17A (F) or IL-22 (G). (H) Quantification of different myeloid subsets isolated from inflamed colonic mucosae of colitic mice. Depicted data are pooled from two independent experiments with n = 12 and n = 13 for antagomir-148a- and antagomir-Scr-treated mice, respectively.

    Techniques Used: Mouse Assay, Expressing, Adoptive Transfer Assay, Isolation

    25) Product Images from "Deletion of F4L (ribonucleotide reductase) in vaccinia virus produces a selective oncolytic virus and promotes anti‐tumor immunity with superior safety in bladder cancer models"

    Article Title: Deletion of F4L (ribonucleotide reductase) in vaccinia virus produces a selective oncolytic virus and promotes anti‐tumor immunity with superior safety in bladder cancer models

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201607296

    VACV  activates immune responses in rats bearing  AY ‐27 bladder tumors VACV‐neutralizing antibodies were measured in virus‐treated rats 15 and 35 days after implantation ( n  = 4–5 rats per group). Protection from subcutaneous tumor challenge after virus‐induced tumor clearance. AY‐27 cells were implanted in the flanks of cured rats ( n  = 6) and naïve age‐matched control rats ( n  = 4). T‐cell proliferation after co‐culturing with bone marrow‐derived dendritic cells (BMDCs). CD4 +  and CD8 +  cells were co‐cultured with BMDCs and proliferation assayed with CellTrace Violet. The representative plots show CD4 +  and CD8 +  T‐cell proliferation after co‐culture with either mock‐pulsed (C) or with tumor‐lysate‐pulsed BMDCs (D). Panel (E) shows the percentage of CD4 +  and CD8 +  T cells that proliferated in response to BMDC stimulation ( n  = 3). Ex vivo  upregulation of CD107a by CD8 +  T cells from challenged rats. (F) CD3 +  cells were incubated +/− BMDCs for 1 h in the presence of anti‐CD107a antibody, incubated for 5 h with monensin and brefeldin A, and then stained with anti‐CD4 and anti‐CD8 antibodies. Events were gated for viable CD8 +  T cells. Panel (G) shows the percentage of CD107a +  CD8 +  T cells +/− BMDC stimulation ( n  = 3). IFN‐γ released after 24‐h co‐culture of CD3 +  cells with BMDCs ( n  = 3–5). T cells activated  ex vivo  by tumor‐lysate‐pulsed DCs are cytotoxic. After 6 days of co‐culture with BMDC, CD3 +  cells were incubated for 18 h with 10,000 target cells and at different effector‐to‐target ratios. Lysis was determined by LDH assay. RK3E are normal rat kidney cells ( n  = 2–3 performed in duplicate). Data information: Mean ± SEM is shown. Two‐way ANOVA followed by Tukey's multiple comparison test was used in (A), (B), and (H). For (A), significance was determined against the ∆ J2R  group. Two‐tailed Student's  t‐ test was used in (E) and (G).
    Figure Legend Snippet: VACV activates immune responses in rats bearing AY ‐27 bladder tumors VACV‐neutralizing antibodies were measured in virus‐treated rats 15 and 35 days after implantation ( n  = 4–5 rats per group). Protection from subcutaneous tumor challenge after virus‐induced tumor clearance. AY‐27 cells were implanted in the flanks of cured rats ( n  = 6) and naïve age‐matched control rats ( n  = 4). T‐cell proliferation after co‐culturing with bone marrow‐derived dendritic cells (BMDCs). CD4 + and CD8 + cells were co‐cultured with BMDCs and proliferation assayed with CellTrace Violet. The representative plots show CD4 + and CD8 + T‐cell proliferation after co‐culture with either mock‐pulsed (C) or with tumor‐lysate‐pulsed BMDCs (D). Panel (E) shows the percentage of CD4 + and CD8 + T cells that proliferated in response to BMDC stimulation ( n  = 3). Ex vivo upregulation of CD107a by CD8 + T cells from challenged rats. (F) CD3 + cells were incubated +/− BMDCs for 1 h in the presence of anti‐CD107a antibody, incubated for 5 h with monensin and brefeldin A, and then stained with anti‐CD4 and anti‐CD8 antibodies. Events were gated for viable CD8 + T cells. Panel (G) shows the percentage of CD107a + CD8 + T cells +/− BMDC stimulation ( n  = 3). IFN‐γ released after 24‐h co‐culture of CD3 + cells with BMDCs ( n  = 3–5). T cells activated ex vivo by tumor‐lysate‐pulsed DCs are cytotoxic. After 6 days of co‐culture with BMDC, CD3 + cells were incubated for 18 h with 10,000 target cells and at different effector‐to‐target ratios. Lysis was determined by LDH assay. RK3E are normal rat kidney cells ( n  = 2–3 performed in duplicate). Data information: Mean ± SEM is shown. Two‐way ANOVA followed by Tukey's multiple comparison test was used in (A), (B), and (H). For (A), significance was determined against the ∆ J2R group. Two‐tailed Student's t‐ test was used in (E) and (G).

    Techniques Used: Derivative Assay, Cell Culture, Co-Culture Assay, Ex Vivo, Incubation, Staining, Lysis, Lactate Dehydrogenase Assay, Two Tailed Test

    26) Product Images from "Selective targeting of pro-inflammatory Th1 cells by microRNA-148a-specific antagomirs in vivo"

    Article Title: Selective targeting of pro-inflammatory Th1 cells by microRNA-148a-specific antagomirs in vivo

    Journal: Journal of Autoimmunity

    doi: 10.1016/j.jaut.2017.11.005

    Systemic antagomir-148a treatment of colitic mice depletes IFN-γ-expressing Th1 cells selectively in the inflamed colon.  (A) Schematic overview depicting the experimental procedure for inducing colitis by adoptive transfer of repeatedly activated Th1 cells into  Rag1 −/−  mice and subsequent antagomir treatment. (B) Representative dot plots showing the frequencies of CD3 + CD4 +  Th cells isolated from inflamed colons of colitic mice that were treated with antagomir-Scr or antagomir-148a. Displayed frequencies are percentages of Th cells among isolated viable cells. (C) Total cell numbers of viable CD3 + CD4 +  Th cells that were isolated from the colons and spleens of colitic mice following antagomir-148a or antagomir-Scr treatment as shown in (A). (D–G) Lymphocytes from colitic mice were isolated from the inflamed colons and stimulated with PMA/ionomycin in the presence of Brefeldin A. Shown are the absolute cell numbers of CD3 + CD4 +  Th cells that expressed IFN-γ (D), IL-10 (E), IL-17A (F) or IL-22 (G). (H) Quantification of different myeloid subsets isolated from inflamed colonic mucosae of colitic mice. Depicted data are pooled from two independent experiments with n = 12 and n = 13 for antagomir-148a- and antagomir-Scr-treated mice, respectively.
    Figure Legend Snippet: Systemic antagomir-148a treatment of colitic mice depletes IFN-γ-expressing Th1 cells selectively in the inflamed colon. (A) Schematic overview depicting the experimental procedure for inducing colitis by adoptive transfer of repeatedly activated Th1 cells into Rag1 −/− mice and subsequent antagomir treatment. (B) Representative dot plots showing the frequencies of CD3 + CD4 + Th cells isolated from inflamed colons of colitic mice that were treated with antagomir-Scr or antagomir-148a. Displayed frequencies are percentages of Th cells among isolated viable cells. (C) Total cell numbers of viable CD3 + CD4 + Th cells that were isolated from the colons and spleens of colitic mice following antagomir-148a or antagomir-Scr treatment as shown in (A). (D–G) Lymphocytes from colitic mice were isolated from the inflamed colons and stimulated with PMA/ionomycin in the presence of Brefeldin A. Shown are the absolute cell numbers of CD3 + CD4 + Th cells that expressed IFN-γ (D), IL-10 (E), IL-17A (F) or IL-22 (G). (H) Quantification of different myeloid subsets isolated from inflamed colonic mucosae of colitic mice. Depicted data are pooled from two independent experiments with n = 12 and n = 13 for antagomir-148a- and antagomir-Scr-treated mice, respectively.

    Techniques Used: Mouse Assay, Expressing, Adoptive Transfer Assay, Isolation

    27) Product Images from "ATP release drives heightened immune responses associated with hypertension"

    Article Title: ATP release drives heightened immune responses associated with hypertension

    Journal: Science immunology

    doi: 10.1126/sciimmunol.aau6426

    Blocking CD86 eliminates the overactivation of immune responses in hypertensive mice. C57BL/6 mice were sham-treated (normotensive) or Ang Il-treated (hypertensive) for 2 weeks. ( A ) Splenic DCs were purified. Some of the cells were pulsed with SIINFEKL peptide. Some cells were also treated with an anti-CD80 antibody or an anti-CD86 antibody or both. They were then coincubated with OT-I T cells, and the expression of T cell CD69 and IFN-γ was measured. ( B  and  C ) Mice were regularly treated with an anti-CD86 antibody or an isotype control antibody from 3 days before being immunized with OVA until sacrifice. Seven days after immunization, the numbers of OVA-specific CD8 +  T cells in the blood (B) and splenocyte IL-2 production after SIINFEKL restimulation were measured (C). ( D  and  E ) Acute hepatitis was induced by intravenous injection of ConA (5 mg/kg) into normotensive and hypertensive mice. Some hypertensive mice were injected intraperitoneally with either CD86 blocking antibody (α-CD86) or an isotype control antibody (IgG) every other day for three times before ConA administration. (D) Blood ALT levels were measured 6 hours after ConA injection. (E) Leukocytes in the liver were isolated with Percoll gradients. Cells were cultured in medium for 6 hours in the presence of brefeldin A, and then intra-cellular staining was performed to examine the production of IFN-γ and TNF-γ by CD4 +  T cells. Data are means ± SEM.  *P
    Figure Legend Snippet: Blocking CD86 eliminates the overactivation of immune responses in hypertensive mice. C57BL/6 mice were sham-treated (normotensive) or Ang Il-treated (hypertensive) for 2 weeks. ( A ) Splenic DCs were purified. Some of the cells were pulsed with SIINFEKL peptide. Some cells were also treated with an anti-CD80 antibody or an anti-CD86 antibody or both. They were then coincubated with OT-I T cells, and the expression of T cell CD69 and IFN-γ was measured. ( B and C ) Mice were regularly treated with an anti-CD86 antibody or an isotype control antibody from 3 days before being immunized with OVA until sacrifice. Seven days after immunization, the numbers of OVA-specific CD8 + T cells in the blood (B) and splenocyte IL-2 production after SIINFEKL restimulation were measured (C). ( D and E ) Acute hepatitis was induced by intravenous injection of ConA (5 mg/kg) into normotensive and hypertensive mice. Some hypertensive mice were injected intraperitoneally with either CD86 blocking antibody (α-CD86) or an isotype control antibody (IgG) every other day for three times before ConA administration. (D) Blood ALT levels were measured 6 hours after ConA injection. (E) Leukocytes in the liver were isolated with Percoll gradients. Cells were cultured in medium for 6 hours in the presence of brefeldin A, and then intra-cellular staining was performed to examine the production of IFN-γ and TNF-γ by CD4 + T cells. Data are means ± SEM. *P

    Techniques Used: Blocking Assay, Mouse Assay, Purification, Expressing, Injection, Isolation, Cell Culture, Staining

    Hypertensive mice are predisposed to autoimmune diseases. ( A  to  C ) RIP-mOVA mice were made hypertensive by treatment with either Ang II or L-NAME. After 2 to 3 weeks, when hypertension was established, 5 × 10 6  OT-I T cells (CD8 +  T cells from OT-I transgenic mice) were transferred intravenously into normotensive and hypertensive RIP-mOVA mice. (A) Blood glucose levels were measured for 2 weeks after OT-I T cell transfer. (B and C) Two weeks after OT-I T cell transfer, the percentages of OT-I T cells among CD8 +  T cells in the blood, spleen, and pancreatic draining lymph nodes (DLN) were quantified by tetramer analysis (B), and the numbers of inflamed islets, identified by anti-CD3 staining, were counted in a blinded fashion (C). ( D  to  F ) ConA (5 mg/kg) was injected intravenously into normotensive and hypertensive mice. Blood ALT levels were measured after 6 hours. (D) Hepatic inflammatory cells were prepared by tissue enzymatic digestion followed by Percoll centrifugation. (E) Cells were cultured in medium for 6 hours in the presence of brefeldin A, and then intracellular staining was performed to examine the production of IFN-γ and TNF-α by CD4 +  T cells. Liver necrosis area was measured after hematoxylin and eosin staining. MFI, mean fluorescence intensity. (F) For each liver, necrosis area represents the average of 10 separate fields. In (A) to (C),  n  = 17, 6, and 12 for normotensive, Ang II-treated, and L-NAME-treated mice, respectively. Data are means ± SEM.  *P
    Figure Legend Snippet: Hypertensive mice are predisposed to autoimmune diseases. ( A to C ) RIP-mOVA mice were made hypertensive by treatment with either Ang II or L-NAME. After 2 to 3 weeks, when hypertension was established, 5 × 10 6 OT-I T cells (CD8 + T cells from OT-I transgenic mice) were transferred intravenously into normotensive and hypertensive RIP-mOVA mice. (A) Blood glucose levels were measured for 2 weeks after OT-I T cell transfer. (B and C) Two weeks after OT-I T cell transfer, the percentages of OT-I T cells among CD8 + T cells in the blood, spleen, and pancreatic draining lymph nodes (DLN) were quantified by tetramer analysis (B), and the numbers of inflamed islets, identified by anti-CD3 staining, were counted in a blinded fashion (C). ( D to F ) ConA (5 mg/kg) was injected intravenously into normotensive and hypertensive mice. Blood ALT levels were measured after 6 hours. (D) Hepatic inflammatory cells were prepared by tissue enzymatic digestion followed by Percoll centrifugation. (E) Cells were cultured in medium for 6 hours in the presence of brefeldin A, and then intracellular staining was performed to examine the production of IFN-γ and TNF-α by CD4 + T cells. Liver necrosis area was measured after hematoxylin and eosin staining. MFI, mean fluorescence intensity. (F) For each liver, necrosis area represents the average of 10 separate fields. In (A) to (C), n = 17, 6, and 12 for normotensive, Ang II-treated, and L-NAME-treated mice, respectively. Data are means ± SEM. *P

    Techniques Used: Mouse Assay, Transgenic Assay, Staining, Injection, Centrifugation, Cell Culture, Fluorescence

    APCs from hypertensive mice present antigens more efficiently. ( A  and  B ) Splenic DCs and peritoneal macrophages (PM) from normotensive or Ang Il-induced hypertensive C57BL/6 mice were pulsed with 10 μM OVA (A) or 50 pM SIINFEKL peptide (SKL) (B) in vitro and were then coincubated with OT-I T cells. The percentages of CD69 +  (after 4 hours) or IFN-γ +  cells (after 6 hours in the presence of brefeldin A) among OT-I T cells were measured by flow cytometry. Representative flow cytometry dot plots of IFN-γ and CD69 expression are shown. ( C ) Splenic DCs from normotensive or L-NAME-induced hypertensive mice were pulsed with SIINFEKL and then coincubated with OT-I T cells. The percentages of CD69 +  or IFN-γ +  cells among all CD8 +  T cells were measured. ( D ) Splenic DCs from normotensive or Ang II-induced hypertensive mice were loaded with SIINFEKL for 4 hours ex vivo. Cells were then transferred intravenously into naїve C57BL/6 mice. Seven days later, splenocytes from the recipients were restimulated with SIINFEKL and the secretion of IL-2 and IFN-γ was measured. Data are means ± SEM. * P
    Figure Legend Snippet: APCs from hypertensive mice present antigens more efficiently. ( A and B ) Splenic DCs and peritoneal macrophages (PM) from normotensive or Ang Il-induced hypertensive C57BL/6 mice were pulsed with 10 μM OVA (A) or 50 pM SIINFEKL peptide (SKL) (B) in vitro and were then coincubated with OT-I T cells. The percentages of CD69 + (after 4 hours) or IFN-γ + cells (after 6 hours in the presence of brefeldin A) among OT-I T cells were measured by flow cytometry. Representative flow cytometry dot plots of IFN-γ and CD69 expression are shown. ( C ) Splenic DCs from normotensive or L-NAME-induced hypertensive mice were pulsed with SIINFEKL and then coincubated with OT-I T cells. The percentages of CD69 + or IFN-γ + cells among all CD8 + T cells were measured. ( D ) Splenic DCs from normotensive or Ang II-induced hypertensive mice were loaded with SIINFEKL for 4 hours ex vivo. Cells were then transferred intravenously into naїve C57BL/6 mice. Seven days later, splenocytes from the recipients were restimulated with SIINFEKL and the secretion of IL-2 and IFN-γ was measured. Data are means ± SEM. * P

    Techniques Used: Mouse Assay, In Vitro, Flow Cytometry, Cytometry, Expressing, Ex Vivo

    28) Product Images from "Restoration of the type I IFN–IL-1 balance through targeted blockade of PTGER4 inhibits autoimmunity in NOD mice"

    Article Title: Restoration of the type I IFN–IL-1 balance through targeted blockade of PTGER4 inhibits autoimmunity in NOD mice

    Journal: JCI Insight

    doi: 10.1172/jci.insight.97843

    Lower IFN response genes correlate with diabetes pathogenesis, and blocking IFNAR in prediabetic NOD mice accelerates diabetes and increases Th1 responses. ( A ) Type I IFN gene expression in sorted splenic moDCs from age-matched NOD and B6.g7 mice. ( B ) NOD mice were treated i.p. with anti-IFNAR1 antibody or control monoclonal antibody 2 times/week from 6–8 weeks of age. The percentage of diabetes-free mice was recorded and compared between the anti–IFNAR antibody–treated group and untreated control group. Statistical analysis was performed with log-rank test. Summation of 2 experiments with n = 16 mice ( C ) Intracellular IFN-γ staining in lymph node–derived CD4 + T cells, after PMA plus ionomycin (Ion) stimulation (total 6 hours), where brefeldin A was added for the last 4 hours. ( D ) Counts of intracellular CD4 + IFN-γ + T cells in lymph node after PMA plus Ion stimulation. Data were pooled from 2 independent experiments, with 5–6 mice per group ( A , C , and D ). Values show individual mice, with mean ± SD. P values in A , C and D were calculated using t test. *P
    Figure Legend Snippet: Lower IFN response genes correlate with diabetes pathogenesis, and blocking IFNAR in prediabetic NOD mice accelerates diabetes and increases Th1 responses. ( A ) Type I IFN gene expression in sorted splenic moDCs from age-matched NOD and B6.g7 mice. ( B ) NOD mice were treated i.p. with anti-IFNAR1 antibody or control monoclonal antibody 2 times/week from 6–8 weeks of age. The percentage of diabetes-free mice was recorded and compared between the anti–IFNAR antibody–treated group and untreated control group. Statistical analysis was performed with log-rank test. Summation of 2 experiments with n = 16 mice ( C ) Intracellular IFN-γ staining in lymph node–derived CD4 + T cells, after PMA plus ionomycin (Ion) stimulation (total 6 hours), where brefeldin A was added for the last 4 hours. ( D ) Counts of intracellular CD4 + IFN-γ + T cells in lymph node after PMA plus Ion stimulation. Data were pooled from 2 independent experiments, with 5–6 mice per group ( A , C , and D ). Values show individual mice, with mean ± SD. P values in A , C and D were calculated using t test. *P

    Techniques Used: Blocking Assay, Mouse Assay, Expressing, Staining, Derivative Assay

    29) Product Images from "CD84 regulates PD-1/PD-L1 expression and function in chronic lymphocytic leukemia"

    Article Title: CD84 regulates PD-1/PD-L1 expression and function in chronic lymphocytic leukemia

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI96610

    Reduced abundance of exhausted CD8 + T cells in the CD84 –/– environment. Eμ-TCL1 splenocytes (4 × 10 7 ) were injected i.v. into the tail vein of C57BL/6 WT or CD84 –/– mice. ( A – D ) After 14 to 21 days, the mice were sacrificed, and expression of the exhaustion markers PD-1, LAG-3, CTLA-4, 2B4, and KLRG1 were determined on CD8 + T cells in ( A ) spleen ( n = 6–11) histograms show representative results; ( B ) PB ( n = 5–11); ( C ) the peritoneal cavity ( n = 6–10); and ( D ) BM ( n = 4–7). ( E ) After 14 to 21 days, the mice were sacrificed, and splenic cells were cultured for 24 hours with anti-CD3 and then with brefeldin A for the last 2 hours of culture. CD8 + T cells were then analyzed for IFN-γ, IL-2, GZMB, and LAMP-1 expression ( n = 7–8). Dot plot show representative results of granzyme b expression. ( F and G ) BM cells (5 × 10 6 ) derived from 8-week-old Eμ-TCL1 mice or negative control littermates (WT) were injected into lethally irradiated C57BL/6 (WT) or CD84-deficient ( CD84 −/− ) mice. After 6 months, the mice were euthanized, and the expression of PD-1 was determined on CD8 + T cells in the peritoneum ( F ) and spleen ( G ) ( n = 3–4). * P
    Figure Legend Snippet: Reduced abundance of exhausted CD8 + T cells in the CD84 –/– environment. Eμ-TCL1 splenocytes (4 × 10 7 ) were injected i.v. into the tail vein of C57BL/6 WT or CD84 –/– mice. ( A – D ) After 14 to 21 days, the mice were sacrificed, and expression of the exhaustion markers PD-1, LAG-3, CTLA-4, 2B4, and KLRG1 were determined on CD8 + T cells in ( A ) spleen ( n = 6–11) histograms show representative results; ( B ) PB ( n = 5–11); ( C ) the peritoneal cavity ( n = 6–10); and ( D ) BM ( n = 4–7). ( E ) After 14 to 21 days, the mice were sacrificed, and splenic cells were cultured for 24 hours with anti-CD3 and then with brefeldin A for the last 2 hours of culture. CD8 + T cells were then analyzed for IFN-γ, IL-2, GZMB, and LAMP-1 expression ( n = 7–8). Dot plot show representative results of granzyme b expression. ( F and G ) BM cells (5 × 10 6 ) derived from 8-week-old Eμ-TCL1 mice or negative control littermates (WT) were injected into lethally irradiated C57BL/6 (WT) or CD84-deficient ( CD84 −/− ) mice. After 6 months, the mice were euthanized, and the expression of PD-1 was determined on CD8 + T cells in the peritoneum ( F ) and spleen ( G ) ( n = 3–4). * P

    Techniques Used: Injection, Mouse Assay, Expressing, Cell Culture, Derivative Assay, Negative Control, Irradiation

    30) Product Images from "The contribution of arachidonate 15-lipoxygenase in tissue macrophages to adipose tissue remodeling"

    Article Title: The contribution of arachidonate 15-lipoxygenase in tissue macrophages to adipose tissue remodeling

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.190

    In vitro co-culture recapitulates adipocyte clearance by M2-like adipose tissue macrophages. ( a ) Immunoblot analysis of caspase-3 activation and perilipin1 expression in differentiated adipocytes from C3H10T1/2 cultures treated with Brefeldin A (5 μ M) up to 24 h. β -actin was used as a loading control. ( b ). Analysis of ability of adipose tissue macrophages to clear apoptotic adipocytes in in vitro co-culture system. Representative images and quantitative analysis of fluorescence intensity of green (DiO-macrophages) and red (BODIPY-lipid) are shown. ( c ) Quantitative analysis of fluorescence intensity of red (BODIPY-lipid) and green (DiO-macrophages) to compare adipocyte clearance rates between groups. Quantifications are representative of three individual experiments. ( d ) qPCR analysis of efferocytosis-related genes in adipose tissue macrophages co-cultured with dying or live adipocytes, and macrophages cultured without adipocytes (no AC) . Error bars indicated S.E.M. of three individual experiments (in comparison to GM + dying adipocytes controls, * P
    Figure Legend Snippet: In vitro co-culture recapitulates adipocyte clearance by M2-like adipose tissue macrophages. ( a ) Immunoblot analysis of caspase-3 activation and perilipin1 expression in differentiated adipocytes from C3H10T1/2 cultures treated with Brefeldin A (5 μ M) up to 24 h. β -actin was used as a loading control. ( b ). Analysis of ability of adipose tissue macrophages to clear apoptotic adipocytes in in vitro co-culture system. Representative images and quantitative analysis of fluorescence intensity of green (DiO-macrophages) and red (BODIPY-lipid) are shown. ( c ) Quantitative analysis of fluorescence intensity of red (BODIPY-lipid) and green (DiO-macrophages) to compare adipocyte clearance rates between groups. Quantifications are representative of three individual experiments. ( d ) qPCR analysis of efferocytosis-related genes in adipose tissue macrophages co-cultured with dying or live adipocytes, and macrophages cultured without adipocytes (no AC) . Error bars indicated S.E.M. of three individual experiments (in comparison to GM + dying adipocytes controls, * P

    Techniques Used: In Vitro, Co-Culture Assay, Activation Assay, Expressing, Fluorescence, Real-time Polymerase Chain Reaction, Cell Culture

    31) Product Images from "Endogenous IL-22 Plays a Dual Role in Arthritis: Regulation of Established Arthritis via IFN-? Responses"

    Article Title: Endogenous IL-22 Plays a Dual Role in Arthritis: Regulation of Established Arthritis via IFN-? Responses

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093279

    Neutralization of IL-22 prior to onset of arthritis increases incidence and severity of arthritis. A: 16 DBA mice were immunized with collagen and CFA. On day 18 following immunization, mice were divided into 2 groups (8 mice per group) to receive either anti-IL-22 antibody (100 ug/mice/day) or isotype control (100 ug/mouse/day) intraperitoneally for 8–10 days. Mice were scored for clinical arthritis every other day. Data shows incidence and cumulative arthritis score in 2 groups over time. B: Histologic scoring of paws for various aspects of joint damage and inflammation assessed after H E staining. Data is from 4 mice per group. C: Hematoxylin and eosin staining of representative paws from the two groups of mice, magnification of 10×. D: Draining inguinal lymph nodes cells from mice receiving anti-IL-22 antibody or isotype control were briefly stimulated ex-vivo with PMA/ionomycin and Brefeldin A for 6 hours followed by fluorescent labeling for surface anti-CD4 antibody and intra-cellular anti-IL-17A and anti-IFN-γ antibody. Representative dot plots shows IFN-γ and IL-17A staining on CD4 gated cells. E: Percentages of CD4 + IL-17 + cells or CD4 + IFN-γ + cells from lymph nodes from the two groups of mice were plotted as a dot plot with each dot representing an individual mouse. F: Single cell suspension of joint cells from mice receiving anti-IL-22 antibody or isotype control were briefly stimulated ex-vivo with PMA/ionomycin and Brefeldin A for 6 hours followed by fluorescent labeling for surface anti-CD4 antibody and intra-cellular anti-IL-17A and anti-IFN-γ antibody. Representative zebra plots show IFN-γ and IL-17A staining on gated CD4 cells from paws. G: Single cell suspensions of the joint cells from mice receiving anti-IL22 or isotype control were stimulated with anti-CD3 (5 ug/ml for 3 days) and IL-17A was measured in culture supernatants by ELISA. Each dot represents an individual mouse.
    Figure Legend Snippet: Neutralization of IL-22 prior to onset of arthritis increases incidence and severity of arthritis. A: 16 DBA mice were immunized with collagen and CFA. On day 18 following immunization, mice were divided into 2 groups (8 mice per group) to receive either anti-IL-22 antibody (100 ug/mice/day) or isotype control (100 ug/mouse/day) intraperitoneally for 8–10 days. Mice were scored for clinical arthritis every other day. Data shows incidence and cumulative arthritis score in 2 groups over time. B: Histologic scoring of paws for various aspects of joint damage and inflammation assessed after H E staining. Data is from 4 mice per group. C: Hematoxylin and eosin staining of representative paws from the two groups of mice, magnification of 10×. D: Draining inguinal lymph nodes cells from mice receiving anti-IL-22 antibody or isotype control were briefly stimulated ex-vivo with PMA/ionomycin and Brefeldin A for 6 hours followed by fluorescent labeling for surface anti-CD4 antibody and intra-cellular anti-IL-17A and anti-IFN-γ antibody. Representative dot plots shows IFN-γ and IL-17A staining on CD4 gated cells. E: Percentages of CD4 + IL-17 + cells or CD4 + IFN-γ + cells from lymph nodes from the two groups of mice were plotted as a dot plot with each dot representing an individual mouse. F: Single cell suspension of joint cells from mice receiving anti-IL-22 antibody or isotype control were briefly stimulated ex-vivo with PMA/ionomycin and Brefeldin A for 6 hours followed by fluorescent labeling for surface anti-CD4 antibody and intra-cellular anti-IL-17A and anti-IFN-γ antibody. Representative zebra plots show IFN-γ and IL-17A staining on gated CD4 cells from paws. G: Single cell suspensions of the joint cells from mice receiving anti-IL22 or isotype control were stimulated with anti-CD3 (5 ug/ml for 3 days) and IL-17A was measured in culture supernatants by ELISA. Each dot represents an individual mouse.

    Techniques Used: Neutralization, Mouse Assay, Staining, Ex Vivo, Labeling, Enzyme-linked Immunosorbent Assay

    Induction of IL-22 response during arthritis. A: DBA mice were immunized with collagen and CFA. Splenocytes (5×10 6 /ml) from naïve mice, mice during initiation phase (2 weeks following immunization with collagen and CFA) or arthritic mice were stimulated with collagen (100 ug/ml) for 7 days or unstimulated. Supernatants were analyzed for IL-22 by ELISA. Data shown is representative of 3 independent experiments with 3 mice per experiment. B: Splenocytes (5×10 6 /ml) from arthritic mice were stimulated with varying concentrations of collagen (0, 10, 50 or 100 ug/ml) for 7 days and IL-22 was measured in supernatant by ELISA. Data shown is representative of 3 independent experiments with 3 mice per experiment. C: Splenocytes from arthritic mice were stimulated with PMA/ionomycin and Brefeldin A for 6 hours, stained intra-cellularly for IL-17, IFN-γ or IL-22 and analyzed by flow-cytometry. Data shown is gated on mononuclear cells based on forward and side scatter. Data is representative of 3 independent experiments with 3 mice per experiment.
    Figure Legend Snippet: Induction of IL-22 response during arthritis. A: DBA mice were immunized with collagen and CFA. Splenocytes (5×10 6 /ml) from naïve mice, mice during initiation phase (2 weeks following immunization with collagen and CFA) or arthritic mice were stimulated with collagen (100 ug/ml) for 7 days or unstimulated. Supernatants were analyzed for IL-22 by ELISA. Data shown is representative of 3 independent experiments with 3 mice per experiment. B: Splenocytes (5×10 6 /ml) from arthritic mice were stimulated with varying concentrations of collagen (0, 10, 50 or 100 ug/ml) for 7 days and IL-22 was measured in supernatant by ELISA. Data shown is representative of 3 independent experiments with 3 mice per experiment. C: Splenocytes from arthritic mice were stimulated with PMA/ionomycin and Brefeldin A for 6 hours, stained intra-cellularly for IL-17, IFN-γ or IL-22 and analyzed by flow-cytometry. Data shown is gated on mononuclear cells based on forward and side scatter. Data is representative of 3 independent experiments with 3 mice per experiment.

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

    Neutralization of IL-22 after onset of arthritis preferentially increases the frequency of Th1 cells in lymphoid organs but not in joints. Experimental design is similar to figure 2 . Draining inguinal lymph node cells and single cell suspensions of the paws from mice receiving anti-IL-22 antibody or isotype control were briefly stimulated ex-vivo with PMA/ionomycin and Brefeldin A for 6 hours followed by fluorescent labeling for surface anti-CD4 antibody and intra-cellular anti-IL-17A and anti-IFN-γ antibody. A: Representative dot plots shows IFN-γ and IL-17A staining on gated CD4 cells from lymph nodes. B: Percentages of CD4 + IL-17 + or CD4 + IFN-γ + lymph node cells from the two groups of mice were plotted as a dot plot with each dot representing an individual mouse. C: Single cell suspension of joint cells from mice receiving anti-IL-22 antibody or isotype control were briefly stimulated ex-vivo with PMA/ionomycin and Brefeldin A for 6 hours followed by fluorescent labeling for surface anti-CD4 antibody and intra-cellular anti-IL-17A and anti-IFN-γ antibody. Representative zebra plots show IFN-γ and IL-17A staining on gated CD4 cells from paws. D: Percentages of CD4 + IL-17 + cells from the paws of two groups of mice were plotted as a dot plot with each dot representing an individual mouse.
    Figure Legend Snippet: Neutralization of IL-22 after onset of arthritis preferentially increases the frequency of Th1 cells in lymphoid organs but not in joints. Experimental design is similar to figure 2 . Draining inguinal lymph node cells and single cell suspensions of the paws from mice receiving anti-IL-22 antibody or isotype control were briefly stimulated ex-vivo with PMA/ionomycin and Brefeldin A for 6 hours followed by fluorescent labeling for surface anti-CD4 antibody and intra-cellular anti-IL-17A and anti-IFN-γ antibody. A: Representative dot plots shows IFN-γ and IL-17A staining on gated CD4 cells from lymph nodes. B: Percentages of CD4 + IL-17 + or CD4 + IFN-γ + lymph node cells from the two groups of mice were plotted as a dot plot with each dot representing an individual mouse. C: Single cell suspension of joint cells from mice receiving anti-IL-22 antibody or isotype control were briefly stimulated ex-vivo with PMA/ionomycin and Brefeldin A for 6 hours followed by fluorescent labeling for surface anti-CD4 antibody and intra-cellular anti-IL-17A and anti-IFN-γ antibody. Representative zebra plots show IFN-γ and IL-17A staining on gated CD4 cells from paws. D: Percentages of CD4 + IL-17 + cells from the paws of two groups of mice were plotted as a dot plot with each dot representing an individual mouse.

    Techniques Used: Neutralization, Mouse Assay, Ex Vivo, Labeling, Staining

    32) Product Images from "The development of steady-state activation hubs between adult LTi ILC3s and primed macrophages in small intestine"

    Article Title: The development of steady-state activation hubs between adult LTi ILC3s and primed macrophages in small intestine

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

    doi: 10.4049/jimmunol.1700155

    Small intestine LTi cells are constitutively activated at SILT. ( A ) Il22 C22/+ murine small intestine was fixed and stained by whole mount for Catch22 (red) and B220 (green) (representative of n=2). Scale bar represents 1 mm. ( B,C ) Detail of the indicated regions from A to demonstrate small ( B ) and large ( C ) SILT structures. ( D ) Small intestine from Il22 C22/+ mice was fixed, sectioned, and stained for Catch22 (red), CD11c (green), VCAM1 (light blue), and DAPI (gray) (representative of n=5). Scale bar represents 100 μm. ( E ) Lymphocytes were isolated from the proximal third of small intestine lamina propria and analyzed for Catch22 fluorescence among CD45 + cells (representative of n=5). ( F ) ILC (Thy1 + CD3 − ) subsets were analyzed for Catch22 and CD69 (representative of n=4–7). ( G ) Small intestine lamina propria cells were isolated in the presence of Brefeldin A and stained for intracellular IL-22 without exogenous stimulation. Data connected by line are from the same mouse (n=5).
    Figure Legend Snippet: Small intestine LTi cells are constitutively activated at SILT. ( A ) Il22 C22/+ murine small intestine was fixed and stained by whole mount for Catch22 (red) and B220 (green) (representative of n=2). Scale bar represents 1 mm. ( B,C ) Detail of the indicated regions from A to demonstrate small ( B ) and large ( C ) SILT structures. ( D ) Small intestine from Il22 C22/+ mice was fixed, sectioned, and stained for Catch22 (red), CD11c (green), VCAM1 (light blue), and DAPI (gray) (representative of n=5). Scale bar represents 100 μm. ( E ) Lymphocytes were isolated from the proximal third of small intestine lamina propria and analyzed for Catch22 fluorescence among CD45 + cells (representative of n=5). ( F ) ILC (Thy1 + CD3 − ) subsets were analyzed for Catch22 and CD69 (representative of n=4–7). ( G ) Small intestine lamina propria cells were isolated in the presence of Brefeldin A and stained for intracellular IL-22 without exogenous stimulation. Data connected by line are from the same mouse (n=5).

    Techniques Used: Staining, Mouse Assay, Isolation, Fluorescence

    33) Product Images from "Butyrate Attenuates Lung Inflammation by Negatively Modulating Th9 Cells"

    Article Title: Butyrate Attenuates Lung Inflammation by Negatively Modulating Th9 Cells

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00067

    Butyrate treatment negatively impact Th9 cells in the lungs of OVA-challenged mice. Male C57BL/6 mice were intraperitoneally (IP) injected with OVA (30 μg) + Al(OH)3 (1.6 mg) at days 0 and 7. Mice also received 250 μl of either PBS or butyrate (1M) (IP) at days 0, 1, 2, 7, 8, and 9. OVA-sensitized mice were nebulized with an OVA solution (3%) for 15 min at days 14, 15, and 16. The group that received butyrate during sensitization was also treated during challenge. A control group was sensitized and challenged without OVA. Euthanasia was performed 24 h after the last challenge (A) . Lungs were digested, cells stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml) and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO 2 , and stained with monoclonal antibodies to determine the frequencies of IL-9+, IL-13+, and FOXP3+ CD4+ T cells by flow cytometry (B) . Bar graphs show the frequencies of IL-9+ (C) , IL-13+ (D) and FOXP3+ (E) CD4+ T cells in the different groups. Data are shown as mean ± SD. One-way ANOVA followed by Tukey's multiple comparison test were used for statistical analysis. n = 5–7 mice per group. NS, not significant.
    Figure Legend Snippet: Butyrate treatment negatively impact Th9 cells in the lungs of OVA-challenged mice. Male C57BL/6 mice were intraperitoneally (IP) injected with OVA (30 μg) + Al(OH)3 (1.6 mg) at days 0 and 7. Mice also received 250 μl of either PBS or butyrate (1M) (IP) at days 0, 1, 2, 7, 8, and 9. OVA-sensitized mice were nebulized with an OVA solution (3%) for 15 min at days 14, 15, and 16. The group that received butyrate during sensitization was also treated during challenge. A control group was sensitized and challenged without OVA. Euthanasia was performed 24 h after the last challenge (A) . Lungs were digested, cells stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml) and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO 2 , and stained with monoclonal antibodies to determine the frequencies of IL-9+, IL-13+, and FOXP3+ CD4+ T cells by flow cytometry (B) . Bar graphs show the frequencies of IL-9+ (C) , IL-13+ (D) and FOXP3+ (E) CD4+ T cells in the different groups. Data are shown as mean ± SD. One-way ANOVA followed by Tukey's multiple comparison test were used for statistical analysis. n = 5–7 mice per group. NS, not significant.

    Techniques Used: Mouse Assay, Injection, Staining, Flow Cytometry, Cytometry

    Adoptive transfer of Th9 cells restores lung inflammation in butyrate-treated OVA-challenged mice. Male C57BL/6 mice were intraperitoneally (IP) injected with OVA (30 μg) + Al(OH)3 (1.6 mg) at days 0 and 7. Mice also received 250 μl of either PBS or butyrate (1 M) (IP) at days 0, 1, 2, 7, 8, and 9. OVA-sensitized mice were nebulized with an OVA solution (3%) for 15 min at days 14, 15, and 16. The groups that received butyrate during sensitization were treated during challenge. Butyrate-treated mice also received an adoptive transfer (IP) of OT-II Th0, Th2, or Th9 cells 24 h before challenge initiation. Euthanasia was performed 24 h after the last challenge (A) . Lungs were digested and cells stained with monoclonal antibodies to determine the frequency of eosinophils as shown by the bar graph (B) . Alternatively, cells were stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO 2 , and stained with monoclonal antibodies to determine the frequency of IL-9+CD4+ and IL-13+CD4+ T cells as shown by the bar graphs (C,D) , respectively. Lung tissues were also stained with hematoxylin/eosin (H E) and periodic acid–Schiff (PAS), scale bars: 100 μm. Thick and thin arrows indicate inflammatory infiltrates and mucus production, respectively (E) . Data are shown as mean ± SD. One-way ANOVA followed by Tukey's multiple comparison test were used for statistical analysis. n = 5–7 mice per group. NS, not significant.
    Figure Legend Snippet: Adoptive transfer of Th9 cells restores lung inflammation in butyrate-treated OVA-challenged mice. Male C57BL/6 mice were intraperitoneally (IP) injected with OVA (30 μg) + Al(OH)3 (1.6 mg) at days 0 and 7. Mice also received 250 μl of either PBS or butyrate (1 M) (IP) at days 0, 1, 2, 7, 8, and 9. OVA-sensitized mice were nebulized with an OVA solution (3%) for 15 min at days 14, 15, and 16. The groups that received butyrate during sensitization were treated during challenge. Butyrate-treated mice also received an adoptive transfer (IP) of OT-II Th0, Th2, or Th9 cells 24 h before challenge initiation. Euthanasia was performed 24 h after the last challenge (A) . Lungs were digested and cells stained with monoclonal antibodies to determine the frequency of eosinophils as shown by the bar graph (B) . Alternatively, cells were stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO 2 , and stained with monoclonal antibodies to determine the frequency of IL-9+CD4+ and IL-13+CD4+ T cells as shown by the bar graphs (C,D) , respectively. Lung tissues were also stained with hematoxylin/eosin (H E) and periodic acid–Schiff (PAS), scale bars: 100 μm. Thick and thin arrows indicate inflammatory infiltrates and mucus production, respectively (E) . Data are shown as mean ± SD. One-way ANOVA followed by Tukey's multiple comparison test were used for statistical analysis. n = 5–7 mice per group. NS, not significant.

    Techniques Used: Adoptive Transfer Assay, Mouse Assay, Injection, Staining

    IL-9 treatment has no impact on IL-9+, IL-13+, and FOXP3+ CD4+ T cells in the lungs of butyrate-treated OVA-challenged mice. Male C57BL/6 mice were intraperitoneally (IP) injected with OVA (30 μg) + Al(OH)3 (1.6 mg) at days 0 and 7. Mice also received 250 μl of either PBS or butyrate (1 M) (IP) at days 0, 1, 2, 7, 8, and 9. OVA-sensitized mice were nebulized with an OVA solution (3%) for 15 min at days 14, 15, and 16. The groups that received butyrate during sensitization were also treated during challenge. Butyrate-treated mice also received either PBS or recombinant IL-9 (IP) at days 14 and 15. Euthanasia was performed 24 h after the last challenge. Lungs were digested, cells stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO 2 , and stained with monoclonal antibodies to determine the frequencies of IL-9+ (A) , IL-13+ (B) , and FOXP3+ (C) CD4+ T cells in the different groups. Data are shown as mean ± SD. One-way ANOVA followed by Tukey's multiple comparison test were used for statistical analysis. n = 5–7 mice per group. NS, not significant.
    Figure Legend Snippet: IL-9 treatment has no impact on IL-9+, IL-13+, and FOXP3+ CD4+ T cells in the lungs of butyrate-treated OVA-challenged mice. Male C57BL/6 mice were intraperitoneally (IP) injected with OVA (30 μg) + Al(OH)3 (1.6 mg) at days 0 and 7. Mice also received 250 μl of either PBS or butyrate (1 M) (IP) at days 0, 1, 2, 7, 8, and 9. OVA-sensitized mice were nebulized with an OVA solution (3%) for 15 min at days 14, 15, and 16. The groups that received butyrate during sensitization were also treated during challenge. Butyrate-treated mice also received either PBS or recombinant IL-9 (IP) at days 14 and 15. Euthanasia was performed 24 h after the last challenge. Lungs were digested, cells stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO 2 , and stained with monoclonal antibodies to determine the frequencies of IL-9+ (A) , IL-13+ (B) , and FOXP3+ (C) CD4+ T cells in the different groups. Data are shown as mean ± SD. One-way ANOVA followed by Tukey's multiple comparison test were used for statistical analysis. n = 5–7 mice per group. NS, not significant.

    Techniques Used: Mouse Assay, Injection, Recombinant, Staining

    Butyrate enhances FOXP3 expression in CD4+ T cells and impairs the differentiation of Th9 cells. Naïve CD4+ T cells (CD62L+CD44low) were sorted from spleens of FOXP3 GFP mice and cultured in 96-well flat bottom plates with plate bound anti-CD3 (2 μg/ml) and soluble anti-CD28 (0.5 μg/ml) in the presence of Th9 (TGF-β 2 ng/ml, IL-4 10 ng/ml and anti-IFNγ 10 μg/ml) differentiation condition for 4 days. Butyrate and propionate (250 μM) were added in the culture at day 0. At day 4, cells were restimulated with with PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO 2 , and analyzed by flow cytometry (A) . The frequencies of FOXP3+ and/or IL-9+ CD4+ T cells are represented by bar graphs (B) . A correlation analysis of IL-9 and FOXP3 expression was performed (C) . The frequencies of IL-9+ and/or IL-10+ CD4+ T cells (D) and FOXP3+ and/or IL-10+ CD4+ T cells (E) were determined and are represented by bar graphs. Data are shown as mean ± S.D. Two-way ANOVA and Tukey's multiple comparison test were used for statistical analysis. NS, not significant.
    Figure Legend Snippet: Butyrate enhances FOXP3 expression in CD4+ T cells and impairs the differentiation of Th9 cells. Naïve CD4+ T cells (CD62L+CD44low) were sorted from spleens of FOXP3 GFP mice and cultured in 96-well flat bottom plates with plate bound anti-CD3 (2 μg/ml) and soluble anti-CD28 (0.5 μg/ml) in the presence of Th9 (TGF-β 2 ng/ml, IL-4 10 ng/ml and anti-IFNγ 10 μg/ml) differentiation condition for 4 days. Butyrate and propionate (250 μM) were added in the culture at day 0. At day 4, cells were restimulated with with PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO 2 , and analyzed by flow cytometry (A) . The frequencies of FOXP3+ and/or IL-9+ CD4+ T cells are represented by bar graphs (B) . A correlation analysis of IL-9 and FOXP3 expression was performed (C) . The frequencies of IL-9+ and/or IL-10+ CD4+ T cells (D) and FOXP3+ and/or IL-10+ CD4+ T cells (E) were determined and are represented by bar graphs. Data are shown as mean ± S.D. Two-way ANOVA and Tukey's multiple comparison test were used for statistical analysis. NS, not significant.

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

    Butyrate sustains cytokine production by differentiated Th9 cells. Naïve CD4+ T cells (CD62L+CD44low) were sorted from spleens of FOXP3 GFP mice and cultured in 96-well flat bottom plates with plate bound anti-CD3 (2 μg/ml) and soluble anti-CD28 (0.5 μg/ml) in the presence of Th9 (TGF-β 2 ng/ml, IL-4 10 ng/ml, and anti-IFNγ 10 μg/ml) differentiation condition for 4 days. At day 4, the medium was replaced by fresh R-10 and butyrate (250 μM) was added in the culture. At day 8, cells were restimulated with with PMA (50 ng/ml), ionomycin (500 ng/ml) and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO 2 , and analyzed by flow cytometry (A) . The frequencies of FOXP3+ and/or IL-9+ CD4+ T cells (B) , FOXP3+ and/or IL-10+ CD4+ T cells (C) and IL-9+ and/or IL-10+ CD4+ T cells (D) are represented by bar graphs. Data are shown as mean ± S.D. Two-way ANOVA and Sidak's multiple comparison test were used for statistical analysis. NS, not significant.
    Figure Legend Snippet: Butyrate sustains cytokine production by differentiated Th9 cells. Naïve CD4+ T cells (CD62L+CD44low) were sorted from spleens of FOXP3 GFP mice and cultured in 96-well flat bottom plates with plate bound anti-CD3 (2 μg/ml) and soluble anti-CD28 (0.5 μg/ml) in the presence of Th9 (TGF-β 2 ng/ml, IL-4 10 ng/ml, and anti-IFNγ 10 μg/ml) differentiation condition for 4 days. At day 4, the medium was replaced by fresh R-10 and butyrate (250 μM) was added in the culture. At day 8, cells were restimulated with with PMA (50 ng/ml), ionomycin (500 ng/ml) and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO 2 , and analyzed by flow cytometry (A) . The frequencies of FOXP3+ and/or IL-9+ CD4+ T cells (B) , FOXP3+ and/or IL-10+ CD4+ T cells (C) and IL-9+ and/or IL-10+ CD4+ T cells (D) are represented by bar graphs. Data are shown as mean ± S.D. Two-way ANOVA and Sidak's multiple comparison test were used for statistical analysis. NS, not significant.

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

    SCFAs have opposite effects on IL-13-expressing T cells. Naïve CD4+ T cells (CD62L+CD44low) were sorted from spleens of FOXP3 GFP mice and cultured in 96-well flat bottom plates with plate bound anti-CD3 (2 μg/ml) and soluble anti-CD28 (0.5 μg/ml) in the presence of Th2 (IL-4 10 ng/ml, anti-IL-12 10 μg/ml, and anti-IFNγ 10 μg/ml) differentiation condition for 4 days. Butyrate and propionate (250 μM) were added in the culture at day 0. At day 4, cells were restimulated with with PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO 2 , and analyzed by flow cytometry (A) . The frequencies of FOXP3+ and/or IL-13+ CD4+ T cells (B) , IL-13+ and/or IL-5+ CD4+ T cells (C) and IL-13+ and/or IL-4+ CD4+ T cells (D) are represented by bar graphs. Data are shown as mean ± S.D. Two-way ANOVA and Tukey's multiple comparison test were used for statistical analysis. NS, not significant.
    Figure Legend Snippet: SCFAs have opposite effects on IL-13-expressing T cells. Naïve CD4+ T cells (CD62L+CD44low) were sorted from spleens of FOXP3 GFP mice and cultured in 96-well flat bottom plates with plate bound anti-CD3 (2 μg/ml) and soluble anti-CD28 (0.5 μg/ml) in the presence of Th2 (IL-4 10 ng/ml, anti-IL-12 10 μg/ml, and anti-IFNγ 10 μg/ml) differentiation condition for 4 days. Butyrate and propionate (250 μM) were added in the culture at day 0. At day 4, cells were restimulated with with PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO 2 , and analyzed by flow cytometry (A) . The frequencies of FOXP3+ and/or IL-13+ CD4+ T cells (B) , IL-13+ and/or IL-5+ CD4+ T cells (C) and IL-13+ and/or IL-4+ CD4+ T cells (D) are represented by bar graphs. Data are shown as mean ± S.D. Two-way ANOVA and Tukey's multiple comparison test were used for statistical analysis. NS, not significant.

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

    Butyrate and propionate enhances FOXP3 expression in CD4+ T cells differentiated into Tregs. Naïve CD4+ T cells (CD62L+CD44low) were sorted from spleens of FOXP3 GFP mice and cultured in 96-well flat bottom plates with plate bound anti-CD3 (2 μg/ml) and soluble anti-CD28 (0.5 μg/ml) in the presence of Treg (TGF-β 2 ng/ml and IL-2 10 ng/ml) differentiation condition for 4 days. Butyrate and propionate (250 μM) were added in the culture at day 0. At day 4, cells were restimulated with with PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO 2 , and analyzed by flow cytometry (A) . The frequencies of FOXP3+ and/or IL-9+ CD4+ T cells (B) and FOXP3+ and/or IL-10+ CD4+ T cells (C) are represented by bar graphs. Data are shown as mean ± S.D. Linear regression, Two-way ANOVA and Tukey's multiple comparison test were used for statistical analysis. NS, not significant.
    Figure Legend Snippet: Butyrate and propionate enhances FOXP3 expression in CD4+ T cells differentiated into Tregs. Naïve CD4+ T cells (CD62L+CD44low) were sorted from spleens of FOXP3 GFP mice and cultured in 96-well flat bottom plates with plate bound anti-CD3 (2 μg/ml) and soluble anti-CD28 (0.5 μg/ml) in the presence of Treg (TGF-β 2 ng/ml and IL-2 10 ng/ml) differentiation condition for 4 days. Butyrate and propionate (250 μM) were added in the culture at day 0. At day 4, cells were restimulated with with PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO 2 , and analyzed by flow cytometry (A) . The frequencies of FOXP3+ and/or IL-9+ CD4+ T cells (B) and FOXP3+ and/or IL-10+ CD4+ T cells (C) are represented by bar graphs. Data are shown as mean ± S.D. Linear regression, Two-way ANOVA and Tukey's multiple comparison test were used for statistical analysis. NS, not significant.

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

    34) Product Images from "Type I interferons suppress viral replication but contribute to T cell depletion and dysfunction during chronic HIV-1 infection"

    Article Title: Type I interferons suppress viral replication but contribute to T cell depletion and dysfunction during chronic HIV-1 infection

    Journal: JCI Insight

    doi: 10.1172/jci.insight.94366

    IFNAR1 blockade during persistent HIV-1 infection rescues the function of human T cells, including HIV-1–specific T cells. Humanized mice infected with HIV-1 were treated with a mAb against IFN-α/β receptor 1 (IFNAR1) or isotype control (mouse IgG2a) twice per week from 6 to 10 weeks postinfection (wpi). Mice were sacrificed at 10 wpi. ( A – C ) Splenocytes were stimulated ex vivo with PMA plus ionomycin for 4 hours followed by intracellular cytokine staining. Representative dot plots ( A ) and summarized data show the percentages of CD8 +  T cells ( B ) and CD4 +  T cells ( C ) producing IFN-γ (mock,  n  = 11; HIV-1 + mIgG2a,  n  = 15; HIV-1 + anti-IFNAR1,  n  = 13, combined data from 4 independent experiments with mean values ± SEM) or IL-2 (mock,  n  = 8; HIV-1 + mIgG2a,  n  = 10; HIV-1 + anti-IFNAR1,  n  = 10, combined data from 3 independent experiments with mean values ± SEM). ( D – F ) Splenocytes were stimulated ex vivo with peptide pools of HIV-1 Gag protein for 8 hours (brefeldin A was added at 3 hours) followed by intracellular cytokine staining. Representative dot plots ( D ) and summarized data show the percentages of CD8 +  T cells ( E ) and CD4 +  T cells ( F ) producing IFN-γ and IL-2 (mock,  n  = 4; HIV-1 + mIgG2a,  n  = 6; HIV-1 + anti-IFNAR1,  n  = 6, combined data from 2 independent experiment with mean values ± SEM). * P
    Figure Legend Snippet: IFNAR1 blockade during persistent HIV-1 infection rescues the function of human T cells, including HIV-1–specific T cells. Humanized mice infected with HIV-1 were treated with a mAb against IFN-α/β receptor 1 (IFNAR1) or isotype control (mouse IgG2a) twice per week from 6 to 10 weeks postinfection (wpi). Mice were sacrificed at 10 wpi. ( A – C ) Splenocytes were stimulated ex vivo with PMA plus ionomycin for 4 hours followed by intracellular cytokine staining. Representative dot plots ( A ) and summarized data show the percentages of CD8 + T cells ( B ) and CD4 + T cells ( C ) producing IFN-γ (mock, n = 11; HIV-1 + mIgG2a, n = 15; HIV-1 + anti-IFNAR1, n = 13, combined data from 4 independent experiments with mean values ± SEM) or IL-2 (mock, n = 8; HIV-1 + mIgG2a, n = 10; HIV-1 + anti-IFNAR1, n = 10, combined data from 3 independent experiments with mean values ± SEM). ( D – F ) Splenocytes were stimulated ex vivo with peptide pools of HIV-1 Gag protein for 8 hours (brefeldin A was added at 3 hours) followed by intracellular cytokine staining. Representative dot plots ( D ) and summarized data show the percentages of CD8 + T cells ( E ) and CD4 + T cells ( F ) producing IFN-γ and IL-2 (mock, n = 4; HIV-1 + mIgG2a, n = 6; HIV-1 + anti-IFNAR1, n = 6, combined data from 2 independent experiment with mean values ± SEM). * P

    Techniques Used: Infection, Mouse Assay, Ex Vivo, Staining

    35) Product Images from "Age-dependent metabolic and immunosuppressive effects of Tacrolimus"

    Article Title: Age-dependent metabolic and immunosuppressive effects of Tacrolimus

    Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

    doi: 10.1111/ajt.14087

    More pronounced suppression of IL-2 in old CD4 +  T cells subsequent to TAC treatment Splenocytes (0.5 × 10 6 ) from young and old C57BL/6 recipients of DBA/2 skin allografts were collected 6 days after transplantation; mice received trough level adjusted TAC (young mice received 0.5mg/kg TAC and old mice 0.25mg/kg TAC). Splenocytes were co-cultured with anergic DBA/2 splenocytes. By 48 hrs, IL-2 secretion was assessed by ELISpot  (A) . CD4 +  T cells were isolated and stimulated with PMA/Ionomycin/Brefeldin A for 4 hrs. IL-2 secretion was measured by flow cytometry and ELISA  (B) . Statistics: n ≥5; mean±SD; *p
    Figure Legend Snippet: More pronounced suppression of IL-2 in old CD4 + T cells subsequent to TAC treatment Splenocytes (0.5 × 10 6 ) from young and old C57BL/6 recipients of DBA/2 skin allografts were collected 6 days after transplantation; mice received trough level adjusted TAC (young mice received 0.5mg/kg TAC and old mice 0.25mg/kg TAC). Splenocytes were co-cultured with anergic DBA/2 splenocytes. By 48 hrs, IL-2 secretion was assessed by ELISpot (A) . CD4 + T cells were isolated and stimulated with PMA/Ionomycin/Brefeldin A for 4 hrs. IL-2 secretion was measured by flow cytometry and ELISA (B) . Statistics: n ≥5; mean±SD; *p

    Techniques Used: Transplantation Assay, Mouse Assay, Cell Culture, Enzyme-linked Immunospot, Isolation, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    Intracellular concentrations of calcineurin and Ca 2+ in young and old murine and human CD4 + T cells Isolated naïve CD4 + T cells from young and old mice were cultured in Th1-polarizing conditions and in presence of α-CD3/α-CD28. By 96 hrs, intracellular concentration of calcineurin was measured by sandwich ELISA (Cloud-Clone Corp., Houston, USA) ( A ). For measurement of Ca 2+ influx, isolated naïve CD4 + T cells from young and old mice were cultured in Th1-polarizing conditions and in presence of α-CD3/α-CD28 for 96 hrs and treated with and without TAC (5ng/ml). Cells were dyed with a fluorescent Ca 2+ calcium indicator (Fluo-4 NW calcium Assay kit, Molecular Probes, OR, USA) and then stimulated with PMA/Ionomycin including Brefeldin A (Biolegend, CA, USA) and subsequently analyzed with a fluorescence reader at 494 nm and 516 nm ( B ). In addition, isolated naïve CD4 + T cells from young (
    Figure Legend Snippet: Intracellular concentrations of calcineurin and Ca 2+ in young and old murine and human CD4 + T cells Isolated naïve CD4 + T cells from young and old mice were cultured in Th1-polarizing conditions and in presence of α-CD3/α-CD28. By 96 hrs, intracellular concentration of calcineurin was measured by sandwich ELISA (Cloud-Clone Corp., Houston, USA) ( A ). For measurement of Ca 2+ influx, isolated naïve CD4 + T cells from young and old mice were cultured in Th1-polarizing conditions and in presence of α-CD3/α-CD28 for 96 hrs and treated with and without TAC (5ng/ml). Cells were dyed with a fluorescent Ca 2+ calcium indicator (Fluo-4 NW calcium Assay kit, Molecular Probes, OR, USA) and then stimulated with PMA/Ionomycin including Brefeldin A (Biolegend, CA, USA) and subsequently analyzed with a fluorescence reader at 494 nm and 516 nm ( B ). In addition, isolated naïve CD4 + T cells from young (

    Techniques Used: Isolation, Mouse Assay, Cell Culture, Concentration Assay, Sandwich ELISA, Calcium Assay, Fluorescence

    36) Product Images from "Deletion of cannabinoid receptors 1 and 2 exacerbates APC function to increase inflammation and cellular immunity during influenza infection"

    Article Title: Deletion of cannabinoid receptors 1 and 2 exacerbates APC function to increase inflammation and cellular immunity during influenza infection

    Journal: Journal of Leukocyte Biology

    doi: 10.1189/jlb.0511219

    Greater cytokine production by leukocytes from CB 1 −/− CB 2 −/−  mice compared with WT mice. Lungs from mice were mechanically disrupted and passed through a sieve ( n =5). After removal of connective tissue, cells were counted and restimulated with PMA/Io for 5 h in the presence of Brefeldin A. (A) Cells were blocked with FcRs and stained with CD4, CD8, and NK1.1, in addition to cytokine staining with IFN-γ and IL-17 (B). Flow cytometry samples were gated as depicted in A. Cytokine secretors were identified as percent of positive cells within surface delineation, and unstimulated samples were used as controls. (C) Immune cell populations were enumerated for percent cytokine expression in FlowJo (type of cytokine indicated on the left) within effector cell populations (surface marker indicated on the right) in FlowJo, and bar graphs were generated using GraphPad Prism. Friedman's test for percentages was performed, and significance is indicated in figures as  # P
    Figure Legend Snippet: Greater cytokine production by leukocytes from CB 1 −/− CB 2 −/− mice compared with WT mice. Lungs from mice were mechanically disrupted and passed through a sieve ( n =5). After removal of connective tissue, cells were counted and restimulated with PMA/Io for 5 h in the presence of Brefeldin A. (A) Cells were blocked with FcRs and stained with CD4, CD8, and NK1.1, in addition to cytokine staining with IFN-γ and IL-17 (B). Flow cytometry samples were gated as depicted in A. Cytokine secretors were identified as percent of positive cells within surface delineation, and unstimulated samples were used as controls. (C) Immune cell populations were enumerated for percent cytokine expression in FlowJo (type of cytokine indicated on the left) within effector cell populations (surface marker indicated on the right) in FlowJo, and bar graphs were generated using GraphPad Prism. Friedman's test for percentages was performed, and significance is indicated in figures as # P

    Techniques Used: Mouse Assay, Staining, Flow Cytometry, Cytometry, Expressing, Marker, Generated

    37) Product Images from "PTPN22 Acts in a Cell Intrinsic Manner to Restrict the Proliferation and Differentiation of T Cells Following Antibody Lymphodepletion"

    Article Title: PTPN22 Acts in a Cell Intrinsic Manner to Restrict the Proliferation and Differentiation of T Cells Following Antibody Lymphodepletion

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.00052

    Foxp3 + Tregs and IL-10 production are cell-intrinsically controlled by PTPN22 in steady state and LIP. (A) Ab-mediated depletion efficacy of CD4 + Foxp3 + Tregs and CD4 + Foxp3 − conventional CD4 T cells 3 days post anti-CD4 and anti-CD8 injection. Data shown as mean with SD with N = 7 animals per group. Pooled data of two independent experiments. (B) Proportion and absolute numbers of regulatory T cells 2 weeks after T cell depletion in combination with IL-7R blockade and (C) 8 weeks post depletion. Each dot represents one animal. In (B) N = 7–8 animals per group, shown data is pooled of two independent experiments. In (C) N = 4–6 animals per group, one representative experiment out of three independent experiments. Two-way ANOVA with Sidak's multiple comparison test. (D) IL-10 production by CD4 T cells, CD4 + CD25 + Tregs and CD4 + CD25 − conventional T cells 2 weeks post depletion. To analyze IL-10 production cells were re-stimulated in presence of Brefeldin A ex vivo . Each dot represents one animal. Each dot represents one animal. N = 6–8 animals per group, shown data is pooled of two independent experiments. Two-way ANOVA with Sidak's multiple comparison test. (E) Bone marrow from CD45.1 PTPN22 WT and CD45.2 PTPN22 KO donor animals was mixed 1:1 and transplanted in into fully irradiated CD45.1/2 PTPN22 WT recipient mice. The Foxp3 + frequency within PTPN22 WT CD45.1 + CD4 + or PTPN22 KO CD45.2 + CD4 + cell pool was assessed 15 weeks post transfer. Each dot represents one animal ( N = 11), data is pooled of two independent experiments. A paired t -test was performed to compare PTPN22 WT and KO cells within the same recipient. (F,G) Phenotypical analysis of Foxp3 + expressing PTPN22 WT CD45.1 + CD4 + and PTPN22 KO CD45.2 + CD4 + cells from the experiment outlined in (E) . Each dot represents one animal ( N = 11), data is pooled of two independent experiments. A paired t -test was performed to compare PTPN22 WT and KO cells within the same recipient. A p
    Figure Legend Snippet: Foxp3 + Tregs and IL-10 production are cell-intrinsically controlled by PTPN22 in steady state and LIP. (A) Ab-mediated depletion efficacy of CD4 + Foxp3 + Tregs and CD4 + Foxp3 − conventional CD4 T cells 3 days post anti-CD4 and anti-CD8 injection. Data shown as mean with SD with N = 7 animals per group. Pooled data of two independent experiments. (B) Proportion and absolute numbers of regulatory T cells 2 weeks after T cell depletion in combination with IL-7R blockade and (C) 8 weeks post depletion. Each dot represents one animal. In (B) N = 7–8 animals per group, shown data is pooled of two independent experiments. In (C) N = 4–6 animals per group, one representative experiment out of three independent experiments. Two-way ANOVA with Sidak's multiple comparison test. (D) IL-10 production by CD4 T cells, CD4 + CD25 + Tregs and CD4 + CD25 − conventional T cells 2 weeks post depletion. To analyze IL-10 production cells were re-stimulated in presence of Brefeldin A ex vivo . Each dot represents one animal. Each dot represents one animal. N = 6–8 animals per group, shown data is pooled of two independent experiments. Two-way ANOVA with Sidak's multiple comparison test. (E) Bone marrow from CD45.1 PTPN22 WT and CD45.2 PTPN22 KO donor animals was mixed 1:1 and transplanted in into fully irradiated CD45.1/2 PTPN22 WT recipient mice. The Foxp3 + frequency within PTPN22 WT CD45.1 + CD4 + or PTPN22 KO CD45.2 + CD4 + cell pool was assessed 15 weeks post transfer. Each dot represents one animal ( N = 11), data is pooled of two independent experiments. A paired t -test was performed to compare PTPN22 WT and KO cells within the same recipient. (F,G) Phenotypical analysis of Foxp3 + expressing PTPN22 WT CD45.1 + CD4 + and PTPN22 KO CD45.2 + CD4 + cells from the experiment outlined in (E) . Each dot represents one animal ( N = 11), data is pooled of two independent experiments. A paired t -test was performed to compare PTPN22 WT and KO cells within the same recipient. A p

    Techniques Used: Injection, Ex Vivo, Irradiation, Mouse Assay, Expressing

    38) Product Images from "Type 1 Conventional CD103+ Dendritic Cells Control Effector CD8+ T Cell Migration, Survival, and Memory Responses During Influenza Infection"

    Article Title: Type 1 Conventional CD103+ Dendritic Cells Control Effector CD8+ T Cell Migration, Survival, and Memory Responses During Influenza Infection

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.03043

    Impaired effector CD8 + T cell responses in the absence of pulmonary CD103 + cDC1s. (A) NP 366−374 -specific CD8 + T cells in the lungs of uninfected, infected wild type, and Clec9A-DTR mice (day 6, 10, and 15 post infection). (B) Kinetics of total CD8 + T cells and NP 366−374 -specific CD8 + T cells in the lungs of uninfected, infected wild type, and Clec9A-DTR mice. Absolute numbers are shown. (C) Lung virus load was measured by relative quantification of M1 viral protein in infected wild type and Clec9A-DTR mice on day 10 post infection. (D–F) Lung cells were harvested from uninfected, infected wild type, and Clec9A-DTR mice on day 10 post infection and stimulated with PMA/Ionomycin for 3 h followed by Brefeldin A incubation for an additional 3 h. Intracellular IFN-γ and IL-10 staining profiles (d) of pulmonary CD8 + T cells, frequency (E) and total numbers (F) of IFN-γ-producing, IL-10-producing, and IFN-γ/IL-10 double-producing CD8 + T cells in the lungs. (G) IFN-γ and IL-10 BAL levels as measured by sandwich ELISA (H) Relative quantification of IFN-γ and IL-10 transcripts. Data are shown as mean ± SEM. * p
    Figure Legend Snippet: Impaired effector CD8 + T cell responses in the absence of pulmonary CD103 + cDC1s. (A) NP 366−374 -specific CD8 + T cells in the lungs of uninfected, infected wild type, and Clec9A-DTR mice (day 6, 10, and 15 post infection). (B) Kinetics of total CD8 + T cells and NP 366−374 -specific CD8 + T cells in the lungs of uninfected, infected wild type, and Clec9A-DTR mice. Absolute numbers are shown. (C) Lung virus load was measured by relative quantification of M1 viral protein in infected wild type and Clec9A-DTR mice on day 10 post infection. (D–F) Lung cells were harvested from uninfected, infected wild type, and Clec9A-DTR mice on day 10 post infection and stimulated with PMA/Ionomycin for 3 h followed by Brefeldin A incubation for an additional 3 h. Intracellular IFN-γ and IL-10 staining profiles (d) of pulmonary CD8 + T cells, frequency (E) and total numbers (F) of IFN-γ-producing, IL-10-producing, and IFN-γ/IL-10 double-producing CD8 + T cells in the lungs. (G) IFN-γ and IL-10 BAL levels as measured by sandwich ELISA (H) Relative quantification of IFN-γ and IL-10 transcripts. Data are shown as mean ± SEM. * p

    Techniques Used: Infection, Mouse Assay, Incubation, Staining, Sandwich ELISA

    39) Product Images from "Apigenin, a Natural Flavonoid, Attenuates EAE Severity Through the Modulation of Dendritic Cell and Other Immune Cell Functions"

    Article Title: Apigenin, a Natural Flavonoid, Attenuates EAE Severity Through the Modulation of Dendritic Cell and Other Immune Cell Functions

    Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

    doi: 10.1007/s11481-015-9617-x

    Effect of Apigenin on DC activation and antigen presentation. Splenocytes from both C57BL/6 and SJL/J mice (EAE + vehicle and EAE + Apigenin groups) were stimulated with MOG 38–49 and PLP 323–339 peptides respectively for 3 days followed by activation with PMA, ionomycin and brefeldin A for 5 h. These cells were subsequently stained with antibodies against CD11c, CD8α, MHC II, and CD86. Data represents CD11c + dendritic cells from mice expressing MHCI I + CD86 + upon stimulation with MOG 38–49 ( top ) and PLP 323–339 ( bottom ). Each bar is representative of the mean percentage for every marker per group for both C57BL/6 ( n =2/group) and SJL/J mice ( n =5/group). Histogram plots are representative of one animal per group. * p
    Figure Legend Snippet: Effect of Apigenin on DC activation and antigen presentation. Splenocytes from both C57BL/6 and SJL/J mice (EAE + vehicle and EAE + Apigenin groups) were stimulated with MOG 38–49 and PLP 323–339 peptides respectively for 3 days followed by activation with PMA, ionomycin and brefeldin A for 5 h. These cells were subsequently stained with antibodies against CD11c, CD8α, MHC II, and CD86. Data represents CD11c + dendritic cells from mice expressing MHCI I + CD86 + upon stimulation with MOG 38–49 ( top ) and PLP 323–339 ( bottom ). Each bar is representative of the mean percentage for every marker per group for both C57BL/6 ( n =2/group) and SJL/J mice ( n =5/group). Histogram plots are representative of one animal per group. * p

    Techniques Used: Activation Assay, Mouse Assay, Plasmid Purification, Staining, Expressing, Marker

    Impact of Apigenin on antigen uptake and immune cell adhesion. Splenocytes from both the EAE + Vehicle and EAE + Apigenin groups of C57BL/6 ( n =2/group) and SJL/J mice ( n =5/group) were stimulated with MOG 38–49 and PLP 323–339 peptides respectively for 3 days followed by activation with PMA, ionomycin and brefeldin A for 5 h. These cells were subsequently stained with antibodies against CD11c, CD8α, α 4 , and CLEC12A. Data represents dendritic cells from C57BL/6 ( a ) and SJL/J ( b ) mice expressing α 4 integrin ( top ) and CLEC12A ( bottom ) upon stimulation with MOG 38–49 and PLP 323–339 respectively. Each bar is representative of the mean percentage for every marker per group. Histogram plots are representative of one animal per group. * p
    Figure Legend Snippet: Impact of Apigenin on antigen uptake and immune cell adhesion. Splenocytes from both the EAE + Vehicle and EAE + Apigenin groups of C57BL/6 ( n =2/group) and SJL/J mice ( n =5/group) were stimulated with MOG 38–49 and PLP 323–339 peptides respectively for 3 days followed by activation with PMA, ionomycin and brefeldin A for 5 h. These cells were subsequently stained with antibodies against CD11c, CD8α, α 4 , and CLEC12A. Data represents dendritic cells from C57BL/6 ( a ) and SJL/J ( b ) mice expressing α 4 integrin ( top ) and CLEC12A ( bottom ) upon stimulation with MOG 38–49 and PLP 323–339 respectively. Each bar is representative of the mean percentage for every marker per group. Histogram plots are representative of one animal per group. * p

    Techniques Used: Mouse Assay, Plasmid Purification, Activation Assay, Staining, Expressing, Marker

    Effect of Apigenin on Treg and Th17 balance in neuroinflammation. Flow cytometry analysis representing CD4 + cells from C57BL/6 mice ( a ) and SJL/J mice ( b ) expressing IL-17A ( top ) and CD25 + FOXP3 ( bottom ) upon stimulation with MOG 38–49 and PLP 323–339 peptides respectively for 3 days followed by activation with PMA, ionomycin and brefeldin A for 5 h. Each bar is representative of the mean percentage for every marker per group. Contour plots representative of one animal per group are shown on the left. * p
    Figure Legend Snippet: Effect of Apigenin on Treg and Th17 balance in neuroinflammation. Flow cytometry analysis representing CD4 + cells from C57BL/6 mice ( a ) and SJL/J mice ( b ) expressing IL-17A ( top ) and CD25 + FOXP3 ( bottom ) upon stimulation with MOG 38–49 and PLP 323–339 peptides respectively for 3 days followed by activation with PMA, ionomycin and brefeldin A for 5 h. Each bar is representative of the mean percentage for every marker per group. Contour plots representative of one animal per group are shown on the left. * p

    Techniques Used: Flow Cytometry, Cytometry, Mouse Assay, Expressing, Plasmid Purification, Activation Assay, Marker

    40) Product Images from "Expansion With IL-15 Increases Cytotoxicity of Vγ9Vδ2 T Cells and Is Associated With Higher Levels of Cytotoxic Molecules and T-bet"

    Article Title: Expansion With IL-15 Increases Cytotoxicity of Vγ9Vδ2 T Cells and Is Associated With Higher Levels of Cytotoxic Molecules and T-bet

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.01868

    IL-2/IL-15-expanded Vγ9Vδ2 T cells displayed higher levels and release of cytotoxic molecules. Vγ9Vδ2 T cells cryopreserved on day 25 were thawed and co-cultured in the presence of Brefeldin A and anti-CD107a antibody for 5 h in the following conditions: (i) cell culture medium (baseline/negative), (ii) FM-28, or (iii) FM-28/ZOL. (A) Intracellular flow analysis was used to determine baseline levels of perforin, granzyme B, and granulysin in Vγ9Vδ2 T cell cultures comparing both expansion conditions. (B) Absolute release of cytotoxic molecules (perforin, granzyme B, and granulysin) by Vγ9Vδ2 T cells comparing IL-2-expanded and IL-2/IL-15-expanded cells. Absolute release was calculated based on the difference in MFI between FM-28-co-cultured and FM-28/ZOL-co-cultured Vγ9Vδ2 T cells. (C) Relative release of cytotoxic molecules was calculated as the ratio of absolute release compared to the MFI of FM-28-co-cultured Vγ9Vδ2 T cells. (D) Degranulation and cytokine expression of IL-2-expanded vs. IL-2/IL-15-expanded Vγ9Vδ2 T cells was determined by gating on positive cells in FM-28/ZOL-co-cultured Vγ9Vδ2 T cells. Gates were set according to the FM-28-co-cultured control. Since all cultures contained > 90% Vγ9 + cells of CD3 + cells, no γδ T cell marker was included in this panel. Instead, CD3 positivity was used to mark Vγ9Vδ2 T cells. The Wilcoxon matched-pairs signed-rank test was applied to determine statistical significant differences between groups. Non-significant p values are not shown. * p
    Figure Legend Snippet: IL-2/IL-15-expanded Vγ9Vδ2 T cells displayed higher levels and release of cytotoxic molecules. Vγ9Vδ2 T cells cryopreserved on day 25 were thawed and co-cultured in the presence of Brefeldin A and anti-CD107a antibody for 5 h in the following conditions: (i) cell culture medium (baseline/negative), (ii) FM-28, or (iii) FM-28/ZOL. (A) Intracellular flow analysis was used to determine baseline levels of perforin, granzyme B, and granulysin in Vγ9Vδ2 T cell cultures comparing both expansion conditions. (B) Absolute release of cytotoxic molecules (perforin, granzyme B, and granulysin) by Vγ9Vδ2 T cells comparing IL-2-expanded and IL-2/IL-15-expanded cells. Absolute release was calculated based on the difference in MFI between FM-28-co-cultured and FM-28/ZOL-co-cultured Vγ9Vδ2 T cells. (C) Relative release of cytotoxic molecules was calculated as the ratio of absolute release compared to the MFI of FM-28-co-cultured Vγ9Vδ2 T cells. (D) Degranulation and cytokine expression of IL-2-expanded vs. IL-2/IL-15-expanded Vγ9Vδ2 T cells was determined by gating on positive cells in FM-28/ZOL-co-cultured Vγ9Vδ2 T cells. Gates were set according to the FM-28-co-cultured control. Since all cultures contained > 90% Vγ9 + cells of CD3 + cells, no γδ T cell marker was included in this panel. Instead, CD3 positivity was used to mark Vγ9Vδ2 T cells. The Wilcoxon matched-pairs signed-rank test was applied to determine statistical significant differences between groups. Non-significant p values are not shown. * p

    Techniques Used: Cell Culture, Expressing, Marker

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    Centrifugation:

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    Article Snippet: .. Collected cells from the pellet fraction after centrifugation at 1200× g for 5 min were incubated with PMA (10 ng/ml, Sigma), ionomycin (1 μg/ml, Sigma) and brefeldin A (5 μg/ml, BioLegend) for 4 h. Then cells were washed and stained for surface marker expression before fixation and permeabilization using the FOXP3 Fix/Perm Buffer Set (BioLegend) according to the manufacturer’s protocol. .. Thereafter, treated cells were stained with PE conjugated anti-IFN-γ mAb (XMG1.2, BioLegend).

    Positive Control:

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    Article Snippet: .. CD4+ T cells (0.5 × 106 ) suspended in X-Vivo 15 medium supplemented with l -glutamine were added to each well of uninfected, infected, UV-inactivated virus-infected, and positive-control mix-pulsed and pp65 and gB protein-pulsed moDCs in the presence of CD107a-Alexa Fluor 647 (BioLegend) for 1 h. Then, 5 μg/ml brefeldin A and 2 μM monensin (both from BioLegend) were added and the cells were incubated overnight at 37°C in a humidified CO2 atmosphere. .. The cells were harvested, washed, and stained with a combination of surface antibodies, CD45RA-PE-Cy7, CD27-APC-eFluor 780 (eBioscience), CD3-Brilliant Violet 650, CD57-PE-Dazzle, CD28-Alexa Fluor 700, CD14- and CD19-Brilliant Violet 510 (BioLegend), or LIVE/DEAD Fixable Aqua Dead cell stain (Invitrogen)-dump channel at 4°C.

    Infection:

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    Article Snippet: .. CD4+ T cells (0.5 × 106 ) suspended in X-Vivo 15 medium supplemented with l -glutamine were added to each well of uninfected, infected, UV-inactivated virus-infected, and positive-control mix-pulsed and pp65 and gB protein-pulsed moDCs in the presence of CD107a-Alexa Fluor 647 (BioLegend) for 1 h. Then, 5 μg/ml brefeldin A and 2 μM monensin (both from BioLegend) were added and the cells were incubated overnight at 37°C in a humidified CO2 atmosphere. .. The cells were harvested, washed, and stained with a combination of surface antibodies, CD45RA-PE-Cy7, CD27-APC-eFluor 780 (eBioscience), CD3-Brilliant Violet 650, CD57-PE-Dazzle, CD28-Alexa Fluor 700, CD14- and CD19-Brilliant Violet 510 (BioLegend), or LIVE/DEAD Fixable Aqua Dead cell stain (Invitrogen)-dump channel at 4°C.

    Incubation:

    Article Title: Adipocyte-specific CD1d-deficiency mitigates diet-induced obesity and insulin resistance in mice
    Article Snippet: .. Collected cells from the pellet fraction after centrifugation at 1200× g for 5 min were incubated with PMA (10 ng/ml, Sigma), ionomycin (1 μg/ml, Sigma) and brefeldin A (5 μg/ml, BioLegend) for 4 h. Then cells were washed and stained for surface marker expression before fixation and permeabilization using the FOXP3 Fix/Perm Buffer Set (BioLegend) according to the manufacturer’s protocol. .. Thereafter, treated cells were stained with PE conjugated anti-IFN-γ mAb (XMG1.2, BioLegend).

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    other:

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    Article Title: CD36 Ecto-domain Phosphorylation Blocks Thrombospondin-1 Binding: Structure - Function Relationships and Regulation by Protein Kinase C
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    Cell Culture:

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    Article Snippet: .. Intracellular cytokine staining Spleen and brain leukocytes were cultured in medium containing 10 µg/ml Brefeldin A for 2 h before tetramer staining was performed, substituting LIVE/DEAD Aqua and αCD8a-PerCP-Cy5.5 (Biolegend). .. After overnight fixation in 2% formaldehyde at 4°C, the cells were permeabilized using 0.5% saponin and stained with αIFN-γ-FITC (BD) and αGranzymeB-PE-Cy7 (eBiosciences) for 20 min at room temperature.

    Expressing:

    Article Title: Adipocyte-specific CD1d-deficiency mitigates diet-induced obesity and insulin resistance in mice
    Article Snippet: .. Collected cells from the pellet fraction after centrifugation at 1200× g for 5 min were incubated with PMA (10 ng/ml, Sigma), ionomycin (1 μg/ml, Sigma) and brefeldin A (5 μg/ml, BioLegend) for 4 h. Then cells were washed and stained for surface marker expression before fixation and permeabilization using the FOXP3 Fix/Perm Buffer Set (BioLegend) according to the manufacturer’s protocol. .. Thereafter, treated cells were stained with PE conjugated anti-IFN-γ mAb (XMG1.2, BioLegend).

    Marker:

    Article Title: Adipocyte-specific CD1d-deficiency mitigates diet-induced obesity and insulin resistance in mice
    Article Snippet: .. Collected cells from the pellet fraction after centrifugation at 1200× g for 5 min were incubated with PMA (10 ng/ml, Sigma), ionomycin (1 μg/ml, Sigma) and brefeldin A (5 μg/ml, BioLegend) for 4 h. Then cells were washed and stained for surface marker expression before fixation and permeabilization using the FOXP3 Fix/Perm Buffer Set (BioLegend) according to the manufacturer’s protocol. .. Thereafter, treated cells were stained with PE conjugated anti-IFN-γ mAb (XMG1.2, BioLegend).

    Staining:

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    BioLegend anti cd16 32 fcγriii ii pe cyanine 7
    Absence of FcγR in leukocytes of NOG-FcγR −/− mice. (A) Spleen cells from NOG, NOG-FcγR +/− , and NOG-FcγR −/− mice were stained with the indicated antibodies and analyzed by flow cytometry. Representative FACS histograms for mouse FcγRI (CD64), <t>FcγRIII/II</t> <t>(CD16/32),</t> and FcεRIα. Solid line for NOG, dotted line for NOG-FcγR +/− and filled histogram for NOG-FcγR −/− mice. (B) The frequencies of FcR-positive cells among total mononuclear cells are plotted for NOG ( n = 4), NOG-FcγR +/− ( n = 8), and NOG-FcγR −/− mice ( n = 11). (C) Failure of mouse monocytes from NOG-FcγR −/− mice to capture human IgG. Trastuzumab (100 μg anti-HER-2 human IgG) was injected i.p. into NOG-FcγR +/− and NOG-FcγR −/− mice. Mononuclear cells prepared from peripheral blood (PB) 3 days after administration were stained with anti-human IgG, together with anti-mouse CD45 and CD16/32 antibodies for gating. Representative FACS plots are from three independent experiments. (D) Frequencies of mouse Gr-1 + neutrophils and F4/80 + macrophage/monocytes in peripheral blood (PB) and spleen. Means ± SDs are shown for NOG mice ( n = 7 for PB and n = 4 for spleen) and NOG-FcγR −/− mice ( n = 7 for PB and n = 4 for spleen). Asterisks indicate the statistical significance by one-way ANOVA (* p
    Anti Cd16 32 Fcγriii Ii Pe Cyanine 7, supplied by BioLegend, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend brefeldin a
    SQLLNAKYL-specific CD8 + T cells are functional and cytotoxic in vivo A,B. Combined tetramer and intracellular cytokine staining were performed on splenocytes and brain-sequestered leukocytes from PbA-infected mice 7 days p.i. Cells were incubated with <t>Brefeldin</t> A for 2 h without restimulation. (A) Representative IFN-γ and GrB profiles of live CD8 + T cells (CD8a + CD16/32 − ). (B) The indicated IFN-γ + GrB + gate was used to analyze tetramer-labelled CD8 + T cells. Bars represent medians. C. Equal numbers of CFSE hi unpulsed naïve splenocytes and CFSE lo SQLLNAKYL-pulsed splenocytes were transferred into naïve or PbA-infected mice 6 days p.i. The mice were sacrificed 20 h later to analyze the CFSE-labelled cells in the spleens. The infected mouse is representative of n = 4, all with 96–97% specific lysis.
    Brefeldin A, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 429 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Absence of FcγR in leukocytes of NOG-FcγR −/− mice. (A) Spleen cells from NOG, NOG-FcγR +/− , and NOG-FcγR −/− mice were stained with the indicated antibodies and analyzed by flow cytometry. Representative FACS histograms for mouse FcγRI (CD64), FcγRIII/II (CD16/32), and FcεRIα. Solid line for NOG, dotted line for NOG-FcγR +/− and filled histogram for NOG-FcγR −/− mice. (B) The frequencies of FcR-positive cells among total mononuclear cells are plotted for NOG ( n = 4), NOG-FcγR +/− ( n = 8), and NOG-FcγR −/− mice ( n = 11). (C) Failure of mouse monocytes from NOG-FcγR −/− mice to capture human IgG. Trastuzumab (100 μg anti-HER-2 human IgG) was injected i.p. into NOG-FcγR +/− and NOG-FcγR −/− mice. Mononuclear cells prepared from peripheral blood (PB) 3 days after administration were stained with anti-human IgG, together with anti-mouse CD45 and CD16/32 antibodies for gating. Representative FACS plots are from three independent experiments. (D) Frequencies of mouse Gr-1 + neutrophils and F4/80 + macrophage/monocytes in peripheral blood (PB) and spleen. Means ± SDs are shown for NOG mice ( n = 7 for PB and n = 4 for spleen) and NOG-FcγR −/− mice ( n = 7 for PB and n = 4 for spleen). Asterisks indicate the statistical significance by one-way ANOVA (* p

    Journal: Frontiers in Immunology

    Article Title: Improved Detection of in vivo Human NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity Using a Novel NOG-FcγR-Deficient Human IL-15 Transgenic Mouse

    doi: 10.3389/fimmu.2020.532684

    Figure Lengend Snippet: Absence of FcγR in leukocytes of NOG-FcγR −/− mice. (A) Spleen cells from NOG, NOG-FcγR +/− , and NOG-FcγR −/− mice were stained with the indicated antibodies and analyzed by flow cytometry. Representative FACS histograms for mouse FcγRI (CD64), FcγRIII/II (CD16/32), and FcεRIα. Solid line for NOG, dotted line for NOG-FcγR +/− and filled histogram for NOG-FcγR −/− mice. (B) The frequencies of FcR-positive cells among total mononuclear cells are plotted for NOG ( n = 4), NOG-FcγR +/− ( n = 8), and NOG-FcγR −/− mice ( n = 11). (C) Failure of mouse monocytes from NOG-FcγR −/− mice to capture human IgG. Trastuzumab (100 μg anti-HER-2 human IgG) was injected i.p. into NOG-FcγR +/− and NOG-FcγR −/− mice. Mononuclear cells prepared from peripheral blood (PB) 3 days after administration were stained with anti-human IgG, together with anti-mouse CD45 and CD16/32 antibodies for gating. Representative FACS plots are from three independent experiments. (D) Frequencies of mouse Gr-1 + neutrophils and F4/80 + macrophage/monocytes in peripheral blood (PB) and spleen. Means ± SDs are shown for NOG mice ( n = 7 for PB and n = 4 for spleen) and NOG-FcγR −/− mice ( n = 7 for PB and n = 4 for spleen). Asterisks indicate the statistical significance by one-way ANOVA (* p

    Article Snippet: For staining mouse cells, anti-FcεRIα-fluorescent isothiocyanate (FITC; clone, Mar-1), anti-CD64 (FcγRI)-Phycoerythrin (PE; X54-5/7.1), anti-F4/80-PE (BM8), anti-CD16/32 (FcγRIII/II)-PE/Cyanine 7 (Cy7; 93), anti-Gr-1-Allophycocyanin (APC; RB6-8C5), and anti-CD45- APC/Cy7 (30-F11) were from Biolegend (San Diego, CA).

    Techniques: Mouse Assay, Staining, Flow Cytometry, FACS, Injection

    SQLLNAKYL-specific CD8 + T cells are functional and cytotoxic in vivo A,B. Combined tetramer and intracellular cytokine staining were performed on splenocytes and brain-sequestered leukocytes from PbA-infected mice 7 days p.i. Cells were incubated with Brefeldin A for 2 h without restimulation. (A) Representative IFN-γ and GrB profiles of live CD8 + T cells (CD8a + CD16/32 − ). (B) The indicated IFN-γ + GrB + gate was used to analyze tetramer-labelled CD8 + T cells. Bars represent medians. C. Equal numbers of CFSE hi unpulsed naïve splenocytes and CFSE lo SQLLNAKYL-pulsed splenocytes were transferred into naïve or PbA-infected mice 6 days p.i. The mice were sacrificed 20 h later to analyze the CFSE-labelled cells in the spleens. The infected mouse is representative of n = 4, all with 96–97% specific lysis.

    Journal: EMBO Molecular Medicine

    Article Title: Brain microvessel cross-presentation is a hallmark of experimental cerebral malaria

    doi: 10.1002/emmm.201202273

    Figure Lengend Snippet: SQLLNAKYL-specific CD8 + T cells are functional and cytotoxic in vivo A,B. Combined tetramer and intracellular cytokine staining were performed on splenocytes and brain-sequestered leukocytes from PbA-infected mice 7 days p.i. Cells were incubated with Brefeldin A for 2 h without restimulation. (A) Representative IFN-γ and GrB profiles of live CD8 + T cells (CD8a + CD16/32 − ). (B) The indicated IFN-γ + GrB + gate was used to analyze tetramer-labelled CD8 + T cells. Bars represent medians. C. Equal numbers of CFSE hi unpulsed naïve splenocytes and CFSE lo SQLLNAKYL-pulsed splenocytes were transferred into naïve or PbA-infected mice 6 days p.i. The mice were sacrificed 20 h later to analyze the CFSE-labelled cells in the spleens. The infected mouse is representative of n = 4, all with 96–97% specific lysis.

    Article Snippet: Intracellular cytokine staining Spleen and brain leukocytes were cultured in medium containing 10 µg/ml Brefeldin A for 2 h before tetramer staining was performed, substituting LIVE/DEAD Aqua and αCD8a-PerCP-Cy5.5 (Biolegend).

    Techniques: Functional Assay, In Vivo, Staining, Infection, Mouse Assay, Incubation, Lysis

    A portion of the HCMV-specific CD4 +  T cells has a cytotoxic capacity and can secrete MIP-1β. PBMCs were stimulated overnight with HCMV peptide pools in the presence of an anti-CD107a antibody, brefeldin A, and monensin to measure the degranulation

    Journal: Journal of Virology

    Article Title: Human Cytomegalovirus (HCMV)-Specific CD4+ T Cells Are Polyfunctional and Can Respond to HCMV-Infected Dendritic Cells In Vitro

    doi: 10.1128/JVI.02128-16

    Figure Lengend Snippet: A portion of the HCMV-specific CD4 + T cells has a cytotoxic capacity and can secrete MIP-1β. PBMCs were stimulated overnight with HCMV peptide pools in the presence of an anti-CD107a antibody, brefeldin A, and monensin to measure the degranulation

    Article Snippet: CD4+ T cells (0.5 × 106 ) suspended in X-Vivo 15 medium supplemented with l -glutamine were added to each well of uninfected, infected, UV-inactivated virus-infected, and positive-control mix-pulsed and pp65 and gB protein-pulsed moDCs in the presence of CD107a-Alexa Fluor 647 (BioLegend) for 1 h. Then, 5 μg/ml brefeldin A and 2 μM monensin (both from BioLegend) were added and the cells were incubated overnight at 37°C in a humidified CO2 atmosphere.

    Techniques:

    HCMV-specific CD4 +  T cells have a predominantly effector memory cell phenotype and are not highly differentiated. PBMCs were stimulated overnight with HCMV protein peptide pools in the presence of brefeldin A. HCMV-specific CD4 +  T cell responses were

    Journal: Journal of Virology

    Article Title: Human Cytomegalovirus (HCMV)-Specific CD4+ T Cells Are Polyfunctional and Can Respond to HCMV-Infected Dendritic Cells In Vitro

    doi: 10.1128/JVI.02128-16

    Figure Lengend Snippet: HCMV-specific CD4 + T cells have a predominantly effector memory cell phenotype and are not highly differentiated. PBMCs were stimulated overnight with HCMV protein peptide pools in the presence of brefeldin A. HCMV-specific CD4 + T cell responses were

    Article Snippet: CD4+ T cells (0.5 × 106 ) suspended in X-Vivo 15 medium supplemented with l -glutamine were added to each well of uninfected, infected, UV-inactivated virus-infected, and positive-control mix-pulsed and pp65 and gB protein-pulsed moDCs in the presence of CD107a-Alexa Fluor 647 (BioLegend) for 1 h. Then, 5 μg/ml brefeldin A and 2 μM monensin (both from BioLegend) were added and the cells were incubated overnight at 37°C in a humidified CO2 atmosphere.

    Techniques:

    IFN-γ enhances CD1d expression in 3T3-L1 adipocytes. ( a–c ) IFN-γ expression by NKT cells and NK cells in adipose tissue from WT and Jα18 −/− mice fed on SFD or HFD (n = 3 in each group). Representative flow cytometric data are shown for NKT cells (gated on TCRβ + NK1.1 + ) in WT mice ( a ), the proportion and cell number (/g adipose tissue) of IFN-γ + NKT cells or NK cells (gated on TCRβ - NK1.1 + ) in WT mice ( b ), and in Jα18 −/− mice ( c ). Intracellular staining was performed after stromal vascular fraction was incubated with PMA/ionomycin in the presence of brefeldin A for 4 h. ( d,e ) The gene expression in 3T3-L1 adipocytes stimulated with IFN-γ (0, 30, 150 ng/ml) for 3 days was quantified by qPCR. ( f ) The expression of Cd1d1 in 3T3-L1 adipocytes stimulated with the supernatant from 3T3-L1 adipocytes + iNKT cells from WT mice or NKT cells from Jα18 −/− mice. ( g,h ) The expression of CD1d, CD80 and CD86 ( g ) and α-GalCer:CD1d complex ( h ), in 3T3-L1 adipocytes stimulated with IFN-γ (gray: isotype, black line: no treatment, red line: IFN−γ treatment) was analyzed by flow cytometry. Numbers are shown as MFI. Representative data from at least 2 independent experiments are shown. Data are shown as mean ± s.d. Statistical analysis was performed according to the Student’s t -test and the Tukey-Kramer test. * P

    Journal: Scientific Reports

    Article Title: Adipocyte-specific CD1d-deficiency mitigates diet-induced obesity and insulin resistance in mice

    doi: 10.1038/srep28473

    Figure Lengend Snippet: IFN-γ enhances CD1d expression in 3T3-L1 adipocytes. ( a–c ) IFN-γ expression by NKT cells and NK cells in adipose tissue from WT and Jα18 −/− mice fed on SFD or HFD (n = 3 in each group). Representative flow cytometric data are shown for NKT cells (gated on TCRβ + NK1.1 + ) in WT mice ( a ), the proportion and cell number (/g adipose tissue) of IFN-γ + NKT cells or NK cells (gated on TCRβ - NK1.1 + ) in WT mice ( b ), and in Jα18 −/− mice ( c ). Intracellular staining was performed after stromal vascular fraction was incubated with PMA/ionomycin in the presence of brefeldin A for 4 h. ( d,e ) The gene expression in 3T3-L1 adipocytes stimulated with IFN-γ (0, 30, 150 ng/ml) for 3 days was quantified by qPCR. ( f ) The expression of Cd1d1 in 3T3-L1 adipocytes stimulated with the supernatant from 3T3-L1 adipocytes + iNKT cells from WT mice or NKT cells from Jα18 −/− mice. ( g,h ) The expression of CD1d, CD80 and CD86 ( g ) and α-GalCer:CD1d complex ( h ), in 3T3-L1 adipocytes stimulated with IFN-γ (gray: isotype, black line: no treatment, red line: IFN−γ treatment) was analyzed by flow cytometry. Numbers are shown as MFI. Representative data from at least 2 independent experiments are shown. Data are shown as mean ± s.d. Statistical analysis was performed according to the Student’s t -test and the Tukey-Kramer test. * P

    Article Snippet: Collected cells from the pellet fraction after centrifugation at 1200× g for 5 min were incubated with PMA (10 ng/ml, Sigma), ionomycin (1 μg/ml, Sigma) and brefeldin A (5 μg/ml, BioLegend) for 4 h. Then cells were washed and stained for surface marker expression before fixation and permeabilization using the FOXP3 Fix/Perm Buffer Set (BioLegend) according to the manufacturer’s protocol.

    Techniques: Expressing, Mouse Assay, Flow Cytometry, Staining, Incubation, Real-time Polymerase Chain Reaction, Cytometry