serum cytokine immunoassay cytokine measurement  (BioLegend)

 
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    Structured Review

    BioLegend serum cytokine immunoassay cytokine measurement
    T cell-specific STAT3 deficiency affects the production of inflammatory cytokines. (A) Representative FACS plot of a multiplex bead-based immunoassay showing the distribution of <t>cytokine</t> production. (B) Bar graphs for the amounts of IFN-γ, IL-6, IL-10, and IL-17A in the serum from control and DSS-treated WT and KO mice (n=5). (C, D) Bar graphs for the mRNA levels of IFN-γ, IL-6, IL-10, IL-17A, and Foxp3 in colon tissues from control and DSS-treated WT and KO mice (n=5). Relative mRNA levels were normalized to those of GAPDH. Data are presented as the means±SEM. Representative data from 3 independent experiments are shown. * p
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    Images

    1) Product Images from "T Cell-Specific Knockout of STAT3 Ameliorates Dextran Sulfate Sodium-Induced Colitis by Reducing the Inflammatory Response"

    Article Title: T Cell-Specific Knockout of STAT3 Ameliorates Dextran Sulfate Sodium-Induced Colitis by Reducing the Inflammatory Response

    Journal: Immune Network

    doi: 10.4110/in.2018.18.e30

    T cell-specific STAT3 deficiency affects the production of inflammatory cytokines. (A) Representative FACS plot of a multiplex bead-based immunoassay showing the distribution of cytokine production. (B) Bar graphs for the amounts of IFN-γ, IL-6, IL-10, and IL-17A in the serum from control and DSS-treated WT and KO mice (n=5). (C, D) Bar graphs for the mRNA levels of IFN-γ, IL-6, IL-10, IL-17A, and Foxp3 in colon tissues from control and DSS-treated WT and KO mice (n=5). Relative mRNA levels were normalized to those of GAPDH. Data are presented as the means±SEM. Representative data from 3 independent experiments are shown. * p
    Figure Legend Snippet: T cell-specific STAT3 deficiency affects the production of inflammatory cytokines. (A) Representative FACS plot of a multiplex bead-based immunoassay showing the distribution of cytokine production. (B) Bar graphs for the amounts of IFN-γ, IL-6, IL-10, and IL-17A in the serum from control and DSS-treated WT and KO mice (n=5). (C, D) Bar graphs for the mRNA levels of IFN-γ, IL-6, IL-10, IL-17A, and Foxp3 in colon tissues from control and DSS-treated WT and KO mice (n=5). Relative mRNA levels were normalized to those of GAPDH. Data are presented as the means±SEM. Representative data from 3 independent experiments are shown. * p

    Techniques Used: FACS, Multiplex Assay, Bead-based Assay, Mouse Assay

    2) Product Images from "Farrerol Relieve Lipopolysaccharide (LPS)-Induced Mastitis by Inhibiting AKT/NF-κB p65, ERK1/2 and P38 Signaling Pathway"

    Article Title: Farrerol Relieve Lipopolysaccharide (LPS)-Induced Mastitis by Inhibiting AKT/NF-κB p65, ERK1/2 and P38 Signaling Pathway

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19061770

    The effect of farrerol on pro-inflammatory mediator’s production in LPS-stimulated mMECs. MMECs were divided into six groups: NT group, Farrerol group, LPS group, LPS + farrerol (90, 110, 130 μM) group. The mRNA levels of TNF-α ( A ); IL-6 ( B ) and IL-1β ( C ) were measured by real-time PCR. The protein levels of iNOS ( D , E ) and COX-2 ( D , F ) were measured by Western blot. Data are presented as mean ± SEM ( n = 3). # p
    Figure Legend Snippet: The effect of farrerol on pro-inflammatory mediator’s production in LPS-stimulated mMECs. MMECs were divided into six groups: NT group, Farrerol group, LPS group, LPS + farrerol (90, 110, 130 μM) group. The mRNA levels of TNF-α ( A ); IL-6 ( B ) and IL-1β ( C ) were measured by real-time PCR. The protein levels of iNOS ( D , E ) and COX-2 ( D , F ) were measured by Western blot. Data are presented as mean ± SEM ( n = 3). # p

    Techniques Used: Real-time Polymerase Chain Reaction, Western Blot

    The levels of TNF-α ( A ); IL-6 ( B ) and IL-1β ( C ) in the homogenate of mouse mammary glands including NT group, LPS group and LPS + farrerol (20, 30, 40 mg/kg) group; The protein levels of iNOS ( D , E ) and COX-2 ( D , F ) were measured by Western blot. Data are presented as mean ± SEM ( n = 3). # p
    Figure Legend Snippet: The levels of TNF-α ( A ); IL-6 ( B ) and IL-1β ( C ) in the homogenate of mouse mammary glands including NT group, LPS group and LPS + farrerol (20, 30, 40 mg/kg) group; The protein levels of iNOS ( D , E ) and COX-2 ( D , F ) were measured by Western blot. Data are presented as mean ± SEM ( n = 3). # p

    Techniques Used: Western Blot

    3) Product Images from "Interaction Between Macrophage Migration Inhibitory Factor and CD74 in Human Immunodeficiency Virus Type I Infected Primary Monocyte-Derived Macrophages Triggers the Production of Proinflammatory Mediators and Enhances Infection of Unactivated CD4+ T Cells"

    Article Title: Interaction Between Macrophage Migration Inhibitory Factor and CD74 in Human Immunodeficiency Virus Type I Infected Primary Monocyte-Derived Macrophages Triggers the Production of Proinflammatory Mediators and Enhances Infection of Unactivated CD4+ T Cells

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01494

    Effect of macrophage migration inhibitory factor (MIF) neutralization in the expression of cytokines from primary human immunodeficiency virus-infected and uninfected monocyte-derived macrophages (MDMs). Serial dilutions of a neutralizing αMIF antibody (clone NIHlllD.9) (A) , and the MIF antagonist MIF098 (B) were used to inhibit MIF activity in infected (In, red lines) and uninfected (Un, gray lines) MDMs at a constant concentration of this cytokine (25 ng/ml). Data represent three independent experiments (donors), each evaluated in duplicate. Data represent the mean ± SD. Data were analyzed by two-way ANOVA followed by Tukey’s post-test (intragroup analysis, In group only; **** p
    Figure Legend Snippet: Effect of macrophage migration inhibitory factor (MIF) neutralization in the expression of cytokines from primary human immunodeficiency virus-infected and uninfected monocyte-derived macrophages (MDMs). Serial dilutions of a neutralizing αMIF antibody (clone NIHlllD.9) (A) , and the MIF antagonist MIF098 (B) were used to inhibit MIF activity in infected (In, red lines) and uninfected (Un, gray lines) MDMs at a constant concentration of this cytokine (25 ng/ml). Data represent three independent experiments (donors), each evaluated in duplicate. Data represent the mean ± SD. Data were analyzed by two-way ANOVA followed by Tukey’s post-test (intragroup analysis, In group only; **** p

    Techniques Used: Migration, Neutralization, Expressing, Infection, Derivative Assay, Activity Assay, Concentration Assay

    Expression of cytokines after macrophage migration inhibitory factor (MIF) stimulation in primary human immunodeficiency virus (HIV)-infected and uninfected monocyte-derived macrophages (MDMs). (A) Expression of IL-8, IL-6, IL-1β, TNF-α, sICAM, and IL-10 in supernatants from HIV-infected (In) and uninfected (Un) MDMs obtained from one representative healthy donor. (B) Data combined from six independent experiments (donors), each evaluated in triplicate. Here, data are shown as the ratio between cytokine concentrations found under the infection condition versus the uninfected counterpart. Cells were stimulated with MIF as follows: 0, 1, 10, or 25 ng/ml. Data shown in the gray boxes depict CD74 blocking (10 ng/ml of αCD74 or the corresponding isotype control) followed by MIF stimulation (1 or 25 ng/ml as denoted). Data represent the mean ± SD. Data were analyzed by one-way ANOVA followed by Tukey’s post-test. * p
    Figure Legend Snippet: Expression of cytokines after macrophage migration inhibitory factor (MIF) stimulation in primary human immunodeficiency virus (HIV)-infected and uninfected monocyte-derived macrophages (MDMs). (A) Expression of IL-8, IL-6, IL-1β, TNF-α, sICAM, and IL-10 in supernatants from HIV-infected (In) and uninfected (Un) MDMs obtained from one representative healthy donor. (B) Data combined from six independent experiments (donors), each evaluated in triplicate. Here, data are shown as the ratio between cytokine concentrations found under the infection condition versus the uninfected counterpart. Cells were stimulated with MIF as follows: 0, 1, 10, or 25 ng/ml. Data shown in the gray boxes depict CD74 blocking (10 ng/ml of αCD74 or the corresponding isotype control) followed by MIF stimulation (1 or 25 ng/ml as denoted). Data represent the mean ± SD. Data were analyzed by one-way ANOVA followed by Tukey’s post-test. * p

    Techniques Used: Expressing, Migration, Infection, Derivative Assay, Blocking Assay

    4) Product Images from "Interaction Between Macrophage Migration Inhibitory Factor and CD74 in Human Immunodeficiency Virus Type I Infected Primary Monocyte-Derived Macrophages Triggers the Production of Proinflammatory Mediators and Enhances Infection of Unactivated CD4+ T Cells"

    Article Title: Interaction Between Macrophage Migration Inhibitory Factor and CD74 in Human Immunodeficiency Virus Type I Infected Primary Monocyte-Derived Macrophages Triggers the Production of Proinflammatory Mediators and Enhances Infection of Unactivated CD4+ T Cells

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01494

    Infection of monocyte-derived macrophages (MDMs) and unactivated CD4 + T-cells with T/F viruses reproduce the results obtained with the R5-tropic (BAL) and the X4-tropic (NL4-3) laboratory strains. (A) Expression of IL-1β, IL-8, IL-6, and IL-10 in supernatants from uninfected (Un) and R5-tropic T/F-infected (In) MDMs. Data represent mean ± SD from three independent donors. Data were analyzed by two-way ANOVA followed by Tukey’s post-test. * p
    Figure Legend Snippet: Infection of monocyte-derived macrophages (MDMs) and unactivated CD4 + T-cells with T/F viruses reproduce the results obtained with the R5-tropic (BAL) and the X4-tropic (NL4-3) laboratory strains. (A) Expression of IL-1β, IL-8, IL-6, and IL-10 in supernatants from uninfected (Un) and R5-tropic T/F-infected (In) MDMs. Data represent mean ± SD from three independent donors. Data were analyzed by two-way ANOVA followed by Tukey’s post-test. * p

    Techniques Used: Infection, Derivative Assay, Expressing

    Identification of cytokines as responsible for enhancing human immunodeficiency virus type I (HIV-1) infection in unactivated CD4 + T-cells. (A) Unactivated CD4 + T-cells were stimulated with different combinations of cytokines for 72 h. Then, cells were infected and p24 antigen production was evaluated at days 4 and 7 post-infection. Each condition was compared with the corresponding RPMI condition (negative control). As a positive control, PHA stimulation was used. Percentage of living CD4 + T-cells (B) and percentage of infected (GFP + ) CD4 + T-cells (C) after stimulation with the denoted treatments are shown. Data represent mean ± SD from four independent donors evaluated in duplicate. Concentrations of cytokines used in these experiments corresponded to the average concentrations found in monocyte-derived macrophage (MDM) supernatants stimulated with 25 ng/ml macrophage migration inhibitory factor (MIF) (peak effect) as follows: 250 pg/ml IL-6, 9,000 pg/ml IL-8, 1,400 pg/ml TNF-α, and 20 pg/ml IL-1β. (D) Neutralization of IL-8, IL-6, IL-1 β, and TNFα biological activity with monoclonal neutralizing antibodies. Primary CD4 + T-cells were incubated with supernatants derived from the 25 ng/ml MIF-treated HIV-infected MDM neutralized previously with 18 µg/ml anti-IL-8, 20 ng/ml anti-IL-6, 2 µg/ml anti-IL-1β, and 2 µg/ml anti-TNFα antibodies. Non-neutralized and isotype control antibody conditions were tested for comparison. Also, RPMI and PHA controls were included. Viral production was evaluated at day 4 post-infection. Data were analyzed by one-way ANOVA followed by Dunnett’s post-test (all conditions versus the corresponding RMPI control) in (A) and Tukey’s post-test in (D) . * p
    Figure Legend Snippet: Identification of cytokines as responsible for enhancing human immunodeficiency virus type I (HIV-1) infection in unactivated CD4 + T-cells. (A) Unactivated CD4 + T-cells were stimulated with different combinations of cytokines for 72 h. Then, cells were infected and p24 antigen production was evaluated at days 4 and 7 post-infection. Each condition was compared with the corresponding RPMI condition (negative control). As a positive control, PHA stimulation was used. Percentage of living CD4 + T-cells (B) and percentage of infected (GFP + ) CD4 + T-cells (C) after stimulation with the denoted treatments are shown. Data represent mean ± SD from four independent donors evaluated in duplicate. Concentrations of cytokines used in these experiments corresponded to the average concentrations found in monocyte-derived macrophage (MDM) supernatants stimulated with 25 ng/ml macrophage migration inhibitory factor (MIF) (peak effect) as follows: 250 pg/ml IL-6, 9,000 pg/ml IL-8, 1,400 pg/ml TNF-α, and 20 pg/ml IL-1β. (D) Neutralization of IL-8, IL-6, IL-1 β, and TNFα biological activity with monoclonal neutralizing antibodies. Primary CD4 + T-cells were incubated with supernatants derived from the 25 ng/ml MIF-treated HIV-infected MDM neutralized previously with 18 µg/ml anti-IL-8, 20 ng/ml anti-IL-6, 2 µg/ml anti-IL-1β, and 2 µg/ml anti-TNFα antibodies. Non-neutralized and isotype control antibody conditions were tested for comparison. Also, RPMI and PHA controls were included. Viral production was evaluated at day 4 post-infection. Data were analyzed by one-way ANOVA followed by Dunnett’s post-test (all conditions versus the corresponding RMPI control) in (A) and Tukey’s post-test in (D) . * p

    Techniques Used: Infection, Negative Control, Positive Control, Derivative Assay, Migration, Neutralization, Activity Assay, Incubation

    Expression of cytokines after macrophage migration inhibitory factor (MIF) stimulation in primary human immunodeficiency virus (HIV)-infected and uninfected monocyte-derived macrophages (MDMs). (A) Expression of IL-8, IL-6, IL-1β, TNF-α, sICAM, and IL-10 in supernatants from HIV-infected (In) and uninfected (Un) MDMs obtained from one representative healthy donor. (B) Data combined from six independent experiments (donors), each evaluated in triplicate. Here, data are shown as the ratio between cytokine concentrations found under the infection condition versus the uninfected counterpart. Cells were stimulated with MIF as follows: 0, 1, 10, or 25 ng/ml. Data shown in the gray boxes depict CD74 blocking (10 ng/ml of αCD74 or the corresponding isotype control) followed by MIF stimulation (1 or 25 ng/ml as denoted). Data represent the mean ± SD. Data were analyzed by one-way ANOVA followed by Tukey’s post-test. * p
    Figure Legend Snippet: Expression of cytokines after macrophage migration inhibitory factor (MIF) stimulation in primary human immunodeficiency virus (HIV)-infected and uninfected monocyte-derived macrophages (MDMs). (A) Expression of IL-8, IL-6, IL-1β, TNF-α, sICAM, and IL-10 in supernatants from HIV-infected (In) and uninfected (Un) MDMs obtained from one representative healthy donor. (B) Data combined from six independent experiments (donors), each evaluated in triplicate. Here, data are shown as the ratio between cytokine concentrations found under the infection condition versus the uninfected counterpart. Cells were stimulated with MIF as follows: 0, 1, 10, or 25 ng/ml. Data shown in the gray boxes depict CD74 blocking (10 ng/ml of αCD74 or the corresponding isotype control) followed by MIF stimulation (1 or 25 ng/ml as denoted). Data represent the mean ± SD. Data were analyzed by one-way ANOVA followed by Tukey’s post-test. * p

    Techniques Used: Expressing, Migration, Infection, Derivative Assay, Blocking Assay

    5) Product Images from "Differential roles of TNFα-TNFR1 and TNFα-TNFR2 in the differentiation and function of CD4+Foxp3+ induced Treg cells in vitro and in vivo periphery in autoimmune diseases"

    Article Title: Differential roles of TNFα-TNFR1 and TNFα-TNFR2 in the differentiation and function of CD4+Foxp3+ induced Treg cells in vitro and in vivo periphery in autoimmune diseases

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1266-6

    The effect of TNFR1 and TNFR2 on Treg, Th1, Th17 cells in EAE mouse model. a EAE model were induced on WT, TNFR1 −/− , and TNFR2 −/− Foxp3-reporter mice, the clinical symptom scores were evaluated blindly. b 30 days after the first immunization, part of the brain and spinal cord were stained with H E (×100, scale bars = 50 μm). c , d , g , h The proportions of Treg cells in brain and spinal cord (SC) were detected by FACS. e , f , i , j The proportions of Th1 and Th17 cells in brain and spinal cord were detected by FACS. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative data is from six independent experiments
    Figure Legend Snippet: The effect of TNFR1 and TNFR2 on Treg, Th1, Th17 cells in EAE mouse model. a EAE model were induced on WT, TNFR1 −/− , and TNFR2 −/− Foxp3-reporter mice, the clinical symptom scores were evaluated blindly. b 30 days after the first immunization, part of the brain and spinal cord were stained with H E (×100, scale bars = 50 μm). c , d , g , h The proportions of Treg cells in brain and spinal cord (SC) were detected by FACS. e , f , i , j The proportions of Th1 and Th17 cells in brain and spinal cord were detected by FACS. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative data is from six independent experiments

    Techniques Used: Mouse Assay, Staining, FACS

    rmTNFα enhances iTreg suppressive function via TNFR2 in vitro and TNFR2 mediated the suppressive capability of iTreg in vivo. a EAE was induced on WT mice. 9 days after the first immunization, iTreg derived from WT, TNFR1 −/− , and TNFR2 −/− mice were adoptively transferred into EAE model, respectively. The clinical scores were monitored. b 30 days after the first immunization, the brain and spinal cord (SC) were subjected to H E staining (×100, scale bars = 50 μm). Foxp3, IL-17A, and IFN-γ expression in brains ( c – e ) and SC ( f – h ) were detected. * P ≤ 0.05; ** P ≤ 0.01, error bars denote SD. Representative data is from six independent experiments
    Figure Legend Snippet: rmTNFα enhances iTreg suppressive function via TNFR2 in vitro and TNFR2 mediated the suppressive capability of iTreg in vivo. a EAE was induced on WT mice. 9 days after the first immunization, iTreg derived from WT, TNFR1 −/− , and TNFR2 −/− mice were adoptively transferred into EAE model, respectively. The clinical scores were monitored. b 30 days after the first immunization, the brain and spinal cord (SC) were subjected to H E staining (×100, scale bars = 50 μm). Foxp3, IL-17A, and IFN-γ expression in brains ( c – e ) and SC ( f – h ) were detected. * P ≤ 0.05; ** P ≤ 0.01, error bars denote SD. Representative data is from six independent experiments

    Techniques Used: In Vitro, In Vivo, Mouse Assay, Derivative Assay, Staining, Expressing

    TNFR2 is a critical signaling for Treg expansion following EAE induction and in vitro. a–d We detected the proportions of Treg in spleen and LNs from naive WT, TNFR1 −/− , and TNFR2 −/− mice. e–h 30 days after immunized with MOG, the proportion of Treg in spleen and LNs from the three groups were detected. i–k Treg isolated from LNs of WT, TNFR1 −/− , and TNFR2 −/− mice were expanded in vitro. Ki-67 was detected after 3 day culture. * P ≤ 0.05; ** P ≤ 0.01, error bars denote SD. Representative data is from six independent experiments
    Figure Legend Snippet: TNFR2 is a critical signaling for Treg expansion following EAE induction and in vitro. a–d We detected the proportions of Treg in spleen and LNs from naive WT, TNFR1 −/− , and TNFR2 −/− mice. e–h 30 days after immunized with MOG, the proportion of Treg in spleen and LNs from the three groups were detected. i–k Treg isolated from LNs of WT, TNFR1 −/− , and TNFR2 −/− mice were expanded in vitro. Ki-67 was detected after 3 day culture. * P ≤ 0.05; ** P ≤ 0.01, error bars denote SD. Representative data is from six independent experiments

    Techniques Used: In Vitro, Mouse Assay, Isolation

    rmTNFα enhances iTreg impressive function via TNFR2 in vitro and TNFR2 mediated the Suppressive capability of iTreg in vivo. a , b iTreg were induced with or without rmTNFα. After 3 days, iTreg were co-cultured with CFSE-labeled Teffs. c , d iTreg were induced without rmTNFα, and then were co-cultured with Teffs with or without rmTNFα in the culture medium. e , f iTreg induced from WT, TNFR1 −/− , and TNFR2 −/− mice were co-cultured with Teffs. ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative data is from six independent experiments
    Figure Legend Snippet: rmTNFα enhances iTreg impressive function via TNFR2 in vitro and TNFR2 mediated the Suppressive capability of iTreg in vivo. a , b iTreg were induced with or without rmTNFα. After 3 days, iTreg were co-cultured with CFSE-labeled Teffs. c , d iTreg were induced without rmTNFα, and then were co-cultured with Teffs with or without rmTNFα in the culture medium. e , f iTreg induced from WT, TNFR1 −/− , and TNFR2 −/− mice were co-cultured with Teffs. ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative data is from six independent experiments

    Techniques Used: In Vitro, In Vivo, Cell Culture, Labeling, Mouse Assay

    rmTNFα increases iTreg differentiation via TNFR2 in vitro. a , b Naive CD4 + T cells were induced into iTreg with different doses of rmTNFα for three days. The percentages of Foxp3 + T cells were determined. c During iTreg induction, the same dose of rmTNFα was added on day 0, 1, 2, or 3. All the cells were harvested after 4 days. The percentages of Foxp3 + T cells were determined. d , e iTreg induced for three days and reseeded with or without rmTNFα for another three days. The percentages of Foxp3 + T cells were determined. f , g , h Naive CD4 + T cells isolated from WT, TNFR1 −/− , and TNFR2 −/− mice were induced to iTreg. The percentages of Foxp3 + T cells, the total cell numbers and Foxp3 + cell numbers were determined, respectively. i Naive CD4 + T cells isolated from WT, TNFR1 −/− , and TNFR2 −/− mice were induced to iTreg with or without rmTNFα for three days. The percentages of Foxp3 + T cells were determined. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative result is from five independent experiments
    Figure Legend Snippet: rmTNFα increases iTreg differentiation via TNFR2 in vitro. a , b Naive CD4 + T cells were induced into iTreg with different doses of rmTNFα for three days. The percentages of Foxp3 + T cells were determined. c During iTreg induction, the same dose of rmTNFα was added on day 0, 1, 2, or 3. All the cells were harvested after 4 days. The percentages of Foxp3 + T cells were determined. d , e iTreg induced for three days and reseeded with or without rmTNFα for another three days. The percentages of Foxp3 + T cells were determined. f , g , h Naive CD4 + T cells isolated from WT, TNFR1 −/− , and TNFR2 −/− mice were induced to iTreg. The percentages of Foxp3 + T cells, the total cell numbers and Foxp3 + cell numbers were determined, respectively. i Naive CD4 + T cells isolated from WT, TNFR1 −/− , and TNFR2 −/− mice were induced to iTreg with or without rmTNFα for three days. The percentages of Foxp3 + T cells were determined. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative result is from five independent experiments

    Techniques Used: In Vitro, Isolation, Mouse Assay

    TNFR2 gene knockout enhanced inflammatory responses and TNFR1 gene knockout was resistant to the development of colitis. a Naive CD4 + T cells isolated from WT, TNFR1 −/− , and TNFR2 −/− mice were injected into Rag1 −/− mice intraperitoneally. b Weights of the recipient mice were monitored after the cell transfer. c , d The colon was subjected to staining with H E (×40, scale bars = 200 μm), and the development of colitis was evaluated blindly by two pathologists. e The morphology and size of the colon, spleen and mLNs from the recipient mice were compared. No.1 was to WT group; No.2 was to TNFR1 −/− group; No.3 was to TNFR2 −/− group. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative result is from six independent experiments
    Figure Legend Snippet: TNFR2 gene knockout enhanced inflammatory responses and TNFR1 gene knockout was resistant to the development of colitis. a Naive CD4 + T cells isolated from WT, TNFR1 −/− , and TNFR2 −/− mice were injected into Rag1 −/− mice intraperitoneally. b Weights of the recipient mice were monitored after the cell transfer. c , d The colon was subjected to staining with H E (×40, scale bars = 200 μm), and the development of colitis was evaluated blindly by two pathologists. e The morphology and size of the colon, spleen and mLNs from the recipient mice were compared. No.1 was to WT group; No.2 was to TNFR1 −/− group; No.3 was to TNFR2 −/− group. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative result is from six independent experiments

    Techniques Used: Gene Knockout, Isolation, Mouse Assay, Injection, Staining

    The effect of TNFR1 and TNFR2 on iTreg stability and function in vivo. iTreg were induced from three strains (Thy1.2 + ) as above protocols. 3 days later, these cells (0.6 × 10 6 ) were i.v. injected into Rag1 −/− mice that has received WT Thy1.1 + naive CD4 + T cells (0.6 × 10 6 ) through i.p. injection on the same day. a Weights of the recipient mice were monitored after the cell transfer. b 56 days after the transfer, spleens, mLNs, and cLP were harvested and the proportions of Foxp3 + T cells gated on Thy1.2 + cells were determined. c The proportions of IL-17A + or IFN-γ + T cells gated on Thy1.1 + cells in cLP were determined. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative data is from six similar independent experiments
    Figure Legend Snippet: The effect of TNFR1 and TNFR2 on iTreg stability and function in vivo. iTreg were induced from three strains (Thy1.2 + ) as above protocols. 3 days later, these cells (0.6 × 10 6 ) were i.v. injected into Rag1 −/− mice that has received WT Thy1.1 + naive CD4 + T cells (0.6 × 10 6 ) through i.p. injection on the same day. a Weights of the recipient mice were monitored after the cell transfer. b 56 days after the transfer, spleens, mLNs, and cLP were harvested and the proportions of Foxp3 + T cells gated on Thy1.2 + cells were determined. c The proportions of IL-17A + or IFN-γ + T cells gated on Thy1.1 + cells in cLP were determined. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative data is from six similar independent experiments

    Techniques Used: In Vivo, Injection, Mouse Assay

    The effect of TNFR1 and TNFR2 on iTreg, Th1, Th17 cell differentiation in vivo. a Naive CD4 + T cells isolated from WT, TNFR1 −/− , and TNFR2 −/− mice were injected into Rag1 −/− mice intraperitoneally. a–i 21 days and 28 days after the transfer, spleens, mLNs, and cLP were harvested and the proportions of Foxp3 + T cells were determined. j–l The proportions of IL-17A + or IFN-γ + T cells in cLP were determined. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative data is from six independent experiments
    Figure Legend Snippet: The effect of TNFR1 and TNFR2 on iTreg, Th1, Th17 cell differentiation in vivo. a Naive CD4 + T cells isolated from WT, TNFR1 −/− , and TNFR2 −/− mice were injected into Rag1 −/− mice intraperitoneally. a–i 21 days and 28 days after the transfer, spleens, mLNs, and cLP were harvested and the proportions of Foxp3 + T cells were determined. j–l The proportions of IL-17A + or IFN-γ + T cells in cLP were determined. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative data is from six independent experiments

    Techniques Used: Cell Differentiation, In Vivo, Isolation, Mouse Assay, Injection

    6) Product Images from "Allograft Inflammatory Factor-1 Governs Hematopoietic Stem Cell Differentiation Into cDC1 and Monocyte-Derived Dendritic Cells Through IRF8 and RelB in vitro"

    Article Title: Allograft Inflammatory Factor-1 Governs Hematopoietic Stem Cell Differentiation Into cDC1 and Monocyte-Derived Dendritic Cells Through IRF8 and RelB in vitro

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00173

    AIF1 is expressed in Lin − CD117 + SCA1 + bone marrow hematopoietic stem cells and in spleen myeloid-derived subsets. Bone marrow cells were assessed for hematopoietic subsets. Dot plots show SSC vs. FSC and Lineage (Lin) vs. CD117 (c-Kit) populations. Expression of AIF1 was assessed in Lin − CD117 + (top row) and Lin + CD117 − (bottom row) cells co-expressed with either (A) SCA1 or (B) CD34. Splenocytes were evaluated for AIF1 expression in subsets of B cells, T cells, macrophages, plasmacytoid DC, and conventional DC; cell populations were, respectively, identified by: (C) CD45R (B220) + gated from IgD + CD19 + cells; (D) CD4 + subsets gated from CD3 + TCR + cells; (E) F4/80 + subsets gated from CD11c − CD11b + cells; (F) CD11c + subsets gated on B220 + PDCA1 + ; and (G) CD8α + gated from MHC class II + CD11c + conventional dendritic cells. (H) IRF8 + cells gated from CD11c + MHC class II + subsets. (I) AIF1 expression percentage in B220 + B cells gated from CD19 + IgD + subsets, TCRβ + T cells, F4/80 + macrophages gated from CD11c − CD11b + subsets, PDCA1 + pDC, CD8a + cDC gated from CD11c + MHC class II + subsets, and IRF8 + cDC gated from CD11c + MHC class II + subsets. All gates were established using respective isotype controls. Isotype used for determining AIF1 expression is shown in all groups. Data is representative of three independent experiments.
    Figure Legend Snippet: AIF1 is expressed in Lin − CD117 + SCA1 + bone marrow hematopoietic stem cells and in spleen myeloid-derived subsets. Bone marrow cells were assessed for hematopoietic subsets. Dot plots show SSC vs. FSC and Lineage (Lin) vs. CD117 (c-Kit) populations. Expression of AIF1 was assessed in Lin − CD117 + (top row) and Lin + CD117 − (bottom row) cells co-expressed with either (A) SCA1 or (B) CD34. Splenocytes were evaluated for AIF1 expression in subsets of B cells, T cells, macrophages, plasmacytoid DC, and conventional DC; cell populations were, respectively, identified by: (C) CD45R (B220) + gated from IgD + CD19 + cells; (D) CD4 + subsets gated from CD3 + TCR + cells; (E) F4/80 + subsets gated from CD11c − CD11b + cells; (F) CD11c + subsets gated on B220 + PDCA1 + ; and (G) CD8α + gated from MHC class II + CD11c + conventional dendritic cells. (H) IRF8 + cells gated from CD11c + MHC class II + subsets. (I) AIF1 expression percentage in B220 + B cells gated from CD19 + IgD + subsets, TCRβ + T cells, F4/80 + macrophages gated from CD11c − CD11b + subsets, PDCA1 + pDC, CD8a + cDC gated from CD11c + MHC class II + subsets, and IRF8 + cDC gated from CD11c + MHC class II + subsets. All gates were established using respective isotype controls. Isotype used for determining AIF1 expression is shown in all groups. Data is representative of three independent experiments.

    Techniques Used: Derivative Assay, Expressing

    7) Product Images from "The Human CD4+ T Cell Response against Mumps Virus Targets a Broadly Recognized Nucleoprotein Epitope"

    Article Title: The Human CD4+ T Cell Response against Mumps Virus Targets a Broadly Recognized Nucleoprotein Epitope

    Journal: Journal of Virology

    doi: 10.1128/JVI.01883-18

    GTYR-specific activation of MuTER.1 is restricted by HLA-DR*04. (A) MuTER.1 cells were stimulated with autologous BLCL pulsed with GTYR peptide in the presence of anti-HLA-DR, anti-HLA-DQ, or anti-HLA-DP blocking antibodies. T cell activation was determined by expression of CD137 and IFN-γ secretion in the supernatant, as indicated. (B) BLCL with various HLA-DRB1 types as indicated were pulsed with GTYR peptide. T cell activation by these peptide-loaded BLCL was measured by IFN-γ secretion in the supernatant. Left bar represents autologous BLCL. T cell response, measured by CD137 expression or IFN-γ secretion (A), was significantly blocked with anti-HLA-DR antibodies ( P
    Figure Legend Snippet: GTYR-specific activation of MuTER.1 is restricted by HLA-DR*04. (A) MuTER.1 cells were stimulated with autologous BLCL pulsed with GTYR peptide in the presence of anti-HLA-DR, anti-HLA-DQ, or anti-HLA-DP blocking antibodies. T cell activation was determined by expression of CD137 and IFN-γ secretion in the supernatant, as indicated. (B) BLCL with various HLA-DRB1 types as indicated were pulsed with GTYR peptide. T cell activation by these peptide-loaded BLCL was measured by IFN-γ secretion in the supernatant. Left bar represents autologous BLCL. T cell response, measured by CD137 expression or IFN-γ secretion (A), was significantly blocked with anti-HLA-DR antibodies ( P

    Techniques Used: Activation Assay, Blocking Assay, Expressing

    The CD4 + T cell clone is specific for MuV. MuTER.1 T cells were cocultured at a 1:1, 10:1, or 100:1 effector/target (E:T) ratio, as indicated, with autologous BLCL that were pulsed with no protein, mock, recombinant N, live MuV JL, or genotype G. After 20 h, T cell responses were analyzed by measuring the levels of secreted IFN-γ in the supernatant (A) or by staining for the activation marker CD137 (B and C). Data shown are from three individual experiments, with means ± the standard deviations (SD), and flow cytometry data are from one representative experiment (C). The T cell response, measured by IFN-γ secretion or CD137 expression (A and B), was significantly higher after stimulation with recombinant N, live MuV JL, or genotype G compared to stimulation with no protein or mock at all effector/target ratios ( P
    Figure Legend Snippet: The CD4 + T cell clone is specific for MuV. MuTER.1 T cells were cocultured at a 1:1, 10:1, or 100:1 effector/target (E:T) ratio, as indicated, with autologous BLCL that were pulsed with no protein, mock, recombinant N, live MuV JL, or genotype G. After 20 h, T cell responses were analyzed by measuring the levels of secreted IFN-γ in the supernatant (A) or by staining for the activation marker CD137 (B and C). Data shown are from three individual experiments, with means ± the standard deviations (SD), and flow cytometry data are from one representative experiment (C). The T cell response, measured by IFN-γ secretion or CD137 expression (A and B), was significantly higher after stimulation with recombinant N, live MuV JL, or genotype G compared to stimulation with no protein or mock at all effector/target ratios ( P

    Techniques Used: Recombinant, Staining, Activation Assay, Marker, Flow Cytometry, Cytometry, Expressing

    MuV-specific CD4 + T cell clone MuTER.1 display a cytotoxic/Th1 phenotype. MuTER.1 cells were stimulated by autologous BLCL that were pulsed with GTYR peptide for activation and functional analysis. (A) T cell activation to a dose range of GTYR peptide was determined by IFN-γ secretion in the supernatant, and expression of the activation marker CD137 or degranulation marker CD107a. (B) Levels of secreted inflammatory and cytotoxicity-associated molecules were determined in the supernatant. Background cytokine levels in MuTER.1 cocultures with nonpulsed BLCL were subtracted. The levels of the cytokines indicated are averages of triplicate wells. (C) BLCL were pulsed with GTYR peptide and cocultured with MuTER.1 at various E:T ratios, as indicated. Killing of BLCL was determined after 24 h by PI staining.
    Figure Legend Snippet: MuV-specific CD4 + T cell clone MuTER.1 display a cytotoxic/Th1 phenotype. MuTER.1 cells were stimulated by autologous BLCL that were pulsed with GTYR peptide for activation and functional analysis. (A) T cell activation to a dose range of GTYR peptide was determined by IFN-γ secretion in the supernatant, and expression of the activation marker CD137 or degranulation marker CD107a. (B) Levels of secreted inflammatory and cytotoxicity-associated molecules were determined in the supernatant. Background cytokine levels in MuTER.1 cocultures with nonpulsed BLCL were subtracted. The levels of the cytokines indicated are averages of triplicate wells. (C) BLCL were pulsed with GTYR peptide and cocultured with MuTER.1 at various E:T ratios, as indicated. Killing of BLCL was determined after 24 h by PI staining.

    Techniques Used: Activation Assay, Functional Assay, Expressing, Marker, Staining

    MuTER.1 clone responds to peptides with the core sequence YRLIPNAR. MuTER.1 cells were stimulated by peptide-pulsed autologous BLCL. (A) After 6 h, T cell activation by 25 different peptide pools was determined by expression of CD137 of CD4 + T cells, in a single experiment. (B and C) BLCL were pulsed with peptides MuV-N 105–119 or MuV-N 109–123 (from pools 2, 3, and 16) or a nonstimulating control peptide MuV-N 401–415 . Clone MuTER.1 was stimulated at a 1:1, 10:1, or 100:1 ratio, as indicated, with pulsed BLCL, and T cell activation was determined from the expression of CD137 (B) or IFN-γ secretion (C). (D) MuTER.1 cells were stimulated with BLCL pulsed with 15-mer peptides representing the MuV-N 101–127 sequence with 14-mer amino acid overlap in 1:1, 10:1 or 100:1 ratio, as indicated. Peptides MuV-N 105–119 or MuV-N 109–123 are underlined. The red bars present the 15-mer peptide MuV-N 110–124 , GTYRLIPNARANLTA, that was selected for further characterization of the MuTER.1 clone. Data shown are triplicates, with means ± the SD, from one representative experiment of two to three individual experiments. The MuTER.1 clone showed significantly higher CD137 + expression upon stimulation with peptide pools 2, 3, 4, and 16 compared to medium control ( P
    Figure Legend Snippet: MuTER.1 clone responds to peptides with the core sequence YRLIPNAR. MuTER.1 cells were stimulated by peptide-pulsed autologous BLCL. (A) After 6 h, T cell activation by 25 different peptide pools was determined by expression of CD137 of CD4 + T cells, in a single experiment. (B and C) BLCL were pulsed with peptides MuV-N 105–119 or MuV-N 109–123 (from pools 2, 3, and 16) or a nonstimulating control peptide MuV-N 401–415 . Clone MuTER.1 was stimulated at a 1:1, 10:1, or 100:1 ratio, as indicated, with pulsed BLCL, and T cell activation was determined from the expression of CD137 (B) or IFN-γ secretion (C). (D) MuTER.1 cells were stimulated with BLCL pulsed with 15-mer peptides representing the MuV-N 101–127 sequence with 14-mer amino acid overlap in 1:1, 10:1 or 100:1 ratio, as indicated. Peptides MuV-N 105–119 or MuV-N 109–123 are underlined. The red bars present the 15-mer peptide MuV-N 110–124 , GTYRLIPNARANLTA, that was selected for further characterization of the MuTER.1 clone. Data shown are triplicates, with means ± the SD, from one representative experiment of two to three individual experiments. The MuTER.1 clone showed significantly higher CD137 + expression upon stimulation with peptide pools 2, 3, 4, and 16 compared to medium control ( P

    Techniques Used: Sequencing, Activation Assay, Expressing

    GTYR-specific CD4 + T cell responses in multiple mumps cases with different HLA-DR types. PBMCs from different mumps cases (1 to 2 months after clinical onset) with known HLA-DRB1 types, as indicated, were stimulated with GTYR peptide for 12 days in the presence of IL-2 to expand GTYR-specific CD4 + T cells. After 12 days, cells were rested for 24 h and restimulated with GTYR peptide in absence or presence of anti-HLA-DR blocking antibodies. GTYR-specific CD4 + T cells were determined after 6 h by intracellular cytokine staining for IFN-γ (A) and CD107a (B) surface expression. The data are from two experiments with eight and five different mumps cases, respectively.
    Figure Legend Snippet: GTYR-specific CD4 + T cell responses in multiple mumps cases with different HLA-DR types. PBMCs from different mumps cases (1 to 2 months after clinical onset) with known HLA-DRB1 types, as indicated, were stimulated with GTYR peptide for 12 days in the presence of IL-2 to expand GTYR-specific CD4 + T cells. After 12 days, cells were rested for 24 h and restimulated with GTYR peptide in absence or presence of anti-HLA-DR blocking antibodies. GTYR-specific CD4 + T cells were determined after 6 h by intracellular cytokine staining for IFN-γ (A) and CD107a (B) surface expression. The data are from two experiments with eight and five different mumps cases, respectively.

    Techniques Used: Blocking Assay, Staining, Expressing

    8) Product Images from "Combining Calcium Phosphates with Polysaccharides: A Bone-Inspired Material Modulating Monocyte/Macrophage Early Inflammatory Response"

    Article Title: Combining Calcium Phosphates with Polysaccharides: A Bone-Inspired Material Modulating Monocyte/Macrophage Early Inflammatory Response

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19113458

    Cytokine, chemokine and growth factors production. Released IL-1β ( A ), TNF-α ( B ), MCP-1 ( C ), IL-6 ( D ), IL-8 ( E ), IL-10 ( F ), VEGF ( G ), TGF-β ( H ) quantified by ELISA, indicating the secretion of pro-inflammatory cytokines/chemokines; concomitant with the secretion of IL-10 anti-inflammatory cytokine by THP-1 in contact with the build-up substrates. Mean TNF-α/mean IL-10 index ( I ), confirming a weak inflammatory profile of THP-1 in contact with the build-up substrates ( n = 10, Mann Whitney statistical test).
    Figure Legend Snippet: Cytokine, chemokine and growth factors production. Released IL-1β ( A ), TNF-α ( B ), MCP-1 ( C ), IL-6 ( D ), IL-8 ( E ), IL-10 ( F ), VEGF ( G ), TGF-β ( H ) quantified by ELISA, indicating the secretion of pro-inflammatory cytokines/chemokines; concomitant with the secretion of IL-10 anti-inflammatory cytokine by THP-1 in contact with the build-up substrates. Mean TNF-α/mean IL-10 index ( I ), confirming a weak inflammatory profile of THP-1 in contact with the build-up substrates ( n = 10, Mann Whitney statistical test).

    Techniques Used: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    9) Product Images from "Combining Calcium Phosphates with Polysaccharides: A Bone-Inspired Material Modulating Monocyte/Macrophage Early Inflammatory Response"

    Article Title: Combining Calcium Phosphates with Polysaccharides: A Bone-Inspired Material Modulating Monocyte/Macrophage Early Inflammatory Response

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19113458

    Cytokine, chemokine and growth factors production. Released IL-1β ( A ), TNF-α ( B ), MCP-1 ( C ), IL-6 ( D ), IL-8 ( E ), IL-10 ( F ), VEGF ( G ), TGF-β ( H ) quantified by ELISA, indicating the secretion of pro-inflammatory cytokines/chemokines; concomitant with the secretion of IL-10 anti-inflammatory cytokine by THP-1 in contact with the build-up substrates. Mean TNF-α/mean IL-10 index ( I ), confirming a weak inflammatory profile of THP-1 in contact with the build-up substrates ( n = 10, Mann Whitney statistical test).
    Figure Legend Snippet: Cytokine, chemokine and growth factors production. Released IL-1β ( A ), TNF-α ( B ), MCP-1 ( C ), IL-6 ( D ), IL-8 ( E ), IL-10 ( F ), VEGF ( G ), TGF-β ( H ) quantified by ELISA, indicating the secretion of pro-inflammatory cytokines/chemokines; concomitant with the secretion of IL-10 anti-inflammatory cytokine by THP-1 in contact with the build-up substrates. Mean TNF-α/mean IL-10 index ( I ), confirming a weak inflammatory profile of THP-1 in contact with the build-up substrates ( n = 10, Mann Whitney statistical test).

    Techniques Used: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    10) Product Images from "Distinct characteristics of Tregs of newborns of healthy and allergic mothers"

    Article Title: Distinct characteristics of Tregs of newborns of healthy and allergic mothers

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0207998

    Expression of surface markers of Treg function in Tregs from cord blood of children of allergic and healthy mothers. Samples of cord blood of children of healthy (H, n = 112) and allergic (A, n = 98) mothers were stained and analysed by flow cytometry. (A) Flow cytometry analysis showing the proportion of PD-1 + cells among CD4 + CD25 + CD127 low Tregs in cord blood (p = 0.0382). (B) Flow cytometry analysis showing the proportion of CTLA-4 + cells among CD4 + CD25 + CD127 low Tregs in cord blood. (C) Flow cytometry analysis showing the proportion of GITR + cells among CD4 + CD25 + CD127 low Tregs in cord blood. p values were calculated using unpaired Student’s t-test.
    Figure Legend Snippet: Expression of surface markers of Treg function in Tregs from cord blood of children of allergic and healthy mothers. Samples of cord blood of children of healthy (H, n = 112) and allergic (A, n = 98) mothers were stained and analysed by flow cytometry. (A) Flow cytometry analysis showing the proportion of PD-1 + cells among CD4 + CD25 + CD127 low Tregs in cord blood (p = 0.0382). (B) Flow cytometry analysis showing the proportion of CTLA-4 + cells among CD4 + CD25 + CD127 low Tregs in cord blood. (C) Flow cytometry analysis showing the proportion of GITR + cells among CD4 + CD25 + CD127 low Tregs in cord blood. p values were calculated using unpaired Student’s t-test.

    Techniques Used: Expressing, Staining, Flow Cytometry, Cytometry

    11) Product Images from "Selective ORAI1 inhibition ameliorates autoimmune CNS inflammation by suppressing effector but not regulatory T cell function"

    Article Title: Selective ORAI1 inhibition ameliorates autoimmune CNS inflammation by suppressing effector but not regulatory T cell function

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

    doi: 10.4049/jimmunol.1501406

    Progression and severity of EAE are attenuated by treatment of mice with CRAC channel inhibitor ( A ) Clinical EAE score in WT mice injected with MOG 35-55 in CFA. Mice were treated with 6 mg/kg of the CRAC channel inhibitor AMG1 or vehicle control for 10 days starting when EAE scores were ≥ 1. Graphs represent the average ± SEM of 8 mice per group. ( B ) Ca 2+ influx was measured in splenocytes isolated at the end of 10 day treatment with inhibitor or vehicle. Cells were loaded with Fura2-AM and stimulated with 1 μM thapsigargin (TG) in Ca 2+ free buffer, followed by readdition of 1 mM extracellular Ca 2+ to induce SOCE. ( C ) Absolute numbers of CD4 + and CD8 + T cells and CD11b + Gr-1 + polymorphonuclear (PMN) cells, CD11b + CD11c - Gr-1 - macrophages and CD11c + Gr-1 - DC isolated from the CNS of mice at day 23 after EAE induction and analyzed by flow cytometry. ( D ) Frequencies of CD4 + Foxp3 + Treg cells in the CNS at day 23 after EAE induction. ( E ) Expression of IFN-γ, IL-17 and GM-CSF by CD4 + T cells isolated from the CNS at day 23 after EAE induction and stimulated with PMA and ionomycin for 6 h. Data represent the average ± SEM of 8 mice per group. Statistical analysis of EAE scores in A (days 13–23) was performed using a Mann-Whitney test; data in panels B–E were analyzed using an unpaired Student’s t test. * p
    Figure Legend Snippet: Progression and severity of EAE are attenuated by treatment of mice with CRAC channel inhibitor ( A ) Clinical EAE score in WT mice injected with MOG 35-55 in CFA. Mice were treated with 6 mg/kg of the CRAC channel inhibitor AMG1 or vehicle control for 10 days starting when EAE scores were ≥ 1. Graphs represent the average ± SEM of 8 mice per group. ( B ) Ca 2+ influx was measured in splenocytes isolated at the end of 10 day treatment with inhibitor or vehicle. Cells were loaded with Fura2-AM and stimulated with 1 μM thapsigargin (TG) in Ca 2+ free buffer, followed by readdition of 1 mM extracellular Ca 2+ to induce SOCE. ( C ) Absolute numbers of CD4 + and CD8 + T cells and CD11b + Gr-1 + polymorphonuclear (PMN) cells, CD11b + CD11c - Gr-1 - macrophages and CD11c + Gr-1 - DC isolated from the CNS of mice at day 23 after EAE induction and analyzed by flow cytometry. ( D ) Frequencies of CD4 + Foxp3 + Treg cells in the CNS at day 23 after EAE induction. ( E ) Expression of IFN-γ, IL-17 and GM-CSF by CD4 + T cells isolated from the CNS at day 23 after EAE induction and stimulated with PMA and ionomycin for 6 h. Data represent the average ± SEM of 8 mice per group. Statistical analysis of EAE scores in A (days 13–23) was performed using a Mann-Whitney test; data in panels B–E were analyzed using an unpaired Student’s t test. * p

    Techniques Used: Mouse Assay, Injection, Isolation, Flow Cytometry, Cytometry, Expressing, MANN-WHITNEY

    Inducible deletion of Orai1 gene in T cells during ongoing EAE ameliorates disease severity ( A,B ) In vitro deletion of Orai1 in CD4 + T cells isolated from Orai1 fl/fl Cre-ERT2 , WT and Orai1 fl/fl Cd4-Cre control mice. Cells were stimulated with anti-CD3/CD28 for 3 days in the presence of 1 μM tamoxifen (TAM) and analyzed for SOCE after stimulation with thapsigargin and addition of 1 mM Ca 2+ ( A ) and Orai1 mRNA expression by RT PCR ( B ). ( C–F ) For passive induction of EAE, Orai1 fl/fl Cre-ERT2 and WT mice were immunized with MOG peptide. T cells were isolated from spleen and LNs 12 days later and restimulated in vitro with MOG peptide in the presence of IL-23 for 3 days. 4 × 10 6 CD4 + T cells were transferred i.v. into sublethally irradiated CD45.1 recipient mice. After EAE symptoms developed, recipient mice were injected with tamoxifen or vehicle control from day 10–14 after T cell transfer. ( C ) Clinical EAE scores. ( D–F ) Absolute numbers of CD4 + and CD8 + T cells ( D ) as well as CD11b + Gr-1 + polymorphonuclear cells (PMN), CD11b + CD11c - Gr-1 - macrophages and CD11c + Gr-1 - dendritic cells ( E ) in the CNS 18 days after adoptive transfer. ( F ) Frequencies of CD4 + IFN-γ + and CD4 + IL-17 + T cells isolated from the CNS and restimulated in vitro with PMA and ionomycin for 6h. Data represent the average ± SEM of 3–4 mice per group. Statistical analysis of Orai1 expression in B was performed using a one-way ANOVA test. Statistical analysis of EAE scores (days 10–18) in C was performed using a Mann-Whitney test. Data in panels D–F were analyzed using an unpaired Student’s t test. * p
    Figure Legend Snippet: Inducible deletion of Orai1 gene in T cells during ongoing EAE ameliorates disease severity ( A,B ) In vitro deletion of Orai1 in CD4 + T cells isolated from Orai1 fl/fl Cre-ERT2 , WT and Orai1 fl/fl Cd4-Cre control mice. Cells were stimulated with anti-CD3/CD28 for 3 days in the presence of 1 μM tamoxifen (TAM) and analyzed for SOCE after stimulation with thapsigargin and addition of 1 mM Ca 2+ ( A ) and Orai1 mRNA expression by RT PCR ( B ). ( C–F ) For passive induction of EAE, Orai1 fl/fl Cre-ERT2 and WT mice were immunized with MOG peptide. T cells were isolated from spleen and LNs 12 days later and restimulated in vitro with MOG peptide in the presence of IL-23 for 3 days. 4 × 10 6 CD4 + T cells were transferred i.v. into sublethally irradiated CD45.1 recipient mice. After EAE symptoms developed, recipient mice were injected with tamoxifen or vehicle control from day 10–14 after T cell transfer. ( C ) Clinical EAE scores. ( D–F ) Absolute numbers of CD4 + and CD8 + T cells ( D ) as well as CD11b + Gr-1 + polymorphonuclear cells (PMN), CD11b + CD11c - Gr-1 - macrophages and CD11c + Gr-1 - dendritic cells ( E ) in the CNS 18 days after adoptive transfer. ( F ) Frequencies of CD4 + IFN-γ + and CD4 + IL-17 + T cells isolated from the CNS and restimulated in vitro with PMA and ionomycin for 6h. Data represent the average ± SEM of 3–4 mice per group. Statistical analysis of Orai1 expression in B was performed using a one-way ANOVA test. Statistical analysis of EAE scores (days 10–18) in C was performed using a Mann-Whitney test. Data in panels D–F were analyzed using an unpaired Student’s t test. * p

    Techniques Used: In Vitro, Isolation, Mouse Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Irradiation, Injection, Adoptive Transfer Assay, MANN-WHITNEY

    Experimental autoimmune encephalomyelitis (EAE) is ameliorated in Orai1 fl/fl Cd4-Cre mice ( A ) RNA from wildtype and Orai1 fl/fl Cd4-Cre was isolated from CD4 + T cells and relative mRNA expression was measured by RT PCR. ( B ) SOCE was measured in Fura2-AM loaded freshly isolated CD4 + T cells from wildtype and Orai1 fl/fl Cd4-Cre mice after stimulation with thapsigargin (TG) followed by readdition of 1 mM Ca 2+ to the extracellular medium. ( C ) Clinical EAE scores in WT and Orai1 fl/fl Cd4-Cre mice injected with MOG 35-55 in CFA. ( D ) Summary of EAE with days to onset (±SEM), incidence of EAE and maximal EAE scores (±SEM). ( E ) H E and Luxol fast blue staining of spinal cords from WT and Orai1 fl/fl Cd4-Cre mice at 27 after EAE induction. Arrows indicate infiltrating cells and demyelination, respectively. Shown is one histological stain representative of 4 mice per group and 6 examined levels of spinal cord per mouse. ( F–I ) Cells were isolated from the spinal cord (CNS) of WT and Orai1 fl/fl Cd4-Cre mice at day 27 after EAE induction and analyzed by flow cytometry. ( F–H ) Absolute numbers of CD4 + and CD8 + T cells ( F ), frequencies of Foxp3 + CD4 + T cells ( G ) and absolute numbers of CD11b + Gr-1 + polymorphonuclear cells (PMN), CD11b + CD11c - Gr-1 - macrophages and CD11c + Gr-1 - dendritic cells in the CNS ( H ). ( I ) Cytokine production of T cells isolated from the CNS was analyzed by flow cytometry after stimulation with PMA and ionomycin in the presence of Brefeldin A for 6 h. Shown are absolute numbers of CD4 + T cells expressing IFN-γ, IL-17 or GM-CSF. Data in panels A and B represent the average ± SEM of 3 mice per group. Data in panels C–D and F–H represent the average ± SEM of 14–16 mice per group. Statistical analysis of EAE scores (starting at the first clinical signs of EAE) was performed using a Mann-Whitney test (panel C); data in panels B, F–H were analyzed using an unpaired Student’s t test. * p
    Figure Legend Snippet: Experimental autoimmune encephalomyelitis (EAE) is ameliorated in Orai1 fl/fl Cd4-Cre mice ( A ) RNA from wildtype and Orai1 fl/fl Cd4-Cre was isolated from CD4 + T cells and relative mRNA expression was measured by RT PCR. ( B ) SOCE was measured in Fura2-AM loaded freshly isolated CD4 + T cells from wildtype and Orai1 fl/fl Cd4-Cre mice after stimulation with thapsigargin (TG) followed by readdition of 1 mM Ca 2+ to the extracellular medium. ( C ) Clinical EAE scores in WT and Orai1 fl/fl Cd4-Cre mice injected with MOG 35-55 in CFA. ( D ) Summary of EAE with days to onset (±SEM), incidence of EAE and maximal EAE scores (±SEM). ( E ) H E and Luxol fast blue staining of spinal cords from WT and Orai1 fl/fl Cd4-Cre mice at 27 after EAE induction. Arrows indicate infiltrating cells and demyelination, respectively. Shown is one histological stain representative of 4 mice per group and 6 examined levels of spinal cord per mouse. ( F–I ) Cells were isolated from the spinal cord (CNS) of WT and Orai1 fl/fl Cd4-Cre mice at day 27 after EAE induction and analyzed by flow cytometry. ( F–H ) Absolute numbers of CD4 + and CD8 + T cells ( F ), frequencies of Foxp3 + CD4 + T cells ( G ) and absolute numbers of CD11b + Gr-1 + polymorphonuclear cells (PMN), CD11b + CD11c - Gr-1 - macrophages and CD11c + Gr-1 - dendritic cells in the CNS ( H ). ( I ) Cytokine production of T cells isolated from the CNS was analyzed by flow cytometry after stimulation with PMA and ionomycin in the presence of Brefeldin A for 6 h. Shown are absolute numbers of CD4 + T cells expressing IFN-γ, IL-17 or GM-CSF. Data in panels A and B represent the average ± SEM of 3 mice per group. Data in panels C–D and F–H represent the average ± SEM of 14–16 mice per group. Statistical analysis of EAE scores (starting at the first clinical signs of EAE) was performed using a Mann-Whitney test (panel C); data in panels B, F–H were analyzed using an unpaired Student’s t test. * p

    Techniques Used: Mouse Assay, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Injection, Staining, Flow Cytometry, Cytometry, MANN-WHITNEY

    12) Product Images from "Cathelicidin Antimicrobial Peptide Expression Is Not Induced or Required for Bacterial Clearance during Salmonella enterica Infection of Human Monocyte-Derived Macrophages"

    Article Title: Cathelicidin Antimicrobial Peptide Expression Is Not Induced or Required for Bacterial Clearance during Salmonella enterica Infection of Human Monocyte-Derived Macrophages

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00672-12

    Levels of proinflammatory cytokines released from S . Typhi- or S . Typhimurium-infected MDMs. Shown are levels of TNF-α (A) and IL-6 (B) released from MDMs after 2.5 and 24 h of S . Typhi infection and TNF-α (C) and IL-6 (D) released from MDMs after 2.5 and 24 h of S . Typhimurium infection. Statistical significance was determined by Student's t test comparing cytokine levels between LPS modification mutant-infected samples and the WT-infected sample collected at that time point. *, P
    Figure Legend Snippet: Levels of proinflammatory cytokines released from S . Typhi- or S . Typhimurium-infected MDMs. Shown are levels of TNF-α (A) and IL-6 (B) released from MDMs after 2.5 and 24 h of S . Typhi infection and TNF-α (C) and IL-6 (D) released from MDMs after 2.5 and 24 h of S . Typhimurium infection. Statistical significance was determined by Student's t test comparing cytokine levels between LPS modification mutant-infected samples and the WT-infected sample collected at that time point. *, P

    Techniques Used: Infection, Modification, Mutagenesis

    13) Product Images from "A TIM-3 Oligonucleotide Aptamer Enhances T Cell Functions and Potentiates Tumor Immunity in Mice"

    Article Title: A TIM-3 Oligonucleotide Aptamer Enhances T Cell Functions and Potentiates Tumor Immunity in Mice

    Journal: Molecular Therapy

    doi: 10.1016/j.ymthe.2017.06.023

    Binding of Aptamers to Murine TIM-3 . (A) Six aptamers that bind to recombinant TIM-3-Fc fusion protein. (B) b0 aptamer binding to TIM-3-expressing CHO cells. (C) b0 aptamer binding to activated BALB/c lymphocytes. (A–C) Black, b0 aptamer incubated to naked beads, parental CHO cells, or non-activated lymphocytes. Blue, scrambled aptamer binding to TIM-3 targets. Red, TIM-3-specific aptamers. (D–F) TIM-3 antibody binding to TIM-3-Fc fusion-coated beads (D), TIM-3 expressing CHO cells (E) and activated lymphocytes (F). Blue, binding of isotype control antibody. Red, TIM-3-specific antibody.
    Figure Legend Snippet: Binding of Aptamers to Murine TIM-3 . (A) Six aptamers that bind to recombinant TIM-3-Fc fusion protein. (B) b0 aptamer binding to TIM-3-expressing CHO cells. (C) b0 aptamer binding to activated BALB/c lymphocytes. (A–C) Black, b0 aptamer incubated to naked beads, parental CHO cells, or non-activated lymphocytes. Blue, scrambled aptamer binding to TIM-3 targets. Red, TIM-3-specific aptamers. (D–F) TIM-3 antibody binding to TIM-3-Fc fusion-coated beads (D), TIM-3 expressing CHO cells (E) and activated lymphocytes (F). Blue, binding of isotype control antibody. Red, TIM-3-specific antibody.

    Techniques Used: Binding Assay, Recombinant, Expressing, Incubation

    14) Product Images from "Inflammatory Th1 and Th17 cells in small intestine are each driven by functionally-specialized dendritic cells with distinct requirements for MyD88"

    Article Title: Inflammatory Th1 and Th17 cells in small intestine are each driven by functionally-specialized dendritic cells with distinct requirements for MyD88

    Journal: Cell reports

    doi: 10.1016/j.celrep.2016.09.091

    In small intestine inflammation CD103 + CD11b + DCs and CD103 − CD11b + DCs expand inflammatory Th17 cells ( a ) Representative flow plot of SI-LP DCs in co-housed, littermate A20 cko -DTA − mice and A20 cko -DTA + mice at 9-12 weeks of age. ( b ) Absolute number of each SI-LP DC subset in A20 wt or A20 cko mice, either DTA − or DTA + . ( c-f ) Absolute number of SI-LP CD4 T cells, either IL-17 + ( c ), IFNγ + ( d ), IL-17 + IFNγ + ( e ) or Foxp3 + CD4 T cells ( f ) in co-housed A20 wt or A20 cko mice, additionally either DTA − or DTA + . Results were combined from 3 independent experiments including at least one mouse of each genotype. Each dot represents a mouse. Error bars represent mean ± SEM. *, P
    Figure Legend Snippet: In small intestine inflammation CD103 + CD11b + DCs and CD103 − CD11b + DCs expand inflammatory Th17 cells ( a ) Representative flow plot of SI-LP DCs in co-housed, littermate A20 cko -DTA − mice and A20 cko -DTA + mice at 9-12 weeks of age. ( b ) Absolute number of each SI-LP DC subset in A20 wt or A20 cko mice, either DTA − or DTA + . ( c-f ) Absolute number of SI-LP CD4 T cells, either IL-17 + ( c ), IFNγ + ( d ), IL-17 + IFNγ + ( e ) or Foxp3 + CD4 T cells ( f ) in co-housed A20 wt or A20 cko mice, additionally either DTA − or DTA + . Results were combined from 3 independent experiments including at least one mouse of each genotype. Each dot represents a mouse. Error bars represent mean ± SEM. *, P

    Techniques Used: Flow Cytometry, Mouse Assay

    In A20 cko and A20/Myd88 cko mice, intestinal DCs are phenotypically mature and expand pathological mucosal T cells that cause small intestine inflammation ( a ) Representative flow cytometry gating strategy in A20 wt mice to identify the three bona fide DC populations of SI-LP. ( b ) Expression of A20 mRNA by the indicated SI-LP DC subset or macrophages from wild-type mice, relative to hprt . ( c-f ) Cell surface expression of maturation markers CD80 ( c ), CD86 ( d ) CD40 ( e ) and total cell number ( f ) of SI-LP DCs and macrophages. Cellular expression of maturation markers is represented as mean fluorescence intensity (MFI) relative to that of the same population in wild-type (WT) mice. ( g ) Cell number of SI-LP CD4 + T cells, IFNγ + , IL-17 + , IFNγ + IL-17 + and Foxp3 + CD4 + T cells from mice of the indicated genotype. Data in ( c-g ) was combined from at least 3 independent experiments with mice between 10-14 weeks of age, including at least one mouse of each genotype per experiment. Each dot represents one mouse. ( h ) Organ weights of small intestine from Rag1 -/- mice of the indicated genotypes (n = 9-11 mice of each genotype, aged 12-14 weeks). Error bars represent mean ± SEM, *, P
    Figure Legend Snippet: In A20 cko and A20/Myd88 cko mice, intestinal DCs are phenotypically mature and expand pathological mucosal T cells that cause small intestine inflammation ( a ) Representative flow cytometry gating strategy in A20 wt mice to identify the three bona fide DC populations of SI-LP. ( b ) Expression of A20 mRNA by the indicated SI-LP DC subset or macrophages from wild-type mice, relative to hprt . ( c-f ) Cell surface expression of maturation markers CD80 ( c ), CD86 ( d ) CD40 ( e ) and total cell number ( f ) of SI-LP DCs and macrophages. Cellular expression of maturation markers is represented as mean fluorescence intensity (MFI) relative to that of the same population in wild-type (WT) mice. ( g ) Cell number of SI-LP CD4 + T cells, IFNγ + , IL-17 + , IFNγ + IL-17 + and Foxp3 + CD4 + T cells from mice of the indicated genotype. Data in ( c-g ) was combined from at least 3 independent experiments with mice between 10-14 weeks of age, including at least one mouse of each genotype per experiment. Each dot represents one mouse. ( h ) Organ weights of small intestine from Rag1 -/- mice of the indicated genotypes (n = 9-11 mice of each genotype, aged 12-14 weeks). Error bars represent mean ± SEM, *, P

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Expressing, Fluorescence

    Microbiota are required for small intestinal inflammation and expansion of pathological mucosal T cells in A20 cko and A20/Myd88 cko mice ( a ) Organ weights of small intestine from mice of the indicated genotypes, treated with or without broad-spectrum antibiotics (Abx) for 9-10 weeks. Mice from these treatment groups were analyzed for cell number of SI-LP IL-17 + ( b ), IFNγ + ( c ) and IFNγ + IL-17 + CD4 T cells ( d ). Each dot represents one mouse. Results are combined from 4 independent experiments. Error bars show mean ± SEM, **, P
    Figure Legend Snippet: Microbiota are required for small intestinal inflammation and expansion of pathological mucosal T cells in A20 cko and A20/Myd88 cko mice ( a ) Organ weights of small intestine from mice of the indicated genotypes, treated with or without broad-spectrum antibiotics (Abx) for 9-10 weeks. Mice from these treatment groups were analyzed for cell number of SI-LP IL-17 + ( b ), IFNγ + ( c ) and IFNγ + IL-17 + CD4 T cells ( d ). Each dot represents one mouse. Results are combined from 4 independent experiments. Error bars show mean ± SEM, **, P

    Techniques Used: Mouse Assay

    Each SI-LP DC subset possesses unique APC functions, and differential requirements for MyD88 signals SI-LP CD103 + CD11b − DCs were isolated from mice of the indicated genotype and co-cultured 1:1 with naïve OT-II T cells for 4 days. ( a ) Representative flow plots of IFNγ + and IL-17 + T cells in each CD103 + CD11b − DC–T cell co-culture. The percentages of IFNγ + or IL-17 + CD4 T cells ( b ) and ELISA quantification of IFNγ and IL-17 protein ( c ) in co-cultures of CD103 + CD11b − DCs from A20 wt , A20 cko and A20/Myd88 cko mice. OT-II co-cultures with CD103 + CD11b + DCs ( d-f ) or CD103 − CD11b + DCs ( g-i ) were assayed as above. Data are combined from 3 independent experiments. Each dot represents one experiment, or replicates within an experiment, using DCs pooled from at least 3 mice of each genotype, and including all 3 genotypes for each experiment. ( j-k ) CD103 − CD11b + DCs isolated from co-housed Myd88 wt and Myd88 cko mice were co-cultured with naïve OT-II T cells, as above. The percentages ( j ) and total cell number ( k ) of IFNγ + or IL-17 + T cells, or IFNγ or IL-17A protein in culture supernatant ( l ) assayed from two independent experiments, with at least 5 mice per genotype, and including both genotypes for each experiment. Each dot represents replicates within an experiment. Error bars represent mean ± SEM, *, P
    Figure Legend Snippet: Each SI-LP DC subset possesses unique APC functions, and differential requirements for MyD88 signals SI-LP CD103 + CD11b − DCs were isolated from mice of the indicated genotype and co-cultured 1:1 with naïve OT-II T cells for 4 days. ( a ) Representative flow plots of IFNγ + and IL-17 + T cells in each CD103 + CD11b − DC–T cell co-culture. The percentages of IFNγ + or IL-17 + CD4 T cells ( b ) and ELISA quantification of IFNγ and IL-17 protein ( c ) in co-cultures of CD103 + CD11b − DCs from A20 wt , A20 cko and A20/Myd88 cko mice. OT-II co-cultures with CD103 + CD11b + DCs ( d-f ) or CD103 − CD11b + DCs ( g-i ) were assayed as above. Data are combined from 3 independent experiments. Each dot represents one experiment, or replicates within an experiment, using DCs pooled from at least 3 mice of each genotype, and including all 3 genotypes for each experiment. ( j-k ) CD103 − CD11b + DCs isolated from co-housed Myd88 wt and Myd88 cko mice were co-cultured with naïve OT-II T cells, as above. The percentages ( j ) and total cell number ( k ) of IFNγ + or IL-17 + T cells, or IFNγ or IL-17A protein in culture supernatant ( l ) assayed from two independent experiments, with at least 5 mice per genotype, and including both genotypes for each experiment. Each dot represents replicates within an experiment. Error bars represent mean ± SEM, *, P

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

    15) Product Images from "Endocytosis of Albumin by Podocytes Elicits an Inflammatory Response and Induces Apoptotic Cell Death"

    Article Title: Endocytosis of Albumin by Podocytes Elicits an Inflammatory Response and Induces Apoptotic Cell Death

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0054817

    Albumin exposure modulates pro-inflammatory cytokine expression and release in human podocytes. A , Time course of IL-1β RNA levels in podocytes treated with 5 mg/ml recombinant low endotoxin human albumin (closed bars) or 5 mg/ml dextran (open bars). * denotes P = 0.0002 compared to dextran controls. B , Time course of TNF RNA expression in podocytes treated with 5 mg/ml human albumin (closed bars) or 5 mg/ml dextran (open bars). * denotes P = 0.01 compared to dextran control. C , IL-6 RNA levels in podocytes treated with 5 mg/ml human albumin (closed bars) or 5 mg/ml dextran (open bars). * denotes P = 0.008 compared to dextran control. D, Amount of IL-1β normalized to total cellular protein released into the medium after treatment of podocytes with 5 mg/ml recombinant human albumin (closed bars) or 5 mg/ml dextran (open bars) for varying amounts of time. * denotes P = 0.005 compared to dextran treated controls. E , Amount of TNF normalized to total cellular protein released into the medium by podocytes after treatment with albumin (closed bars) or dextran (open bars) for varying amounts of time. * denotes P = 0.003 compared to dextran treated control cells. F , Levels of IL-6 normalized to total cellular protein released into the medium by podocytes treated with albumin (closed bars) or dextran (open bars) for varying amounts of time. * denotes P
    Figure Legend Snippet: Albumin exposure modulates pro-inflammatory cytokine expression and release in human podocytes. A , Time course of IL-1β RNA levels in podocytes treated with 5 mg/ml recombinant low endotoxin human albumin (closed bars) or 5 mg/ml dextran (open bars). * denotes P = 0.0002 compared to dextran controls. B , Time course of TNF RNA expression in podocytes treated with 5 mg/ml human albumin (closed bars) or 5 mg/ml dextran (open bars). * denotes P = 0.01 compared to dextran control. C , IL-6 RNA levels in podocytes treated with 5 mg/ml human albumin (closed bars) or 5 mg/ml dextran (open bars). * denotes P = 0.008 compared to dextran control. D, Amount of IL-1β normalized to total cellular protein released into the medium after treatment of podocytes with 5 mg/ml recombinant human albumin (closed bars) or 5 mg/ml dextran (open bars) for varying amounts of time. * denotes P = 0.005 compared to dextran treated controls. E , Amount of TNF normalized to total cellular protein released into the medium by podocytes after treatment with albumin (closed bars) or dextran (open bars) for varying amounts of time. * denotes P = 0.003 compared to dextran treated control cells. F , Levels of IL-6 normalized to total cellular protein released into the medium by podocytes treated with albumin (closed bars) or dextran (open bars) for varying amounts of time. * denotes P

    Techniques Used: Expressing, Recombinant, RNA Expression

    Albumin overload induces proteinuria and upregulates pro-inflammatory cytokine expression in isolated mouse glomeruli. A , Urinary protein normalized to urinary creatinine in mice injected with saline (open bar) or low endotoxin bovine serum albumin (BSA; closed bar; n = 4 animals per group). * denotes P = 0.004 compared to saline injected controls. B , IL-1β expression in isolated glomeruli from mice injected with saline (open bar) or BSA (closed bar; n = 4 animals per group). * denotes P = 0.02 compared to controls. C , TNF expression in isolated glomeruli from mice injected with saline (open bar) or BSA (closed bar; n = 4 animals per group). * denotes P = 0.02 compared to controls. D , IL-6 expression in isolated glomeruli from mice injected with saline (open bar) or BSA (closed bar; n = 4 animals per group).
    Figure Legend Snippet: Albumin overload induces proteinuria and upregulates pro-inflammatory cytokine expression in isolated mouse glomeruli. A , Urinary protein normalized to urinary creatinine in mice injected with saline (open bar) or low endotoxin bovine serum albumin (BSA; closed bar; n = 4 animals per group). * denotes P = 0.004 compared to saline injected controls. B , IL-1β expression in isolated glomeruli from mice injected with saline (open bar) or BSA (closed bar; n = 4 animals per group). * denotes P = 0.02 compared to controls. C , TNF expression in isolated glomeruli from mice injected with saline (open bar) or BSA (closed bar; n = 4 animals per group). * denotes P = 0.02 compared to controls. D , IL-6 expression in isolated glomeruli from mice injected with saline (open bar) or BSA (closed bar; n = 4 animals per group).

    Techniques Used: Expressing, Isolation, Mouse Assay, Injection

    16) Product Images from "Lithium Controls Central Nervous System Autoimmunity through Modulation of IFN-? Signaling"

    Article Title: Lithium Controls Central Nervous System Autoimmunity through Modulation of IFN-? Signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0052658

    Lithium inhibits Th1-induced, but not Th17-induced EAE. (A) Th1 cells (4–6×10 6 ), (B) Th17 cells (4–6×10 6 ), or (C and D) IL-17F-Thy1.1 cells (3–6×10 5 ) were adoptively transferred i.v. into naïve untreated or lithium–treated recipient mice to induce EAE (mean ± SEM, n = 8–11 mice/group, * p
    Figure Legend Snippet: Lithium inhibits Th1-induced, but not Th17-induced EAE. (A) Th1 cells (4–6×10 6 ), (B) Th17 cells (4–6×10 6 ), or (C and D) IL-17F-Thy1.1 cells (3–6×10 5 ) were adoptively transferred i.v. into naïve untreated or lithium–treated recipient mice to induce EAE (mean ± SEM, n = 8–11 mice/group, * p

    Techniques Used: Mouse Assay

    17) Product Images from "Enhanced Insight into the Autoimmune Component of Glaucoma: IgG Autoantibody Accumulation and Pro-Inflammatory Conditions in Human Glaucomatous Retina"

    Article Title: Enhanced Insight into the Autoimmune Component of Glaucoma: IgG Autoantibody Accumulation and Pro-Inflammatory Conditions in Human Glaucomatous Retina

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0057557

    Levels of pro-inflammatory cytokines in glaucomatous retinal tissue. Figure 1 depicts the altered concentrations of the pro-inflammatory cytokines TNF-α, IL-1ß, IL-6, IL-8 and IFN-γ in detail. The arrowheads indicate the relative intensity (U) on the y-axis of each individual subject as sample means and the black bar summarizes the group means. All investigated pro-inflammatory cytokines were up-regulated in the glaucomatous group (glaucoma) compared to the healthy subjects (ctrl). (In some box-plots, the arrowheads of sample means overlaid.).
    Figure Legend Snippet: Levels of pro-inflammatory cytokines in glaucomatous retinal tissue. Figure 1 depicts the altered concentrations of the pro-inflammatory cytokines TNF-α, IL-1ß, IL-6, IL-8 and IFN-γ in detail. The arrowheads indicate the relative intensity (U) on the y-axis of each individual subject as sample means and the black bar summarizes the group means. All investigated pro-inflammatory cytokines were up-regulated in the glaucomatous group (glaucoma) compared to the healthy subjects (ctrl). (In some box-plots, the arrowheads of sample means overlaid.).

    Techniques Used:

    18) Product Images from "Involvement of DNA-PKcs in the IL-6 and IL-12 Response to CpG-ODN Is Mediated by Its Interaction with TRAF6 in Dendritic Cells"

    Article Title: Involvement of DNA-PKcs in the IL-6 and IL-12 Response to CpG-ODN Is Mediated by Its Interaction with TRAF6 in Dendritic Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0058072

    DNA-PKcs is involved in the IL-6 and IL-12 response to CpG-ODN. Bone marrow-derived dendritic cells (DCs) from WT, DNA-PKcs −/− , TLR9 −/− and DNA-PKcs −/−/ TLR9 −/− mice were harvested based on their natural status: adhesion and suspension. ( A ). Combined DCs (adhesion and suspension) at day 7.5 were subjected to flow cytometry. The levels of CD11b, CD11c and MHC-II expression on DCs were determined. ( B–M ). Adhesion or suspension DCs at day 6.5 were seeded in 96-well plates at 1×10 5 /well in triplicate, and then treated with CpG-ODN (CpG, 0.2, 0.73, 2.2 or 7.3 µg/ml), LPS (0.1 µg/ml), R848 (0.125 µg/ml), or left untreated for indicated time durations. The levels of IL-6 ( B–G ) and IL-12p40 ( H–M ) were determined by ELISA using IL-6 or IL-12p40 ELISA kits. Similar results were obtained in at least 3 independent experiments. Note: bars represent the average of triplicates ± SD. * p
    Figure Legend Snippet: DNA-PKcs is involved in the IL-6 and IL-12 response to CpG-ODN. Bone marrow-derived dendritic cells (DCs) from WT, DNA-PKcs −/− , TLR9 −/− and DNA-PKcs −/−/ TLR9 −/− mice were harvested based on their natural status: adhesion and suspension. ( A ). Combined DCs (adhesion and suspension) at day 7.5 were subjected to flow cytometry. The levels of CD11b, CD11c and MHC-II expression on DCs were determined. ( B–M ). Adhesion or suspension DCs at day 6.5 were seeded in 96-well plates at 1×10 5 /well in triplicate, and then treated with CpG-ODN (CpG, 0.2, 0.73, 2.2 or 7.3 µg/ml), LPS (0.1 µg/ml), R848 (0.125 µg/ml), or left untreated for indicated time durations. The levels of IL-6 ( B–G ) and IL-12p40 ( H–M ) were determined by ELISA using IL-6 or IL-12p40 ELISA kits. Similar results were obtained in at least 3 independent experiments. Note: bars represent the average of triplicates ± SD. * p

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

    19) Product Images from "Identification of Potential Cytokine Pathways for Therapeutic Intervention in Murine Primary Biliary Cirrhosis"

    Article Title: Identification of Potential Cytokine Pathways for Therapeutic Intervention in Murine Primary Biliary Cirrhosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074225

    Inflammatory cytokine production in mice immunized with 2OA-BSA. (A) Serum levels of IL-17A, IFN-γ, TNF-α and IL-6 in WT, IL-17A −/− , IL-17F −/− and IL-22 −/− mice, respectively, at 2 weeks following 2OA-BSA immunization. (B) Production of IFN-γ in supernatant fluids of cultured splenic and hepatic MNCs (n = 4) with anti-CD3/CD28 mAbs at 3 days. (C) The level of inflammatory cytokines in extracted liver protein from WT, IL-17A −/− , IL-17F −/− and IL-22 −/− mice, respectively. Each group n = 8. *p
    Figure Legend Snippet: Inflammatory cytokine production in mice immunized with 2OA-BSA. (A) Serum levels of IL-17A, IFN-γ, TNF-α and IL-6 in WT, IL-17A −/− , IL-17F −/− and IL-22 −/− mice, respectively, at 2 weeks following 2OA-BSA immunization. (B) Production of IFN-γ in supernatant fluids of cultured splenic and hepatic MNCs (n = 4) with anti-CD3/CD28 mAbs at 3 days. (C) The level of inflammatory cytokines in extracted liver protein from WT, IL-17A −/− , IL-17F −/− and IL-22 −/− mice, respectively. Each group n = 8. *p

    Techniques Used: Mouse Assay, Cell Culture

    Pathological changes in liver of mice immunized with 2OA-BSA. (A) Representative H E staining profiles of liver from IL-17A −/− , IL-17F −/− and IL-22 −/− mice 8 weeks after immunization. Portal inflammatory changes with interlobular bile duct damage (red arrow) were observed. IL-17F −/− demonstrating more epithelioid granulomas (red arrowhead) in liver compared to IL-17A −/− and IL-22 −/− mice. (B) Scoring of portal inflammation and bile duct damage in liver from WT (n = 25), IL-17A −/− (n = 19), IL-17F −/− (n = 13) and IL-22 −/− (n = 14) mice.
    Figure Legend Snippet: Pathological changes in liver of mice immunized with 2OA-BSA. (A) Representative H E staining profiles of liver from IL-17A −/− , IL-17F −/− and IL-22 −/− mice 8 weeks after immunization. Portal inflammatory changes with interlobular bile duct damage (red arrow) were observed. IL-17F −/− demonstrating more epithelioid granulomas (red arrowhead) in liver compared to IL-17A −/− and IL-22 −/− mice. (B) Scoring of portal inflammation and bile duct damage in liver from WT (n = 25), IL-17A −/− (n = 19), IL-17F −/− (n = 13) and IL-22 −/− (n = 14) mice.

    Techniques Used: Mouse Assay, Staining

    20) Product Images from "BHBA Suppresses LPS-Induced Inflammation in BV-2 Cells by Inhibiting NF-κB Activation"

    Article Title: BHBA Suppresses LPS-Induced Inflammation in BV-2 Cells by Inhibiting NF-κB Activation

    Journal: Mediators of Inflammation

    doi: 10.1155/2014/983401

    Effects of BHBA on LPS-induced the gene expression and protein secretion of proinflammatory cytokines (TNF- α , IL-1 β , and IL-6) in BV-2 cells. Cells were pretreated with BHBA (0.5, 1.0, and 1.5 mM) 1 h prior to incubation of LPS (1 μ g/mL) for 4 h (mRNA) or 24 h (protein). Proteins and mRNA of TNF- α ((a), (b)), IL-1 β ((c), (d)), and IL-6 ((e), (f)) were determined by ELISA and quantitative real-time PCR as described in Section 2 . The relative mRNA level was normalized to β -actin mRNA. Results are expressed as mean ± SD for each group from three independent experiments. # Significantly different when compared with control alone, P
    Figure Legend Snippet: Effects of BHBA on LPS-induced the gene expression and protein secretion of proinflammatory cytokines (TNF- α , IL-1 β , and IL-6) in BV-2 cells. Cells were pretreated with BHBA (0.5, 1.0, and 1.5 mM) 1 h prior to incubation of LPS (1 μ g/mL) for 4 h (mRNA) or 24 h (protein). Proteins and mRNA of TNF- α ((a), (b)), IL-1 β ((c), (d)), and IL-6 ((e), (f)) were determined by ELISA and quantitative real-time PCR as described in Section 2 . The relative mRNA level was normalized to β -actin mRNA. Results are expressed as mean ± SD for each group from three independent experiments. # Significantly different when compared with control alone, P

    Techniques Used: Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

    Effects of BHBA mediated by GPR109A. BV-2 cells were pretreated with vehicle or PTX for 1 h. Medium from each condition was removed and replaced with vehicle, BHBA (1.5 mM), PTX (100 ng/mL), LPS (1 μ g/mL), BHBA + LPS, or BHBA + LPS + PTX. BV-2 cells were sampled at 4 h. The mRNA of proinflammatory enzymes and proinflammatory cytokines were determined by quantitative real-time PCR. Attenuation by BHBA of induced mRNA of iNOS (a), COX-2 (b), TNF- α (c), IL-1 β (d), and IL-6 (e) from BV-2 cells; this effect is abolished with pretreatment with PTX (** P
    Figure Legend Snippet: Effects of BHBA mediated by GPR109A. BV-2 cells were pretreated with vehicle or PTX for 1 h. Medium from each condition was removed and replaced with vehicle, BHBA (1.5 mM), PTX (100 ng/mL), LPS (1 μ g/mL), BHBA + LPS, or BHBA + LPS + PTX. BV-2 cells were sampled at 4 h. The mRNA of proinflammatory enzymes and proinflammatory cytokines were determined by quantitative real-time PCR. Attenuation by BHBA of induced mRNA of iNOS (a), COX-2 (b), TNF- α (c), IL-1 β (d), and IL-6 (e) from BV-2 cells; this effect is abolished with pretreatment with PTX (** P

    Techniques Used: Real-time Polymerase Chain Reaction

    21) Product Images from "Zinc finger protein Zfp335 is required for the formation of the naïve T cell compartment"

    Article Title: Zinc finger protein Zfp335 is required for the formation of the naïve T cell compartment

    Journal: eLife

    doi: 10.7554/eLife.03549

    Impaired late-stage SP thymocyte development and early post-thymic peripheral T cell maturation in blt/blt mice. ( A ) Turnover of DN, DP, semi-mature, and mature CD4SP thymocytes assessed after 2–4 days of continuous in vivo BrdU labeling (mean ± s.d., n = 4). ( B ) Input-normalized fraction of donor CD45.2 + CD4 + CD62L hi CD44 lo naïve T cells (closed symbols) or CD4SP thymocytes (open symbols) that were blt/blt , recovered from spleen at indicated time points after transfer of blt/blt and control peripheral lymphocytes or thymocytes to lymphoreplete CD45.1 + hosts (mean ± s.d., n = 2–4). ( C ) (left) GFP signal in CD62L hi CD44 lo CD4 + and CD8 + T cells from spleen of Rag1-GFP transgenic blt /+ vs. blt / blt mice. (right) Percentage of GFP hi naïve T cells, gated as shown in histograms (mean ± s.d., n = 6–8). ( D ) Percentage of total naïve T cells, GFP hi RTEs and GFP lo mature naïve (MN) T cells from spleens of blt/blt mice relative to matched blt /+ littermate controls. Data obtained from mice analyzed between 7 to 10 weeks of age (mean ± s.d., n = 10). DOI: http://dx.doi.org/10.7554/eLife.03549.011
    Figure Legend Snippet: Impaired late-stage SP thymocyte development and early post-thymic peripheral T cell maturation in blt/blt mice. ( A ) Turnover of DN, DP, semi-mature, and mature CD4SP thymocytes assessed after 2–4 days of continuous in vivo BrdU labeling (mean ± s.d., n = 4). ( B ) Input-normalized fraction of donor CD45.2 + CD4 + CD62L hi CD44 lo naïve T cells (closed symbols) or CD4SP thymocytes (open symbols) that were blt/blt , recovered from spleen at indicated time points after transfer of blt/blt and control peripheral lymphocytes or thymocytes to lymphoreplete CD45.1 + hosts (mean ± s.d., n = 2–4). ( C ) (left) GFP signal in CD62L hi CD44 lo CD4 + and CD8 + T cells from spleen of Rag1-GFP transgenic blt /+ vs. blt / blt mice. (right) Percentage of GFP hi naïve T cells, gated as shown in histograms (mean ± s.d., n = 6–8). ( D ) Percentage of total naïve T cells, GFP hi RTEs and GFP lo mature naïve (MN) T cells from spleens of blt/blt mice relative to matched blt /+ littermate controls. Data obtained from mice analyzed between 7 to 10 weeks of age (mean ± s.d., n = 10). DOI: http://dx.doi.org/10.7554/eLife.03549.011

    Techniques Used: Mouse Assay, In Vivo, Labeling, Transgenic Assay

    Normal expression of IL-7 receptor and Bcl2 family members. ( A ) Normalized expression levels of indicated Bcl2 family genes from Affymetrix array analysis of mRNA from sorted WT and blt / blt mature CD4SP thymocytes (mean ± s.d., n = 3). ( B ) Quantitative RT-PCR analysis of Il7r transcript in FACS-purified mature SP thymocytes and naïve T cells (mean ± s.d., n = 3). ( C ) Surface expression of IL-7Rα on WT (CD45.1 + CD45.2 + ) and blt / blt (CD45.2 + ) mature SP thymocytes and naïve T cells from mixed chimeras. Data are representative of eight mice. DOI: http://dx.doi.org/10.7554/eLife.03549.014
    Figure Legend Snippet: Normal expression of IL-7 receptor and Bcl2 family members. ( A ) Normalized expression levels of indicated Bcl2 family genes from Affymetrix array analysis of mRNA from sorted WT and blt / blt mature CD4SP thymocytes (mean ± s.d., n = 3). ( B ) Quantitative RT-PCR analysis of Il7r transcript in FACS-purified mature SP thymocytes and naïve T cells (mean ± s.d., n = 3). ( C ) Surface expression of IL-7Rα on WT (CD45.1 + CD45.2 + ) and blt / blt (CD45.2 + ) mature SP thymocytes and naïve T cells from mixed chimeras. Data are representative of eight mice. DOI: http://dx.doi.org/10.7554/eLife.03549.014

    Techniques Used: Expressing, Quantitative RT-PCR, FACS, Purification, Mouse Assay

    Mice heterozygous for the bloto mutation do not exhibit a T cell phenotype. ( A ) Number of CD4 + and CD8 + naïve T cells from spleens of WT ( n = 6) and blt /+ ( n = 4) mice. ( B ) Ratio of blt /+ (CD45.2 + ) vs WT (CD45.1 + CD45.2 + ) cells in splenic NK cells and indicated thymocyte and T cell populations from irradiation chimeras reconstituted with a mix of blt /+ and WT bone marrow as in Figure 1F . DOI: http://dx.doi.org/10.7554/eLife.03549.005
    Figure Legend Snippet: Mice heterozygous for the bloto mutation do not exhibit a T cell phenotype. ( A ) Number of CD4 + and CD8 + naïve T cells from spleens of WT ( n = 6) and blt /+ ( n = 4) mice. ( B ) Ratio of blt /+ (CD45.2 + ) vs WT (CD45.1 + CD45.2 + ) cells in splenic NK cells and indicated thymocyte and T cell populations from irradiation chimeras reconstituted with a mix of blt /+ and WT bone marrow as in Figure 1F . DOI: http://dx.doi.org/10.7554/eLife.03549.005

    Techniques Used: Mouse Assay, Mutagenesis, Irradiation

    22) Product Images from "Matrix Metalloprotease 9 Mediates Neutrophil Migration into the Airways in Response to Influenza Virus-Induced Toll-Like Receptor Signaling"

    Article Title: Matrix Metalloprotease 9 Mediates Neutrophil Migration into the Airways in Response to Influenza Virus-Induced Toll-Like Receptor Signaling

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002641

    Morbidity, inflammation, and neutrophil number are increased after infection with high dose influenza virus. (A) Kinetics of weight loss as a percentage of starting weight in C57BL/6 mice infected i.n. with 125 ( light-grey squares ), 1250 ( dark-grey triangles ), or 12500 ( black circles ) EID 50 PR8 virus. Mean ± SEM (n = 5, representative of four independent experiments). (B) Cytokine and chemokine levels in the BAL of uninfected mice (‘control’, clear bars ) or mice infected 6 days earlier with 125 ( light-grey bars ), 1250 ( grey bars ), or 12500 ( dark-grey bars ) EID 50 PR8 virus. Mean ± SEM (n = 3–4, representative of two independent experiments). (C) Histological examination of uninfected lungs (control, left ) or lungs 6 days after infection with 125 ( middle ) or 12500 ( right ) EID 50 PR8 virus. Perfused lungs were fixed in formalin and stained for hematoxylin and eosin (scalebar = 100 µm). Images are representative of multiple mice. b. bronchiole, a. arteriole, av. alveole, and arrowhead. venule. (D) Neutrophil numbers in the BAL and lung of uninfected mice (‘control’, clear bars ) or mice infected 6 days earlier with 125 ( light-grey bars ) or 12500 ( dark-grey bars ) EID 50 PR8 virus. Mean ± SEM (n = 3, representative of two independent experiments). (E) The percentage of blood neutrophils from LysM-GFP mice infected with 125 ( light-grey squares ) or 12500 ( black circles ) EID 50 PR8 virus compared to control (PBS, open squares ) or allantoic fluid at the same dilution as the highest viral dose ( open triangles ). Mean ± SEM (n = 4–5).
    Figure Legend Snippet: Morbidity, inflammation, and neutrophil number are increased after infection with high dose influenza virus. (A) Kinetics of weight loss as a percentage of starting weight in C57BL/6 mice infected i.n. with 125 ( light-grey squares ), 1250 ( dark-grey triangles ), or 12500 ( black circles ) EID 50 PR8 virus. Mean ± SEM (n = 5, representative of four independent experiments). (B) Cytokine and chemokine levels in the BAL of uninfected mice (‘control’, clear bars ) or mice infected 6 days earlier with 125 ( light-grey bars ), 1250 ( grey bars ), or 12500 ( dark-grey bars ) EID 50 PR8 virus. Mean ± SEM (n = 3–4, representative of two independent experiments). (C) Histological examination of uninfected lungs (control, left ) or lungs 6 days after infection with 125 ( middle ) or 12500 ( right ) EID 50 PR8 virus. Perfused lungs were fixed in formalin and stained for hematoxylin and eosin (scalebar = 100 µm). Images are representative of multiple mice. b. bronchiole, a. arteriole, av. alveole, and arrowhead. venule. (D) Neutrophil numbers in the BAL and lung of uninfected mice (‘control’, clear bars ) or mice infected 6 days earlier with 125 ( light-grey bars ) or 12500 ( dark-grey bars ) EID 50 PR8 virus. Mean ± SEM (n = 3, representative of two independent experiments). (E) The percentage of blood neutrophils from LysM-GFP mice infected with 125 ( light-grey squares ) or 12500 ( black circles ) EID 50 PR8 virus compared to control (PBS, open squares ) or allantoic fluid at the same dilution as the highest viral dose ( open triangles ). Mean ± SEM (n = 4–5).

    Techniques Used: Infection, Mouse Assay, Staining

    Depletion of neutrophils abrogates MMP9 secretion after influenza virus infection. Neutrophils were depleted by injecting C57BL/6 mice with 400 µg anti-Ly6G antibody (αLy6G) or isotype control (IgG) one day before infection and every other day thereafter. (A) Depletion of Ly6G+ cells in αLy6G-treated C57BL/6 mice ( bottom panel ) compared to IgG ( top panel ) was verified by flow cytometric analysis 6 days after infection. (B) Kinetics of weight loss as a percentage of starting weight in uninfected (control, open symbols ) and infected ( closed symbols ) mice treated with the αLy6G ( squares ) or IgG isotype ( circles ). (C) MMP9 secretion by cells from BAL and lung from infected mice that were treated with αLy6G ( clear bars ) or IgG isotype ( grey bars ) was measured by ELISPOT. (D) Inflammatory cytokine release in airways after neutrophil depletion. BALs were collected 6 days after infection and supernatants assayed by bead array. (B–D) Mean ± SEM (n = 3, representative of two independent experiments). (B–C) Considered significant at * P
    Figure Legend Snippet: Depletion of neutrophils abrogates MMP9 secretion after influenza virus infection. Neutrophils were depleted by injecting C57BL/6 mice with 400 µg anti-Ly6G antibody (αLy6G) or isotype control (IgG) one day before infection and every other day thereafter. (A) Depletion of Ly6G+ cells in αLy6G-treated C57BL/6 mice ( bottom panel ) compared to IgG ( top panel ) was verified by flow cytometric analysis 6 days after infection. (B) Kinetics of weight loss as a percentage of starting weight in uninfected (control, open symbols ) and infected ( closed symbols ) mice treated with the αLy6G ( squares ) or IgG isotype ( circles ). (C) MMP9 secretion by cells from BAL and lung from infected mice that were treated with αLy6G ( clear bars ) or IgG isotype ( grey bars ) was measured by ELISPOT. (D) Inflammatory cytokine release in airways after neutrophil depletion. BALs were collected 6 days after infection and supernatants assayed by bead array. (B–D) Mean ± SEM (n = 3, representative of two independent experiments). (B–C) Considered significant at * P

    Techniques Used: Infection, Mouse Assay, Flow Cytometry, Enzyme-linked Immunospot

    23) Product Images from "IL-33-Mediated Protection against Experimental Cerebral Malaria Is Linked to Induction of Type 2 Innate Lymphoid Cells, M2 Macrophages and Regulatory T Cells"

    Article Title: IL-33-Mediated Protection against Experimental Cerebral Malaria Is Linked to Induction of Type 2 Innate Lymphoid Cells, M2 Macrophages and Regulatory T Cells

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004607

    IL-33 expands ILC2 which adoptively protect mice against ECM. C57BL/6 mice were not infected (NI) or infected with PbA and treated daily with PBS or IL-33 from day 0. Splenic ILC2 were analysed by FACS on day 3. (A) Representative FACS showing % of ICOS + ST2 + cells gated on Lin − CD45 + cells. Cumulative percentage (B) and number (C) of ILC2 per spleen are shown. (D) Percentage of Ki67 + ILC2. Data are mean ± SEM (n = 5 per group) and representative of 2 independent experiments. *P
    Figure Legend Snippet: IL-33 expands ILC2 which adoptively protect mice against ECM. C57BL/6 mice were not infected (NI) or infected with PbA and treated daily with PBS or IL-33 from day 0. Splenic ILC2 were analysed by FACS on day 3. (A) Representative FACS showing % of ICOS + ST2 + cells gated on Lin − CD45 + cells. Cumulative percentage (B) and number (C) of ILC2 per spleen are shown. (D) Percentage of Ki67 + ILC2. Data are mean ± SEM (n = 5 per group) and representative of 2 independent experiments. *P

    Techniques Used: Mouse Assay, Infection, FACS

    IL-33, ILC2 and M2 induce Tregs. (A-C) C57BL/6 mice were infected i.v. with PbA and treated daily with PBS or IL-33 from day 0. (A) Relative expression (% of Hprt1 ) of Foxp3 + mRNA in purified CD4 + cells from the spleen. (B) percentage of Foxp3 + cells gated on CD4 + cells and number of Foxp3 + CD4 + cells in the spleen. Data are mean ± SEM (n = 5 per group), representative of at least 2 independent experiments. *P
    Figure Legend Snippet: IL-33, ILC2 and M2 induce Tregs. (A-C) C57BL/6 mice were infected i.v. with PbA and treated daily with PBS or IL-33 from day 0. (A) Relative expression (% of Hprt1 ) of Foxp3 + mRNA in purified CD4 + cells from the spleen. (B) percentage of Foxp3 + cells gated on CD4 + cells and number of Foxp3 + CD4 + cells in the spleen. Data are mean ± SEM (n = 5 per group), representative of at least 2 independent experiments. *P

    Techniques Used: Mouse Assay, Infection, Expressing, Purification

    ILC2 promote M2 macrophage polarization. (A) BMDM from C57BL/6 mice were cultured in the lower chamber of a 24-transwell plate in complete medium alone (M0) or supplemented with IL-4 (M2). In some experiments ILC2, sorted from naïve WT mice pre-treated with IL-33, were added to the upper chamber. After 48 h, BMDM were collected and assayed for the expression of M2 markers by qPCR (relative to Hprt1 ). (B) ST2-deficient BMDM were co-cultured in transwell plates as above with WT ILC2 in the presence of IL-33 alone or in combination with IL-7. After 48 h, BMDM were collected and assayed for the expression of M2 markers by qPCR (relative to Hprt1 ). Type 2 cytokines in the supernatants of ILC2 cultured in the presence of IL-33 or IL-33 + IL-7 were determined by ELISA (C), or by FACS (D, E). Data are mean ± SEM (n = 3 per group), representative of two independent experiments, *P
    Figure Legend Snippet: ILC2 promote M2 macrophage polarization. (A) BMDM from C57BL/6 mice were cultured in the lower chamber of a 24-transwell plate in complete medium alone (M0) or supplemented with IL-4 (M2). In some experiments ILC2, sorted from naïve WT mice pre-treated with IL-33, were added to the upper chamber. After 48 h, BMDM were collected and assayed for the expression of M2 markers by qPCR (relative to Hprt1 ). (B) ST2-deficient BMDM were co-cultured in transwell plates as above with WT ILC2 in the presence of IL-33 alone or in combination with IL-7. After 48 h, BMDM were collected and assayed for the expression of M2 markers by qPCR (relative to Hprt1 ). Type 2 cytokines in the supernatants of ILC2 cultured in the presence of IL-33 or IL-33 + IL-7 were determined by ELISA (C), or by FACS (D, E). Data are mean ± SEM (n = 3 per group), representative of two independent experiments, *P

    Techniques Used: Mouse Assay, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, FACS

    24) Product Images from "Bridelia ferruginea Produces Antineuroinflammatory Activity through Inhibition of Nuclear Factor-kappa B and p38 MAPK Signalling"

    Article Title: Bridelia ferruginea Produces Antineuroinflammatory Activity through Inhibition of Nuclear Factor-kappa B and p38 MAPK Signalling

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2012/546873

    (a) BFE inhibited COX-2 protein expression in LPS-stimulated primary microglia. Cells were stimulated with LPS (100 ng/mL) in the presence or absence of BFE (25–200 μ g/mL) for 24 h. At the end of incubation period, COX-2 protein levels were measured using western blot using specific antibodies for each protein. (b) Quantitative densitometric analysis of COX-2 protein expression normalized to actin loading control. All values are expressed as mean ± SEM for 3 independent experiments. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P
    Figure Legend Snippet: (a) BFE inhibited COX-2 protein expression in LPS-stimulated primary microglia. Cells were stimulated with LPS (100 ng/mL) in the presence or absence of BFE (25–200 μ g/mL) for 24 h. At the end of incubation period, COX-2 protein levels were measured using western blot using specific antibodies for each protein. (b) Quantitative densitometric analysis of COX-2 protein expression normalized to actin loading control. All values are expressed as mean ± SEM for 3 independent experiments. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P

    Techniques Used: Expressing, Incubation, Western Blot

    (a) BFE inhibited iNOS protein expression in LPS-stimulated primary microglia. Cells were stimulated with LPS (100 ng/mL) in the presence or absence of BFE (25–200 μ g/mL) for 24 h. At the end of incubation period, iNOS protein levels were measured using western blot using specific antibodies for each protein. (b) Quantitative densitometric analysis of iNOS protein expression normalized to actin loading control. All values are expressed as mean ± SEM for 3 independent experiments. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P
    Figure Legend Snippet: (a) BFE inhibited iNOS protein expression in LPS-stimulated primary microglia. Cells were stimulated with LPS (100 ng/mL) in the presence or absence of BFE (25–200 μ g/mL) for 24 h. At the end of incubation period, iNOS protein levels were measured using western blot using specific antibodies for each protein. (b) Quantitative densitometric analysis of iNOS protein expression normalized to actin loading control. All values are expressed as mean ± SEM for 3 independent experiments. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P

    Techniques Used: Expressing, Incubation, Western Blot

    Reduction of nitrite production in LPS-stimulated microglia. Cells were stimulated with LPS (100 ng/mL) in the presence or absence of BFE (25–200 μ g/mL) for 24 h. At the end of the incubation period, supernatants were collected for nitrite measurement with the Griess assay. All values are expressed as mean ± SEM for 3 independent experiments. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P
    Figure Legend Snippet: Reduction of nitrite production in LPS-stimulated microglia. Cells were stimulated with LPS (100 ng/mL) in the presence or absence of BFE (25–200 μ g/mL) for 24 h. At the end of the incubation period, supernatants were collected for nitrite measurement with the Griess assay. All values are expressed as mean ± SEM for 3 independent experiments. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P

    Techniques Used: Incubation, Griess Assay

    BFE inhibited p38, but not p42/44 or JNK MAPK in LPS-stimulated BV-2 microglia. Lysates from LPS-stimulated microglia were analysed using ELISA for phospho-p38, phospho-42/44, and phospho-JNK. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P
    Figure Legend Snippet: BFE inhibited p38, but not p42/44 or JNK MAPK in LPS-stimulated BV-2 microglia. Lysates from LPS-stimulated microglia were analysed using ELISA for phospho-p38, phospho-42/44, and phospho-JNK. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Reduction of TNF α ((a) and (b)) and IL-6 ((a) and (c)) production in LPS-stimulated BV-2 microglia. Cells were stimulated with LPS (100 ng/mL) in the presence or absence of BFE (25–200 μ g/mL) for 24 h. At the end of the incubation period, supernatants were collected for TNF α and IL-6 measurement according to the manufacturer's instructions. All values are expressed as mean ± SEM for 3 independent experiments. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P
    Figure Legend Snippet: Reduction of TNF α ((a) and (b)) and IL-6 ((a) and (c)) production in LPS-stimulated BV-2 microglia. Cells were stimulated with LPS (100 ng/mL) in the presence or absence of BFE (25–200 μ g/mL) for 24 h. At the end of the incubation period, supernatants were collected for TNF α and IL-6 measurement according to the manufacturer's instructions. All values are expressed as mean ± SEM for 3 independent experiments. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P

    Techniques Used: Incubation

    Reduction of PGE 2 production in LPS-stimulated microglia. Cells were stimulated with LPS (100 ng/mL) in the presence or absence of BFE (25–200 μ g/mL) for 24 h. At the end of the incubation period, supernatants were collected for PGE 2 measurement. All values are expressed as mean ± SEM for 3 independent experiments. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P
    Figure Legend Snippet: Reduction of PGE 2 production in LPS-stimulated microglia. Cells were stimulated with LPS (100 ng/mL) in the presence or absence of BFE (25–200 μ g/mL) for 24 h. At the end of the incubation period, supernatants were collected for PGE 2 measurement. All values are expressed as mean ± SEM for 3 independent experiments. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P

    Techniques Used: Incubation

    25) Product Images from "RAGE deficiency predisposes mice to virus-induced paucigranulocytic asthma"

    Article Title: RAGE deficiency predisposes mice to virus-induced paucigranulocytic asthma

    Journal: eLife

    doi: 10.7554/eLife.21199

    Neonatal RAGE deficient mice do not display a pronounced T H 2 response to an early life infection with PVM. RAGE deficient and WT (RAGE + ) mice were infected with 10 PFU of PVM or mock at seven days of age and the inflammatory profile was assessed seven days post-infection ( A ) Inflammatory cells in the bronchoalveolar lavage fluid were enumerated by differential counting following Geimsa staining. ( B ) Cytokine levels in the bronchoalveolar lavage fluid of neonatal mice seven days post-PVM or mock infection (i.e. at 14-days of age). ( C ) Cytokine levels in the lungs of neonatal mice seven days post-PVM or mock infection (i.e. at 14-days of age). Statistical significance was determined by a one-way ANOVA with Tukey’s multiple comparisons test ( A ) or a two-way ANOVA ( B and C ). Statistical significance is denoted by asterisks (*p
    Figure Legend Snippet: Neonatal RAGE deficient mice do not display a pronounced T H 2 response to an early life infection with PVM. RAGE deficient and WT (RAGE + ) mice were infected with 10 PFU of PVM or mock at seven days of age and the inflammatory profile was assessed seven days post-infection ( A ) Inflammatory cells in the bronchoalveolar lavage fluid were enumerated by differential counting following Geimsa staining. ( B ) Cytokine levels in the bronchoalveolar lavage fluid of neonatal mice seven days post-PVM or mock infection (i.e. at 14-days of age). ( C ) Cytokine levels in the lungs of neonatal mice seven days post-PVM or mock infection (i.e. at 14-days of age). Statistical significance was determined by a one-way ANOVA with Tukey’s multiple comparisons test ( A ) or a two-way ANOVA ( B and C ). Statistical significance is denoted by asterisks (*p

    Techniques Used: Mouse Assay, Infection, Staining

    RAGE deficient mice do not display a pronounced T H 2 response upon re-infection with PVM. Neonatal WT (RAGE + ), TLR7 deficient and RAGE deficient mice were infected with PVM (10 PFU) or mock at seven days of age and later re-infected with PVM (100 PFU) or mock 42 days after the primary infection. ( A ) Inflammatory cells in the bronchoalveolar lavage fluid were enumerated by differential counting following Geimsa staining seven days post-reinfection (i.e. at 56-days of age). ( B ) Cytokine levels in the bronchoalveolar lavage fluid of neonatal mice seven days post-re infection (i.e. at 56-days of age). ( C ) Cytokine levels in the lungs of neonatal mice seven days post- reinfection (i.e. at 56-days of age). Periostin levels were determined by immunohistochemistry and quantified as a percentage of the epithelial basement membrane length. Statistical significance was determined by a Student’s t-test ( A ) and a two-way ANOVA with Tukey’s multiple comparisons test ( B and D ). Statistical significance is denoted by asterisks (*p
    Figure Legend Snippet: RAGE deficient mice do not display a pronounced T H 2 response upon re-infection with PVM. Neonatal WT (RAGE + ), TLR7 deficient and RAGE deficient mice were infected with PVM (10 PFU) or mock at seven days of age and later re-infected with PVM (100 PFU) or mock 42 days after the primary infection. ( A ) Inflammatory cells in the bronchoalveolar lavage fluid were enumerated by differential counting following Geimsa staining seven days post-reinfection (i.e. at 56-days of age). ( B ) Cytokine levels in the bronchoalveolar lavage fluid of neonatal mice seven days post-re infection (i.e. at 56-days of age). ( C ) Cytokine levels in the lungs of neonatal mice seven days post- reinfection (i.e. at 56-days of age). Periostin levels were determined by immunohistochemistry and quantified as a percentage of the epithelial basement membrane length. Statistical significance was determined by a Student’s t-test ( A ) and a two-way ANOVA with Tukey’s multiple comparisons test ( B and D ). Statistical significance is denoted by asterisks (*p

    Techniques Used: Mouse Assay, Infection, Staining, Immunohistochemistry

    26) Product Images from "Adjuvant-dependent regulation of interleukin-17 expressing γδ T cells and inhibition of Th2 responses in allergic airways disease"

    Article Title: Adjuvant-dependent regulation of interleukin-17 expressing γδ T cells and inhibition of Th2 responses in allergic airways disease

    Journal: Respiratory Research

    doi: 10.1186/s12931-014-0090-5

    OVA/CFA sensitized mice have more γδ + IL-17 + T cells in the BAL fluid. BAL fluid cells were stimulated with PMA/ionomycin and triple stained with α-CD4, α-γδ TCR and α-IL-17 antibodies. (A) Representative flow cytometry plots of cells in BAL fluid (top panel) and lung (bottom panel) gated first to identify IL-17 + cells and subsequently to identify frequencies of CD4 and γδ T cells. (B) The frequency distribution of IL-17 + cells within the CD4 and γδ T cell populations is presented as the ratio (γδ/CD4) of these cells. One-way ANOVA, Holm-Sidak. (C) Total numbers of CD4 + IL-17 + and γδ + IL-17 + populations calculated from the frequency of these cells and the total cell counts are shown. Two-way ANOVA, Holm-Sidak. (D) The distribution of total γδ + IL-17 + and CD4 + IL-17 + T cells is presented as the ratio (γδ/CD4) of these cells. One-way ANOVA, Holm Sidak. (B-D) Data are from 7–11 total mice per group from at least 2 independent experiments. *p
    Figure Legend Snippet: OVA/CFA sensitized mice have more γδ + IL-17 + T cells in the BAL fluid. BAL fluid cells were stimulated with PMA/ionomycin and triple stained with α-CD4, α-γδ TCR and α-IL-17 antibodies. (A) Representative flow cytometry plots of cells in BAL fluid (top panel) and lung (bottom panel) gated first to identify IL-17 + cells and subsequently to identify frequencies of CD4 and γδ T cells. (B) The frequency distribution of IL-17 + cells within the CD4 and γδ T cell populations is presented as the ratio (γδ/CD4) of these cells. One-way ANOVA, Holm-Sidak. (C) Total numbers of CD4 + IL-17 + and γδ + IL-17 + populations calculated from the frequency of these cells and the total cell counts are shown. Two-way ANOVA, Holm-Sidak. (D) The distribution of total γδ + IL-17 + and CD4 + IL-17 + T cells is presented as the ratio (γδ/CD4) of these cells. One-way ANOVA, Holm Sidak. (B-D) Data are from 7–11 total mice per group from at least 2 independent experiments. *p

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

    Frequencies of total IL-17 expressing cells and of IL-17-γδ cells are increased in OVA/CFA sensitized mice treated with a γδ TCR stimulatory antibody. OVA/CFA sensitized mice were IV injected with a γδ TCR stimulatory antibody (UC7-13D5) or isotype control before airway challenge. BAL fluid cells were stimulated with PMA/ionomycin and stained with α-γδ TCR and α-IL-17 antibodies. (A) Representative flow cytometry plots of BAL fluid cells. The mean frequencies (left panels) and numbers (right panels) of (B) IL-17-γδ and (C) IL-17 + BAL fluid cells are presented for 5 total mice per group from 2 independent experiments. (B-C) Unpaired, two-tailed t-test. *p
    Figure Legend Snippet: Frequencies of total IL-17 expressing cells and of IL-17-γδ cells are increased in OVA/CFA sensitized mice treated with a γδ TCR stimulatory antibody. OVA/CFA sensitized mice were IV injected with a γδ TCR stimulatory antibody (UC7-13D5) or isotype control before airway challenge. BAL fluid cells were stimulated with PMA/ionomycin and stained with α-γδ TCR and α-IL-17 antibodies. (A) Representative flow cytometry plots of BAL fluid cells. The mean frequencies (left panels) and numbers (right panels) of (B) IL-17-γδ and (C) IL-17 + BAL fluid cells are presented for 5 total mice per group from 2 independent experiments. (B-C) Unpaired, two-tailed t-test. *p

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

    OVA/CFA sensitized mice receiving a γδ TCR stimulatory antibody, have reduced airway eosinophilia and AHR. OVA/CFA sensitized mice were IV injected with a γδ TCR (UC7-13D5) stimulatory antibody or isotype control before airway challenge. The mean (A) total cell counts and (B) frequencies of macrophages, eosinophils, neutrophils and lymphocytes (+SEM) are shown for 5 total mice per group from 2 independent experiments. (C) Total lung resistance and elastance were assessed 24 h after the last airway OVA challenge. Mean (±SEM) respiratory system resistance and elastance to increasing concentrations of methacholine are shown. (A-C) Unpaired, two-tailed t-test. *p
    Figure Legend Snippet: OVA/CFA sensitized mice receiving a γδ TCR stimulatory antibody, have reduced airway eosinophilia and AHR. OVA/CFA sensitized mice were IV injected with a γδ TCR (UC7-13D5) stimulatory antibody or isotype control before airway challenge. The mean (A) total cell counts and (B) frequencies of macrophages, eosinophils, neutrophils and lymphocytes (+SEM) are shown for 5 total mice per group from 2 independent experiments. (C) Total lung resistance and elastance were assessed 24 h after the last airway OVA challenge. Mean (±SEM) respiratory system resistance and elastance to increasing concentrations of methacholine are shown. (A-C) Unpaired, two-tailed t-test. *p

    Techniques Used: Mouse Assay, Injection, Two Tailed Test

    27) Product Images from "Impairment of autophagy in the central nervous system during lipopolysaccharide-induced inflammatory stress in mice"

    Article Title: Impairment of autophagy in the central nervous system during lipopolysaccharide-induced inflammatory stress in mice

    Journal: Molecular Brain

    doi: 10.1186/s13041-014-0056-z

    Pro-inflammatory cytokine levels after chronic LPS-induced inflammatory stress. IL1-β (A) , TNF-α (B) and IL-6 (C) levels in cortex (CO) and hippocampus (HI) from LPS-treated mice (0.5 mg/kg of LPS every 3 days for 3 months) or control (0.9% NaCl every 3 days for 3 months) by using ELISA assay. Treatment started at 3 months and mice were sacrificed at 6 months of age. Cytokine levels were expressed in pg/mg protein. Results are mean ± SEM for 6 mice in each group. ** p
    Figure Legend Snippet: Pro-inflammatory cytokine levels after chronic LPS-induced inflammatory stress. IL1-β (A) , TNF-α (B) and IL-6 (C) levels in cortex (CO) and hippocampus (HI) from LPS-treated mice (0.5 mg/kg of LPS every 3 days for 3 months) or control (0.9% NaCl every 3 days for 3 months) by using ELISA assay. Treatment started at 3 months and mice were sacrificed at 6 months of age. Cytokine levels were expressed in pg/mg protein. Results are mean ± SEM for 6 mice in each group. ** p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Pro-inflammatory cytokine levels after acute LPS-induced inflammatory stress. IL-1β, TNF-α and IL-6 levels in cortex ( A , B , C , respectively) and hippocampus ( D , E , F , respectively) of mice treated with a single () or two () or three () i.p. injections of LPS at 10 mg/kg or vehicle (0.9% NaCl) were measured by ELISA assay. Mice were sacrificed 2, 4, 6 or 12 h after a single injection or 24 h after two or three injections. Cytokine levels were expressed in pg/mg protein. Results are mean ± SEM for 6 mice in each group. *p
    Figure Legend Snippet: Pro-inflammatory cytokine levels after acute LPS-induced inflammatory stress. IL-1β, TNF-α and IL-6 levels in cortex ( A , B , C , respectively) and hippocampus ( D , E , F , respectively) of mice treated with a single () or two () or three () i.p. injections of LPS at 10 mg/kg or vehicle (0.9% NaCl) were measured by ELISA assay. Mice were sacrificed 2, 4, 6 or 12 h after a single injection or 24 h after two or three injections. Cytokine levels were expressed in pg/mg protein. Results are mean ± SEM for 6 mice in each group. *p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Injection

    28) Product Images from "Control of Methicillin-Resistant Staphylococcus aureus Pneumonia Utilizing TLR2 Agonist Pam3CSK4"

    Article Title: Control of Methicillin-Resistant Staphylococcus aureus Pneumonia Utilizing TLR2 Agonist Pam3CSK4

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0149233

    Pam3CSK4-pretreated mice have less neutrophil recruitment in MRSA-induced pneumonia. Infiltrating neutrophils were digested from lung tissues or washed out from the bronchus 6 and 12 h post infection. Cells were incubated with PE/CY5.5-anti-Gr-1, PE-anti-CD11b and analyzed by flow cytometry. Numbers of neutrophils in lung and bronchial lavage (BAL) in naïve and MRSA-challenged mice at 6 h (A) and 12 h (B) post infection were detected. The absolute numbers of neutrophils in the lungs and bronchus from mice 12 h post infection were calculated (C). Sections from MRSA-infected mice and naïve mice were stained by H E (D). (The representative percentages of neutrophils in lung or bronchus came from one mice per group (n = 5)), *P
    Figure Legend Snippet: Pam3CSK4-pretreated mice have less neutrophil recruitment in MRSA-induced pneumonia. Infiltrating neutrophils were digested from lung tissues or washed out from the bronchus 6 and 12 h post infection. Cells were incubated with PE/CY5.5-anti-Gr-1, PE-anti-CD11b and analyzed by flow cytometry. Numbers of neutrophils in lung and bronchial lavage (BAL) in naïve and MRSA-challenged mice at 6 h (A) and 12 h (B) post infection were detected. The absolute numbers of neutrophils in the lungs and bronchus from mice 12 h post infection were calculated (C). Sections from MRSA-infected mice and naïve mice were stained by H E (D). (The representative percentages of neutrophils in lung or bronchus came from one mice per group (n = 5)), *P

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

    29) Product Images from "Downregulating Galectin-3 Inhibits Proinflammatory Cytokine Production by Human Monocyte-Derived Dendritic Cells"

    Article Title: Downregulating Galectin-3 Inhibits Proinflammatory Cytokine Production by Human Monocyte-Derived Dendritic Cells

    Journal: Cellular immunology

    doi: 10.1016/j.cellimm.2015.01.017

    Effect of anti-Gal-3 antibodies and Gal-3 antagonists on IL-23 p19 versus IL-12 p35 production by MoDCs. MoDCs were stimulated with LPS (250 ng/ml) and/or R848 (5 µg/ml) in the presence of anti-Gal-3 B2C10 (25 µg/ml), polyclonal anti-Gal-3 (20 µg/ml), Gal-3 antagonists, e.g., NAC lactosamine, galactose. Supernatants were harvested 2 days later and evaluated for levels of IL-12 p35 ( A ) and IL-23 p19 ( B ) by ELISAs. I 125 -Gal-3 (1.2 × 10 6 cpm) was preincubated with B2C10 (0.1 to 25 µg/ml) at rt for 2 h or β-galactosides, NAC lactosamine (1–1000 ug/ml) or galactose (10 µg to 10 mg/ml) at rt for 2 h, and then added to NP-specific IgE coated on NP-BSA solid phase for 2 h. The radiolabeled Gal-3 bound to IgE on the solid phase was then evaluated by a γ-counter ( C ). MoDCs treated with Gal-3 siRNA or control scRNA were stimulated with LPS with recombinant human Gal-3 added back to cultures at 20 µg/ml for 48 h. Supernatants were then harvested and evaluated for IL-23 p19 and IL-12 p35 by ELISAs ( D ). Experiments were repeated three times from different donors. The results of the representative experiments in triplicates as means+/−SE, and the p value computed.
    Figure Legend Snippet: Effect of anti-Gal-3 antibodies and Gal-3 antagonists on IL-23 p19 versus IL-12 p35 production by MoDCs. MoDCs were stimulated with LPS (250 ng/ml) and/or R848 (5 µg/ml) in the presence of anti-Gal-3 B2C10 (25 µg/ml), polyclonal anti-Gal-3 (20 µg/ml), Gal-3 antagonists, e.g., NAC lactosamine, galactose. Supernatants were harvested 2 days later and evaluated for levels of IL-12 p35 ( A ) and IL-23 p19 ( B ) by ELISAs. I 125 -Gal-3 (1.2 × 10 6 cpm) was preincubated with B2C10 (0.1 to 25 µg/ml) at rt for 2 h or β-galactosides, NAC lactosamine (1–1000 ug/ml) or galactose (10 µg to 10 mg/ml) at rt for 2 h, and then added to NP-specific IgE coated on NP-BSA solid phase for 2 h. The radiolabeled Gal-3 bound to IgE on the solid phase was then evaluated by a γ-counter ( C ). MoDCs treated with Gal-3 siRNA or control scRNA were stimulated with LPS with recombinant human Gal-3 added back to cultures at 20 µg/ml for 48 h. Supernatants were then harvested and evaluated for IL-23 p19 and IL-12 p35 by ELISAs ( D ). Experiments were repeated three times from different donors. The results of the representative experiments in triplicates as means+/−SE, and the p value computed.

    Techniques Used: Recombinant

    Altered cytokine messages in Gal-3 siRNA-treated MoDCs. MoDCs, matured in a full cocktail (TNF-α, PGE2, IL-1β, and IL-6) were transfected with 1 nmol siRNA-4 or treated with scramble control, non-targeting RNA control (sc/snRNA) for 2 h and then stimulated with LPS (250 ng/ml) and/or R848 (5 µg/ml) for 48 h. Messages for IL-12 p19 (A), IL-12 p35 (B), IL-10 (C), IL-12 p40 (D) and OX40L (E) in Gal-3 siRNA-4 and scRNA-treated MoDCs were evaluated by qRT-PCR as described. Primers have been titrated and optimized for reliable performance. Experiments were repeated three times from different donors. The results of the representative experiments in triplicates as means+/−SE, and the p value computed.
    Figure Legend Snippet: Altered cytokine messages in Gal-3 siRNA-treated MoDCs. MoDCs, matured in a full cocktail (TNF-α, PGE2, IL-1β, and IL-6) were transfected with 1 nmol siRNA-4 or treated with scramble control, non-targeting RNA control (sc/snRNA) for 2 h and then stimulated with LPS (250 ng/ml) and/or R848 (5 µg/ml) for 48 h. Messages for IL-12 p19 (A), IL-12 p35 (B), IL-10 (C), IL-12 p40 (D) and OX40L (E) in Gal-3 siRNA-4 and scRNA-treated MoDCs were evaluated by qRT-PCR as described. Primers have been titrated and optimized for reliable performance. Experiments were repeated three times from different donors. The results of the representative experiments in triplicates as means+/−SE, and the p value computed.

    Techniques Used: Transfection, Quantitative RT-PCR

    Differential IL-23 p19 versus IL-12 p35 production in Gal-3 siRNA-treated MoDCs stimulated with TLR/NOD and innate immunity ligands. GM-CSF/IL-4 cultured MoDCs were matured with a full cocktail (TNF-α, PGE2, IL-1β, and IL-6). MoDCs were treated with Gal-3 siRNA-4 or scRNA for 2 h and then stimulated for 48 h with different innate immunity ligands, e.g., LPS (250 ng/ml) and/or R848 (5 µg/ml), Pam3CSK4 (10 µg/ml), MDP (2 µg/ml), zymosan (25 µg/ml), or house dust mites (HDM, 25 µg/ml). Levels of secreted IL-23 p19 (Panel A) and IL-12 p35 (Panel B) were evaluated by ELISAs. Experiments were repeated three times. The results of the representative experiments in triplicates as means+/−SE, and the p value computed.
    Figure Legend Snippet: Differential IL-23 p19 versus IL-12 p35 production in Gal-3 siRNA-treated MoDCs stimulated with TLR/NOD and innate immunity ligands. GM-CSF/IL-4 cultured MoDCs were matured with a full cocktail (TNF-α, PGE2, IL-1β, and IL-6). MoDCs were treated with Gal-3 siRNA-4 or scRNA for 2 h and then stimulated for 48 h with different innate immunity ligands, e.g., LPS (250 ng/ml) and/or R848 (5 µg/ml), Pam3CSK4 (10 µg/ml), MDP (2 µg/ml), zymosan (25 µg/ml), or house dust mites (HDM, 25 µg/ml). Levels of secreted IL-23 p19 (Panel A) and IL-12 p35 (Panel B) were evaluated by ELISAs. Experiments were repeated three times. The results of the representative experiments in triplicates as means+/−SE, and the p value computed.

    Techniques Used: Cell Culture

    30) Product Images from "Sirt6 regulates dendritic cell differentiation, maturation, and function"

    Article Title: Sirt6 regulates dendritic cell differentiation, maturation, and function

    Journal: Aging (Albany NY)

    doi:

    Sirt6 deletion skews cytokine production in BMDCs ( A - E ) WT and Sirt6KO BMDCs were harvested at day 8 and stimulated for 24 h with or without different TLR ligands. ( A - C ) Cells were harvested, stained for CD11c, CD86 and intracellular TNF-α, IL-6 or IL-12. CD11c + CD86 + TNF-α-, IL-6-, or IL-12-producing cells were quantified by flow cytometry. Results are means ± SEM of three-to-five separate experiments, n=3-10 for each genotype. In ( D , E ), cytokine secretion into cell supernatants was measured by ELISA. Results are means ± SEM of 3-10 separate experiments; *: p
    Figure Legend Snippet: Sirt6 deletion skews cytokine production in BMDCs ( A - E ) WT and Sirt6KO BMDCs were harvested at day 8 and stimulated for 24 h with or without different TLR ligands. ( A - C ) Cells were harvested, stained for CD11c, CD86 and intracellular TNF-α, IL-6 or IL-12. CD11c + CD86 + TNF-α-, IL-6-, or IL-12-producing cells were quantified by flow cytometry. Results are means ± SEM of three-to-five separate experiments, n=3-10 for each genotype. In ( D , E ), cytokine secretion into cell supernatants was measured by ELISA. Results are means ± SEM of 3-10 separate experiments; *: p

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

    31) Product Images from "Sirt6 regulates dendritic cell differentiation, maturation, and function"

    Article Title: Sirt6 regulates dendritic cell differentiation, maturation, and function

    Journal: Aging (Albany NY)

    doi:

    Sirt6 deletion skews cytokine production in BMDCs ( A - E ) WT and Sirt6KO BMDCs were harvested at day 8 and stimulated for 24 h with or without different TLR ligands. ( A - C ) Cells were harvested, stained for CD11c, CD86 and intracellular TNF-α, IL-6 or IL-12. CD11c + CD86 + TNF-α-, IL-6-, or IL-12-producing cells were quantified by flow cytometry. Results are means ± SEM of three-to-five separate experiments, n=3-10 for each genotype. In ( D , E ), cytokine secretion into cell supernatants was measured by ELISA. Results are means ± SEM of 3-10 separate experiments; *: p
    Figure Legend Snippet: Sirt6 deletion skews cytokine production in BMDCs ( A - E ) WT and Sirt6KO BMDCs were harvested at day 8 and stimulated for 24 h with or without different TLR ligands. ( A - C ) Cells were harvested, stained for CD11c, CD86 and intracellular TNF-α, IL-6 or IL-12. CD11c + CD86 + TNF-α-, IL-6-, or IL-12-producing cells were quantified by flow cytometry. Results are means ± SEM of three-to-five separate experiments, n=3-10 for each genotype. In ( D , E ), cytokine secretion into cell supernatants was measured by ELISA. Results are means ± SEM of 3-10 separate experiments; *: p

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

    Sirt6 deletion hampers the spontaneous maturation of BMDCs ( A , B ) BM cells from WT and Sirt6KO mice were cultured for 6 days with 20 ng/ml GM-CSF. At day 6, cells were re-seeded in the presence of 5 ng/ml GM-CSF and CD86, MHCII, and CD11c expression was determined at day 7 by flow cytometry. ( A , B ) One representative experiment out of eight is presented. ( B ) lower inset, results are means ± SEM of eight separate experiments; n=13 for each genotype; *: p
    Figure Legend Snippet: Sirt6 deletion hampers the spontaneous maturation of BMDCs ( A , B ) BM cells from WT and Sirt6KO mice were cultured for 6 days with 20 ng/ml GM-CSF. At day 6, cells were re-seeded in the presence of 5 ng/ml GM-CSF and CD86, MHCII, and CD11c expression was determined at day 7 by flow cytometry. ( A , B ) One representative experiment out of eight is presented. ( B ) lower inset, results are means ± SEM of eight separate experiments; n=13 for each genotype; *: p

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

    In vitro generated Sirt6KO BMDCs show increased endocytic activity and impaired allostimulatory capacity ( A , B ) WT and Sirt6KO BMDCs were harvested at day 7 and incubated with dextran-FITC for 30 min at 37°C or at 4°C. Thereafter, cells were stained for CD11c and finally analyzed by flow cytometry. ( A ) Gating was done on CD11c + cells; one representative experiment out of six is presented. ( B ) Dextran-FITC + /CD11c + Sirt6KO BMDCs were enumerated and their frequency was normalized to that of dextran-FITC + /CD11c + WT BMDCs. Results are means ± SEM of six separate experiments, n=6 for each genotype. ( C ) purified allogeneic (BALB-c) CD4 + splenocytes (responders) were stained with CFSE and incubated with 5 μg/ml phythoemagglutinin (PHA) or with sorted, WT or Sirt6KO, CD11c + MHCII + BMDCs at the indicated S:R ratios. Proliferation of alive (propidium-iodide negative) CD3 + CD4 + CD11c − cells was evaluated by carboxyfluorescein succinimidyl ester (CFSE) dilution after a 5-day incubation. One representative experiment out of three is presented, n=4 for each genotype.
    Figure Legend Snippet: In vitro generated Sirt6KO BMDCs show increased endocytic activity and impaired allostimulatory capacity ( A , B ) WT and Sirt6KO BMDCs were harvested at day 7 and incubated with dextran-FITC for 30 min at 37°C or at 4°C. Thereafter, cells were stained for CD11c and finally analyzed by flow cytometry. ( A ) Gating was done on CD11c + cells; one representative experiment out of six is presented. ( B ) Dextran-FITC + /CD11c + Sirt6KO BMDCs were enumerated and their frequency was normalized to that of dextran-FITC + /CD11c + WT BMDCs. Results are means ± SEM of six separate experiments, n=6 for each genotype. ( C ) purified allogeneic (BALB-c) CD4 + splenocytes (responders) were stained with CFSE and incubated with 5 μg/ml phythoemagglutinin (PHA) or with sorted, WT or Sirt6KO, CD11c + MHCII + BMDCs at the indicated S:R ratios. Proliferation of alive (propidium-iodide negative) CD3 + CD4 + CD11c − cells was evaluated by carboxyfluorescein succinimidyl ester (CFSE) dilution after a 5-day incubation. One representative experiment out of three is presented, n=4 for each genotype.

    Techniques Used: In Vitro, Generated, Activity Assay, Incubation, Staining, Flow Cytometry, Cytometry, Purification

    Sirt6KO BMDCs show impaired maturation and CCR7 expression in response to TLR ligands ( A - C ) WT and Sirt6KO BMDCs were harvested at day 7 and stimulated with or without LPS or with CpG for 24 h. Thereafter, cells were harvested, washed, and MHCII, CD86, CD80, CD40 and CCR7 expression on CD11c + cells was analyzed by flow cytometry. ( A , B ) One representative experiment out of ten ( A ) or out of four ( B ) is presented (n=4-21 for each genotype). ( C ) CCR7 mean fluorescence intensity (MFI) was normalized to that of WT BMDCs. Results are means ± SEM of four separate experiments, n=11 for each genotype; ***: p
    Figure Legend Snippet: Sirt6KO BMDCs show impaired maturation and CCR7 expression in response to TLR ligands ( A - C ) WT and Sirt6KO BMDCs were harvested at day 7 and stimulated with or without LPS or with CpG for 24 h. Thereafter, cells were harvested, washed, and MHCII, CD86, CD80, CD40 and CCR7 expression on CD11c + cells was analyzed by flow cytometry. ( A , B ) One representative experiment out of ten ( A ) or out of four ( B ) is presented (n=4-21 for each genotype). ( C ) CCR7 mean fluorescence intensity (MFI) was normalized to that of WT BMDCs. Results are means ± SEM of four separate experiments, n=11 for each genotype; ***: p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Fluorescence

    32) Product Images from "CYLD Limits Lys63- and Met1-Linked Ubiquitin at Receptor Complexes to Regulate Innate Immune Signaling"

    Article Title: CYLD Limits Lys63- and Met1-Linked Ubiquitin at Receptor Complexes to Regulate Innate Immune Signaling

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2016.02.062

    Inhibition of IAPs Reveals Extensive Regulation of RIPK2 Ubiquitination by CYLD and OTULIN (A and B) Purification of endogenous Ub conjugates from U2OS/NOD2 cells depleted for CYLD or OTULIN by siRNA. Cells were pre-treated with DMSO (control) or with 1 μM compound A (CpA) for 30 min before stimulation with (A) L18-MDP (200 ng/ml; 1 hr) or (B) TNF (10 ng/ml; 10 min). (C and D) Intracellular flow cytometry analysis of IL-8 in control (MM) U2OS/NOD2 cells or cells depleted for CYLD or OTULIN by siRNA pre-treated with 1 μM CpA for 30 min before stimulation with (C) L18-MDP (200 ng/ml; 4 hr) or (D) combination of phorbol myristate acetate (PMA) (50 ng/ml) and ionomycin (500 μM) for 4 hr. Data in (C) and (D) represent the mean ± SEM of at least three independent experiments, each performed in duplicate. ∗∗ p
    Figure Legend Snippet: Inhibition of IAPs Reveals Extensive Regulation of RIPK2 Ubiquitination by CYLD and OTULIN (A and B) Purification of endogenous Ub conjugates from U2OS/NOD2 cells depleted for CYLD or OTULIN by siRNA. Cells were pre-treated with DMSO (control) or with 1 μM compound A (CpA) for 30 min before stimulation with (A) L18-MDP (200 ng/ml; 1 hr) or (B) TNF (10 ng/ml; 10 min). (C and D) Intracellular flow cytometry analysis of IL-8 in control (MM) U2OS/NOD2 cells or cells depleted for CYLD or OTULIN by siRNA pre-treated with 1 μM CpA for 30 min before stimulation with (C) L18-MDP (200 ng/ml; 4 hr) or (D) combination of phorbol myristate acetate (PMA) (50 ng/ml) and ionomycin (500 μM) for 4 hr. Data in (C) and (D) represent the mean ± SEM of at least three independent experiments, each performed in duplicate. ∗∗ p

    Techniques Used: Inhibition, Purification, Flow Cytometry, Cytometry

    CYLD Restricts NOD2 Signaling and Cytokine Production (A) Relative levels of TNF and CXCL8 transcripts from U2OS/NOD2 control (siMM) and CYLD-depleted (siCYLD) cells treated with L18-MDP (200 ng/ml; 3 hr) normalized to untreated siMM. (B) Intracellular flow cytometry analysis of IL-8 in shRNA control (shMM) U2OS/NOD2 cells or cells stably depleted for OTULIN (shOTLN) or CYLD (shCYLD) in response to L18-MDP (200 ng/ml; 4 hr) or TNF (10 ng/ml; 4 hr). (C) WT and Cyld −/− BMDCs were stimulated with MDP (10 μg/ml; 24 hr), and secreted cytokines were measured in culture supernatants. (D) Intracellular flow cytometry analysis of IL-8 in U2OS/NOD2 cells depleted for OTULIN (siOTLN) and CYLD (siCYLD) or control (siMM) using siRNA oligos in response to L18-MDP (200 ng/ml; 4 hr). (E) Immunoblot of HOIP levels in control HCT-116 cells and in CRISPR/Cas9 HOIP KO cells. (F) Relative levels of CXCL8 transcripts from HCT-116 WT and HOIP KO cells treated with L18-MDP (200 ng/ml) and TNF (10 ng/ml) for the indicated times and normalized to untreated control cells. Data are shown on a two-segmented y axis. Data in (A), (B), (D), and (F) represent the mean ± SEM of at least three independent experiments, each performed in duplicate. ∗∗ p
    Figure Legend Snippet: CYLD Restricts NOD2 Signaling and Cytokine Production (A) Relative levels of TNF and CXCL8 transcripts from U2OS/NOD2 control (siMM) and CYLD-depleted (siCYLD) cells treated with L18-MDP (200 ng/ml; 3 hr) normalized to untreated siMM. (B) Intracellular flow cytometry analysis of IL-8 in shRNA control (shMM) U2OS/NOD2 cells or cells stably depleted for OTULIN (shOTLN) or CYLD (shCYLD) in response to L18-MDP (200 ng/ml; 4 hr) or TNF (10 ng/ml; 4 hr). (C) WT and Cyld −/− BMDCs were stimulated with MDP (10 μg/ml; 24 hr), and secreted cytokines were measured in culture supernatants. (D) Intracellular flow cytometry analysis of IL-8 in U2OS/NOD2 cells depleted for OTULIN (siOTLN) and CYLD (siCYLD) or control (siMM) using siRNA oligos in response to L18-MDP (200 ng/ml; 4 hr). (E) Immunoblot of HOIP levels in control HCT-116 cells and in CRISPR/Cas9 HOIP KO cells. (F) Relative levels of CXCL8 transcripts from HCT-116 WT and HOIP KO cells treated with L18-MDP (200 ng/ml) and TNF (10 ng/ml) for the indicated times and normalized to untreated control cells. Data are shown on a two-segmented y axis. Data in (A), (B), (D), and (F) represent the mean ± SEM of at least three independent experiments, each performed in duplicate. ∗∗ p

    Techniques Used: Flow Cytometry, Cytometry, shRNA, Stable Transfection, CRISPR

    Lys63-Ub and Met1-Ub Are Individually Indispensable for NOD2 Signaling (A) Schematic representation of the GFP-tagged Ub-binding constructs used for transient expression in cells (GFP-M1-SUB: UBAN domain from NEMO; GFP-K63-SUB: three UIMs from RAP80 in tandem). (B) Immunofluorescence analysis of nuclear translocation of RelA (red) in response to L18-MDP stimulation (1 μg/ml; 1 hr) or no treatment (NT) in U2OS/NOD2 cells transfected with GFP, GFP-K63-SUB, or GFP-M1-SUB (green) for 24 hr. The scale bar represents 10 μm. (C) Quantification of (B). (D) Intracellular flow cytometry analysis of IL-8 in U2OS/NOD2 cells transfected as indicated for 48 hr before L18-MDP stimulation (200 ng/ml; 4 hr). (E) Gating of cells in (D) based on GFP levels (relative fluorescence units [RFUs]). RFU values on x axis indicate the maximal RFU in each gate with the previous value defining the lower limit, except of the “zero” RFU population, which includes cells with values up to 100 RFUs. (F) As in (D) except that GFP, GFP-K63-SUB, or GFP-M1-SUB contains a nuclear localization signal (NLS). (G) Intracellular flow cytometry analysis of IL-8 in control (MM) U2OS/NOD2 cells or cells stably depleted for CYLD or OTULIN; depleted for HOIP, Ubc13, or both by siRNA as indicated; and treated with L18-MDP (200 ng/ml; 4 hr) or not treated (NT). Data in (C)–(G) represent the mean ± SEM of at least three independent experiments, each performed in duplicate. ∗∗ p
    Figure Legend Snippet: Lys63-Ub and Met1-Ub Are Individually Indispensable for NOD2 Signaling (A) Schematic representation of the GFP-tagged Ub-binding constructs used for transient expression in cells (GFP-M1-SUB: UBAN domain from NEMO; GFP-K63-SUB: three UIMs from RAP80 in tandem). (B) Immunofluorescence analysis of nuclear translocation of RelA (red) in response to L18-MDP stimulation (1 μg/ml; 1 hr) or no treatment (NT) in U2OS/NOD2 cells transfected with GFP, GFP-K63-SUB, or GFP-M1-SUB (green) for 24 hr. The scale bar represents 10 μm. (C) Quantification of (B). (D) Intracellular flow cytometry analysis of IL-8 in U2OS/NOD2 cells transfected as indicated for 48 hr before L18-MDP stimulation (200 ng/ml; 4 hr). (E) Gating of cells in (D) based on GFP levels (relative fluorescence units [RFUs]). RFU values on x axis indicate the maximal RFU in each gate with the previous value defining the lower limit, except of the “zero” RFU population, which includes cells with values up to 100 RFUs. (F) As in (D) except that GFP, GFP-K63-SUB, or GFP-M1-SUB contains a nuclear localization signal (NLS). (G) Intracellular flow cytometry analysis of IL-8 in control (MM) U2OS/NOD2 cells or cells stably depleted for CYLD or OTULIN; depleted for HOIP, Ubc13, or both by siRNA as indicated; and treated with L18-MDP (200 ng/ml; 4 hr) or not treated (NT). Data in (C)–(G) represent the mean ± SEM of at least three independent experiments, each performed in duplicate. ∗∗ p

    Techniques Used: Binding Assay, Construct, Expressing, Immunofluorescence, Translocation Assay, Transfection, Flow Cytometry, Cytometry, Fluorescence, Stable Transfection

    CYLD Catalytic Activity Inhibits the NOD2 Pathway Upstream of Nuclear Translocation of NF-κB (A) Immunofluorescence analysis of nuclear translocation of the NF-κB subunit RelA/p65 (red) in response to L18-MDP (1 μg/ml; 1 hr) in U2OS/NOD2 cells transfected with FLAG-CYLD variants and stained with anti-FLAG (green; scale bar, 10 μm). (B) Quantification of cells with nuclear RelA treated as in (A). (C) Intracellular flow cytometry analysis of IL-8 in U2OS/NOD2 cells transfected with FLAG-CYLD variants in response to L18-MDP (200 ng/ml; 4 hr). Cells were cotransfected with a GFP vector (ratio 1:10) as a marker of transfection. (D) NF-κB activity in HEK293FT cell lysates transfected with dual luciferase reporters, XIAP, CYLD, and OTULIN as indicated. Values are expressed relative to XIAP transfection. (E) NF-κB activity in HEK293T cell lysates transfected with luciferase reporters, vector, or XIAP and depleted for Ubc13 using two different siRNAs. Values are expressed relative to XIAP transfection. Data in (B)–(E) represent the mean ± SEM of at least three independent experiments, each performed in duplicate. ∗∗ p
    Figure Legend Snippet: CYLD Catalytic Activity Inhibits the NOD2 Pathway Upstream of Nuclear Translocation of NF-κB (A) Immunofluorescence analysis of nuclear translocation of the NF-κB subunit RelA/p65 (red) in response to L18-MDP (1 μg/ml; 1 hr) in U2OS/NOD2 cells transfected with FLAG-CYLD variants and stained with anti-FLAG (green; scale bar, 10 μm). (B) Quantification of cells with nuclear RelA treated as in (A). (C) Intracellular flow cytometry analysis of IL-8 in U2OS/NOD2 cells transfected with FLAG-CYLD variants in response to L18-MDP (200 ng/ml; 4 hr). Cells were cotransfected with a GFP vector (ratio 1:10) as a marker of transfection. (D) NF-κB activity in HEK293FT cell lysates transfected with dual luciferase reporters, XIAP, CYLD, and OTULIN as indicated. Values are expressed relative to XIAP transfection. (E) NF-κB activity in HEK293T cell lysates transfected with luciferase reporters, vector, or XIAP and depleted for Ubc13 using two different siRNAs. Values are expressed relative to XIAP transfection. Data in (B)–(E) represent the mean ± SEM of at least three independent experiments, each performed in duplicate. ∗∗ p

    Techniques Used: Activity Assay, Translocation Assay, Immunofluorescence, Transfection, Staining, Flow Cytometry, Cytometry, Plasmid Preparation, Marker, Luciferase

    33) Product Images from "Frequency of circulating topoisomerase-I-specific CD4 T cells predicts presence and progression of interstitial lung disease in scleroderma"

    Article Title: Frequency of circulating topoisomerase-I-specific CD4 T cells predicts presence and progression of interstitial lung disease in scleroderma

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-016-0993-2

    Detection of circulating topoisomerase-I-specific CD4+ T cells by CD154 and CD69 expression and upregulation. After incubation (15 min) with anti-CD40 blocking antibodies, freshly isolated peripheral blood mononuclear cells (PBMCs) were stimulated for 18 h in presence of 5 % autologous serum as indicated below. Gating was set on lymphocytes, singlets, live, CD3+ and CD4+ cells. Numbers in quadrants indicate the percent (%) cells in the parent CD4+ population. a PBMCs from a healthy donor recently immunized with combined tetanus, diphtheria, and pertussis (Tdap) vaccine were stimulated with PRMT6 (negative control), SEA + SEB (positive control) and tetanus toxoid (TT). b PBMCs from a topo-I+ SSc patient were stimulated with PRMT6, PAD4 (negative controls), or topo-I. c Representative experiments on two anti-topo-I-negative and two anti-topo-I-positive SSc patients. The gating strategy for this experiment is shown in Additional file 1 : Figure S1. PRMT6 protein arginine methyltransferase 6, PAD4 human peptidyl arginine deiminase type 4
    Figure Legend Snippet: Detection of circulating topoisomerase-I-specific CD4+ T cells by CD154 and CD69 expression and upregulation. After incubation (15 min) with anti-CD40 blocking antibodies, freshly isolated peripheral blood mononuclear cells (PBMCs) were stimulated for 18 h in presence of 5 % autologous serum as indicated below. Gating was set on lymphocytes, singlets, live, CD3+ and CD4+ cells. Numbers in quadrants indicate the percent (%) cells in the parent CD4+ population. a PBMCs from a healthy donor recently immunized with combined tetanus, diphtheria, and pertussis (Tdap) vaccine were stimulated with PRMT6 (negative control), SEA + SEB (positive control) and tetanus toxoid (TT). b PBMCs from a topo-I+ SSc patient were stimulated with PRMT6, PAD4 (negative controls), or topo-I. c Representative experiments on two anti-topo-I-negative and two anti-topo-I-positive SSc patients. The gating strategy for this experiment is shown in Additional file 1 : Figure S1. PRMT6 protein arginine methyltransferase 6, PAD4 human peptidyl arginine deiminase type 4

    Techniques Used: Expressing, Incubation, Blocking Assay, Isolation, Negative Control, Positive Control

    Increased frequencies of topoisomerase-I-specific CD4+ T cells in the blood of anti-topo-I-positive SSc patients. Topo-I-specific CD4+ T cells frequency was measured in the peripheral blood of 15 anti-topo-I-positive, 12 anti-topo-I-negative SSc patients, and 4 healthy donors (HD). Values represent the percentage of CD154 + CD69+ cells within the CD4+ population. To minimize any noise from the background, the frequency of topo-I-reactive T cells was calculated after subtracting the small percentage of CD154 + CD69 + CD4+ T cells detected within unstimulated PBMCs from the same subject. Horizontal lines indicate the mean frequency for each group
    Figure Legend Snippet: Increased frequencies of topoisomerase-I-specific CD4+ T cells in the blood of anti-topo-I-positive SSc patients. Topo-I-specific CD4+ T cells frequency was measured in the peripheral blood of 15 anti-topo-I-positive, 12 anti-topo-I-negative SSc patients, and 4 healthy donors (HD). Values represent the percentage of CD154 + CD69+ cells within the CD4+ population. To minimize any noise from the background, the frequency of topo-I-reactive T cells was calculated after subtracting the small percentage of CD154 + CD69 + CD4+ T cells detected within unstimulated PBMCs from the same subject. Horizontal lines indicate the mean frequency for each group

    Techniques Used:

    Topoisomerase-I-specific CD4+ T cells activation is HLA-DR restricted. PBMC from topo-I-positive SSc patients were cultured for 18 h with topo-I and anti-HLA-DR or anti-HLA-DP blocking antibodies or isotype control (1 ug/ml). Values are represented as percent of response relative to the frequency of CD154 + CD69 + CD4+ cells detected after stimulation with topo-I alone as assessed by flow cytometry. Data are representative of three separate experiments (mean ± SD)
    Figure Legend Snippet: Topoisomerase-I-specific CD4+ T cells activation is HLA-DR restricted. PBMC from topo-I-positive SSc patients were cultured for 18 h with topo-I and anti-HLA-DR or anti-HLA-DP blocking antibodies or isotype control (1 ug/ml). Values are represented as percent of response relative to the frequency of CD154 + CD69 + CD4+ cells detected after stimulation with topo-I alone as assessed by flow cytometry. Data are representative of three separate experiments (mean ± SD)

    Techniques Used: Activation Assay, Cell Culture, Blocking Assay, Flow Cytometry, Cytometry

    34) Product Images from "Induction of IL-25 secretion from tumour-associated fibroblasts suppresses mammary tumour metastasis"

    Article Title: Induction of IL-25 secretion from tumour-associated fibroblasts suppresses mammary tumour metastasis

    Journal: Nature Communications

    doi: 10.1038/ncomms11311

    Q2-3 treatment significantly upregulates IL-25 expression in tumour-associated fibroblasts, as evaluated by 3D cell co-culture system. ( a ) Effect of Q2-3 on population change of FSP-1 + ER-TR7 + cells and their IL-25 expression level in lungs of test mice, quantified using flow cytometry. The percentage of IL-25 + fibroblasts in gated FSP-1 + ER-TR7 + cells were compared with corresponding IL-25 + fibroblast population in PBS group. Data are reported as mean±s.d. ( n =3). * P
    Figure Legend Snippet: Q2-3 treatment significantly upregulates IL-25 expression in tumour-associated fibroblasts, as evaluated by 3D cell co-culture system. ( a ) Effect of Q2-3 on population change of FSP-1 + ER-TR7 + cells and their IL-25 expression level in lungs of test mice, quantified using flow cytometry. The percentage of IL-25 + fibroblasts in gated FSP-1 + ER-TR7 + cells were compared with corresponding IL-25 + fibroblast population in PBS group. Data are reported as mean±s.d. ( n =3). * P

    Techniques Used: Expressing, Co-Culture Assay, Mouse Assay, Flow Cytometry, Cytometry

    In vivo neutralization of IL-25 activity can effectively invalidate the anti-metastatic activity of Q2-3. ( a ) Representative bioluminescent images of tumour-resected mice ( n =8 per group) after in vivo treatment with PBS (0.1% DMSO in saline), Q2-3 (100 μg kg −1 ; 3 injections per week), anti-mouse IL-25 Ab (100 μg per mice; 2 injections per week), Q2-3+IL-25 Ab, or Q2-3+isotype IgG, at 3 weeks post tumour resection. The label ‘D' in the photograph denotes the mice died before 3 weeks post tumour resection. ( b ) Quantification of tumour metastasis by measuring luciferase activity photons s −1 cm −2 sr −1 in mice, as revealed along the indicated time course (12 weeks). ( c ) Survival of test mice after different treatments. NS, no significant difference between the ‘Q2-3' and ‘Q2-3+Anti-IgG' groups. ** P
    Figure Legend Snippet: In vivo neutralization of IL-25 activity can effectively invalidate the anti-metastatic activity of Q2-3. ( a ) Representative bioluminescent images of tumour-resected mice ( n =8 per group) after in vivo treatment with PBS (0.1% DMSO in saline), Q2-3 (100 μg kg −1 ; 3 injections per week), anti-mouse IL-25 Ab (100 μg per mice; 2 injections per week), Q2-3+IL-25 Ab, or Q2-3+isotype IgG, at 3 weeks post tumour resection. The label ‘D' in the photograph denotes the mice died before 3 weeks post tumour resection. ( b ) Quantification of tumour metastasis by measuring luciferase activity photons s −1 cm −2 sr −1 in mice, as revealed along the indicated time course (12 weeks). ( c ) Survival of test mice after different treatments. NS, no significant difference between the ‘Q2-3' and ‘Q2-3+Anti-IgG' groups. ** P

    Techniques Used: In Vivo, Neutralization, Activity Assay, Mouse Assay, Luciferase

    IL-25 secreted by Q2-3-treated fibroblasts suppresses the growth of mammary tumour cells. ( a ) Western blot analysis of the IL-25 secretion activity of mouse (3T3) and human (WI38) fibroblasts in response to Q2-3 treatment. Different fibroblast-conditioned media (CM), including 3T3-CM and WI38-CM, were collected from the 3D cultures and were stained with coomassie blue, revealing that the total protein level in tested CM was normalized. Aliquots of 3T3-CM and WI38-CM were immunodepleted for IL-25. Rabbit IgG (isotype control) was used as a negative control. Amounts of IL-25 (relative staining intensity) were further normalized with the value detected for Q2-3-treated 3T3-CM (blue) or Q2-3-treated WI38-CM samples (purple). ( b ) Reduction in cytotoxicity of 3T3-CM on 4T1 cells after immunodepletion of IL-25. The control (fresh) medium, 3T3-CM, Q2-3-treated 3T3-CM, Q2-3-treated 3T3-CM with added IL-25 protein, Q2-3-treated 3T3-CM with the immunodepletion of IL-25 and Q2-3-treated 3T3-CM with control IgG-mediated immunodepletion, were compared for their suppressive effect on growth of 4T1 cells. ( c ) Reduction in cytotoxicity of WI38-CM on MDA-MB-231 after immunodepletion of IL-25. Similarly, WI38-CM with added IL-25 protein, WI38-CM with the immunodepletion of IL-25, or Q2-3-treated WI38-CM with control IgG-mediated immunodepletion, were compared for their effect on growth of MDA-MB-231 cells. The growth activity of 4T1 cells or MDA-MB-231 cells was determined using MTT assay at 5 days post cultivation, and was normalized to the control group (with fresh medium only). Data are reported as mean±s.d. ( n =3). * P
    Figure Legend Snippet: IL-25 secreted by Q2-3-treated fibroblasts suppresses the growth of mammary tumour cells. ( a ) Western blot analysis of the IL-25 secretion activity of mouse (3T3) and human (WI38) fibroblasts in response to Q2-3 treatment. Different fibroblast-conditioned media (CM), including 3T3-CM and WI38-CM, were collected from the 3D cultures and were stained with coomassie blue, revealing that the total protein level in tested CM was normalized. Aliquots of 3T3-CM and WI38-CM were immunodepleted for IL-25. Rabbit IgG (isotype control) was used as a negative control. Amounts of IL-25 (relative staining intensity) were further normalized with the value detected for Q2-3-treated 3T3-CM (blue) or Q2-3-treated WI38-CM samples (purple). ( b ) Reduction in cytotoxicity of 3T3-CM on 4T1 cells after immunodepletion of IL-25. The control (fresh) medium, 3T3-CM, Q2-3-treated 3T3-CM, Q2-3-treated 3T3-CM with added IL-25 protein, Q2-3-treated 3T3-CM with the immunodepletion of IL-25 and Q2-3-treated 3T3-CM with control IgG-mediated immunodepletion, were compared for their suppressive effect on growth of 4T1 cells. ( c ) Reduction in cytotoxicity of WI38-CM on MDA-MB-231 after immunodepletion of IL-25. Similarly, WI38-CM with added IL-25 protein, WI38-CM with the immunodepletion of IL-25, or Q2-3-treated WI38-CM with control IgG-mediated immunodepletion, were compared for their effect on growth of MDA-MB-231 cells. The growth activity of 4T1 cells or MDA-MB-231 cells was determined using MTT assay at 5 days post cultivation, and was normalized to the control group (with fresh medium only). Data are reported as mean±s.d. ( n =3). * P

    Techniques Used: Western Blot, Activity Assay, Staining, Negative Control, Multiple Displacement Amplification, MTT Assay

    In vivo treatment of Q2-3 confers comparable anti-metastatic activity with IL-25 administration. ( a ) Tumour-resected mice ( n =8 per group) were treated with PBS (0.1% DMSO in saline), IL-25 (200 ng per mice), Q2-3 (100 μg kg −1 ) or co-treated with IL-25 and Q2-3 for 3 weeks. Quantification of tumour metastasis by measuring luciferase activity in photons s −1 cm −2 sr −1 in mice revealed along the indicated time course. ( b ) Survival of test mice after different treatments. NS, no significant difference between the Q2-3 and co-treatment groups (Kaplan–Meier results were analysed by log-rank test). Similar results were obtained from three independent experiments.
    Figure Legend Snippet: In vivo treatment of Q2-3 confers comparable anti-metastatic activity with IL-25 administration. ( a ) Tumour-resected mice ( n =8 per group) were treated with PBS (0.1% DMSO in saline), IL-25 (200 ng per mice), Q2-3 (100 μg kg −1 ) or co-treated with IL-25 and Q2-3 for 3 weeks. Quantification of tumour metastasis by measuring luciferase activity in photons s −1 cm −2 sr −1 in mice revealed along the indicated time course. ( b ) Survival of test mice after different treatments. NS, no significant difference between the Q2-3 and co-treatment groups (Kaplan–Meier results were analysed by log-rank test). Similar results were obtained from three independent experiments.

    Techniques Used: In Vivo, Activity Assay, Mouse Assay, Luciferase

    35) Product Images from "GM-CSF Signalling Boosts Dramatically IL-1Production"

    Article Title: GM-CSF Signalling Boosts Dramatically IL-1Production

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0023025

    GM-CSF boosts LPS-induced IL-1 secretion. (A) CD11b + fraction of FLT3L derived DCs was stimulated 24 h with a wide range of different LPS concentrations (0.001–10 µg/ml) in absence (white circles) or presence of 5 ng/ml GM-CSF (black circles). 5 mM ATP was added as a danger signal. Released IL-1β, IL-1α, TNF-α and IL-6 were measured in the culture supernatants by standard ELISA and each value represents the mean of triplicates +/− SD. (B) CD11b + fraction of FLT3L generated DCs was primed for 24 h with 100 ng/ml LPS with (back bars) or without (white bars) 5 ng/ml GM-CSF and stimulated with different danger signals (5 mM ATP, 1 µM nigericin, 100 µg/ml MSU, 200 µg/ml Alu). Each bar represents the mean of triplicates +/− SD. (C) CD11b + fraction of FLT3L generated DCs was primed with TLR agonists (100 ng/ml LPS and Pam 3 CSK 4 ), Dectin agonist, Curdlan (100 µg/ml) and pro-inflammatory cytokine TNF-α (100 ng/ml) in absence (white bars) or presence (back bars) of 5 ng/ml GM-CSF and stimulated subsequently with ATP. Each bar represents the mean of triplicates +/− SD. (D) GM-CSF derived BM DCs, M-CSF-derived BM MØ as well as L929-derived BM MØ were compared to the CD11b + fraction of FLT3L-derived DCs for their capacity to secrete IL-1β upon 24 h LPS stimulation (100 ng/ml) in absence or in presence of GM-CSF (5 ng/ml). ATP was added as danger signal. Both, WT (black bars) and GM-CSF R−/− cells (white bars) were tested. Each bar represents the mean of triplicates +/− SD. All results are representative of at least two independent experiments.
    Figure Legend Snippet: GM-CSF boosts LPS-induced IL-1 secretion. (A) CD11b + fraction of FLT3L derived DCs was stimulated 24 h with a wide range of different LPS concentrations (0.001–10 µg/ml) in absence (white circles) or presence of 5 ng/ml GM-CSF (black circles). 5 mM ATP was added as a danger signal. Released IL-1β, IL-1α, TNF-α and IL-6 were measured in the culture supernatants by standard ELISA and each value represents the mean of triplicates +/− SD. (B) CD11b + fraction of FLT3L generated DCs was primed for 24 h with 100 ng/ml LPS with (back bars) or without (white bars) 5 ng/ml GM-CSF and stimulated with different danger signals (5 mM ATP, 1 µM nigericin, 100 µg/ml MSU, 200 µg/ml Alu). Each bar represents the mean of triplicates +/− SD. (C) CD11b + fraction of FLT3L generated DCs was primed with TLR agonists (100 ng/ml LPS and Pam 3 CSK 4 ), Dectin agonist, Curdlan (100 µg/ml) and pro-inflammatory cytokine TNF-α (100 ng/ml) in absence (white bars) or presence (back bars) of 5 ng/ml GM-CSF and stimulated subsequently with ATP. Each bar represents the mean of triplicates +/− SD. (D) GM-CSF derived BM DCs, M-CSF-derived BM MØ as well as L929-derived BM MØ were compared to the CD11b + fraction of FLT3L-derived DCs for their capacity to secrete IL-1β upon 24 h LPS stimulation (100 ng/ml) in absence or in presence of GM-CSF (5 ng/ml). ATP was added as danger signal. Both, WT (black bars) and GM-CSF R−/− cells (white bars) were tested. Each bar represents the mean of triplicates +/− SD. All results are representative of at least two independent experiments.

    Techniques Used: Derivative Assay, Enzyme-linked Immunosorbent Assay, Generated

    CD11b + fraction of FLT3L derived DCs were stimulated for 3, 6, 9 and 24 h respectively with LPS alone (white bars) or in combination with GM-CSF (black bars). For the detection of IL-1β, 4×10 5 cells were stimulated for 1 h with ATP, for IL-6 and TNF-α the cytokine release of 1×10 5 cells was analysed without ATP treatment. Released cytokines were measured by ELISA. Each bar represents the mean of triplicates +/− SD.
    Figure Legend Snippet: CD11b + fraction of FLT3L derived DCs were stimulated for 3, 6, 9 and 24 h respectively with LPS alone (white bars) or in combination with GM-CSF (black bars). For the detection of IL-1β, 4×10 5 cells were stimulated for 1 h with ATP, for IL-6 and TNF-α the cytokine release of 1×10 5 cells was analysed without ATP treatment. Released cytokines were measured by ELISA. Each bar represents the mean of triplicates +/− SD.

    Techniques Used: Derivative Assay, Enzyme-linked Immunosorbent Assay

    GM-CSF R−/− mice survive LPS-induced septic shock. (A) Survival of WT and GM-CSFR−/− mice ( n = 12 each group) injected i.p. with 50 µg/g body weight LPS. (B) WT and GM-CSFR−/− mice were bled from the retro-orbital plexus 3 h after LPS treatment. Pro-inflammatory cytokines such as IL-1α, IL-1β, IL-6 and TNF-α were measured in the serum by ELISA. The data represent the mean +/− SD of three pooled independent experiments. *
    Figure Legend Snippet: GM-CSF R−/− mice survive LPS-induced septic shock. (A) Survival of WT and GM-CSFR−/− mice ( n = 12 each group) injected i.p. with 50 µg/g body weight LPS. (B) WT and GM-CSFR−/− mice were bled from the retro-orbital plexus 3 h after LPS treatment. Pro-inflammatory cytokines such as IL-1α, IL-1β, IL-6 and TNF-α were measured in the serum by ELISA. The data represent the mean +/− SD of three pooled independent experiments. *

    Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

    36) Product Images from "Caveolin-1 Expression Increases upon Maturation in Dendritic Cells and Promotes Their Migration to Lymph Nodes Thereby Favoring the Induction of CD8+ T Cell Responses"

    Article Title: Caveolin-1 Expression Increases upon Maturation in Dendritic Cells and Promotes Their Migration to Lymph Nodes Thereby Favoring the Induction of CD8+ T Cell Responses

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01794

    Caveolin-1 (CAV1) is expressed in dendritic cells (DCs) and upregulated upon maturation. CAV1 expression in DCs was assessed by Western blotting. GAPDH and actin were used as loading controls. CAV1 protein expression was quantified by densitometry analysis and standardized to loading control. Values normalized to untreated controls (NT) are shown. (A) Spleen DCs purified from wild-type mice were treated with LPS (100 ng/ml) for 6 h. (B,C) Bone marrow-derived DCs (BM-DCs) were stimulated with LPS (100 ng/ml) or TNF-α (20 ng/ml) for 0, 1, 3, 6, 12, or 24 h. (D) BM-DCs were treated with LPS (100 ng/ml) alone or in combination with a TNF-α blocking antibody (5 µg/ml) for 24 h. Values are presented as the mean ± SEM. * p
    Figure Legend Snippet: Caveolin-1 (CAV1) is expressed in dendritic cells (DCs) and upregulated upon maturation. CAV1 expression in DCs was assessed by Western blotting. GAPDH and actin were used as loading controls. CAV1 protein expression was quantified by densitometry analysis and standardized to loading control. Values normalized to untreated controls (NT) are shown. (A) Spleen DCs purified from wild-type mice were treated with LPS (100 ng/ml) for 6 h. (B,C) Bone marrow-derived DCs (BM-DCs) were stimulated with LPS (100 ng/ml) or TNF-α (20 ng/ml) for 0, 1, 3, 6, 12, or 24 h. (D) BM-DCs were treated with LPS (100 ng/ml) alone or in combination with a TNF-α blocking antibody (5 µg/ml) for 24 h. Values are presented as the mean ± SEM. * p

    Techniques Used: Expressing, Western Blot, Purification, Mouse Assay, Derivative Assay, Blocking Assay

    37) Product Images from "Escherichia coli Maltose-Binding Protein Induces M1 Polarity of RAW264.7 Macrophage Cells via a TLR2- and TLR4-Dependent Manner"

    Article Title: Escherichia coli Maltose-Binding Protein Induces M1 Polarity of RAW264.7 Macrophage Cells via a TLR2- and TLR4-Dependent Manner

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms16059896

    Effects of MBP on expression of TLR2 and TLR4 in RAW264.7 macrophages. RAW264.7 cells were treated with 5 μg/mL of MBP or 1 μg/mL of LPS for 48 h in the presence or absence of 5 μg/mL of polymyxin B (PB). Expression of TLR2 and TLR4 was analyzed by flow cytometry. Representative flow plots are shown, and results from three independent experiments are presented as mean ± SD.
    Figure Legend Snippet: Effects of MBP on expression of TLR2 and TLR4 in RAW264.7 macrophages. RAW264.7 cells were treated with 5 μg/mL of MBP or 1 μg/mL of LPS for 48 h in the presence or absence of 5 μg/mL of polymyxin B (PB). Expression of TLR2 and TLR4 was analyzed by flow cytometry. Representative flow plots are shown, and results from three independent experiments are presented as mean ± SD.

    Techniques Used: Expressing, Flow Cytometry, Cytometry

    Effects of MBP on activation of NF-κB and p38 MAPK via TLR2 and TLR4. ( A ) RAW264.7 cells were treated with MBP (1, 5, 10 μg/mL) for 6 h. MyD88 expression was determined by Western blotting using whole cell lysates. MyD88 protein levels relative to the endogenous control β-actin were quantified and presented in bar graph. * p
    Figure Legend Snippet: Effects of MBP on activation of NF-κB and p38 MAPK via TLR2 and TLR4. ( A ) RAW264.7 cells were treated with MBP (1, 5, 10 μg/mL) for 6 h. MyD88 expression was determined by Western blotting using whole cell lysates. MyD88 protein levels relative to the endogenous control β-actin were quantified and presented in bar graph. * p

    Techniques Used: Activation Assay, Expressing, Western Blot

    Effects of MBP-induced activation and polarization of RAW264.7 cells via TLR2 and TLR4. RAW264.7 cells were pre-incubated with anti-TLR2 or anti-TLR4 (20 μg/mL) antibody for 2 h prior to the addition of MBP or LPS (5 μg/mL) for 48 h. Mouse IgG2a (20 µg/mL) was used as isotype control. ( A ) MBP-induced NO production in RAW264.7 cells was partially inhibited by either anti-TLR2 or anti-TLR4; ( B ) MBP-induced M1 polarization of RAW264.7 cells was greatly inhibited by either anti-TLR2 or anti-TLR4. Results were presented as mean ± SD from three independent experiments, each performed in triplicate. * p
    Figure Legend Snippet: Effects of MBP-induced activation and polarization of RAW264.7 cells via TLR2 and TLR4. RAW264.7 cells were pre-incubated with anti-TLR2 or anti-TLR4 (20 μg/mL) antibody for 2 h prior to the addition of MBP or LPS (5 μg/mL) for 48 h. Mouse IgG2a (20 µg/mL) was used as isotype control. ( A ) MBP-induced NO production in RAW264.7 cells was partially inhibited by either anti-TLR2 or anti-TLR4; ( B ) MBP-induced M1 polarization of RAW264.7 cells was greatly inhibited by either anti-TLR2 or anti-TLR4. Results were presented as mean ± SD from three independent experiments, each performed in triplicate. * p

    Techniques Used: Activation Assay, Incubation

    38) Product Images from "Galectin-8 as an immunosuppressor in experimental autoimmune encephalomyelitis and a target of human early prognostic antibodies in multiple sclerosis"

    Article Title: Galectin-8 as an immunosuppressor in experimental autoimmune encephalomyelitis and a target of human early prognostic antibodies in multiple sclerosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0177472

    Gal-8 deficit favors selective Th17 cell differentiation upon polyclonal activation. Splenocytes isolated from Lgals8 +/+ (WT) and Lgals8 -/- (KO) mice were analyzed by FACS: (A) Dendritic cells (CD11c + ), B cells (CD19 + ), CD8 + T cells and different CD4 + T cells subsets, naïve (CD44 - CD62L + ), effector (CD44 + CD62L + ), memory (CD44 + CD62L - ) and total cells analyzed in the subset of viable CD4 + CD25 - T-cells show no differences between WT and KO mice. Graphics of frequency +/-SD (n = 5). (B) T cell activation by 72 h incubation with anti-CD3 (anti-CD3) and anti-CD28 (anti-CD28) antibodies show Th17 increased frequency in KO mice while Th1 and Th2 cells are similar in WT and KO mice. Graph shows frequency +/-SD (*p
    Figure Legend Snippet: Gal-8 deficit favors selective Th17 cell differentiation upon polyclonal activation. Splenocytes isolated from Lgals8 +/+ (WT) and Lgals8 -/- (KO) mice were analyzed by FACS: (A) Dendritic cells (CD11c + ), B cells (CD19 + ), CD8 + T cells and different CD4 + T cells subsets, naïve (CD44 - CD62L + ), effector (CD44 + CD62L + ), memory (CD44 + CD62L - ) and total cells analyzed in the subset of viable CD4 + CD25 - T-cells show no differences between WT and KO mice. Graphics of frequency +/-SD (n = 5). (B) T cell activation by 72 h incubation with anti-CD3 (anti-CD3) and anti-CD28 (anti-CD28) antibodies show Th17 increased frequency in KO mice while Th1 and Th2 cells are similar in WT and KO mice. Graph shows frequency +/-SD (*p

    Techniques Used: Cell Differentiation, Activation Assay, Isolation, Mouse Assay, FACS, Incubation

    Gal-8 deficit favors Th17 polarization during MOGp-induced EAE and ex-vivo re-stimulation. Th17 and Th1 subpopulations in splenocytes from Lgals8 -/- (KO) and Lgals8 +/+ (WT) mice obtained after 10 days of EAE induction were analyzed either immediately or after 72 h of ex vivo MOGp re-stimulation, in the absence or presence of Gal-8. Gal-8 KO mice show higher frequency of Th17 cells both at steady state and after MOGp re-stimulation. Incubation with Gal-8 reduced Th17 cells only in Gal-8 KO. Graph shows frequency +/-SD (*p
    Figure Legend Snippet: Gal-8 deficit favors Th17 polarization during MOGp-induced EAE and ex-vivo re-stimulation. Th17 and Th1 subpopulations in splenocytes from Lgals8 -/- (KO) and Lgals8 +/+ (WT) mice obtained after 10 days of EAE induction were analyzed either immediately or after 72 h of ex vivo MOGp re-stimulation, in the absence or presence of Gal-8. Gal-8 KO mice show higher frequency of Th17 cells both at steady state and after MOGp re-stimulation. Incubation with Gal-8 reduced Th17 cells only in Gal-8 KO. Graph shows frequency +/-SD (*p

    Techniques Used: Ex Vivo, Mouse Assay, Incubation

    Gal-8 ameliorates EAE and induces Th17 cell death in vitro . (A) Gal-8 treatment ameliorates MOGp-induced EAE in C57BL/6 mice. The mice were injected daily by intraperitoneal injection of either PBS (Control) or Gal-8 100 μg/ml. Gal-8-treated animals tend to start the disease later and show lower EAE scores during the acute and chronic phases of the disease (*p
    Figure Legend Snippet: Gal-8 ameliorates EAE and induces Th17 cell death in vitro . (A) Gal-8 treatment ameliorates MOGp-induced EAE in C57BL/6 mice. The mice were injected daily by intraperitoneal injection of either PBS (Control) or Gal-8 100 μg/ml. Gal-8-treated animals tend to start the disease later and show lower EAE scores during the acute and chronic phases of the disease (*p

    Techniques Used: In Vitro, Mouse Assay, Injection

    39) Product Images from "Human multipotent stromal cells attenuate lipopolysaccharide-induced acute lung injury in mice via secretion of tumor necrosis factor-?-induced protein 6"

    Article Title: Human multipotent stromal cells attenuate lipopolysaccharide-induced acute lung injury in mice via secretion of tumor necrosis factor-?-induced protein 6

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/scrt68

    Proinflammatory cytokine and chemokine expression is decreased by hMSC administration . Twenty-four hours after LPS exposure, IL-1β, IL-6, IL-17, IP-10, MCP-1, MIP-1α and RANTES levels were measured by multiplex immunoassay in BAL fluid. Data are expressed as mean ± SEM (n = 6 per group). * P
    Figure Legend Snippet: Proinflammatory cytokine and chemokine expression is decreased by hMSC administration . Twenty-four hours after LPS exposure, IL-1β, IL-6, IL-17, IP-10, MCP-1, MIP-1α and RANTES levels were measured by multiplex immunoassay in BAL fluid. Data are expressed as mean ± SEM (n = 6 per group). * P

    Techniques Used: Expressing, Multiplex Assay

    40) Product Images from "Impact of HIV-ART on the restoration of Th17 and Treg cells in blood and female genital mucosa"

    Article Title: Impact of HIV-ART on the restoration of Th17 and Treg cells in blood and female genital mucosa

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-38547-1

    Functionality of CMCs (related to Th17 and Treg-cks) was associated with local mucosal genital chemokine levels. Chemokine levels were quantified by CBA (pg/ml) in ectocervix samples from the different groups as described in methodology (Column a ): Row I: CXCL5 (ENA-78), (II) CXCL1 (GRO-α), (III) CCL17/CCL20 ratio (TARC/MIP-3α ratio) and (IV) CCL17 (TARC) quantities are shown. (Column b rows I to III): HIV+ ART+ group was subdivided in two sub-groups according to have “at least three” or “one or less” Th17-cks (≥3Th17-cks vs ≤1 Th17-ck) in the CMCs samples. (Column b row IV): HIV+ ART+ group was subdivided in two subgroups according to have two Treg- cks vs one or less Treg-ck (2 Treg-cks vs ≤1 Treg-ck). CXCL5, CXCL1, CCL17/CCL20 ratio and CCL17 levels are shown for HIV-neg and HIV+ ART+ sub-divided groups. (Column c rows I to IV): HIV+ ART+ group was subdivided in two subgroups according to have at least three Th17-cks plus two Treg-cks vs one or less Th17-cks plus one or less Treg-cks (≥3Th17-cks + 2Treg-cks vs ≤1 Th17-ck + ≤1 Treg-ck). CXCL5, CXCL1, CCL17/CCL20 ratio and CCL17 levels are shown for HIV-neg and HIV+ ART+ sub-divided groups. (Column d ): Correlations found among HIV+ ART+ patients: (row I) log CXCL5 versus (vs) log IL-17A, (row II) log CXCL1 vs log IL-17A, (row III) log CCL17/CCL20 ratio vs log IL-17A and (row IV) log CCL17 vs log IL-10. Lines indicate median and IQR. Symbols represent individual patients: HIV-neg (○), HIV+ ART− (●), HIV+ ART+ (◉). The p values obtained are depicted as *p
    Figure Legend Snippet: Functionality of CMCs (related to Th17 and Treg-cks) was associated with local mucosal genital chemokine levels. Chemokine levels were quantified by CBA (pg/ml) in ectocervix samples from the different groups as described in methodology (Column a ): Row I: CXCL5 (ENA-78), (II) CXCL1 (GRO-α), (III) CCL17/CCL20 ratio (TARC/MIP-3α ratio) and (IV) CCL17 (TARC) quantities are shown. (Column b rows I to III): HIV+ ART+ group was subdivided in two sub-groups according to have “at least three” or “one or less” Th17-cks (≥3Th17-cks vs ≤1 Th17-ck) in the CMCs samples. (Column b row IV): HIV+ ART+ group was subdivided in two subgroups according to have two Treg- cks vs one or less Treg-ck (2 Treg-cks vs ≤1 Treg-ck). CXCL5, CXCL1, CCL17/CCL20 ratio and CCL17 levels are shown for HIV-neg and HIV+ ART+ sub-divided groups. (Column c rows I to IV): HIV+ ART+ group was subdivided in two subgroups according to have at least three Th17-cks plus two Treg-cks vs one or less Th17-cks plus one or less Treg-cks (≥3Th17-cks + 2Treg-cks vs ≤1 Th17-ck + ≤1 Treg-ck). CXCL5, CXCL1, CCL17/CCL20 ratio and CCL17 levels are shown for HIV-neg and HIV+ ART+ sub-divided groups. (Column d ): Correlations found among HIV+ ART+ patients: (row I) log CXCL5 versus (vs) log IL-17A, (row II) log CXCL1 vs log IL-17A, (row III) log CCL17/CCL20 ratio vs log IL-17A and (row IV) log CCL17 vs log IL-10. Lines indicate median and IQR. Symbols represent individual patients: HIV-neg (○), HIV+ ART− (●), HIV+ ART+ (◉). The p values obtained are depicted as *p

    Techniques Used: Crocin Bleaching Assay

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    Cell Culture:

    Article Title: Macrophage β2-Integrins Regulate IL-22 by ILC3s and Protect from Lethal Citrobacter rodentium-Induced Colitis
    Article Snippet: .. For intracellular detection of cytokines in ILC3s, cells were first cultured in the presence of mouse recombinant IL-1β and IL-23 each at a concentration of 20 ng/ml for 2h (Biolegend) and then stimulated with phorbol myristate acetate, ionomycin and brefeldin A for 4h, as previously described ( ). .. The cells were then fixed and permeabilized using an intracellular fixation and permeabilization kit (ThermoFisher) and stained using antibodies against IL-17 (TC11–18H10.1), IFN-γ (XMG1.2) or IL-22 (1H8PWSR).

    Cytometry:

    Article Title: Nrf2 signaling and inflammation are key events in physical plasma-spurred wound healing
    Article Snippet: .. Cytokine levels in serum were measured using bead-based multiplex cytokine analysis (BioLegend, San Diego, USA) according to the vendor's protocol, acquired on a CytoFlex S flow cytometer (Beckman-Coulter, Indianapolis, IN, USA) and analyzed using LegendPlex software 8.0 (VigineTech, San Diego, CA, USA). .. Cell culture and knockdown of NRF2 and KEAP1 by short interfering RNA (siRNA) To evaluate the effect of cold plasma on cellular translocation of Nrf2, dermal fibroblasts and epidermal keratinocytes ( Figure A ) were isolated from SKH1 skin (n = 6) and cultivated over 14 days in a keratinocytes or fibroblasts EMEM medium (PromoCell, Heidelberg, Germany) at 37°C with 5% CO2 in a humidified incubator.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Farrerol Relieve Lipopolysaccharide (LPS)-Induced Mastitis by Inhibiting AKT/NF-κB p65, ERK1/2 and P38 Signaling Pathway
    Article Snippet: .. The levels of TNF-α, IL-6 and IL-1β in mammary glands were determined using the mouse ELISA kits (Biolegend, San Diego, CA, USA) according to the manufacturer’s instructions. .. Cell Culture The mMECs were purchased from the American Type Culture Collection (ATCC, ATCC® CRL-3063™, Rockville, MD, USA), and these cells were cultured in DMEM medium (Gibco, Grand Island, NY 14072, USA) containing 10% FBS (Clark Bioscience, Richmond, VA, USA) at 37 °C in a humidified incubator with 5% CO2 .

    Article Title: Interaction Between Macrophage Migration Inhibitory Factor and CD74 in Human Immunodeficiency Virus Type I Infected Primary Monocyte-Derived Macrophages Triggers the Production of Proinflammatory Mediators and Enhances Infection of Unactivated CD4+ T Cells
    Article Snippet: .. Cytokine QuantitationThe levels of the following cytokines were evaluated in MDM supernatants using commercially available ELISA sets: IL-8, IL-6, IL-1β, TNFα, IL-10 (ELISA MAX Deluxe kits, BioLegend) and sICAM (DouSet ELISA, R & D Systems). .. MIF plasma levels were evaluated using an in-house ELISA constructed with an anti-human MIF antibody pair and an MIF standard obtained from BioLegend.

    Concentration Assay:

    Article Title: Macrophage β2-Integrins Regulate IL-22 by ILC3s and Protect from Lethal Citrobacter rodentium-Induced Colitis
    Article Snippet: .. For intracellular detection of cytokines in ILC3s, cells were first cultured in the presence of mouse recombinant IL-1β and IL-23 each at a concentration of 20 ng/ml for 2h (Biolegend) and then stimulated with phorbol myristate acetate, ionomycin and brefeldin A for 4h, as previously described ( ). .. The cells were then fixed and permeabilized using an intracellular fixation and permeabilization kit (ThermoFisher) and stained using antibodies against IL-17 (TC11–18H10.1), IFN-γ (XMG1.2) or IL-22 (1H8PWSR).

    Recombinant:

    Article Title: Macrophage β2-Integrins Regulate IL-22 by ILC3s and Protect from Lethal Citrobacter rodentium-Induced Colitis
    Article Snippet: .. For intracellular detection of cytokines in ILC3s, cells were first cultured in the presence of mouse recombinant IL-1β and IL-23 each at a concentration of 20 ng/ml for 2h (Biolegend) and then stimulated with phorbol myristate acetate, ionomycin and brefeldin A for 4h, as previously described ( ). .. The cells were then fixed and permeabilized using an intracellular fixation and permeabilization kit (ThermoFisher) and stained using antibodies against IL-17 (TC11–18H10.1), IFN-γ (XMG1.2) or IL-22 (1H8PWSR).

    Software:

    Article Title: Nrf2 signaling and inflammation are key events in physical plasma-spurred wound healing
    Article Snippet: .. Cytokine levels in serum were measured using bead-based multiplex cytokine analysis (BioLegend, San Diego, USA) according to the vendor's protocol, acquired on a CytoFlex S flow cytometer (Beckman-Coulter, Indianapolis, IN, USA) and analyzed using LegendPlex software 8.0 (VigineTech, San Diego, CA, USA). .. Cell culture and knockdown of NRF2 and KEAP1 by short interfering RNA (siRNA) To evaluate the effect of cold plasma on cellular translocation of Nrf2, dermal fibroblasts and epidermal keratinocytes ( Figure A ) were isolated from SKH1 skin (n = 6) and cultivated over 14 days in a keratinocytes or fibroblasts EMEM medium (PromoCell, Heidelberg, Germany) at 37°C with 5% CO2 in a humidified incubator.

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    BioLegend interleukin 6
    Levels of proinflammatory cytokines released from S . Typhi- or S . Typhimurium-infected MDMs. Shown are levels of TNF-α (A) and <t>IL-6</t> (B) released from MDMs after 2.5 and 24 h of S . Typhi infection and TNF-α (C) and IL-6 (D) released from MDMs after 2.5 and 24 h of S . Typhimurium infection. Statistical significance was determined by Student's t test comparing cytokine levels between LPS modification mutant-infected samples and the WT-infected sample collected at that time point. *, P
    Interleukin 6, supplied by BioLegend, used in various techniques. Bioz Stars score: 92/100, based on 149 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/interleukin 6/product/BioLegend
    Average 92 stars, based on 149 article reviews
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    interleukin 6 - by Bioz Stars, 2021-01
    92/100 stars
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    92
    BioLegend mouse il 6
    Proinflammatory cytokine and chemokine expression is decreased by hMSC administration . Twenty-four hours after LPS exposure, IL-1β, <t>IL-6,</t> IL-17, IP-10, MCP-1, MIP-1α and RANTES levels were measured by multiplex immunoassay in BAL fluid. Data are expressed as mean ± SEM (n = 6 per group). * P
    Mouse Il 6, supplied by BioLegend, used in various techniques. Bioz Stars score: 92/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse il 6/product/BioLegend
    Average 92 stars, based on 47 article reviews
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    mouse il 6 - by Bioz Stars, 2021-01
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    BioLegend anti il 6
    Pullulan induces pro-inflammatory cytokine production on DCs BMDCs were incubated with 5 μg/mL pullulan or LPS for 2 or 24 hours. A. mRNA levels of <t>IL-6,</t> IL-12p40 and TNF-α in BMDCs were measured 2 hours after treatment B. IL-6, IL-12p70 and TNF-α levels in culture supernatant 24 hours after treatment. C. Cytokine mRNA levels in splenocytes were measured 2 hours after 25 mg/kg pullulan and LPS injection. D. Cytokine concentrations in sera from pullulan- or LPS-treated mice are shown. E. Intracellular cytokine production levels were measured in spleen DCs. All data are representative of or the average of analyses of 6 individual mice each group (2 mice per experiment, 3 independent experiments); * p
    Anti Il 6, supplied by BioLegend, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti il 6/product/BioLegend
    Average 92 stars, based on 10 article reviews
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    anti il 6 - by Bioz Stars, 2021-01
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    LEGENDplex NHP IL 6 Capture Bead A6 13X Apps Multiplex Size 100 tests 270 µl
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    Image Search Results


    Levels of proinflammatory cytokines released from S . Typhi- or S . Typhimurium-infected MDMs. Shown are levels of TNF-α (A) and IL-6 (B) released from MDMs after 2.5 and 24 h of S . Typhi infection and TNF-α (C) and IL-6 (D) released from MDMs after 2.5 and 24 h of S . Typhimurium infection. Statistical significance was determined by Student's t test comparing cytokine levels between LPS modification mutant-infected samples and the WT-infected sample collected at that time point. *, P

    Journal: Infection and Immunity

    Article Title: Cathelicidin Antimicrobial Peptide Expression Is Not Induced or Required for Bacterial Clearance during Salmonella enterica Infection of Human Monocyte-Derived Macrophages

    doi: 10.1128/IAI.00672-12

    Figure Lengend Snippet: Levels of proinflammatory cytokines released from S . Typhi- or S . Typhimurium-infected MDMs. Shown are levels of TNF-α (A) and IL-6 (B) released from MDMs after 2.5 and 24 h of S . Typhi infection and TNF-α (C) and IL-6 (D) released from MDMs after 2.5 and 24 h of S . Typhimurium infection. Statistical significance was determined by Student's t test comparing cytokine levels between LPS modification mutant-infected samples and the WT-infected sample collected at that time point. *, P

    Article Snippet: The proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) were detected in cell culture supernatant by enzyme-linked immunosorbent assay (ELISA) (Biolegend, San Diego, CA, and R & D Systems, Minneapolis, MN, respectively).

    Techniques: Infection, Modification, Mutagenesis

    Production of inflammatory cytokines by the LNP and alum in vivo. Mice were treated intraperitoneally with the SV alone, SV plus LNP, or SV plus alum. After 4 h, the concentrations of IL-6 and granulocyte-colony stimulating factor (G-CSF) in peritoneal lavage fluid were measured by using ELISA. n = 5 per group. Data are means ± SD. ††† p

    Journal: Vaccines

    Article Title: Lipid Nanoparticle Acts as a Potential Adjuvant for Influenza Split Vaccine without Inducing Inflammatory Responses

    doi: 10.3390/vaccines8030433

    Figure Lengend Snippet: Production of inflammatory cytokines by the LNP and alum in vivo. Mice were treated intraperitoneally with the SV alone, SV plus LNP, or SV plus alum. After 4 h, the concentrations of IL-6 and granulocyte-colony stimulating factor (G-CSF) in peritoneal lavage fluid were measured by using ELISA. n = 5 per group. Data are means ± SD. ††† p

    Article Snippet: The concentrations of IL-6 and granulocyte-colony stimulating factor (G-CSF) in the supernatants of lavage fluid after centrifugation were analyzed by ELISA in accordance with the manufacturers’ instructions (IL-6: BioLegend; G-CSF: R & D Systems).

    Techniques: In Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Cytokine production by mouse-derived dendritic cells (DCs) in response to the lipid nanoparticle (LNP) and LNP-cytosine–phosphate–guanine (LNP-CpG) in vitro. Mouse-derived DCs were treated with the LNP (13.3 or 4.4 μg lipid/mL) or LNP-CpG (13.3 μg lipid with 1.0 μg CpG oligodeoxynucleotide (ODN)/mL or 4.4 μg lipid with 0.33 μg CpG ODN/mL) for 24 h in vitro. ( a ) Levels of interleukin (IL)-6, IL-12 p40, tumor necrosis factor (TNF)-α and interferon (IFN)-α in the supernatants were measured by using ELISA. ( b ) Expression levels of CD80 and CD86 on DCs were measured by flow cytometry; percentages of positive DCs are shown. ( a , b ) n = 5 per group. Data are means ± SD. **** p

    Journal: Vaccines

    Article Title: Lipid Nanoparticle Acts as a Potential Adjuvant for Influenza Split Vaccine without Inducing Inflammatory Responses

    doi: 10.3390/vaccines8030433

    Figure Lengend Snippet: Cytokine production by mouse-derived dendritic cells (DCs) in response to the lipid nanoparticle (LNP) and LNP-cytosine–phosphate–guanine (LNP-CpG) in vitro. Mouse-derived DCs were treated with the LNP (13.3 or 4.4 μg lipid/mL) or LNP-CpG (13.3 μg lipid with 1.0 μg CpG oligodeoxynucleotide (ODN)/mL or 4.4 μg lipid with 0.33 μg CpG ODN/mL) for 24 h in vitro. ( a ) Levels of interleukin (IL)-6, IL-12 p40, tumor necrosis factor (TNF)-α and interferon (IFN)-α in the supernatants were measured by using ELISA. ( b ) Expression levels of CD80 and CD86 on DCs were measured by flow cytometry; percentages of positive DCs are shown. ( a , b ) n = 5 per group. Data are means ± SD. **** p

    Article Snippet: The concentrations of IL-6 and granulocyte-colony stimulating factor (G-CSF) in the supernatants of lavage fluid after centrifugation were analyzed by ELISA in accordance with the manufacturers’ instructions (IL-6: BioLegend; G-CSF: R & D Systems).

    Techniques: Derivative Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry

    Proinflammatory cytokine and chemokine expression is decreased by hMSC administration . Twenty-four hours after LPS exposure, IL-1β, IL-6, IL-17, IP-10, MCP-1, MIP-1α and RANTES levels were measured by multiplex immunoassay in BAL fluid. Data are expressed as mean ± SEM (n = 6 per group). * P

    Journal: Stem Cell Research & Therapy

    Article Title: Human multipotent stromal cells attenuate lipopolysaccharide-induced acute lung injury in mice via secretion of tumor necrosis factor-?-induced protein 6

    doi: 10.1186/scrt68

    Figure Lengend Snippet: Proinflammatory cytokine and chemokine expression is decreased by hMSC administration . Twenty-four hours after LPS exposure, IL-1β, IL-6, IL-17, IP-10, MCP-1, MIP-1α and RANTES levels were measured by multiplex immunoassay in BAL fluid. Data are expressed as mean ± SEM (n = 6 per group). * P

    Article Snippet: The quantification of mouse IL-6 was performed with mouse IL-6 ELISA MAX™ Deluxe Set from BioLegend (San Diego, CA, USA) according to the manufacturer's instruction.

    Techniques: Expressing, Multiplex Assay

    Pullulan induces pro-inflammatory cytokine production on DCs BMDCs were incubated with 5 μg/mL pullulan or LPS for 2 or 24 hours. A. mRNA levels of IL-6, IL-12p40 and TNF-α in BMDCs were measured 2 hours after treatment B. IL-6, IL-12p70 and TNF-α levels in culture supernatant 24 hours after treatment. C. Cytokine mRNA levels in splenocytes were measured 2 hours after 25 mg/kg pullulan and LPS injection. D. Cytokine concentrations in sera from pullulan- or LPS-treated mice are shown. E. Intracellular cytokine production levels were measured in spleen DCs. All data are representative of or the average of analyses of 6 individual mice each group (2 mice per experiment, 3 independent experiments); * p

    Journal: Oncotarget

    Article Title: Maturation of dendritic cells by pullulan promotes anti-cancer effect

    doi: 10.18632/oncotarget.10183

    Figure Lengend Snippet: Pullulan induces pro-inflammatory cytokine production on DCs BMDCs were incubated with 5 μg/mL pullulan or LPS for 2 or 24 hours. A. mRNA levels of IL-6, IL-12p40 and TNF-α in BMDCs were measured 2 hours after treatment B. IL-6, IL-12p70 and TNF-α levels in culture supernatant 24 hours after treatment. C. Cytokine mRNA levels in splenocytes were measured 2 hours after 25 mg/kg pullulan and LPS injection. D. Cytokine concentrations in sera from pullulan- or LPS-treated mice are shown. E. Intracellular cytokine production levels were measured in spleen DCs. All data are representative of or the average of analyses of 6 individual mice each group (2 mice per experiment, 3 independent experiments); * p

    Article Snippet: Antibodies Isotype control antibodies (Abs) (IgG1, IgG2a or IgG2b), CD11c (HL3), CD4 (GK1.5), CD8α (YTS169.4), CD40 (3/23), CD80 (16-10A1), CD86 (GL-1), anti-IL-4 (11B11), anti-IL-6 (MP5-20F3) and anti-IL-12/23p40 (C17.8) were obtained from BioLegend; anti-MHC class I (AF6-88.5.3), anti-MHC class II (M5/114.15.2), anti-IFN-γ (XMG1.2), anti-IL-17 (TCC11-18H10.1) and anti-TNF-α (MP6-XT22) were obtained from eBioscience.

    Techniques: Incubation, Injection, Mouse Assay