tnfα Biolegend Search Results


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  • 94
    BioLegend tnfα
    Reduction of <t>TNFα</t> expression in CD4+ or CD8+ T cells after co-culture with HIV-1-exposed B cells. Intracellular expression of TNFα by CD4+ and CD8+ T cells was analyzed after co-culture of effector cells (EC) and non-treated (NT) or treated B cells (ratio Breg:EC=2:1). Correlation between the ratio of CD4/CD8 and the percentage of reduction in TNFα expression in co-culture experiments when ( a ) HIV-1 NL4-3 -exposed, ( b ) CD40L/IL-4-exposed, ( c ) CpG/CD40L/LPS-exposed and ( d ) CpG/CD40L/LPS/HIV-1 NL4-3 -exposed B cells were calculated considering the mock-treated B-cell co-culture as the baseline reference level. Black squares and solid lines represent the results from CD4+ T cells, and open squares and dotted black lines represent the results from CD8+ T cells. Each square represents one experiment.
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    BioLegend tnfα clone mp6 xt22 biolegend
    Reduction of <t>TNFα</t> expression in CD4+ or CD8+ T cells after co-culture with HIV-1-exposed B cells. Intracellular expression of TNFα by CD4+ and CD8+ T cells was analyzed after co-culture of effector cells (EC) and non-treated (NT) or treated B cells (ratio Breg:EC=2:1). Correlation between the ratio of CD4/CD8 and the percentage of reduction in TNFα expression in co-culture experiments when ( a ) HIV-1 NL4-3 -exposed, ( b ) CD40L/IL-4-exposed, ( c ) CpG/CD40L/LPS-exposed and ( d ) CpG/CD40L/LPS/HIV-1 NL4-3 -exposed B cells were calculated considering the mock-treated B-cell co-culture as the baseline reference level. Black squares and solid lines represent the results from CD4+ T cells, and open squares and dotted black lines represent the results from CD8+ T cells. Each square represents one experiment.
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    BioLegend tnfα pecy7
    Reduction of <t>TNFα</t> expression in CD4+ or CD8+ T cells after co-culture with HIV-1-exposed B cells. Intracellular expression of TNFα by CD4+ and CD8+ T cells was analyzed after co-culture of effector cells (EC) and non-treated (NT) or treated B cells (ratio Breg:EC=2:1). Correlation between the ratio of CD4/CD8 and the percentage of reduction in TNFα expression in co-culture experiments when ( a ) HIV-1 NL4-3 -exposed, ( b ) CD40L/IL-4-exposed, ( c ) CpG/CD40L/LPS-exposed and ( d ) CpG/CD40L/LPS/HIV-1 NL4-3 -exposed B cells were calculated considering the mock-treated B-cell co-culture as the baseline reference level. Black squares and solid lines represent the results from CD4+ T cells, and open squares and dotted black lines represent the results from CD8+ T cells. Each square represents one experiment.
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    BioLegend murine tnfα
    Sod3 protects Histoplasma yeasts from ROS produced by activated macrophages. ( A–B ) Survival of yeasts after infection of resting ( A ) or cytokine-activated ( B ) murine macrophages. SOD3(+) (OSU45), sod3Δ (OSU15) and Candida albicans yeasts were added to resident peritoneal macrophages at an MOI of 1∶50. Yeast survival was determined by enumeration of viable cfu after 2 and 4 hours of co-incubation of yeasts with macrophages at 37°C. In ( B ), 10 U <t>TNFα</t> and 100 U IFNγ were added to macrophages 24 hours prior to infection to enhance ROS production. Results are plotted as relative yeast survival (mean ± standard deviation of 3 replicates) compared to viable cfu of yeasts incubated in the absence of macrophages. Significantly decreased survival compared to SOD3(+) yeasts is indicated by asterisks (* p
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    91
    BioLegend tnfα apc
    Sod3 protects Histoplasma yeasts from ROS produced by activated macrophages. ( A–B ) Survival of yeasts after infection of resting ( A ) or cytokine-activated ( B ) murine macrophages. SOD3(+) (OSU45), sod3Δ (OSU15) and Candida albicans yeasts were added to resident peritoneal macrophages at an MOI of 1∶50. Yeast survival was determined by enumeration of viable cfu after 2 and 4 hours of co-incubation of yeasts with macrophages at 37°C. In ( B ), 10 U <t>TNFα</t> and 100 U IFNγ were added to macrophages 24 hours prior to infection to enhance ROS production. Results are plotted as relative yeast survival (mean ± standard deviation of 3 replicates) compared to viable cfu of yeasts incubated in the absence of macrophages. Significantly decreased survival compared to SOD3(+) yeasts is indicated by asterisks (* p
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    93
    BioLegend moab tnfα pe
    Sod3 protects Histoplasma yeasts from ROS produced by activated macrophages. ( A–B ) Survival of yeasts after infection of resting ( A ) or cytokine-activated ( B ) murine macrophages. SOD3(+) (OSU45), sod3Δ (OSU15) and Candida albicans yeasts were added to resident peritoneal macrophages at an MOI of 1∶50. Yeast survival was determined by enumeration of viable cfu after 2 and 4 hours of co-incubation of yeasts with macrophages at 37°C. In ( B ), 10 U <t>TNFα</t> and 100 U IFNγ were added to macrophages 24 hours prior to infection to enhance ROS production. Results are plotted as relative yeast survival (mean ± standard deviation of 3 replicates) compared to viable cfu of yeasts incubated in the absence of macrophages. Significantly decreased survival compared to SOD3(+) yeasts is indicated by asterisks (* p
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    BioLegend tnfα pe
    Host pre-conditioning with CTX allows adoptively transferred tumor-specific CD4+ T cells to differentiate into polyfunctional effector cells and regain IL-7Rα expression. ( A ) Kinetics of IL-7Rα expression on donor CD4+ T cells following adoptive transfer. Following the timeline depicted in the schema, mice with established systemic A20HA tumors were divided into two groups. One group of mice were pre-conditioned with CTX while the other group received PBS. All mice received adoptive transfer of HA-specific CD4+ T cells the next day. At the indicated time points, tail blood samples were collected and analyzed for IL-7Rα expression on donor CD4+ T cells by FACS. IL-7Rα expression profiles relative to cell division in transferred CD4+ T cells are shown in representative dot plots. The numbers indicate the percentage of cells in the corresponding quadrant. Results are summarized in graph at right. Data for day 0 and day3 are gated on total donor T cells because cells barely divided at these two time points. ( B ) Differential expression of IL-7Rα in donor CD4+ T cells in mice with or without CTX pre-conditioning. 7 days after T-cell transfer, spleen cells were isolated to examine IL-7Rα expression on donor CD4+ T cells. Donor T cells were also evaluated for expression levels of PD1, Foxp3, CD40L, IFNγ and <t>TNFα.</t> The phenotypes of the divided donor T cells in PBS or CTX-conditioned mice are summarized in ( C ). ( D ) Donor CD4+ T cells transferred into CTX-conditioned mice but not unconditioned mice are capable of producing IL-2. IL-2 expression profiles relative to cell division in transferred CD4+ T cells are revealed by intracellular staining (ICS). ( E ) IL-2 neutralization inhibits IL-7Rα re-expression in donor CD4+ T cells in CTX-conditioned mice. Tumor-bearing mice were treated with CTX followed by CD4+ T cell transfer the next day. Some mice were injected with IL-2 neutralizing mAbs every other day. 7 days after T cell transfer, IL-7Rα expression on donor CD4+ T cells were analyzed by FACS. Results summarized in bar graph are shown as mean ± SD of 3 mice each group. ( F ) Exogenous IL-2 partially restores IL-7Rα expression in donor CD4+ T cells transferred into unconditioned mice. Following the timeline depicted in the schema, A20HA tumor-bearing mice received adoptive transfer of HA-specific CD4+ T cells. A cohort of mice further received IL-2/αIL-2 complex (IL-2C) every other day. IL-7Rα expressions on donor CD4+ T cells were analyzed 7 days after T cell transfer.
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    BioLegend tnfα bv605
    Host pre-conditioning with CTX allows adoptively transferred tumor-specific CD4+ T cells to differentiate into polyfunctional effector cells and regain IL-7Rα expression. ( A ) Kinetics of IL-7Rα expression on donor CD4+ T cells following adoptive transfer. Following the timeline depicted in the schema, mice with established systemic A20HA tumors were divided into two groups. One group of mice were pre-conditioned with CTX while the other group received PBS. All mice received adoptive transfer of HA-specific CD4+ T cells the next day. At the indicated time points, tail blood samples were collected and analyzed for IL-7Rα expression on donor CD4+ T cells by FACS. IL-7Rα expression profiles relative to cell division in transferred CD4+ T cells are shown in representative dot plots. The numbers indicate the percentage of cells in the corresponding quadrant. Results are summarized in graph at right. Data for day 0 and day3 are gated on total donor T cells because cells barely divided at these two time points. ( B ) Differential expression of IL-7Rα in donor CD4+ T cells in mice with or without CTX pre-conditioning. 7 days after T-cell transfer, spleen cells were isolated to examine IL-7Rα expression on donor CD4+ T cells. Donor T cells were also evaluated for expression levels of PD1, Foxp3, CD40L, IFNγ and <t>TNFα.</t> The phenotypes of the divided donor T cells in PBS or CTX-conditioned mice are summarized in ( C ). ( D ) Donor CD4+ T cells transferred into CTX-conditioned mice but not unconditioned mice are capable of producing IL-2. IL-2 expression profiles relative to cell division in transferred CD4+ T cells are revealed by intracellular staining (ICS). ( E ) IL-2 neutralization inhibits IL-7Rα re-expression in donor CD4+ T cells in CTX-conditioned mice. Tumor-bearing mice were treated with CTX followed by CD4+ T cell transfer the next day. Some mice were injected with IL-2 neutralizing mAbs every other day. 7 days after T cell transfer, IL-7Rα expression on donor CD4+ T cells were analyzed by FACS. Results summarized in bar graph are shown as mean ± SD of 3 mice each group. ( F ) Exogenous IL-2 partially restores IL-7Rα expression in donor CD4+ T cells transferred into unconditioned mice. Following the timeline depicted in the schema, A20HA tumor-bearing mice received adoptive transfer of HA-specific CD4+ T cells. A cohort of mice further received IL-2/αIL-2 complex (IL-2C) every other day. IL-7Rα expressions on donor CD4+ T cells were analyzed 7 days after T cell transfer.
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    Becton Dickinson tnfα
    CD8 T-cell memory alters the immune profile in enhanced HLH without altering viral load Prf1 −/− mice were immunized against GP33 or control, rested for 30 days and infected with LCMV. Mice were sacrificed on day 7 post-infection, serum samples were obtained and splenocytes were stimulated in vitro and analyzed. A. Serum cytokine levels in immunized and control mice (n=8-12). B. Total GP33-specific CD8 T cells (CD90+, CD8+, CD44+, IFNγ+) in immunized and control mice determined by IFNγ production following in vitro stimulation (n=6-8). Total number of GP33-specific CD8 T cells determined by GP33 tetramer staining (CD90+, CD8+, CD44+, GP33 tetramer+) (n=6-7). C. Percent of total CD8 T cells specific for GP33 or NP396 determined by IFNγ production following in vitro peptide stimulation with the respective peptide in GP33 immunized and control mice on day 7 post-infection (CD90+, CD8+, CD44+, IFNγ+) (n=4). D. Seven days post LCMV infection, total number of GP33 specific <t>TNFα</t> producing splenic CD8 T cells (CD90+, CD8+, CD44+, TNFα+) following in vitro peptide stimulation (n=6-8). E. LCMV splenic titers (n=6-8). Log scale is shown. *P
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    BioLegend tnfα bv421
    CD8 T-cell memory alters the immune profile in enhanced HLH without altering viral load Prf1 −/− mice were immunized against GP33 or control, rested for 30 days and infected with LCMV. Mice were sacrificed on day 7 post-infection, serum samples were obtained and splenocytes were stimulated in vitro and analyzed. A. Serum cytokine levels in immunized and control mice (n=8-12). B. Total GP33-specific CD8 T cells (CD90+, CD8+, CD44+, IFNγ+) in immunized and control mice determined by IFNγ production following in vitro stimulation (n=6-8). Total number of GP33-specific CD8 T cells determined by GP33 tetramer staining (CD90+, CD8+, CD44+, GP33 tetramer+) (n=6-7). C. Percent of total CD8 T cells specific for GP33 or NP396 determined by IFNγ production following in vitro peptide stimulation with the respective peptide in GP33 immunized and control mice on day 7 post-infection (CD90+, CD8+, CD44+, IFNγ+) (n=4). D. Seven days post LCMV infection, total number of GP33 specific <t>TNFα</t> producing splenic CD8 T cells (CD90+, CD8+, CD44+, TNFα+) following in vitro peptide stimulation (n=6-8). E. LCMV splenic titers (n=6-8). Log scale is shown. *P
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    BioLegend liver tnfα
    LPS induces miR-155 and <t>TNFα</t> expression in hepatic immune cells. Isolated LMNCs and Kupffer cells from C57Bl/6 WT mice were stimulated or not with 100ng/ml LPS for 6 hours in vitro. miR-155 expression (A: LMNCs, C: KCs) and TNF-α protein secretion (B: LMNCs, D: KCs) were measured in the cells and in the supernatant, respectively (n = 8-10/group). RNA was isolated from C57Bl/6 WT mice fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 3, 6 and 8 weeks. miR155 and TNFα mRNA expression was determined (n = 5-6/group, total 32) and correlation was plotted (E); correlation coefficient is shown. (*) indicates p
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    BioLegend tnf α biolegend levels
    LPS induces miR-155 and <t>TNFα</t> expression in hepatic immune cells. Isolated LMNCs and Kupffer cells from C57Bl/6 WT mice were stimulated or not with 100ng/ml LPS for 6 hours in vitro. miR-155 expression (A: LMNCs, C: KCs) and TNF-α protein secretion (B: LMNCs, D: KCs) were measured in the cells and in the supernatant, respectively (n = 8-10/group). RNA was isolated from C57Bl/6 WT mice fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 3, 6 and 8 weeks. miR155 and TNFα mRNA expression was determined (n = 5-6/group, total 32) and correlation was plotted (E); correlation coefficient is shown. (*) indicates p
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    BioLegend tnfα elisa kits
    LPS plus Smac mimetics induces RIPK1-dependent apoptosis that switches to necroptosis upon caspase inhibition. ( a ) WT and Xiap − / − neutrophils were pre-treated with Nec.1 (20 μ M) for 30 min, AT-406 (1 μ M) or Cp.A (500 nM) for 30 min and subsequently incubated with LPS (100 ng/ml) for 3 and 6 h. Lysates were assayed for caspase-3/-7 activity; n ≥4, mean±S.E.M. ( b ) WT and Xiap − / − neutrophils were pre-treated with Nec.1 (20 μ M, 30 min) or Q-VD-OPh (20 μ M, 30 min), then with either AT-406 (1 μ M) or Cp.A (500 nM), and incubated with LPS (100 ng/ml) for 6 h. Lysates were assayed by immunoblot. Presented immunoblots are representative of at least two independent experiments. ( c ) qPCR analysis of <t>Tnfα</t> in WT and Xiap − / − neutrophils pre-treated with Nec.1 (20 μ M, 30 min) followed by either AT-406 (1 μ M) or Cp.A (500 nM), for another 30 min and stimulated with LPS (100 ng/ml) for 4 h. Hprt was used as reference gene; n =3, mean±S.E.M. ( d ) WT and Xiap − / − neutrophils were preincubated with Nec.1 (20 μ M, 30 min), treated with either AT-406 (1 μ M) or Cp.A (500 nM) and stimulated with LPS (100 ng/ml) for 6 h. Supernatants were analyzed for TNF α by <t>ELISA;</t> n ≥4, mean±S.E.M. ( e ) WT and Xiap − / − neutrophils were pre-treated with Q-VD-OPh (20 μ M) or Nec.1 (20 μ M) for 30 min, treated with AT-406 (1 μ M) or Cp.A (500 nM) for 30 min and subsequently stimulated with LPS (100 ng/ml) for different time points. Viability was determined by flow cytometry; n ≥3, mean±S.E.M. Data sets of untreated control from Figure 1a and SM+LPS from Figure 2b are included. ( f ) Ripk3 − / − and ( g ) Ripk3 − / − Xiap − / − neutrophils were pre-treated with Q-VD-OPh (20 μ M) for 30 min, incubated with Cp.A (500 nM) or AT-406 (1 μ M), respectively, and stimulated with LPS (100 ng/ml) for indicated time points. Viability was assessed by flow cytometry; n ≥3, mean±S.E.M. ( h ) WT and Xiap − / − neutrophils were pre-treated with Q-VD (20 μ M) for 30 min followed by administration of Cp.A (500 nM) or AT-406 (1 μ M), respectively, for 30 min and stimulated with LPS for 6 h. Fractionation by phase separation was performed and lysates were assayed by quantitative immunoblot. Presented immunoblots are representative of at least two independent experiments. All experiments were performed with in vitro differentiated neutrophils. * P
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    BioLegend tnfα pacific blue
    LPS plus Smac mimetics induces RIPK1-dependent apoptosis that switches to necroptosis upon caspase inhibition. ( a ) WT and Xiap − / − neutrophils were pre-treated with Nec.1 (20 μ M) for 30 min, AT-406 (1 μ M) or Cp.A (500 nM) for 30 min and subsequently incubated with LPS (100 ng/ml) for 3 and 6 h. Lysates were assayed for caspase-3/-7 activity; n ≥4, mean±S.E.M. ( b ) WT and Xiap − / − neutrophils were pre-treated with Nec.1 (20 μ M, 30 min) or Q-VD-OPh (20 μ M, 30 min), then with either AT-406 (1 μ M) or Cp.A (500 nM), and incubated with LPS (100 ng/ml) for 6 h. Lysates were assayed by immunoblot. Presented immunoblots are representative of at least two independent experiments. ( c ) qPCR analysis of <t>Tnfα</t> in WT and Xiap − / − neutrophils pre-treated with Nec.1 (20 μ M, 30 min) followed by either AT-406 (1 μ M) or Cp.A (500 nM), for another 30 min and stimulated with LPS (100 ng/ml) for 4 h. Hprt was used as reference gene; n =3, mean±S.E.M. ( d ) WT and Xiap − / − neutrophils were preincubated with Nec.1 (20 μ M, 30 min), treated with either AT-406 (1 μ M) or Cp.A (500 nM) and stimulated with LPS (100 ng/ml) for 6 h. Supernatants were analyzed for TNF α by <t>ELISA;</t> n ≥4, mean±S.E.M. ( e ) WT and Xiap − / − neutrophils were pre-treated with Q-VD-OPh (20 μ M) or Nec.1 (20 μ M) for 30 min, treated with AT-406 (1 μ M) or Cp.A (500 nM) for 30 min and subsequently stimulated with LPS (100 ng/ml) for different time points. Viability was determined by flow cytometry; n ≥3, mean±S.E.M. Data sets of untreated control from Figure 1a and SM+LPS from Figure 2b are included. ( f ) Ripk3 − / − and ( g ) Ripk3 − / − Xiap − / − neutrophils were pre-treated with Q-VD-OPh (20 μ M) for 30 min, incubated with Cp.A (500 nM) or AT-406 (1 μ M), respectively, and stimulated with LPS (100 ng/ml) for indicated time points. Viability was assessed by flow cytometry; n ≥3, mean±S.E.M. ( h ) WT and Xiap − / − neutrophils were pre-treated with Q-VD (20 μ M) for 30 min followed by administration of Cp.A (500 nM) or AT-406 (1 μ M), respectively, for 30 min and stimulated with LPS for 6 h. Fractionation by phase separation was performed and lysates were assayed by quantitative immunoblot. Presented immunoblots are representative of at least two independent experiments. All experiments were performed with in vitro differentiated neutrophils. * P
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    BioLegend tnfα apc cy7
    LPS plus Smac mimetics induces RIPK1-dependent apoptosis that switches to necroptosis upon caspase inhibition. ( a ) WT and Xiap − / − neutrophils were pre-treated with Nec.1 (20 μ M) for 30 min, AT-406 (1 μ M) or Cp.A (500 nM) for 30 min and subsequently incubated with LPS (100 ng/ml) for 3 and 6 h. Lysates were assayed for caspase-3/-7 activity; n ≥4, mean±S.E.M. ( b ) WT and Xiap − / − neutrophils were pre-treated with Nec.1 (20 μ M, 30 min) or Q-VD-OPh (20 μ M, 30 min), then with either AT-406 (1 μ M) or Cp.A (500 nM), and incubated with LPS (100 ng/ml) for 6 h. Lysates were assayed by immunoblot. Presented immunoblots are representative of at least two independent experiments. ( c ) qPCR analysis of <t>Tnfα</t> in WT and Xiap − / − neutrophils pre-treated with Nec.1 (20 μ M, 30 min) followed by either AT-406 (1 μ M) or Cp.A (500 nM), for another 30 min and stimulated with LPS (100 ng/ml) for 4 h. Hprt was used as reference gene; n =3, mean±S.E.M. ( d ) WT and Xiap − / − neutrophils were preincubated with Nec.1 (20 μ M, 30 min), treated with either AT-406 (1 μ M) or Cp.A (500 nM) and stimulated with LPS (100 ng/ml) for 6 h. Supernatants were analyzed for TNF α by <t>ELISA;</t> n ≥4, mean±S.E.M. ( e ) WT and Xiap − / − neutrophils were pre-treated with Q-VD-OPh (20 μ M) or Nec.1 (20 μ M) for 30 min, treated with AT-406 (1 μ M) or Cp.A (500 nM) for 30 min and subsequently stimulated with LPS (100 ng/ml) for different time points. Viability was determined by flow cytometry; n ≥3, mean±S.E.M. Data sets of untreated control from Figure 1a and SM+LPS from Figure 2b are included. ( f ) Ripk3 − / − and ( g ) Ripk3 − / − Xiap − / − neutrophils were pre-treated with Q-VD-OPh (20 μ M) for 30 min, incubated with Cp.A (500 nM) or AT-406 (1 μ M), respectively, and stimulated with LPS (100 ng/ml) for indicated time points. Viability was assessed by flow cytometry; n ≥3, mean±S.E.M. ( h ) WT and Xiap − / − neutrophils were pre-treated with Q-VD (20 μ M) for 30 min followed by administration of Cp.A (500 nM) or AT-406 (1 μ M), respectively, for 30 min and stimulated with LPS for 6 h. Fractionation by phase separation was performed and lysates were assayed by quantitative immunoblot. Presented immunoblots are representative of at least two independent experiments. All experiments were performed with in vitro differentiated neutrophils. * P
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    94
    BioLegend human tnfα
    Comparison of BM-DC response to Man and Gal-LPS. A , two LPS used in this study are shown. Man-LPS from H. alvei PCM 1223 has a mannosylated repeating unit, whereas Gal-LPS from S. enterica O66 has a galactosylated repeat. B , HEK293 cells stably transfected with TLR4-MD2 were cultured in the presence of LPS. The TLR4 activation was monitored by measuring alkaline phosphatase activity using the substrate. C , mouse BM-DCs were stimulated with 1 μg/ml Man-LPS or 4 μg/ml Gal-LPS for 7 h. The amount of <t>TNFα</t> and IL-10 in the culture supernatant was analyzed by ELISA. Data are representative of three independent experiments with similar results. Error bars , S.D. Statistical analyses were performed by one-way ANOVA followed by Tukey's test. ***, p
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    Reduction of TNFα expression in CD4+ or CD8+ T cells after co-culture with HIV-1-exposed B cells. Intracellular expression of TNFα by CD4+ and CD8+ T cells was analyzed after co-culture of effector cells (EC) and non-treated (NT) or treated B cells (ratio Breg:EC=2:1). Correlation between the ratio of CD4/CD8 and the percentage of reduction in TNFα expression in co-culture experiments when ( a ) HIV-1 NL4-3 -exposed, ( b ) CD40L/IL-4-exposed, ( c ) CpG/CD40L/LPS-exposed and ( d ) CpG/CD40L/LPS/HIV-1 NL4-3 -exposed B cells were calculated considering the mock-treated B-cell co-culture as the baseline reference level. Black squares and solid lines represent the results from CD4+ T cells, and open squares and dotted black lines represent the results from CD8+ T cells. Each square represents one experiment.

    Journal: Cellular and Molecular Immunology

    Article Title: Human immunodeficiency virus type-1 induces a regulatory B cell-like phenotype in vitro

    doi: 10.1038/cmi.2017.48

    Figure Lengend Snippet: Reduction of TNFα expression in CD4+ or CD8+ T cells after co-culture with HIV-1-exposed B cells. Intracellular expression of TNFα by CD4+ and CD8+ T cells was analyzed after co-culture of effector cells (EC) and non-treated (NT) or treated B cells (ratio Breg:EC=2:1). Correlation between the ratio of CD4/CD8 and the percentage of reduction in TNFα expression in co-culture experiments when ( a ) HIV-1 NL4-3 -exposed, ( b ) CD40L/IL-4-exposed, ( c ) CpG/CD40L/LPS-exposed and ( d ) CpG/CD40L/LPS/HIV-1 NL4-3 -exposed B cells were calculated considering the mock-treated B-cell co-culture as the baseline reference level. Black squares and solid lines represent the results from CD4+ T cells, and open squares and dotted black lines represent the results from CD8+ T cells. Each square represents one experiment.

    Article Snippet: For intracellular labeling of cytokines IL-10 (Miltenyi Biotec) or TNFα (Biolegend), cell cultures were supplemented with PMA (10 ng/ml)+ionomycin (0.25 μg/ml, both from Sigma Aldrich), and GolgiStop (BD biosciences) for the last 5 h of incubation before staining and permeabilization.

    Techniques: Expressing, Co-Culture Assay

    Treatment of NP-specific CD8 + cytotoxic T lymphocyte (CTL) with EF3030-derived lysate inhibits the production of IFNγ and tumor necrosis factor α (TNFα) and the release of cytolytic granules. NP 147–155 -specific CTL was stimulated with peptide in the presence of increasing amounts of Spn lysate. Anti-CD107a antibody was included in the stimulation phase to identify cells releasing lytic granules. Following stimulation, cells were stained for CD8, IFNγ, and TNFα. Data shown are pregated on live CD8 + CTL. The percent inhibition refers to loss of the effector function noted in the legend. (A) Representative flow plots. (B) Averaged data ± SEM from eight independent experiments assessed using two independently generated NP-specific CTL lines.

    Journal: Frontiers in Immunology

    Article Title: A Novel Function for the Streptococcus pneumoniae Aminopeptidase N: Inhibition of T Cell Effector Function through Regulation of TCR Signaling

    doi: 10.3389/fimmu.2017.01610

    Figure Lengend Snippet: Treatment of NP-specific CD8 + cytotoxic T lymphocyte (CTL) with EF3030-derived lysate inhibits the production of IFNγ and tumor necrosis factor α (TNFα) and the release of cytolytic granules. NP 147–155 -specific CTL was stimulated with peptide in the presence of increasing amounts of Spn lysate. Anti-CD107a antibody was included in the stimulation phase to identify cells releasing lytic granules. Following stimulation, cells were stained for CD8, IFNγ, and TNFα. Data shown are pregated on live CD8 + CTL. The percent inhibition refers to loss of the effector function noted in the legend. (A) Representative flow plots. (B) Averaged data ± SEM from eight independent experiments assessed using two independently generated NP-specific CTL lines.

    Article Snippet: Cells were then fixed and permeabilized (Cytofix/Cytoperm kit, BD Biosciences) followed by incubation with antibodies specific for IFNγ (Biolegend) and in some cases TNFα (Biolegend).

    Techniques: CTL Assay, Derivative Assay, Staining, Inhibition, Flow Cytometry, Generated

    EC sensitization preferentially promotes an IL-22 response A-D. serum IL-22 levels (A), IL-22 secretion by OVA stimulated splenocytes (B), Il22 and Tnfa mRNA expression in the lungs (C) and IL-22 and TNFα secretion by OVA stimulated lung cells of mice EC or IP sensitized with OVA and i.n. challenged with OVA. Bars represent mean±SEM (n=3–5 per group). *p

    Journal: The Journal of allergy and clinical immunology

    Article Title: IL-22 promotes allergic airway inflammation in epicutaneously sensitized mice

    doi: 10.1016/j.jaci.2018.05.032

    Figure Lengend Snippet: EC sensitization preferentially promotes an IL-22 response A-D. serum IL-22 levels (A), IL-22 secretion by OVA stimulated splenocytes (B), Il22 and Tnfa mRNA expression in the lungs (C) and IL-22 and TNFα secretion by OVA stimulated lung cells of mice EC or IP sensitized with OVA and i.n. challenged with OVA. Bars represent mean±SEM (n=3–5 per group). *p

    Article Snippet: Cells were then fixed and permeabilized using BD Cytofix/Cytoperm Kit (BD Biosciences) and stained with anti-IL-22 (Biolegend), anti-TNFα (Biolegend), anti-IL-17A (eBioscience) or anti-IFNγ (eBioscience) antibodies..

    Techniques: Expressing, Mouse Assay

    IL-22 synergizes with TNFα to promote neutrophil airway inflammation A-D. Total and differential counts in BALF (A), frequency of neutrophils in lungs (B), H E stained lung sections (C), chemokine mRNA levels in the lungs (D) and lung resistance in response to increasing doses of methacholine (E) in WT mice treated intranasally with saline, rIL-22, rTNFα or rIL-22+rTNFα. Bars represent mean±SEM (n=4–6 per group). *p

    Journal: The Journal of allergy and clinical immunology

    Article Title: IL-22 promotes allergic airway inflammation in epicutaneously sensitized mice

    doi: 10.1016/j.jaci.2018.05.032

    Figure Lengend Snippet: IL-22 synergizes with TNFα to promote neutrophil airway inflammation A-D. Total and differential counts in BALF (A), frequency of neutrophils in lungs (B), H E stained lung sections (C), chemokine mRNA levels in the lungs (D) and lung resistance in response to increasing doses of methacholine (E) in WT mice treated intranasally with saline, rIL-22, rTNFα or rIL-22+rTNFα. Bars represent mean±SEM (n=4–6 per group). *p

    Article Snippet: Cells were then fixed and permeabilized using BD Cytofix/Cytoperm Kit (BD Biosciences) and stained with anti-IL-22 (Biolegend), anti-TNFα (Biolegend), anti-IL-17A (eBioscience) or anti-IFNγ (eBioscience) antibodies..

    Techniques: Staining, Mouse Assay

    EC sensitization elicits a systemic IL-22 response and an antigen-specific IL-22 response in the lungs A-B. IL-22 secretion by OVA stimulated splenocytes (A) and IL-22 serum levels (B). C,D . Il22 mRNA expression in the lungs (C), and IL-22 secretion by OVA stimulated lung cells (D). E. Representative FACS analysis and quantitation of intracelluar expression of IL-22 + cells among CD3 + CD4 + T cells and of IL-17A + and TNFα + cells among CD3 + CD4 + IL-22 + cells in the lung. Mice were EC sensitized with OVA or saline in A and B, followed by i.n. challenged with OVA in C-F. Bars represent mean±SEM (n=5–10 per group). *p

    Journal: The Journal of allergy and clinical immunology

    Article Title: IL-22 promotes allergic airway inflammation in epicutaneously sensitized mice

    doi: 10.1016/j.jaci.2018.05.032

    Figure Lengend Snippet: EC sensitization elicits a systemic IL-22 response and an antigen-specific IL-22 response in the lungs A-B. IL-22 secretion by OVA stimulated splenocytes (A) and IL-22 serum levels (B). C,D . Il22 mRNA expression in the lungs (C), and IL-22 secretion by OVA stimulated lung cells (D). E. Representative FACS analysis and quantitation of intracelluar expression of IL-22 + cells among CD3 + CD4 + T cells and of IL-17A + and TNFα + cells among CD3 + CD4 + IL-22 + cells in the lung. Mice were EC sensitized with OVA or saline in A and B, followed by i.n. challenged with OVA in C-F. Bars represent mean±SEM (n=5–10 per group). *p

    Article Snippet: Cells were then fixed and permeabilized using BD Cytofix/Cytoperm Kit (BD Biosciences) and stained with anti-IL-22 (Biolegend), anti-TNFα (Biolegend), anti-IL-17A (eBioscience) or anti-IFNγ (eBioscience) antibodies..

    Techniques: Expressing, FACS, Quantitation Assay, Mouse Assay

    HIF1α is critical for pro-inflammatory macrophage differentiation in vitro . Sorted PEMs from WT and HIF1α −/− mice were stimulated with LPS (100 ng/mL) or curdlan (100 ng/mL) for 10–12 h, and the indicated mRNA expression was determined with qPCR ( A ). Supernatant was collected and the concentration of the indicated cytokines was determined with an ELISA ( B ). ( C ) Peritoneal exudate cells from WT and HIF1α −/− mice were activated with the indicated stimuli, and the intracellular expression of TNFα in F4/80 + macrophages was determined with flow cytometry; a representative image is shown in the left figure, and data are summarized in the right figure. ( D ) Sorted PEMs were stimulated with LPS or curdlan for 10–12 h, and the mRNA expression of glycolytic pathway associated molecules was determined with qPCR. Data are presented as the means ± SD (n = 3–5). One representative experiment of three independent experiments is shown. * P

    Journal: Scientific Reports

    Article Title: HIF1α-dependent glycolysis promotes macrophage functional activities in protecting against bacterial and fungal infection

    doi: 10.1038/s41598-018-22039-9

    Figure Lengend Snippet: HIF1α is critical for pro-inflammatory macrophage differentiation in vitro . Sorted PEMs from WT and HIF1α −/− mice were stimulated with LPS (100 ng/mL) or curdlan (100 ng/mL) for 10–12 h, and the indicated mRNA expression was determined with qPCR ( A ). Supernatant was collected and the concentration of the indicated cytokines was determined with an ELISA ( B ). ( C ) Peritoneal exudate cells from WT and HIF1α −/− mice were activated with the indicated stimuli, and the intracellular expression of TNFα in F4/80 + macrophages was determined with flow cytometry; a representative image is shown in the left figure, and data are summarized in the right figure. ( D ) Sorted PEMs were stimulated with LPS or curdlan for 10–12 h, and the mRNA expression of glycolytic pathway associated molecules was determined with qPCR. Data are presented as the means ± SD (n = 3–5). One representative experiment of three independent experiments is shown. * P

    Article Snippet: For intracellular staining, TNFα expression (MP6-XT22; Biolegend) was analyzed by flow cytometry according to the manufacturer’s instructions.

    Techniques: In Vitro, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

    HIF1α is required for pro-inflammatory macrophage differentiation following Listeria bacterial infection. C57BL/6 WT or HIF1α −/− mice were i.v. injected with 1 × 10 5 CFU of L. monocytogenes bacteria. 48 h after infection, mouse livers were collected, and the CFU were determined ( A ). Infected mice developed severe infection and inflammatory cell infiltration, as shown by the histological staining of H E ( B ). At the same time point after infection, serum or peritoneal exudate TNFα levels were determined using an ELISA ( C and E ); the sorted splenic macrophages or PEMs were stimulated with LPS for 12 h, the supernatant was collected, and TNFα levels were determined using an ELISA ( D and F ); the glycolytic pathway activity of macrophages was also determined ( H and I ). TNFα expression in F4/80 + macrophages from peritoneal exudates was analyzed with FCM. A representative figure is shown in the left image, and the data are summarized in the right image ( G ). Data are presented as the means ± SD (n = 3–5). One representative experiment of three independent experiments is shown. *** P

    Journal: Scientific Reports

    Article Title: HIF1α-dependent glycolysis promotes macrophage functional activities in protecting against bacterial and fungal infection

    doi: 10.1038/s41598-018-22039-9

    Figure Lengend Snippet: HIF1α is required for pro-inflammatory macrophage differentiation following Listeria bacterial infection. C57BL/6 WT or HIF1α −/− mice were i.v. injected with 1 × 10 5 CFU of L. monocytogenes bacteria. 48 h after infection, mouse livers were collected, and the CFU were determined ( A ). Infected mice developed severe infection and inflammatory cell infiltration, as shown by the histological staining of H E ( B ). At the same time point after infection, serum or peritoneal exudate TNFα levels were determined using an ELISA ( C and E ); the sorted splenic macrophages or PEMs were stimulated with LPS for 12 h, the supernatant was collected, and TNFα levels were determined using an ELISA ( D and F ); the glycolytic pathway activity of macrophages was also determined ( H and I ). TNFα expression in F4/80 + macrophages from peritoneal exudates was analyzed with FCM. A representative figure is shown in the left image, and the data are summarized in the right image ( G ). Data are presented as the means ± SD (n = 3–5). One representative experiment of three independent experiments is shown. *** P

    Article Snippet: For intracellular staining, TNFα expression (MP6-XT22; Biolegend) was analyzed by flow cytometry according to the manufacturer’s instructions.

    Techniques: Infection, Mouse Assay, Injection, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Expressing

    Glycolytic pathway activity was required for pro-inflammatory macrophage differentiation. PEMs with indicated treatments for 10–12 h (2-DG, 1 mmol/L; LPS, 100 ng/mL; curdlan, 100 ng/mL; A – D ). A . HIF1α mRNA expression of PEMs was determined with qPCR (Value of control groups was set to 1). ( B and C) Supernatants were collected, and the indicated cytokine concentration was determined using an ELISA. ( D ) The glycolytic pathway activity was summarized. Mice were i.p. injected with 1 × 10 5 of C. albicans yeast and also injected intraperitoneally with 2-DG (2 g/kg body weight) or solvent alone (PBS; Ctrl) for 9 days. 2-DG or PBS were given daily up until the day before the mice were euthanized ( E – H ). ( E ) Mouse liver and kidneys were collected, and the CFU was evaluated. ( F ) Serum TNFα concentration ( F ). ( G ) TNFα expression of PEMs with FCM. A representative image is shown in the left figure and data are summarized in the right figure. ( H) The glycolytic pathway activity of PEMs. Data are presented as the means ± SD (n = 3–4). One representative experiment of three independent experiments is shown. P

    Journal: Scientific Reports

    Article Title: HIF1α-dependent glycolysis promotes macrophage functional activities in protecting against bacterial and fungal infection

    doi: 10.1038/s41598-018-22039-9

    Figure Lengend Snippet: Glycolytic pathway activity was required for pro-inflammatory macrophage differentiation. PEMs with indicated treatments for 10–12 h (2-DG, 1 mmol/L; LPS, 100 ng/mL; curdlan, 100 ng/mL; A – D ). A . HIF1α mRNA expression of PEMs was determined with qPCR (Value of control groups was set to 1). ( B and C) Supernatants were collected, and the indicated cytokine concentration was determined using an ELISA. ( D ) The glycolytic pathway activity was summarized. Mice were i.p. injected with 1 × 10 5 of C. albicans yeast and also injected intraperitoneally with 2-DG (2 g/kg body weight) or solvent alone (PBS; Ctrl) for 9 days. 2-DG or PBS were given daily up until the day before the mice were euthanized ( E – H ). ( E ) Mouse liver and kidneys were collected, and the CFU was evaluated. ( F ) Serum TNFα concentration ( F ). ( G ) TNFα expression of PEMs with FCM. A representative image is shown in the left figure and data are summarized in the right figure. ( H) The glycolytic pathway activity of PEMs. Data are presented as the means ± SD (n = 3–4). One representative experiment of three independent experiments is shown. P

    Article Snippet: For intracellular staining, TNFα expression (MP6-XT22; Biolegend) was analyzed by flow cytometry according to the manufacturer’s instructions.

    Techniques: Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection

    Pharmacologically targeting HIF1α and glycolytic pathway activity in mouse and human cells. Mouse PEMs ( A – E ) or human macrophages ( F – J ) pulsed with C. albicans yeast (1 × 10 5 ) for 3 days in the absence or presence of CoCl 2 (200 µM), 2-ME (2 µM), or 2-DG (1 mmol/L). ( A and E) The HIF1α mRNA expression of macrophages was determined with qPCR. ( B and F ) The TNFα concentration of supernatants was determined. ( C and G ) The CFU was evaluated. ( D and H) The Glut1 mRNA expression was determined with qPCR. Data are presented as the means ± SD (n = 3–4). One representative experiment of three independent experiments is shown. * P

    Journal: Scientific Reports

    Article Title: HIF1α-dependent glycolysis promotes macrophage functional activities in protecting against bacterial and fungal infection

    doi: 10.1038/s41598-018-22039-9

    Figure Lengend Snippet: Pharmacologically targeting HIF1α and glycolytic pathway activity in mouse and human cells. Mouse PEMs ( A – E ) or human macrophages ( F – J ) pulsed with C. albicans yeast (1 × 10 5 ) for 3 days in the absence or presence of CoCl 2 (200 µM), 2-ME (2 µM), or 2-DG (1 mmol/L). ( A and E) The HIF1α mRNA expression of macrophages was determined with qPCR. ( B and F ) The TNFα concentration of supernatants was determined. ( C and G ) The CFU was evaluated. ( D and H) The Glut1 mRNA expression was determined with qPCR. Data are presented as the means ± SD (n = 3–4). One representative experiment of three independent experiments is shown. * P

    Article Snippet: For intracellular staining, TNFα expression (MP6-XT22; Biolegend) was analyzed by flow cytometry according to the manufacturer’s instructions.

    Techniques: Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Concentration Assay

    Impact of menopause and ET on IL-6 levels and TNFα and IFNγ production. (A) Frequency of CD4 and CD8 T cells that secrete TNFα, IFNγ or both in response to CD3 stimulation was determined by intracellular cytokine staining and FCM using PBMC collected during visit 1 before the administration of the influenza vaccine. (B) Plasma IL-6 levels using samples collected during visit 1 were determined by ELISA.

    Journal: PLoS ONE

    Article Title: Impact of Estrogen Therapy on Lymphocyte Homeostasis and the Response to Seasonal Influenza Vaccine in Post-Menopausal Women

    doi: 10.1371/journal.pone.0149045

    Figure Lengend Snippet: Impact of menopause and ET on IL-6 levels and TNFα and IFNγ production. (A) Frequency of CD4 and CD8 T cells that secrete TNFα, IFNγ or both in response to CD3 stimulation was determined by intracellular cytokine staining and FCM using PBMC collected during visit 1 before the administration of the influenza vaccine. (B) Plasma IL-6 levels using samples collected during visit 1 were determined by ELISA.

    Article Snippet: The cells were then fixed and permeabilized to allow for intracellular staining with anti-IFNγ and TNFα antibodies (Biolegend).

    Techniques: Staining, Enzyme-linked Immunosorbent Assay

    Sod3 protects Histoplasma yeasts from ROS produced by activated macrophages. ( A–B ) Survival of yeasts after infection of resting ( A ) or cytokine-activated ( B ) murine macrophages. SOD3(+) (OSU45), sod3Δ (OSU15) and Candida albicans yeasts were added to resident peritoneal macrophages at an MOI of 1∶50. Yeast survival was determined by enumeration of viable cfu after 2 and 4 hours of co-incubation of yeasts with macrophages at 37°C. In ( B ), 10 U TNFα and 100 U IFNγ were added to macrophages 24 hours prior to infection to enhance ROS production. Results are plotted as relative yeast survival (mean ± standard deviation of 3 replicates) compared to viable cfu of yeasts incubated in the absence of macrophages. Significantly decreased survival compared to SOD3(+) yeasts is indicated by asterisks (* p

    Journal: PLoS Pathogens

    Article Title: Extracellular Superoxide Dismutase Protects Histoplasma Yeast Cells from Host-Derived Oxidative Stress

    doi: 10.1371/journal.ppat.1002713

    Figure Lengend Snippet: Sod3 protects Histoplasma yeasts from ROS produced by activated macrophages. ( A–B ) Survival of yeasts after infection of resting ( A ) or cytokine-activated ( B ) murine macrophages. SOD3(+) (OSU45), sod3Δ (OSU15) and Candida albicans yeasts were added to resident peritoneal macrophages at an MOI of 1∶50. Yeast survival was determined by enumeration of viable cfu after 2 and 4 hours of co-incubation of yeasts with macrophages at 37°C. In ( B ), 10 U TNFα and 100 U IFNγ were added to macrophages 24 hours prior to infection to enhance ROS production. Results are plotted as relative yeast survival (mean ± standard deviation of 3 replicates) compared to viable cfu of yeasts incubated in the absence of macrophages. Significantly decreased survival compared to SOD3(+) yeasts is indicated by asterisks (* p

    Article Snippet: 100 U murine IFNγ and/or 10 U murine TNFα (Biolegend) were added to wells for 24 hours at 37°C in 5% CO2 /95% air for activation of macrophages.

    Techniques: Produced, Infection, Incubation, Standard Deviation

    Protein S (PROS1)-deficient macrophages display reduced reprogramming. Macrophages from Pros1 fl/fl or LysM Cre/+ ; Pros1 fl/fl mice were isolated from peritoneal exudates 66 h after zymosan A injection and incubated for 24 h with vehicle or lipopolysaccharide (LPS, 1 µg/ml). Then, supernatants were collected and analyzed for their content of TNFα [ (A) , ** P = 0.003], IL-6 (B) , CCL3 [ (C) , * P = 0.02], and IL-10 [ (D) ; * P ≤ 0.03] by standard ELISA. (E) IL-10 secretion by untreated or LPS-stimulated control and Pros1 -cKO peritoneal macrophages, or by macrophages supplemented with either PROS1 (25 nM), apoptotic cells (AC) or both (AC + PROS1). Results represent the means ± SEM of at least three independent experiments. Two-way ANOVA, * P = 0.02, *** P ≤ 0.0001.

    Journal: Frontiers in Immunology

    Article Title: Macrophage-Derived Protein S Facilitates Apoptotic Polymorphonuclear Cell Clearance by Resolution Phase Macrophages and Supports Their Reprogramming

    doi: 10.3389/fimmu.2018.00358

    Figure Lengend Snippet: Protein S (PROS1)-deficient macrophages display reduced reprogramming. Macrophages from Pros1 fl/fl or LysM Cre/+ ; Pros1 fl/fl mice were isolated from peritoneal exudates 66 h after zymosan A injection and incubated for 24 h with vehicle or lipopolysaccharide (LPS, 1 µg/ml). Then, supernatants were collected and analyzed for their content of TNFα [ (A) , ** P = 0.003], IL-6 (B) , CCL3 [ (C) , * P = 0.02], and IL-10 [ (D) ; * P ≤ 0.03] by standard ELISA. (E) IL-10 secretion by untreated or LPS-stimulated control and Pros1 -cKO peritoneal macrophages, or by macrophages supplemented with either PROS1 (25 nM), apoptotic cells (AC) or both (AC + PROS1). Results represent the means ± SEM of at least three independent experiments. Two-way ANOVA, * P = 0.02, *** P ≤ 0.0001.

    Article Snippet: ELISA kits for mouse TNFα, IL-10, and IL-6 were obtained from Biolegend; a mouse CCL3 detection kit was obtained from R & D Systems.

    Techniques: Mouse Assay, Isolation, Injection, Incubation, Enzyme-linked Immunosorbent Assay

    Host pre-conditioning with CTX allows adoptively transferred tumor-specific CD4+ T cells to differentiate into polyfunctional effector cells and regain IL-7Rα expression. ( A ) Kinetics of IL-7Rα expression on donor CD4+ T cells following adoptive transfer. Following the timeline depicted in the schema, mice with established systemic A20HA tumors were divided into two groups. One group of mice were pre-conditioned with CTX while the other group received PBS. All mice received adoptive transfer of HA-specific CD4+ T cells the next day. At the indicated time points, tail blood samples were collected and analyzed for IL-7Rα expression on donor CD4+ T cells by FACS. IL-7Rα expression profiles relative to cell division in transferred CD4+ T cells are shown in representative dot plots. The numbers indicate the percentage of cells in the corresponding quadrant. Results are summarized in graph at right. Data for day 0 and day3 are gated on total donor T cells because cells barely divided at these two time points. ( B ) Differential expression of IL-7Rα in donor CD4+ T cells in mice with or without CTX pre-conditioning. 7 days after T-cell transfer, spleen cells were isolated to examine IL-7Rα expression on donor CD4+ T cells. Donor T cells were also evaluated for expression levels of PD1, Foxp3, CD40L, IFNγ and TNFα. The phenotypes of the divided donor T cells in PBS or CTX-conditioned mice are summarized in ( C ). ( D ) Donor CD4+ T cells transferred into CTX-conditioned mice but not unconditioned mice are capable of producing IL-2. IL-2 expression profiles relative to cell division in transferred CD4+ T cells are revealed by intracellular staining (ICS). ( E ) IL-2 neutralization inhibits IL-7Rα re-expression in donor CD4+ T cells in CTX-conditioned mice. Tumor-bearing mice were treated with CTX followed by CD4+ T cell transfer the next day. Some mice were injected with IL-2 neutralizing mAbs every other day. 7 days after T cell transfer, IL-7Rα expression on donor CD4+ T cells were analyzed by FACS. Results summarized in bar graph are shown as mean ± SD of 3 mice each group. ( F ) Exogenous IL-2 partially restores IL-7Rα expression in donor CD4+ T cells transferred into unconditioned mice. Following the timeline depicted in the schema, A20HA tumor-bearing mice received adoptive transfer of HA-specific CD4+ T cells. A cohort of mice further received IL-2/αIL-2 complex (IL-2C) every other day. IL-7Rα expressions on donor CD4+ T cells were analyzed 7 days after T cell transfer.

    Journal: Scientific Reports

    Article Title: Adjuvant IL-7 potentiates adoptive T cell therapy by amplifying and sustaining polyfunctional antitumor CD4+ T cells

    doi: 10.1038/s41598-017-12488-z

    Figure Lengend Snippet: Host pre-conditioning with CTX allows adoptively transferred tumor-specific CD4+ T cells to differentiate into polyfunctional effector cells and regain IL-7Rα expression. ( A ) Kinetics of IL-7Rα expression on donor CD4+ T cells following adoptive transfer. Following the timeline depicted in the schema, mice with established systemic A20HA tumors were divided into two groups. One group of mice were pre-conditioned with CTX while the other group received PBS. All mice received adoptive transfer of HA-specific CD4+ T cells the next day. At the indicated time points, tail blood samples were collected and analyzed for IL-7Rα expression on donor CD4+ T cells by FACS. IL-7Rα expression profiles relative to cell division in transferred CD4+ T cells are shown in representative dot plots. The numbers indicate the percentage of cells in the corresponding quadrant. Results are summarized in graph at right. Data for day 0 and day3 are gated on total donor T cells because cells barely divided at these two time points. ( B ) Differential expression of IL-7Rα in donor CD4+ T cells in mice with or without CTX pre-conditioning. 7 days after T-cell transfer, spleen cells were isolated to examine IL-7Rα expression on donor CD4+ T cells. Donor T cells were also evaluated for expression levels of PD1, Foxp3, CD40L, IFNγ and TNFα. The phenotypes of the divided donor T cells in PBS or CTX-conditioned mice are summarized in ( C ). ( D ) Donor CD4+ T cells transferred into CTX-conditioned mice but not unconditioned mice are capable of producing IL-2. IL-2 expression profiles relative to cell division in transferred CD4+ T cells are revealed by intracellular staining (ICS). ( E ) IL-2 neutralization inhibits IL-7Rα re-expression in donor CD4+ T cells in CTX-conditioned mice. Tumor-bearing mice were treated with CTX followed by CD4+ T cell transfer the next day. Some mice were injected with IL-2 neutralizing mAbs every other day. 7 days after T cell transfer, IL-7Rα expression on donor CD4+ T cells were analyzed by FACS. Results summarized in bar graph are shown as mean ± SD of 3 mice each group. ( F ) Exogenous IL-2 partially restores IL-7Rα expression in donor CD4+ T cells transferred into unconditioned mice. Following the timeline depicted in the schema, A20HA tumor-bearing mice received adoptive transfer of HA-specific CD4+ T cells. A cohort of mice further received IL-2/αIL-2 complex (IL-2C) every other day. IL-7Rα expressions on donor CD4+ T cells were analyzed 7 days after T cell transfer.

    Article Snippet: Antibodies and flow cytometry analysis The following fluorochrome-conjugated antibodies were used for flow cytometry: anti-mouse PD1-PE (RMP1–30), IL-7Rα-APC (A7R34), CD4-APC/Cy7 (RM4–5), CD8-PE/Cy7 (53.6.7), CD45.1-PE (A20), CD40L (MR1), IFNγ-APC (XMG1.2), IFNγ-FITC (XMG1.2), TNFα-PE (MP6-XT22), TNFα-PE/Cy7 (MP6-XT22), IL-2-PE (JES6–5H4), and control IgG mAbs were purchased from Biolegend.

    Techniques: Expressing, Adoptive Transfer Assay, Mouse Assay, FACS, Isolation, Staining, Neutralization, Injection

    CD8 T-cell memory alters the immune profile in enhanced HLH without altering viral load Prf1 −/− mice were immunized against GP33 or control, rested for 30 days and infected with LCMV. Mice were sacrificed on day 7 post-infection, serum samples were obtained and splenocytes were stimulated in vitro and analyzed. A. Serum cytokine levels in immunized and control mice (n=8-12). B. Total GP33-specific CD8 T cells (CD90+, CD8+, CD44+, IFNγ+) in immunized and control mice determined by IFNγ production following in vitro stimulation (n=6-8). Total number of GP33-specific CD8 T cells determined by GP33 tetramer staining (CD90+, CD8+, CD44+, GP33 tetramer+) (n=6-7). C. Percent of total CD8 T cells specific for GP33 or NP396 determined by IFNγ production following in vitro peptide stimulation with the respective peptide in GP33 immunized and control mice on day 7 post-infection (CD90+, CD8+, CD44+, IFNγ+) (n=4). D. Seven days post LCMV infection, total number of GP33 specific TNFα producing splenic CD8 T cells (CD90+, CD8+, CD44+, TNFα+) following in vitro peptide stimulation (n=6-8). E. LCMV splenic titers (n=6-8). Log scale is shown. *P

    Journal: ImmunoHorizons

    Article Title: CD8 T Cell Memory Increases Immunopathology in the Perforin-Deficient Model of Hemophagocytic Lymphohistiocytosis Secondary to TNF-α

    doi: 10.4049/immunohorizons.1800003

    Figure Lengend Snippet: CD8 T-cell memory alters the immune profile in enhanced HLH without altering viral load Prf1 −/− mice were immunized against GP33 or control, rested for 30 days and infected with LCMV. Mice were sacrificed on day 7 post-infection, serum samples were obtained and splenocytes were stimulated in vitro and analyzed. A. Serum cytokine levels in immunized and control mice (n=8-12). B. Total GP33-specific CD8 T cells (CD90+, CD8+, CD44+, IFNγ+) in immunized and control mice determined by IFNγ production following in vitro stimulation (n=6-8). Total number of GP33-specific CD8 T cells determined by GP33 tetramer staining (CD90+, CD8+, CD44+, GP33 tetramer+) (n=6-7). C. Percent of total CD8 T cells specific for GP33 or NP396 determined by IFNγ production following in vitro peptide stimulation with the respective peptide in GP33 immunized and control mice on day 7 post-infection (CD90+, CD8+, CD44+, IFNγ+) (n=4). D. Seven days post LCMV infection, total number of GP33 specific TNFα producing splenic CD8 T cells (CD90+, CD8+, CD44+, TNFα+) following in vitro peptide stimulation (n=6-8). E. LCMV splenic titers (n=6-8). Log scale is shown. *P

    Article Snippet: Staining was performed with LIVE/DEAD fixable viability dye (Life Technologies) and CD4, CD8α, CD44, CD90.2, IFNγ, and TNFα (BD Bioscience, Biolegend).

    Techniques: Mouse Assay, Infection, In Vitro, Staining

    TNFα is produced by memory CD8 T cells and contributes to mortality in immune memory HLH Prf1 −/− mice were immunized against GP33 or with control procedure, rested for 30 days, infected with LCMV and treated with either IFNγ blockade or isotype control beginning day 2 post-infection, and every 3 rd day thereafter. Mice were sacrificed on day 7 post-infection. A. Serum TNFα shows no difference in immune memory compared to control and this is unaffected by IFNγ blockade. Not significant by 2-way ANOVA (n=10-14, pooled two experimental replicates). B. Percent GP33-specific memory CD8 T cells of total splenic CD8 T cells. Expansion is unaffected by IFNγ blockade (CD90, CD8, CD44, IFNγ) however the effect of immune memory is significant by 2-way ANOVA, *P

    Journal: ImmunoHorizons

    Article Title: CD8 T Cell Memory Increases Immunopathology in the Perforin-Deficient Model of Hemophagocytic Lymphohistiocytosis Secondary to TNF-α

    doi: 10.4049/immunohorizons.1800003

    Figure Lengend Snippet: TNFα is produced by memory CD8 T cells and contributes to mortality in immune memory HLH Prf1 −/− mice were immunized against GP33 or with control procedure, rested for 30 days, infected with LCMV and treated with either IFNγ blockade or isotype control beginning day 2 post-infection, and every 3 rd day thereafter. Mice were sacrificed on day 7 post-infection. A. Serum TNFα shows no difference in immune memory compared to control and this is unaffected by IFNγ blockade. Not significant by 2-way ANOVA (n=10-14, pooled two experimental replicates). B. Percent GP33-specific memory CD8 T cells of total splenic CD8 T cells. Expansion is unaffected by IFNγ blockade (CD90, CD8, CD44, IFNγ) however the effect of immune memory is significant by 2-way ANOVA, *P

    Article Snippet: Staining was performed with LIVE/DEAD fixable viability dye (Life Technologies) and CD4, CD8α, CD44, CD90.2, IFNγ, and TNFα (BD Bioscience, Biolegend).

    Techniques: Produced, Mouse Assay, Infection

    LPS induces miR-155 and TNFα expression in hepatic immune cells. Isolated LMNCs and Kupffer cells from C57Bl/6 WT mice were stimulated or not with 100ng/ml LPS for 6 hours in vitro. miR-155 expression (A: LMNCs, C: KCs) and TNF-α protein secretion (B: LMNCs, D: KCs) were measured in the cells and in the supernatant, respectively (n = 8-10/group). RNA was isolated from C57Bl/6 WT mice fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 3, 6 and 8 weeks. miR155 and TNFα mRNA expression was determined (n = 5-6/group, total 32) and correlation was plotted (E); correlation coefficient is shown. (*) indicates p

    Journal: PLoS ONE

    Article Title: MicroRNA-155 Deficiency Attenuates Liver Steatosis and Fibrosis without Reducing Inflammation in a Mouse Model of Steatohepatitis

    doi: 10.1371/journal.pone.0129251

    Figure Lengend Snippet: LPS induces miR-155 and TNFα expression in hepatic immune cells. Isolated LMNCs and Kupffer cells from C57Bl/6 WT mice were stimulated or not with 100ng/ml LPS for 6 hours in vitro. miR-155 expression (A: LMNCs, C: KCs) and TNF-α protein secretion (B: LMNCs, D: KCs) were measured in the cells and in the supernatant, respectively (n = 8-10/group). RNA was isolated from C57Bl/6 WT mice fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 3, 6 and 8 weeks. miR155 and TNFα mRNA expression was determined (n = 5-6/group, total 32) and correlation was plotted (E); correlation coefficient is shown. (*) indicates p

    Article Snippet: Serum and liver TNFα (BioLegend Inc ., San Diego CA , USA) , IL-1β (R&D Systems , Minneapolis , MN , USA) and MCP1 (BioLegend Inc ., San Diego CA , USA) levels were determined by ELISA as described by manufactures.

    Techniques: Expressing, Isolation, Mouse Assay, In Vitro

    miR-155 deficiency does not attenuate hepatic inflammation in MCD-steatohepatitis. Wild type (WT) and miR-155 deficient (KO) mice were fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 5 weeks. Liver TNFα (A top panel: mRNA, A bottom panel: protein), MCP1 (B top panel: mRNA, B bottom panel: protein) and IL1-β (C top panel: mRNA, C bottom panel: protein) mRNA and protein levels were measured by qPCR and ELISA, respectively (n = 6-8/group). NF-κB nuclear binding was evaluated by EMSA using liver nuclear extracts (D, top panel: representative blot, bottom panel: densitometry showing cumulative data of n = 6/group). (*) indicates p

    Journal: PLoS ONE

    Article Title: MicroRNA-155 Deficiency Attenuates Liver Steatosis and Fibrosis without Reducing Inflammation in a Mouse Model of Steatohepatitis

    doi: 10.1371/journal.pone.0129251

    Figure Lengend Snippet: miR-155 deficiency does not attenuate hepatic inflammation in MCD-steatohepatitis. Wild type (WT) and miR-155 deficient (KO) mice were fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 5 weeks. Liver TNFα (A top panel: mRNA, A bottom panel: protein), MCP1 (B top panel: mRNA, B bottom panel: protein) and IL1-β (C top panel: mRNA, C bottom panel: protein) mRNA and protein levels were measured by qPCR and ELISA, respectively (n = 6-8/group). NF-κB nuclear binding was evaluated by EMSA using liver nuclear extracts (D, top panel: representative blot, bottom panel: densitometry showing cumulative data of n = 6/group). (*) indicates p

    Article Snippet: Serum and liver TNFα (BioLegend Inc ., San Diego CA , USA) , IL-1β (R&D Systems , Minneapolis , MN , USA) and MCP1 (BioLegend Inc ., San Diego CA , USA) levels were determined by ELISA as described by manufactures.

    Techniques: Mouse Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Binding Assay

    MCD diet-induced steatohepatitis is associated with increased miR-155 expression in parenchymal and non-parenchymal cells. C57Bl/6 mice were fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 3, 6 and 8 weeks. Serum alanine aminotransferase levels (A) and TNFα mRNA expression (B) were determined (n = 5-8/group). Liver fibrosis was assessed by Sirius Red staining (100x; n = 3-6/group), representative slides are shown (C). miR-155 expression was detected by qPCR in total livers (n = 5-8/group) (D). Primary murine hepatocytes (n = 7/group), Kupffer cells (n = 6, pooled data, 2 datapoints/group) and liver mononuclear cells (LMNC; n = 3-4/group) were isolated from a subset of mice after 6 weeks of MCS or MCD diet feeding and cell-specific miR-155 expression was determined and represented as 1/dCt (E). miR-155* expression was determined in total livers (F). (*) indicates p

    Journal: PLoS ONE

    Article Title: MicroRNA-155 Deficiency Attenuates Liver Steatosis and Fibrosis without Reducing Inflammation in a Mouse Model of Steatohepatitis

    doi: 10.1371/journal.pone.0129251

    Figure Lengend Snippet: MCD diet-induced steatohepatitis is associated with increased miR-155 expression in parenchymal and non-parenchymal cells. C57Bl/6 mice were fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 3, 6 and 8 weeks. Serum alanine aminotransferase levels (A) and TNFα mRNA expression (B) were determined (n = 5-8/group). Liver fibrosis was assessed by Sirius Red staining (100x; n = 3-6/group), representative slides are shown (C). miR-155 expression was detected by qPCR in total livers (n = 5-8/group) (D). Primary murine hepatocytes (n = 7/group), Kupffer cells (n = 6, pooled data, 2 datapoints/group) and liver mononuclear cells (LMNC; n = 3-4/group) were isolated from a subset of mice after 6 weeks of MCS or MCD diet feeding and cell-specific miR-155 expression was determined and represented as 1/dCt (E). miR-155* expression was determined in total livers (F). (*) indicates p

    Article Snippet: Serum and liver TNFα (BioLegend Inc ., San Diego CA , USA) , IL-1β (R&D Systems , Minneapolis , MN , USA) and MCP1 (BioLegend Inc ., San Diego CA , USA) levels were determined by ELISA as described by manufactures.

    Techniques: Expressing, Mouse Assay, Staining, Real-time Polymerase Chain Reaction, Isolation

    LPS plus Smac mimetics induces RIPK1-dependent apoptosis that switches to necroptosis upon caspase inhibition. ( a ) WT and Xiap − / − neutrophils were pre-treated with Nec.1 (20 μ M) for 30 min, AT-406 (1 μ M) or Cp.A (500 nM) for 30 min and subsequently incubated with LPS (100 ng/ml) for 3 and 6 h. Lysates were assayed for caspase-3/-7 activity; n ≥4, mean±S.E.M. ( b ) WT and Xiap − / − neutrophils were pre-treated with Nec.1 (20 μ M, 30 min) or Q-VD-OPh (20 μ M, 30 min), then with either AT-406 (1 μ M) or Cp.A (500 nM), and incubated with LPS (100 ng/ml) for 6 h. Lysates were assayed by immunoblot. Presented immunoblots are representative of at least two independent experiments. ( c ) qPCR analysis of Tnfα in WT and Xiap − / − neutrophils pre-treated with Nec.1 (20 μ M, 30 min) followed by either AT-406 (1 μ M) or Cp.A (500 nM), for another 30 min and stimulated with LPS (100 ng/ml) for 4 h. Hprt was used as reference gene; n =3, mean±S.E.M. ( d ) WT and Xiap − / − neutrophils were preincubated with Nec.1 (20 μ M, 30 min), treated with either AT-406 (1 μ M) or Cp.A (500 nM) and stimulated with LPS (100 ng/ml) for 6 h. Supernatants were analyzed for TNF α by ELISA; n ≥4, mean±S.E.M. ( e ) WT and Xiap − / − neutrophils were pre-treated with Q-VD-OPh (20 μ M) or Nec.1 (20 μ M) for 30 min, treated with AT-406 (1 μ M) or Cp.A (500 nM) for 30 min and subsequently stimulated with LPS (100 ng/ml) for different time points. Viability was determined by flow cytometry; n ≥3, mean±S.E.M. Data sets of untreated control from Figure 1a and SM+LPS from Figure 2b are included. ( f ) Ripk3 − / − and ( g ) Ripk3 − / − Xiap − / − neutrophils were pre-treated with Q-VD-OPh (20 μ M) for 30 min, incubated with Cp.A (500 nM) or AT-406 (1 μ M), respectively, and stimulated with LPS (100 ng/ml) for indicated time points. Viability was assessed by flow cytometry; n ≥3, mean±S.E.M. ( h ) WT and Xiap − / − neutrophils were pre-treated with Q-VD (20 μ M) for 30 min followed by administration of Cp.A (500 nM) or AT-406 (1 μ M), respectively, for 30 min and stimulated with LPS for 6 h. Fractionation by phase separation was performed and lysates were assayed by quantitative immunoblot. Presented immunoblots are representative of at least two independent experiments. All experiments were performed with in vitro differentiated neutrophils. * P

    Journal: Cell Death & Disease

    Article Title: Loss of XIAP facilitates switch to TNFα-induced necroptosis in mouse neutrophils

    doi: 10.1038/cddis.2016.311

    Figure Lengend Snippet: LPS plus Smac mimetics induces RIPK1-dependent apoptosis that switches to necroptosis upon caspase inhibition. ( a ) WT and Xiap − / − neutrophils were pre-treated with Nec.1 (20 μ M) for 30 min, AT-406 (1 μ M) or Cp.A (500 nM) for 30 min and subsequently incubated with LPS (100 ng/ml) for 3 and 6 h. Lysates were assayed for caspase-3/-7 activity; n ≥4, mean±S.E.M. ( b ) WT and Xiap − / − neutrophils were pre-treated with Nec.1 (20 μ M, 30 min) or Q-VD-OPh (20 μ M, 30 min), then with either AT-406 (1 μ M) or Cp.A (500 nM), and incubated with LPS (100 ng/ml) for 6 h. Lysates were assayed by immunoblot. Presented immunoblots are representative of at least two independent experiments. ( c ) qPCR analysis of Tnfα in WT and Xiap − / − neutrophils pre-treated with Nec.1 (20 μ M, 30 min) followed by either AT-406 (1 μ M) or Cp.A (500 nM), for another 30 min and stimulated with LPS (100 ng/ml) for 4 h. Hprt was used as reference gene; n =3, mean±S.E.M. ( d ) WT and Xiap − / − neutrophils were preincubated with Nec.1 (20 μ M, 30 min), treated with either AT-406 (1 μ M) or Cp.A (500 nM) and stimulated with LPS (100 ng/ml) for 6 h. Supernatants were analyzed for TNF α by ELISA; n ≥4, mean±S.E.M. ( e ) WT and Xiap − / − neutrophils were pre-treated with Q-VD-OPh (20 μ M) or Nec.1 (20 μ M) for 30 min, treated with AT-406 (1 μ M) or Cp.A (500 nM) for 30 min and subsequently stimulated with LPS (100 ng/ml) for different time points. Viability was determined by flow cytometry; n ≥3, mean±S.E.M. Data sets of untreated control from Figure 1a and SM+LPS from Figure 2b are included. ( f ) Ripk3 − / − and ( g ) Ripk3 − / − Xiap − / − neutrophils were pre-treated with Q-VD-OPh (20 μ M) for 30 min, incubated with Cp.A (500 nM) or AT-406 (1 μ M), respectively, and stimulated with LPS (100 ng/ml) for indicated time points. Viability was assessed by flow cytometry; n ≥3, mean±S.E.M. ( h ) WT and Xiap − / − neutrophils were pre-treated with Q-VD (20 μ M) for 30 min followed by administration of Cp.A (500 nM) or AT-406 (1 μ M), respectively, for 30 min and stimulated with LPS for 6 h. Fractionation by phase separation was performed and lysates were assayed by quantitative immunoblot. Presented immunoblots are representative of at least two independent experiments. All experiments were performed with in vitro differentiated neutrophils. * P

    Article Snippet: Cytokine ELISA Mouse-specific IL-1β , IL-6 and TNFα ELISA Kits were from BioLegend and cytokine levels in cellular supernatants were measured according to the manufacturer's instructions.

    Techniques: Inhibition, Incubation, Activity Assay, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Fractionation, In Vitro

    Comparison of BM-DC response to Man and Gal-LPS. A , two LPS used in this study are shown. Man-LPS from H. alvei PCM 1223 has a mannosylated repeating unit, whereas Gal-LPS from S. enterica O66 has a galactosylated repeat. B , HEK293 cells stably transfected with TLR4-MD2 were cultured in the presence of LPS. The TLR4 activation was monitored by measuring alkaline phosphatase activity using the substrate. C , mouse BM-DCs were stimulated with 1 μg/ml Man-LPS or 4 μg/ml Gal-LPS for 7 h. The amount of TNFα and IL-10 in the culture supernatant was analyzed by ELISA. Data are representative of three independent experiments with similar results. Error bars , S.D. Statistical analyses were performed by one-way ANOVA followed by Tukey's test. ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: Dectin-2 Recognizes Mannosylated O-antigens of Human Opportunistic Pathogens and Augments Lipopolysaccharide Activation of Myeloid Cells *

    doi: 10.1074/jbc.M116.741256

    Figure Lengend Snippet: Comparison of BM-DC response to Man and Gal-LPS. A , two LPS used in this study are shown. Man-LPS from H. alvei PCM 1223 has a mannosylated repeating unit, whereas Gal-LPS from S. enterica O66 has a galactosylated repeat. B , HEK293 cells stably transfected with TLR4-MD2 were cultured in the presence of LPS. The TLR4 activation was monitored by measuring alkaline phosphatase activity using the substrate. C , mouse BM-DCs were stimulated with 1 μg/ml Man-LPS or 4 μg/ml Gal-LPS for 7 h. The amount of TNFα and IL-10 in the culture supernatant was analyzed by ELISA. Data are representative of three independent experiments with similar results. Error bars , S.D. Statistical analyses were performed by one-way ANOVA followed by Tukey's test. ***, p

    Article Snippet: ELISA kits for mouse and human TNFα and IL-10 were from Biolegend and were used according to the manufacturer's instructions.

    Techniques: Stable Transfection, Transfection, Cell Culture, Activation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay