il-12p70 Search Results


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  • 96
    Thermo Fisher il 12p70
    DAC affects the production of inflammatory cytokines by DCs from active BD patients The protein concentrations of IL-6, IL-1β, IL-23 and <t>IL-12p70</t> in the supernatants of the DCs which were cultured with or without DAC were determined by ELISA. (A) The protein concentrations of IL-6, IL-1β, IL-23 and IL-12p70 were significantly increased in BD patients compared with the normal controls (n=12, *p
    Il 12p70, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 987 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Quanterix il 12p70
    Synergy between human MAIT cell agonists and TLR agonists. ( A ) Bioactive <t>IL-12p70</t> in the supernatant of DCs pulsed with E. coli upernatant in the presence or absence of allogeneic MAIT cells and blocking anti-CD40L or anti-MR1 Abs. ( B ) Bioactive IL-12p70 in the supernatant of DCs pulsed with the indicated concentrations of 5-A-RU/MG (ng/ml), LPS (ng/ml), or R848 (μg/ml) in the presence or absence of allogeneic MAIT cells and blocking anti-CD40L or anti-MR1 Abs. ( C ) IFN-γ levels in the supernatant of MAIT-allogeneic DC cocultures pulsed with E. coli supernatant in the presence or absence of blocking anti-CD40L, anti–IL-12, or anti-MR1 Abs. ( D ) IFN-γ levels in the supernatant of MAIT–allogeneic DC cocultures pulsed with the indicated concentrations of 5-A-RU/MG (ng/ml), LPS (ng/ml), or R848 (μg/ml) in the presence or absence of blocking anti-CD40L or anti-MR1 Abs. Data are from one experiment representative of four; data are mean ± SD. Two more donors are shown in Supplemental Fig. 2 .
    Il 12p70, supplied by Quanterix, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Becton Dickinson il 12p70
    Role of TLR2 in the induction of IL-12 subunits by F. tularensis LVS-stimulated Mφ or DC. Wild-type or TLR2-deficient Mφ or DC were stimulated with F. tularensis LVS at the indicated MOI, and culture fluids were assayed for either <t>IL-12p70</t>
    Il 12p70, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 96/100, based on 2628 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    BioLegend il 12p70
    Day-4 DCs produce the highest levels of <t>IL-12p70</t> and IP-10. Day-2, Day-4, and Day-7 DCs were first pulsed with either UVBL or FTL for 16 h, and then stimulated with LPS (60 EU/ml) and IFN-γ (2000 IU/ml) to determine (A) IL-12p70 and, (B) IP-10 production. Supernatants from the DC-tumor lysate cocultures were collected and evaluated by ELISA as described in Materials and Methods . The results are from 4 different normal healthy donors and are expressed as mean (pg/ml) ± standard errors.
    Il 12p70, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 269 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Mab Technologies il 12p70
    Activation of allogeneic T cells by monocyte-derived dendritic cells matured in the presence of different stimulatory cocktails. Monocytes from three donors were analyzed for the production of <t>IL-12p70</t> after an initial 48 h culture with GM-CSF/IL-4 followed by harvest, wash, and thereafter 18 h with GM-CSF/IL-4/IFNγ/R848 together with combinations of TNFα and GMP-grade poly I:C Hiltonol ® or non-GMP-grade poly I:C from either GE Healthcare or Sigma-Aldrich without ( a ) or with addition of LPS ( b ). The monocyte-derived dendritic cells were harvested, washed, and then co-cultured for 4 days with allogeneic bulk T cells before the analysis of IFNγ production ( c , d ). Note the difference in scales of y -axes
    Il 12p70, supplied by Mab Technologies, used in various techniques. Bioz Stars score: 92/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    R&D Systems il 12p70
    Dexamethasone prevents cocktail induction of <t>IL-12p70</t> and TNFα . Immature DCs from a single donor shows a dexamethasone-mediated dose dependent suppression of IL-12p70 and TNFα secretion. Dexamethasone was pre-incubated with imDCs for 6 hours (A and C) or 24 hours (B and D) with increasing concentration of dexamethasone at 0-0.01-0.1 and 1.0 μM. Dexamethasone treatment without cocktail did not induce IL-12p70 or TNFα (first 4 bars). The cocktails used are indicated below each set of data, and their exact composition is seen in table 1. This shows one representative example out of three. Cell viability was not significantly affected by treatment with cocktail and/or dexamethasone (not shown).
    Il 12p70, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 1048 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Luminex il 12p70
    Dexamethasone prevents cocktail induction of <t>IL-12p70</t> and TNFα . Immature DCs from a single donor shows a dexamethasone-mediated dose dependent suppression of IL-12p70 and TNFα secretion. Dexamethasone was pre-incubated with imDCs for 6 hours (A and C) or 24 hours (B and D) with increasing concentration of dexamethasone at 0-0.01-0.1 and 1.0 μM. Dexamethasone treatment without cocktail did not induce IL-12p70 or TNFα (first 4 bars). The cocktails used are indicated below each set of data, and their exact composition is seen in table 1. This shows one representative example out of three. Cell viability was not significantly affected by treatment with cocktail and/or dexamethasone (not shown).
    Il 12p70, supplied by Luminex, used in various techniques. Bioz Stars score: 92/100, based on 278 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    U-CyTech il 12p70
    Human immature DCs are activated by B. abortus S19. Monocyte-derived immature DCs were infected with B. abortus S19 (MOI, 20) for 1 h, the bacteria were washed out, and the cells were incubated for another 48 h. Control cells were left uninfected. (A) After 48 h, the phenotype of the cells was determined by flow cytometry (bold lines, infected cells; grey areas, uninfected cells; dotted lines, isotype controls). One representative of at least six independent experiments is shown. (B) Supernatants were analyzed for the presence of IL-12/23p40, <t>IL-12p70,</t> IL-10, and TNF-α. Data expressed as median with interquartile range.
    Il 12p70, supplied by U-CyTech, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad il 12p70
    Streptococcus suis -induced type I interferon produced by dendritic cells (DCs) modulates autocrine cytokine production. Production of tumor necrosis factor (A) , interleukin (IL)-6 (B) , <t>IL-12p70</t> (C) , CCL2 (D) , CCL3 (E) , and CXCL1 (F) by DCs 16 h following infection with the different S. suis strains. Data represent the mean ± SEM ( n = 4). ** ( p
    Il 12p70, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 292 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Diaclone il 12p70
    Donor- and cytokine specific effect of neonatal plasma on LPS-induced cytokine production Adult PBMC derived from heparinized blood from three separate donors were stimulated with LPS and IFN-γ in the presence of 10% heterologous adult or neonatal plasma. Each dot represents a different plasma donor. After 24h incubation, the extracellular medium was collected for IL-10 and <t>IL-12p70</t> measurements. Bars represent median values.
    Il 12p70, supplied by Diaclone, used in various techniques. Bioz Stars score: 92/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pharmingen il 12p70
    COX-2 activity influences IFN-γ and <t>IL-12p70</t> production in antigen stimulated cultures of DO11.10 CD4+ T cells. Peritoneal macrophages were isolated and cultured in the presence of medium alone (A) or viable Salmonella (B) . Following exposure for 45 minutes, cells were washed to remove any extracellular bacteria with gentamicin-containing medium. Magnetically separated splenic DO11.10 CD4 + T cells, B cells, and OVA peptide were added. To assess the role of COX-2, some cultures were supplemented with 1 uM celecoxib 20 minutes prior to infection. Levels of IFN-γ (A and B) and IL-12p70 (C) in 48-hour supernatants were measured by enzyme immunoassay, and are reported as mean values of triplicate determinations. These studies were performed three times with similar results.
    Il 12p70, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 92 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Sanquin il 12p70
    IFN-γ in supernatants of IL-23 plus IL-1β stimulated CD56 + cells primes monocytes for <t>IL-12p70</t> production in response to LPS. 1 ·10 5 CD14 + monocytes were stimulated with 100 ng/ml LPS in combination with 2.5 ng/ml IFN-γ, supernatants of unstimulated CD56 + cells, supernatants of IL-23 plus IL-1β stimulated CD56 + cells, or medium alone for 24 hours (A). 1 ·10 5 CD14 + monocytes were stimulated with 100 ng/ml LPS in combination supernatants of IL-23 plus IL-1β stimulated CD56+ cells, plus or minus 2 µg/ml anti-IFN-γ for 24 hours (B). Supernatants were taken and IL-12p70 production was measured by ELISA. Data are means±SD of triplicates from one representative experiment of three.
    Il 12p70, supplied by Sanquin, used in various techniques. Bioz Stars score: 92/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    InvivoGen il 12p70
    Blocking of demyelination in HSV-IL-2 infected mice by <t>IL-12p70</t> HSV-1 recombinant virus BALB/c mice were co-infected with HSV-IL-2 + HSV-IL-12p70 at 1:1, 1:10, or 1:100 ratios of HSV-IL-2 to HSV-IL-12p70. On day 14 post-infection, optic nerves and spinal cords were collected, fixed, sectioned, and stained with LFB. Representative photomicrographs are shown. Arrows indicate areas of demyelination. 10X direct magnification.
    Il 12p70, supplied by InvivoGen, used in various techniques. Bioz Stars score: 92/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Pharmingen il 12p70 kits
    C57Bl/6 transgenic mice harboring the SJL/J variant of the IL-12p40 gene or the C57Bl/6 variant were challenged with a sublethal dose of LPS after which the concentration in serum of <t>IL-12p70</t> and IL-12p40 was measured by ELISA. Bars represent mean ±
    Il 12p70 Kits, supplied by Pharmingen, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems il 12p70 kits
    Interferon (IFN)-γ secretion in response to activation with live bacille Calmette–Guérin (BCG) and Toll-like receptor (TLR) agonists. Results are expressed as IFN-γ secretion (pg/ml) per IFN-γ-producing cells × 10 3 . (a) Monocyte/macrophage and T cell interaction. Whole blood was stimulated with live BCG at a multiplicity of infection (MOI) of 20 BCG/leucocytes, or with BCG plus interleukin <t>(IL)-12p70</t> (20 ng/ml). Production of IFN-γ (pg/ml) was analysed after 48 h of culture. (b) Lipopolysaccharide (LPS) activation. Whole blood was stimulated with LPS from Salmonella minnesota R595 at 10 μg/ml or with LPS plus IL-12p70 (20 ng/ml). Production of IFN-γ (pg/ml) was analysed after 48 h of culture. (c) Heat-killed Staphylococcus aureus (SAC) activation. Whole blood was stimulated with SAC at 5 × 10 6 particles/ml, or with SAC plus IL-12p70 (20 ng/ml). Production of IFN-γ (pg/ml) was analysed after 48 h of culture.
    Il 12p70 Kits, supplied by R&D Systems, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    R&D Systems il 12p70 cytokines
    Interferon (IFN)-γ secretion in response to activation with live bacille Calmette–Guérin (BCG) and Toll-like receptor (TLR) agonists. Results are expressed as IFN-γ secretion (pg/ml) per IFN-γ-producing cells × 10 3 . (a) Monocyte/macrophage and T cell interaction. Whole blood was stimulated with live BCG at a multiplicity of infection (MOI) of 20 BCG/leucocytes, or with BCG plus interleukin <t>(IL)-12p70</t> (20 ng/ml). Production of IFN-γ (pg/ml) was analysed after 48 h of culture. (b) Lipopolysaccharide (LPS) activation. Whole blood was stimulated with LPS from Salmonella minnesota R595 at 10 μg/ml or with LPS plus IL-12p70 (20 ng/ml). Production of IFN-γ (pg/ml) was analysed after 48 h of culture. (c) Heat-killed Staphylococcus aureus (SAC) activation. Whole blood was stimulated with SAC at 5 × 10 6 particles/ml, or with SAC plus IL-12p70 (20 ng/ml). Production of IFN-γ (pg/ml) was analysed after 48 h of culture.
    Il 12p70 Cytokines, supplied by R&D Systems, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend capture il 12p70
    Day-4 DCs produce the highest levels of <t>IL-12p70</t> and IP-10. Day-2, Day-4, and Day-7 DCs were first pulsed with either UVBL or FTL for 16 h, and then stimulated with LPS (60 EU/ml) and IFN-γ (2000 IU/ml) to determine (A) IL-12p70 and, (B) IP-10 production. Supernatants from the DC-tumor lysate cocultures were collected and evaluated by ELISA as described in Materials and Methods . The results are from 4 different normal healthy donors and are expressed as mean (pg/ml) ± standard errors.
    Capture Il 12p70, supplied by BioLegend, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems il 12p70 levels
    Day-4 DCs produce the highest levels of <t>IL-12p70</t> and IP-10. Day-2, Day-4, and Day-7 DCs were first pulsed with either UVBL or FTL for 16 h, and then stimulated with LPS (60 EU/ml) and IFN-γ (2000 IU/ml) to determine (A) IL-12p70 and, (B) IP-10 production. Supernatants from the DC-tumor lysate cocultures were collected and evaluated by ELISA as described in Materials and Methods . The results are from 4 different normal healthy donors and are expressed as mean (pg/ml) ± standard errors.
    Il 12p70 Levels, supplied by R&D Systems, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher murine il 12p70
    Day-4 DCs produce the highest levels of <t>IL-12p70</t> and IP-10. Day-2, Day-4, and Day-7 DCs were first pulsed with either UVBL or FTL for 16 h, and then stimulated with LPS (60 EU/ml) and IFN-γ (2000 IU/ml) to determine (A) IL-12p70 and, (B) IP-10 production. Supernatants from the DC-tumor lysate cocultures were collected and evaluated by ELISA as described in Materials and Methods . The results are from 4 different normal healthy donors and are expressed as mean (pg/ml) ± standard errors.
    Murine Il 12p70, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pharmingen recombinant il 12p70
    COX-2 activity influences IFN-γ and <t>IL-12p70</t> production in antigen stimulated cultures of DO11.10 CD4+ T cells. Peritoneal macrophages were isolated and cultured in the presence of medium alone (A) or viable Salmonella (B) . Following exposure for 45 minutes, cells were washed to remove any extracellular bacteria with gentamicin-containing medium. Magnetically separated splenic DO11.10 CD4 + T cells, B cells, and OVA peptide were added. To assess the role of COX-2, some cultures were supplemented with 1 uM celecoxib 20 minutes prior to infection. Levels of IFN-γ (A and B) and IL-12p70 (C) in 48-hour supernatants were measured by enzyme immunoassay, and are reported as mean values of triplicate determinations. These studies were performed three times with similar results.
    Recombinant Il 12p70, supplied by Pharmingen, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems mouse il 12p70
    COX-2 activity influences IFN-γ and <t>IL-12p70</t> production in antigen stimulated cultures of DO11.10 CD4+ T cells. Peritoneal macrophages were isolated and cultured in the presence of medium alone (A) or viable Salmonella (B) . Following exposure for 45 minutes, cells were washed to remove any extracellular bacteria with gentamicin-containing medium. Magnetically separated splenic DO11.10 CD4 + T cells, B cells, and OVA peptide were added. To assess the role of COX-2, some cultures were supplemented with 1 uM celecoxib 20 minutes prior to infection. Levels of IFN-γ (A and B) and IL-12p70 (C) in 48-hour supernatants were measured by enzyme immunoassay, and are reported as mean values of triplicate determinations. These studies were performed three times with similar results.
    Mouse Il 12p70, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher il 12p70 biotin
    COX-2 activity influences IFN-γ and <t>IL-12p70</t> production in antigen stimulated cultures of DO11.10 CD4+ T cells. Peritoneal macrophages were isolated and cultured in the presence of medium alone (A) or viable Salmonella (B) . Following exposure for 45 minutes, cells were washed to remove any extracellular bacteria with gentamicin-containing medium. Magnetically separated splenic DO11.10 CD4 + T cells, B cells, and OVA peptide were added. To assess the role of COX-2, some cultures were supplemented with 1 uM celecoxib 20 minutes prior to infection. Levels of IFN-γ (A and B) and IL-12p70 (C) in 48-hour supernatants were measured by enzyme immunoassay, and are reported as mean values of triplicate determinations. These studies were performed three times with similar results.
    Il 12p70 Biotin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems recombinant il 12p70
    The impact of CD40 and CD40L deficiency on IL-12 and IFN-γ production. <t>IL-12p70,</t> IL-12p40, and IFN-γ production, measured by classical sandwich ELISA, in a mixture of purified monocytes and T cells, as indicated, upon stimulation with PHA from a healthy donor with (A) CD40L- and (B) CD40-deficient PBMCs. MDDCs obtained from two healthy controls (C2 and C3), P2 and P3, after 24 h of incubation alone or with LPS (1 μg/ml) plus IL-1β (10 ng/ml) as a control of CD40-independent activation, cocultured with L-cells transfected with human CD40L (L-cell-hCD40L) and nontransfected L-cells (L-cell): (C) IL-12p70, IL-12p40, TNF-α, and IL-6 production, measured by classical sandwich ELISA, and (D) FACS analysis of cell surface expression of CD40, CD80, and CD86 costimulatory molecules gating on CD1a-positive MDDCs. (E) Intracellular staining of NEMO protein in MDDCs from P2 and P3, and their respective controls (C2 and C3) in the experimental conditions of Fig. 3 . (F) IgE secretion by B cells in vitro, as measured by ELISA, after the activation of PBMCs from 20 healthy controls (C+, •), P2 (□), and a CD40-deficient patient as the negative control (C−,▵), with soluble CD40L in combination with IL-4. The results shown are representative of two independent experiments.
    Recombinant Il 12p70, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Miltenyi Biotec il 12p70
    Influence of ILT2/ILT5 on the cytokine secretion properties of dendritic cells. (A and B) Secretion of <t>IL-12p70,</t> IL-6, or TNF-α by mDC after stimulation with TLR7/8 ligands in the presence of ILT2 (A) or ILT5 (B) antibodies ( n = 10 study
    Il 12p70, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 92/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend il 12p70 elisa
    Influence of ILT2/ILT5 on the cytokine secretion properties of dendritic cells. (A and B) Secretion of <t>IL-12p70,</t> IL-6, or TNF-α by mDC after stimulation with TLR7/8 ligands in the presence of ILT2 (A) or ILT5 (B) antibodies ( n = 10 study
    Il 12p70 Elisa, supplied by BioLegend, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    DAC affects the production of inflammatory cytokines by DCs from active BD patients The protein concentrations of IL-6, IL-1β, IL-23 and IL-12p70 in the supernatants of the DCs which were cultured with or without DAC were determined by ELISA. (A) The protein concentrations of IL-6, IL-1β, IL-23 and IL-12p70 were significantly increased in BD patients compared with the normal controls (n=12, *p

    Journal: Oncotarget

    Article Title: Ocular Behcet’s disease is associated with aberrant methylation of interferon regulatory factor 8 (IRF8) in monocyte-derived dendritic cells

    doi: 10.18632/oncotarget.17235

    Figure Lengend Snippet: DAC affects the production of inflammatory cytokines by DCs from active BD patients The protein concentrations of IL-6, IL-1β, IL-23 and IL-12p70 in the supernatants of the DCs which were cultured with or without DAC were determined by ELISA. (A) The protein concentrations of IL-6, IL-1β, IL-23 and IL-12p70 were significantly increased in BD patients compared with the normal controls (n=12, *p

    Article Snippet: The expression of IL-12p70 and IL-23 were evaluated by the IL-23 ELISA Ready-SET-GO kit and the IL-12(p70) high sensitivity ELISA kit, respectively (both from eBioscience, San Diego, CA, USA) based on the manufacturer's instructions.

    Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

    Synergy between human MAIT cell agonists and TLR agonists. ( A ) Bioactive IL-12p70 in the supernatant of DCs pulsed with E. coli upernatant in the presence or absence of allogeneic MAIT cells and blocking anti-CD40L or anti-MR1 Abs. ( B ) Bioactive IL-12p70 in the supernatant of DCs pulsed with the indicated concentrations of 5-A-RU/MG (ng/ml), LPS (ng/ml), or R848 (μg/ml) in the presence or absence of allogeneic MAIT cells and blocking anti-CD40L or anti-MR1 Abs. ( C ) IFN-γ levels in the supernatant of MAIT-allogeneic DC cocultures pulsed with E. coli supernatant in the presence or absence of blocking anti-CD40L, anti–IL-12, or anti-MR1 Abs. ( D ) IFN-γ levels in the supernatant of MAIT–allogeneic DC cocultures pulsed with the indicated concentrations of 5-A-RU/MG (ng/ml), LPS (ng/ml), or R848 (μg/ml) in the presence or absence of blocking anti-CD40L or anti-MR1 Abs. Data are from one experiment representative of four; data are mean ± SD. Two more donors are shown in Supplemental Fig. 2 .

    Journal: The Journal of Immunology Author Choice

    Article Title: Activation of Human Mucosal-Associated Invariant T Cells Induces CD40L-Dependent Maturation of Monocyte-Derived and Primary Dendritic Cells

    doi: 10.4049/jimmunol.1700615

    Figure Lengend Snippet: Synergy between human MAIT cell agonists and TLR agonists. ( A ) Bioactive IL-12p70 in the supernatant of DCs pulsed with E. coli upernatant in the presence or absence of allogeneic MAIT cells and blocking anti-CD40L or anti-MR1 Abs. ( B ) Bioactive IL-12p70 in the supernatant of DCs pulsed with the indicated concentrations of 5-A-RU/MG (ng/ml), LPS (ng/ml), or R848 (μg/ml) in the presence or absence of allogeneic MAIT cells and blocking anti-CD40L or anti-MR1 Abs. ( C ) IFN-γ levels in the supernatant of MAIT-allogeneic DC cocultures pulsed with E. coli supernatant in the presence or absence of blocking anti-CD40L, anti–IL-12, or anti-MR1 Abs. ( D ) IFN-γ levels in the supernatant of MAIT–allogeneic DC cocultures pulsed with the indicated concentrations of 5-A-RU/MG (ng/ml), LPS (ng/ml), or R848 (μg/ml) in the presence or absence of blocking anti-CD40L or anti-MR1 Abs. Data are from one experiment representative of four; data are mean ± SD. Two more donors are shown in Supplemental Fig. 2 .

    Article Snippet: Simoa immunoassay To detect very low levels of IL-12p70, supernatants were subjected to automatized ELISA analysis with the Simoa HD-1 Analyzer and Single Molecule Array (Simoa) technology (Quanterix), using the Simoa Human IL-12 p70 kit, following the recommendations of the manufacturer ( ).

    Techniques: Blocking Assay

    ( A ) FACS dot plots showing intracellular expression of IL-12 and TNF-α in monocytes and DCs (gated as in Fig. 6 , Supplemental Fig. 4 ), 16 h after incubation with 5-A-RU/MG in the presence or absence of anti-MR1 or anti-CD40L blocking Abs. Profiles of LPS- and CpG-stimulated cells are shown as positive control. ( B ) Cumulative data for the intracellular TNF-α expression described in (A). Data are depicted as box-and-whisker plots, with all points indicated ( n = 4). ( C ) IL-12p70 detected by Simoa immunoassay in whole blood stimulated for 16 h with 5-A-RU/MG in the presence or absence of anti-MR1 or anti-CD40L blocking Abs. Cumulative data ( n = 6; n = 5 for anti-CD40L) are depicted as box-and-whisker plots, with all points indicated ±SD. ( D ) PBMCs were stimulated with 5-A-RU/MG in the presence or absence of blocking Abs to MR1 ( n = 8), IL-18R ( n = 14), and IL-12 and CD40L ( n = 6), and the percentage of IFN-γ–secreting NK cells was determined by FACS after 16 h. The response with Ag was normalized to 100%; we observed background IFN-γ in the majority of the donors tested. Each symbol represents a donor. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Activation of Human Mucosal-Associated Invariant T Cells Induces CD40L-Dependent Maturation of Monocyte-Derived and Primary Dendritic Cells

    doi: 10.4049/jimmunol.1700615

    Figure Lengend Snippet: ( A ) FACS dot plots showing intracellular expression of IL-12 and TNF-α in monocytes and DCs (gated as in Fig. 6 , Supplemental Fig. 4 ), 16 h after incubation with 5-A-RU/MG in the presence or absence of anti-MR1 or anti-CD40L blocking Abs. Profiles of LPS- and CpG-stimulated cells are shown as positive control. ( B ) Cumulative data for the intracellular TNF-α expression described in (A). Data are depicted as box-and-whisker plots, with all points indicated ( n = 4). ( C ) IL-12p70 detected by Simoa immunoassay in whole blood stimulated for 16 h with 5-A-RU/MG in the presence or absence of anti-MR1 or anti-CD40L blocking Abs. Cumulative data ( n = 6; n = 5 for anti-CD40L) are depicted as box-and-whisker plots, with all points indicated ±SD. ( D ) PBMCs were stimulated with 5-A-RU/MG in the presence or absence of blocking Abs to MR1 ( n = 8), IL-18R ( n = 14), and IL-12 and CD40L ( n = 6), and the percentage of IFN-γ–secreting NK cells was determined by FACS after 16 h. The response with Ag was normalized to 100%; we observed background IFN-γ in the majority of the donors tested. Each symbol represents a donor. * p

    Article Snippet: Simoa immunoassay To detect very low levels of IL-12p70, supernatants were subjected to automatized ELISA analysis with the Simoa HD-1 Analyzer and Single Molecule Array (Simoa) technology (Quanterix), using the Simoa Human IL-12 p70 kit, following the recommendations of the manufacturer ( ).

    Techniques: FACS, Expressing, Incubation, Blocking Assay, Positive Control, Whisker Assay

    Role of TLR2 in the induction of IL-12 subunits by F. tularensis LVS-stimulated Mφ or DC. Wild-type or TLR2-deficient Mφ or DC were stimulated with F. tularensis LVS at the indicated MOI, and culture fluids were assayed for either IL-12p70

    Journal:

    Article Title: Toll-Like Receptor 2 Controls the Gamma Interferon Response to Francisella tularensis by Mouse Liver Lymphocytes ▿

    doi: 10.1128/IAI.00561-07

    Figure Lengend Snippet: Role of TLR2 in the induction of IL-12 subunits by F. tularensis LVS-stimulated Mφ or DC. Wild-type or TLR2-deficient Mφ or DC were stimulated with F. tularensis LVS at the indicated MOI, and culture fluids were assayed for either IL-12p70

    Article Snippet: Paired OptEIA antibody sets and cytokine standards for the detection of mouse IFN-γ, IL-12p40, and IL-12p70 were purchased from BD Biosciences.

    Techniques:

    Day-4 DCs produce the highest levels of IL-12p70 and IP-10. Day-2, Day-4, and Day-7 DCs were first pulsed with either UVBL or FTL for 16 h, and then stimulated with LPS (60 EU/ml) and IFN-γ (2000 IU/ml) to determine (A) IL-12p70 and, (B) IP-10 production. Supernatants from the DC-tumor lysate cocultures were collected and evaluated by ELISA as described in Materials and Methods . The results are from 4 different normal healthy donors and are expressed as mean (pg/ml) ± standard errors.

    Journal: PLoS ONE

    Article Title: Day-4 Myeloid Dendritic Cells Pulsed with Whole Tumor Lysate Are Highly Immunogenic and Elicit Potent Anti-Tumor Responses

    doi: 10.1371/journal.pone.0028732

    Figure Lengend Snippet: Day-4 DCs produce the highest levels of IL-12p70 and IP-10. Day-2, Day-4, and Day-7 DCs were first pulsed with either UVBL or FTL for 16 h, and then stimulated with LPS (60 EU/ml) and IFN-γ (2000 IU/ml) to determine (A) IL-12p70 and, (B) IP-10 production. Supernatants from the DC-tumor lysate cocultures were collected and evaluated by ELISA as described in Materials and Methods . The results are from 4 different normal healthy donors and are expressed as mean (pg/ml) ± standard errors.

    Article Snippet: IL-12p70 and IP-10 production by lysate-pulsed dendritic cells IL-12 produced by DCs is critical for complete priming of naive T cells.

    Techniques: Enzyme-linked Immunosorbent Assay

    Activation of allogeneic T cells by monocyte-derived dendritic cells matured in the presence of different stimulatory cocktails. Monocytes from three donors were analyzed for the production of IL-12p70 after an initial 48 h culture with GM-CSF/IL-4 followed by harvest, wash, and thereafter 18 h with GM-CSF/IL-4/IFNγ/R848 together with combinations of TNFα and GMP-grade poly I:C Hiltonol ® or non-GMP-grade poly I:C from either GE Healthcare or Sigma-Aldrich without ( a ) or with addition of LPS ( b ). The monocyte-derived dendritic cells were harvested, washed, and then co-cultured for 4 days with allogeneic bulk T cells before the analysis of IFNγ production ( c , d ). Note the difference in scales of y -axes

    Journal: Cancer Immunology, Immunotherapy

    Article Title: Enhanced stimulation of human tumor-specific T cells by dendritic cells matured in the presence of interferon-γ and multiple toll-like receptor agonists

    doi: 10.1007/s00262-017-2029-4

    Figure Lengend Snippet: Activation of allogeneic T cells by monocyte-derived dendritic cells matured in the presence of different stimulatory cocktails. Monocytes from three donors were analyzed for the production of IL-12p70 after an initial 48 h culture with GM-CSF/IL-4 followed by harvest, wash, and thereafter 18 h with GM-CSF/IL-4/IFNγ/R848 together with combinations of TNFα and GMP-grade poly I:C Hiltonol ® or non-GMP-grade poly I:C from either GE Healthcare or Sigma-Aldrich without ( a ) or with addition of LPS ( b ). The monocyte-derived dendritic cells were harvested, washed, and then co-cultured for 4 days with allogeneic bulk T cells before the analysis of IFNγ production ( c , d ). Note the difference in scales of y -axes

    Article Snippet: Capacity of DC to stimulate IFNγ production in allogeneic T cells (Fig. c, d) correlated significantly with their ability to produce IL-12p70 (Fig. ).

    Techniques: Activation Assay, Derivative Assay, Cell Culture

    Correlation between IL-12p70 production in dendritic cells and IFNγ production by allogeneic bulk T cells stimulated with these dendritic cells. The amount of IL-12p70 produced by monocyte-derived dendritic cells matured by different maturation cocktails was compared to their ability to, after harvest and wash, induce IFNγ production in allogeneic T cells during a 4-day co-culture. Depicted are cytokine levels for 7 donors and p -values for linear regression

    Journal: Cancer Immunology, Immunotherapy

    Article Title: Enhanced stimulation of human tumor-specific T cells by dendritic cells matured in the presence of interferon-γ and multiple toll-like receptor agonists

    doi: 10.1007/s00262-017-2029-4

    Figure Lengend Snippet: Correlation between IL-12p70 production in dendritic cells and IFNγ production by allogeneic bulk T cells stimulated with these dendritic cells. The amount of IL-12p70 produced by monocyte-derived dendritic cells matured by different maturation cocktails was compared to their ability to, after harvest and wash, induce IFNγ production in allogeneic T cells during a 4-day co-culture. Depicted are cytokine levels for 7 donors and p -values for linear regression

    Article Snippet: Capacity of DC to stimulate IFNγ production in allogeneic T cells (Fig. c, d) correlated significantly with their ability to produce IL-12p70 (Fig. ).

    Techniques: Produced, Derivative Assay, Co-Culture Assay

    Production of cytokines by monocyte-derived dendritic cells matured in the presence of different stimulatory cocktails. Monocytes from two donors were screened for the production of IL-12p70 after an initial 48 h culture with GM-CSF/IL-4 followed by harvest, wash, and thereafter 18 h with GM-CSF/IL-4 together with different combinations of TNFα, IFNγ, R848, and the GMP-grade poly I:C Hiltonol ® ( a ). Monocytes from three donors were matured into mDC with the same stimulators as above and also LPS. The supernatants were screened for the production of a panel of different cytokines ( b ). Numbers represent concentrations (pg/ml) of the respective cytokine and the gray scale represents the lowest ( white ) to highest ( black ) concentrations within each donor. Stars indicate that the measured cytokine was added to some cocktails (~35,000 pg/ml IFNγ or 20,000 pg/ml TNFα). Monocytes from three donors were screened for the production of IL-12p70 after an initial 48 h culture with GM-CSF/IL-4 followed by 18 h with IFNγ, R848, Hiltonol, and LPS or with the “gold standard” (TNFα, IL1β, IL-6, PGE2) and “alpha type-1” (TNFα, IL-1β, IFNα, IFNγ, Hiltonol) DC cocktail ( c )

    Journal: Cancer Immunology, Immunotherapy

    Article Title: Enhanced stimulation of human tumor-specific T cells by dendritic cells matured in the presence of interferon-γ and multiple toll-like receptor agonists

    doi: 10.1007/s00262-017-2029-4

    Figure Lengend Snippet: Production of cytokines by monocyte-derived dendritic cells matured in the presence of different stimulatory cocktails. Monocytes from two donors were screened for the production of IL-12p70 after an initial 48 h culture with GM-CSF/IL-4 followed by harvest, wash, and thereafter 18 h with GM-CSF/IL-4 together with different combinations of TNFα, IFNγ, R848, and the GMP-grade poly I:C Hiltonol ® ( a ). Monocytes from three donors were matured into mDC with the same stimulators as above and also LPS. The supernatants were screened for the production of a panel of different cytokines ( b ). Numbers represent concentrations (pg/ml) of the respective cytokine and the gray scale represents the lowest ( white ) to highest ( black ) concentrations within each donor. Stars indicate that the measured cytokine was added to some cocktails (~35,000 pg/ml IFNγ or 20,000 pg/ml TNFα). Monocytes from three donors were screened for the production of IL-12p70 after an initial 48 h culture with GM-CSF/IL-4 followed by 18 h with IFNγ, R848, Hiltonol, and LPS or with the “gold standard” (TNFα, IL1β, IL-6, PGE2) and “alpha type-1” (TNFα, IL-1β, IFNα, IFNγ, Hiltonol) DC cocktail ( c )

    Article Snippet: Capacity of DC to stimulate IFNγ production in allogeneic T cells (Fig. c, d) correlated significantly with their ability to produce IL-12p70 (Fig. ).

    Techniques: Derivative Assay

    Activation of tyrosinase-specific T cells by tyrosinase-loaded monocyte-derived dendritic cells matured in the presence of different stimulatory cocktails. Monocytes from six donors were analyzed for the production of IL-12p70 after an initial 48 h culture with GM-CSF/IL-4 followed by harvest, wash, and thereafter 18 h with GM-CSF/IL-4 together with the combinations of IFNα, TNFγ, R848, GMP-grade poly I:C Hiltonol ® , different forms of LPS, and/or monophosphoryl Lipid A (MPLA). The mature monocyte-derived dendritic cells from the first three donors were then harvested, washed, and either kept unloaded or pulsed with tyrosinase or HCV peptide and co-cultured with allogeneic tyrosinase-specific TCR-transduced T cells. Specific activation was calculated by subtracting the IFNγ response against the control HCV peptide-loaded DC from the IFNγ response against the tyrosinase peptide-loaded DC ( a ). For the last three donors, tumor cell lysate from either tyrosinase-negative A375 cells or tyrosinase-transduced A375 was added during the maturation from immature to mature dendritic cells. Thereafter the dendritic cells were harvested, washed, and co-cultured with the tyrosinase-specific T cells. Specific activation was calculated by subtracting the IFNγ response against the control A375 lysate-loaded DC from the IFNγ response against the tyrosinase-expressing A375 lysate-loaded DC ( b )

    Journal: Cancer Immunology, Immunotherapy

    Article Title: Enhanced stimulation of human tumor-specific T cells by dendritic cells matured in the presence of interferon-γ and multiple toll-like receptor agonists

    doi: 10.1007/s00262-017-2029-4

    Figure Lengend Snippet: Activation of tyrosinase-specific T cells by tyrosinase-loaded monocyte-derived dendritic cells matured in the presence of different stimulatory cocktails. Monocytes from six donors were analyzed for the production of IL-12p70 after an initial 48 h culture with GM-CSF/IL-4 followed by harvest, wash, and thereafter 18 h with GM-CSF/IL-4 together with the combinations of IFNα, TNFγ, R848, GMP-grade poly I:C Hiltonol ® , different forms of LPS, and/or monophosphoryl Lipid A (MPLA). The mature monocyte-derived dendritic cells from the first three donors were then harvested, washed, and either kept unloaded or pulsed with tyrosinase or HCV peptide and co-cultured with allogeneic tyrosinase-specific TCR-transduced T cells. Specific activation was calculated by subtracting the IFNγ response against the control HCV peptide-loaded DC from the IFNγ response against the tyrosinase peptide-loaded DC ( a ). For the last three donors, tumor cell lysate from either tyrosinase-negative A375 cells or tyrosinase-transduced A375 was added during the maturation from immature to mature dendritic cells. Thereafter the dendritic cells were harvested, washed, and co-cultured with the tyrosinase-specific T cells. Specific activation was calculated by subtracting the IFNγ response against the control A375 lysate-loaded DC from the IFNγ response against the tyrosinase-expressing A375 lysate-loaded DC ( b )

    Article Snippet: Capacity of DC to stimulate IFNγ production in allogeneic T cells (Fig. c, d) correlated significantly with their ability to produce IL-12p70 (Fig. ).

    Techniques: Activation Assay, Derivative Assay, Cell Culture, Expressing

    Dexamethasone prevents cocktail induction of IL-12p70 and TNFα . Immature DCs from a single donor shows a dexamethasone-mediated dose dependent suppression of IL-12p70 and TNFα secretion. Dexamethasone was pre-incubated with imDCs for 6 hours (A and C) or 24 hours (B and D) with increasing concentration of dexamethasone at 0-0.01-0.1 and 1.0 μM. Dexamethasone treatment without cocktail did not induce IL-12p70 or TNFα (first 4 bars). The cocktails used are indicated below each set of data, and their exact composition is seen in table 1. This shows one representative example out of three. Cell viability was not significantly affected by treatment with cocktail and/or dexamethasone (not shown).

    Journal: Journal of Inflammation (London, England)

    Article Title: Differential induction of inflammatory cytokines by dendritic cells treated with novel TLR-agonist and cytokine based cocktails: targeting dendritic cells in autoimmunity

    doi: 10.1186/1476-9255-7-37

    Figure Lengend Snippet: Dexamethasone prevents cocktail induction of IL-12p70 and TNFα . Immature DCs from a single donor shows a dexamethasone-mediated dose dependent suppression of IL-12p70 and TNFα secretion. Dexamethasone was pre-incubated with imDCs for 6 hours (A and C) or 24 hours (B and D) with increasing concentration of dexamethasone at 0-0.01-0.1 and 1.0 μM. Dexamethasone treatment without cocktail did not induce IL-12p70 or TNFα (first 4 bars). The cocktails used are indicated below each set of data, and their exact composition is seen in table 1. This shows one representative example out of three. Cell viability was not significantly affected by treatment with cocktail and/or dexamethasone (not shown).

    Article Snippet: The secretion of IL-12p70 was determined for all DC-batches and the exact protein levels shown for each cocktail in figure .

    Techniques: Incubation, Concentration Assay

    Cocktail screening and donor variation for IL-12p70 and TNFα secretion . A total of 15 different donor-derived imDCs were treated with LPS (0.1 μg/mL) or the 9 cocktails as indicated in table 1. After 24 h of incubation, IL-12p70 and TNFα levels in the conditioned media was determined by ELISA. A) The amount of IL-12p70 protein in each donor is indicated by a dot, and the average of all 15 donors indicated by a horizontal bar. B) Amount of TNFα protein was determined similarly in the 15 donors. Data were analyzed using unpaired, two sided t-test, (***P

    Journal: Journal of Inflammation (London, England)

    Article Title: Differential induction of inflammatory cytokines by dendritic cells treated with novel TLR-agonist and cytokine based cocktails: targeting dendritic cells in autoimmunity

    doi: 10.1186/1476-9255-7-37

    Figure Lengend Snippet: Cocktail screening and donor variation for IL-12p70 and TNFα secretion . A total of 15 different donor-derived imDCs were treated with LPS (0.1 μg/mL) or the 9 cocktails as indicated in table 1. After 24 h of incubation, IL-12p70 and TNFα levels in the conditioned media was determined by ELISA. A) The amount of IL-12p70 protein in each donor is indicated by a dot, and the average of all 15 donors indicated by a horizontal bar. B) Amount of TNFα protein was determined similarly in the 15 donors. Data were analyzed using unpaired, two sided t-test, (***P

    Article Snippet: The secretion of IL-12p70 was determined for all DC-batches and the exact protein levels shown for each cocktail in figure .

    Techniques: Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay

    Human immature DCs are activated by B. abortus S19. Monocyte-derived immature DCs were infected with B. abortus S19 (MOI, 20) for 1 h, the bacteria were washed out, and the cells were incubated for another 48 h. Control cells were left uninfected. (A) After 48 h, the phenotype of the cells was determined by flow cytometry (bold lines, infected cells; grey areas, uninfected cells; dotted lines, isotype controls). One representative of at least six independent experiments is shown. (B) Supernatants were analyzed for the presence of IL-12/23p40, IL-12p70, IL-10, and TNF-α. Data expressed as median with interquartile range.

    Journal: PLoS ONE

    Article Title: The Attenuated Brucella abortus Strain 19 Invades, Persists in, and Activates Human Dendritic Cells, and Induces the Secretion of IL-12p70 but Not IL-23

    doi: 10.1371/journal.pone.0065934

    Figure Lengend Snippet: Human immature DCs are activated by B. abortus S19. Monocyte-derived immature DCs were infected with B. abortus S19 (MOI, 20) for 1 h, the bacteria were washed out, and the cells were incubated for another 48 h. Control cells were left uninfected. (A) After 48 h, the phenotype of the cells was determined by flow cytometry (bold lines, infected cells; grey areas, uninfected cells; dotted lines, isotype controls). One representative of at least six independent experiments is shown. (B) Supernatants were analyzed for the presence of IL-12/23p40, IL-12p70, IL-10, and TNF-α. Data expressed as median with interquartile range.

    Article Snippet: Analysis of cytokine secretion by DCs 48 h cell-free supernatants of DC cultures were harvested and stored at –80°C until analysis by sandwich ELISAs for IL-12/23p40, IL-12p70, IL-10, TNF-α (all U-CyTech, Utrecht, The Netherlands), or IL-23 (eBioscience).

    Techniques: Derivative Assay, Infection, Incubation, Flow Cytometry, Cytometry

    B. abortus S19 does not interfere with the cytokine-induced maturation of immature human DCs. Monocyte-derived immature DCs were infected with B. abortus S19 (MOI, 20) for 1 h, the bacteria were washed out, and the cells incubated in the presence of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, PGE 2 ). Uninfected control cells were incubated alongside in the presence or absence of cytokines. (A) After 48 h, the phenotype of the cells was determined by flow cytometry (bold lines, stimulated cells; grey areas, unstimulated cells; dotted lines, isotype controls). One representative of at least six independent experiments is shown. (B) Supernatants were analyzed for the presence of IL-12/23p40, IL-12p70, IL-23, and IL-10 (data expressed as median with interquartile range; n.s., not significant).

    Journal: PLoS ONE

    Article Title: The Attenuated Brucella abortus Strain 19 Invades, Persists in, and Activates Human Dendritic Cells, and Induces the Secretion of IL-12p70 but Not IL-23

    doi: 10.1371/journal.pone.0065934

    Figure Lengend Snippet: B. abortus S19 does not interfere with the cytokine-induced maturation of immature human DCs. Monocyte-derived immature DCs were infected with B. abortus S19 (MOI, 20) for 1 h, the bacteria were washed out, and the cells incubated in the presence of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, PGE 2 ). Uninfected control cells were incubated alongside in the presence or absence of cytokines. (A) After 48 h, the phenotype of the cells was determined by flow cytometry (bold lines, stimulated cells; grey areas, unstimulated cells; dotted lines, isotype controls). One representative of at least six independent experiments is shown. (B) Supernatants were analyzed for the presence of IL-12/23p40, IL-12p70, IL-23, and IL-10 (data expressed as median with interquartile range; n.s., not significant).

    Article Snippet: Analysis of cytokine secretion by DCs 48 h cell-free supernatants of DC cultures were harvested and stored at –80°C until analysis by sandwich ELISAs for IL-12/23p40, IL-12p70, IL-10, TNF-α (all U-CyTech, Utrecht, The Netherlands), or IL-23 (eBioscience).

    Techniques: Derivative Assay, Infection, Incubation, Flow Cytometry, Cytometry

    Streptococcus suis -induced type I interferon produced by dendritic cells (DCs) modulates autocrine cytokine production. Production of tumor necrosis factor (A) , interleukin (IL)-6 (B) , IL-12p70 (C) , CCL2 (D) , CCL3 (E) , and CXCL1 (F) by DCs 16 h following infection with the different S. suis strains. Data represent the mean ± SEM ( n = 4). ** ( p

    Journal: Frontiers in Immunology

    Article Title: Type I Interferon Induced by Streptococcus suis Serotype 2 is Strain-Dependent and May Be Beneficial for Host Survival

    doi: 10.3389/fimmu.2017.01039

    Figure Lengend Snippet: Streptococcus suis -induced type I interferon produced by dendritic cells (DCs) modulates autocrine cytokine production. Production of tumor necrosis factor (A) , interleukin (IL)-6 (B) , IL-12p70 (C) , CCL2 (D) , CCL3 (E) , and CXCL1 (F) by DCs 16 h following infection with the different S. suis strains. Data represent the mean ± SEM ( n = 4). ** ( p

    Article Snippet: Plasmatic concentrations of TNF, IL-6, IL-12p70, CCL2, CCL3, and CXCL1 were measured using a custom-made cytokine Bio-Plex Pro™ assay (Bio-Rad) according to the manufacturer’s instructions.

    Techniques: Produced, Infection

    Type I interferon modulates plasma pro-inflammatory cytokines involved in Streptococcus suis -induced systemic inflammation. Plasma levels of tumor necrosis factor (A) , interleukin (IL)-6 (B) , IL-12p70 (C) , CCL2 (D) , CCL3 (E) , and CXCL1 (F) in wild-type and IFNAR −/− mice 12 h following infection with the different S. suis strains. Data represent the mean ± SEM ( n = 8). * ( p

    Journal: Frontiers in Immunology

    Article Title: Type I Interferon Induced by Streptococcus suis Serotype 2 is Strain-Dependent and May Be Beneficial for Host Survival

    doi: 10.3389/fimmu.2017.01039

    Figure Lengend Snippet: Type I interferon modulates plasma pro-inflammatory cytokines involved in Streptococcus suis -induced systemic inflammation. Plasma levels of tumor necrosis factor (A) , interleukin (IL)-6 (B) , IL-12p70 (C) , CCL2 (D) , CCL3 (E) , and CXCL1 (F) in wild-type and IFNAR −/− mice 12 h following infection with the different S. suis strains. Data represent the mean ± SEM ( n = 8). * ( p

    Article Snippet: Plasmatic concentrations of TNF, IL-6, IL-12p70, CCL2, CCL3, and CXCL1 were measured using a custom-made cytokine Bio-Plex Pro™ assay (Bio-Rad) according to the manufacturer’s instructions.

    Techniques: Mouse Assay, Infection

    Donor- and cytokine specific effect of neonatal plasma on LPS-induced cytokine production Adult PBMC derived from heparinized blood from three separate donors were stimulated with LPS and IFN-γ in the presence of 10% heterologous adult or neonatal plasma. Each dot represents a different plasma donor. After 24h incubation, the extracellular medium was collected for IL-10 and IL-12p70 measurements. Bars represent median values.

    Journal: Clinical immunology (Orlando, Fla.)

    Article Title: Skewed pattern of Toll-like receptor 4-mediated cytokine production in human neonatal blood: Low LPS-induced IL-12p70 and high IL-10 persist throughout the first month of life

    doi: 10.1016/j.clim.2009.07.003

    Figure Lengend Snippet: Donor- and cytokine specific effect of neonatal plasma on LPS-induced cytokine production Adult PBMC derived from heparinized blood from three separate donors were stimulated with LPS and IFN-γ in the presence of 10% heterologous adult or neonatal plasma. Each dot represents a different plasma donor. After 24h incubation, the extracellular medium was collected for IL-10 and IL-12p70 measurements. Bars represent median values.

    Article Snippet: Addition of exogenous IFN-γ has been shown to increase transcription of IL-12p35 and IL-12p40, and to increase LPS-induced release of IL-12p70 , .

    Techniques: Derivative Assay, Incubation

    Distinct TLR4 responses up until the age of one month Cytokine production in cord blood (n=18), neonatal venous blood at age one month (n=96) and adult venous blood (n=17) was measured after a 24h-incubation with LPS+IFN-γ. Cord blood TLR4 responses were characterized by high levels of IL-10 (A) and low levels of IL-12p70 (B). Although LPS+IFN- γ-induced production of IL-10 and IL-12p70 gradually decreased or increased respectively, TLR4-mediated cytokine responses at the age of one month remained significantly impaired compared to adult responses. *, p

    Journal: Clinical immunology (Orlando, Fla.)

    Article Title: Skewed pattern of Toll-like receptor 4-mediated cytokine production in human neonatal blood: Low LPS-induced IL-12p70 and high IL-10 persist throughout the first month of life

    doi: 10.1016/j.clim.2009.07.003

    Figure Lengend Snippet: Distinct TLR4 responses up until the age of one month Cytokine production in cord blood (n=18), neonatal venous blood at age one month (n=96) and adult venous blood (n=17) was measured after a 24h-incubation with LPS+IFN-γ. Cord blood TLR4 responses were characterized by high levels of IL-10 (A) and low levels of IL-12p70 (B). Although LPS+IFN- γ-induced production of IL-10 and IL-12p70 gradually decreased or increased respectively, TLR4-mediated cytokine responses at the age of one month remained significantly impaired compared to adult responses. *, p

    Article Snippet: Addition of exogenous IFN-γ has been shown to increase transcription of IL-12p35 and IL-12p40, and to increase LPS-induced release of IL-12p70 , .

    Techniques: Incubation

    COX-2 activity influences IFN-γ and IL-12p70 production in antigen stimulated cultures of DO11.10 CD4+ T cells. Peritoneal macrophages were isolated and cultured in the presence of medium alone (A) or viable Salmonella (B) . Following exposure for 45 minutes, cells were washed to remove any extracellular bacteria with gentamicin-containing medium. Magnetically separated splenic DO11.10 CD4 + T cells, B cells, and OVA peptide were added. To assess the role of COX-2, some cultures were supplemented with 1 uM celecoxib 20 minutes prior to infection. Levels of IFN-γ (A and B) and IL-12p70 (C) in 48-hour supernatants were measured by enzyme immunoassay, and are reported as mean values of triplicate determinations. These studies were performed three times with similar results.

    Journal: The Open Microbiology Journal

    Article Title: Cyclooxygenase-2 Inhibition Enhances Activation of T Helper Type 1 Responses During Salmonella Infection

    doi: 10.2174/1874285800903010023

    Figure Lengend Snippet: COX-2 activity influences IFN-γ and IL-12p70 production in antigen stimulated cultures of DO11.10 CD4+ T cells. Peritoneal macrophages were isolated and cultured in the presence of medium alone (A) or viable Salmonella (B) . Following exposure for 45 minutes, cells were washed to remove any extracellular bacteria with gentamicin-containing medium. Magnetically separated splenic DO11.10 CD4 + T cells, B cells, and OVA peptide were added. To assess the role of COX-2, some cultures were supplemented with 1 uM celecoxib 20 minutes prior to infection. Levels of IFN-γ (A and B) and IL-12p70 (C) in 48-hour supernatants were measured by enzyme immunoassay, and are reported as mean values of triplicate determinations. These studies were performed three times with similar results.

    Article Snippet: Quantitation of Cytokines in Culture Supernatants and Tissue Homogenates Culture supernatants were assayed for IFN-γ or IL-12p70 by ELISA using the appropriate capture and detection antibody pairs and recombinant standards (PharMingen).

    Techniques: Activity Assay, Isolation, Cell Culture, Infection, Enzyme-linked Immunosorbent Assay

    IFN-γ in supernatants of IL-23 plus IL-1β stimulated CD56 + cells primes monocytes for IL-12p70 production in response to LPS. 1 ·10 5 CD14 + monocytes were stimulated with 100 ng/ml LPS in combination with 2.5 ng/ml IFN-γ, supernatants of unstimulated CD56 + cells, supernatants of IL-23 plus IL-1β stimulated CD56 + cells, or medium alone for 24 hours (A). 1 ·10 5 CD14 + monocytes were stimulated with 100 ng/ml LPS in combination supernatants of IL-23 plus IL-1β stimulated CD56+ cells, plus or minus 2 µg/ml anti-IFN-γ for 24 hours (B). Supernatants were taken and IL-12p70 production was measured by ELISA. Data are means±SD of triplicates from one representative experiment of three.

    Journal: PLoS ONE

    Article Title: Salmonella Induced IL-23 and IL-1? Allow for IL-12 Production by Monocytes and M?1 through Induction of IFN-? in CD56+ NK/NK-Like T Cells

    doi: 10.1371/journal.pone.0008396

    Figure Lengend Snippet: IFN-γ in supernatants of IL-23 plus IL-1β stimulated CD56 + cells primes monocytes for IL-12p70 production in response to LPS. 1 ·10 5 CD14 + monocytes were stimulated with 100 ng/ml LPS in combination with 2.5 ng/ml IFN-γ, supernatants of unstimulated CD56 + cells, supernatants of IL-23 plus IL-1β stimulated CD56 + cells, or medium alone for 24 hours (A). 1 ·10 5 CD14 + monocytes were stimulated with 100 ng/ml LPS in combination supernatants of IL-23 plus IL-1β stimulated CD56+ cells, plus or minus 2 µg/ml anti-IFN-γ for 24 hours (B). Supernatants were taken and IL-12p70 production was measured by ELISA. Data are means±SD of triplicates from one representative experiment of three.

    Article Snippet: To verify that the Mφ1 used in these experiments are capable of producing IL-12p70 they were stimulated with LPS in combination with IFN-γ.

    Techniques: Enzyme-linked Immunosorbent Assay

    Blocking of demyelination in HSV-IL-2 infected mice by IL-12p70 HSV-1 recombinant virus BALB/c mice were co-infected with HSV-IL-2 + HSV-IL-12p70 at 1:1, 1:10, or 1:100 ratios of HSV-IL-2 to HSV-IL-12p70. On day 14 post-infection, optic nerves and spinal cords were collected, fixed, sectioned, and stained with LFB. Representative photomicrographs are shown. Arrows indicate areas of demyelination. 10X direct magnification.

    Journal: Gene therapy

    Article Title: Use of cytokine immunotherapy to block CNS demyelination induced by a recombinant HSV-1 expressing IL-2

    doi: 10.1038/gt.2011.32

    Figure Lengend Snippet: Blocking of demyelination in HSV-IL-2 infected mice by IL-12p70 HSV-1 recombinant virus BALB/c mice were co-infected with HSV-IL-2 + HSV-IL-12p70 at 1:1, 1:10, or 1:100 ratios of HSV-IL-2 to HSV-IL-12p70. On day 14 post-infection, optic nerves and spinal cords were collected, fixed, sectioned, and stained with LFB. Representative photomicrographs are shown. Arrows indicate areas of demyelination. 10X direct magnification.

    Article Snippet: DNA injection The complete open-reading frames (ORF) for IL-12p70, IL-23, IL-27, and IL-35 (pORF-mIL12, pORF-mIL23, pORF-mIL27, pORF-mIL35) were purchased from InvivoGen (San Diego, CA), while the ORF for IL-12p35 and IL-12p40 was inserted into pVR1055 as we described previously .

    Techniques: Blocking Assay, Infection, Mouse Assay, Recombinant, Staining

    Effects of IL-12 family members on HSV-IL-2-induced demyelination BALB/c mice were injected intramuscularly 3X with IL-12p70 DNA, IL-23 DNA, IL-27 DNA, IL-35 DNA as described in Materials and Methods. Four hours after the third DNA injection, mice were infected ocularly with HSV-IL-2. On day 14 post infection, optic nerves and spinal cords were collected, fixed, sectioned, and stained with LFB. Representative photomicrographs are shown. Arrows indicate areas of demyelination. 10X direct magnification.

    Journal: Gene therapy

    Article Title: Use of cytokine immunotherapy to block CNS demyelination induced by a recombinant HSV-1 expressing IL-2

    doi: 10.1038/gt.2011.32

    Figure Lengend Snippet: Effects of IL-12 family members on HSV-IL-2-induced demyelination BALB/c mice were injected intramuscularly 3X with IL-12p70 DNA, IL-23 DNA, IL-27 DNA, IL-35 DNA as described in Materials and Methods. Four hours after the third DNA injection, mice were infected ocularly with HSV-IL-2. On day 14 post infection, optic nerves and spinal cords were collected, fixed, sectioned, and stained with LFB. Representative photomicrographs are shown. Arrows indicate areas of demyelination. 10X direct magnification.

    Article Snippet: DNA injection The complete open-reading frames (ORF) for IL-12p70, IL-23, IL-27, and IL-35 (pORF-mIL12, pORF-mIL23, pORF-mIL27, pORF-mIL35) were purchased from InvivoGen (San Diego, CA), while the ORF for IL-12p35 and IL-12p40 was inserted into pVR1055 as we described previously .

    Techniques: Mouse Assay, Injection, Infection, Staining

    C57Bl/6 transgenic mice harboring the SJL/J variant of the IL-12p40 gene or the C57Bl/6 variant were challenged with a sublethal dose of LPS after which the concentration in serum of IL-12p70 and IL-12p40 was measured by ELISA. Bars represent mean ±

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

    Article Title: A Polymorphism in the Coding Region of Il12b promotes IL-12p70 and IL-23 Heterodimer Formation

    doi: 10.4049/jimmunol.1001330

    Figure Lengend Snippet: C57Bl/6 transgenic mice harboring the SJL/J variant of the IL-12p40 gene or the C57Bl/6 variant were challenged with a sublethal dose of LPS after which the concentration in serum of IL-12p70 and IL-12p40 was measured by ELISA. Bars represent mean ±

    Article Snippet: The IL-12p40 and IL-12p70 kits were obtained from PharMingen (PharMingen, Alphen a/d Rijn, The Netherlands), the IL-23 kit was purchased from eBioscience (ITK diagnostics, Uithoorn, The Netherlands).

    Techniques: Transgenic Assay, Mouse Assay, Variant Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

    A. IL-12p70 synthesis relative to IL-12p40 synthesis.

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

    Article Title: A Polymorphism in the Coding Region of Il12b promotes IL-12p70 and IL-23 Heterodimer Formation

    doi: 10.4049/jimmunol.1001330

    Figure Lengend Snippet: A. IL-12p70 synthesis relative to IL-12p40 synthesis.

    Article Snippet: The IL-12p40 and IL-12p70 kits were obtained from PharMingen (PharMingen, Alphen a/d Rijn, The Netherlands), the IL-23 kit was purchased from eBioscience (ITK diagnostics, Uithoorn, The Netherlands).

    Techniques:

    Interferon (IFN)-γ secretion in response to activation with live bacille Calmette–Guérin (BCG) and Toll-like receptor (TLR) agonists. Results are expressed as IFN-γ secretion (pg/ml) per IFN-γ-producing cells × 10 3 . (a) Monocyte/macrophage and T cell interaction. Whole blood was stimulated with live BCG at a multiplicity of infection (MOI) of 20 BCG/leucocytes, or with BCG plus interleukin (IL)-12p70 (20 ng/ml). Production of IFN-γ (pg/ml) was analysed after 48 h of culture. (b) Lipopolysaccharide (LPS) activation. Whole blood was stimulated with LPS from Salmonella minnesota R595 at 10 μg/ml or with LPS plus IL-12p70 (20 ng/ml). Production of IFN-γ (pg/ml) was analysed after 48 h of culture. (c) Heat-killed Staphylococcus aureus (SAC) activation. Whole blood was stimulated with SAC at 5 × 10 6 particles/ml, or with SAC plus IL-12p70 (20 ng/ml). Production of IFN-γ (pg/ml) was analysed after 48 h of culture.

    Journal: Clinical and Experimental Immunology

    Article Title: Impaired interferon-? production in response to live bacteria and Toll-like receptor agonists in patients with ataxia telangiectasia

    doi: 10.1111/j.1365-2249.2006.03221.x

    Figure Lengend Snippet: Interferon (IFN)-γ secretion in response to activation with live bacille Calmette–Guérin (BCG) and Toll-like receptor (TLR) agonists. Results are expressed as IFN-γ secretion (pg/ml) per IFN-γ-producing cells × 10 3 . (a) Monocyte/macrophage and T cell interaction. Whole blood was stimulated with live BCG at a multiplicity of infection (MOI) of 20 BCG/leucocytes, or with BCG plus interleukin (IL)-12p70 (20 ng/ml). Production of IFN-γ (pg/ml) was analysed after 48 h of culture. (b) Lipopolysaccharide (LPS) activation. Whole blood was stimulated with LPS from Salmonella minnesota R595 at 10 μg/ml or with LPS plus IL-12p70 (20 ng/ml). Production of IFN-γ (pg/ml) was analysed after 48 h of culture. (c) Heat-killed Staphylococcus aureus (SAC) activation. Whole blood was stimulated with SAC at 5 × 10 6 particles/ml, or with SAC plus IL-12p70 (20 ng/ml). Production of IFN-γ (pg/ml) was analysed after 48 h of culture.

    Article Snippet: Cell culture supernatants were taken at 18, 24 and 48 h and their cytokine concentrations were analysed by ELISA, using the human Quantikine IL-12p40 and IL-12p70 kits from R & D Systems, the human Pelikin or Pelipair IFN-γ kit from Sanquin (Amsterdam, Netherlands) and the human IL-10 kit from Sanquin, according to the manufacturers’ guidelines.

    Techniques: Activation Assay, Infection

    Mitogen-triggered T cell activation. Whole blood of ataxia telangiecasia patients and controls was stimulated with phytohaemagglutinin (PHA) (final dilution 1/700), or with PHA plus recombinant interleukin (IL)-12p70 (20 ng/ml). Secretion of interferon (IFN)-γ (pg/ml) was analysed after 48 h of culture. (a) IFN-γ secretion (pg/ml) after stimulation of whole blood; (b) IFN-γ secretion (pg/ml) after stimulation of whole blood per IFN-γ-producing cells × 10 3 .

    Journal: Clinical and Experimental Immunology

    Article Title: Impaired interferon-? production in response to live bacteria and Toll-like receptor agonists in patients with ataxia telangiectasia

    doi: 10.1111/j.1365-2249.2006.03221.x

    Figure Lengend Snippet: Mitogen-triggered T cell activation. Whole blood of ataxia telangiecasia patients and controls was stimulated with phytohaemagglutinin (PHA) (final dilution 1/700), or with PHA plus recombinant interleukin (IL)-12p70 (20 ng/ml). Secretion of interferon (IFN)-γ (pg/ml) was analysed after 48 h of culture. (a) IFN-γ secretion (pg/ml) after stimulation of whole blood; (b) IFN-γ secretion (pg/ml) after stimulation of whole blood per IFN-γ-producing cells × 10 3 .

    Article Snippet: Cell culture supernatants were taken at 18, 24 and 48 h and their cytokine concentrations were analysed by ELISA, using the human Quantikine IL-12p40 and IL-12p70 kits from R & D Systems, the human Pelikin or Pelipair IFN-γ kit from Sanquin (Amsterdam, Netherlands) and the human IL-10 kit from Sanquin, according to the manufacturers’ guidelines.

    Techniques: Activation Assay, Recombinant

    Day-4 DCs produce the highest levels of IL-12p70 and IP-10. Day-2, Day-4, and Day-7 DCs were first pulsed with either UVBL or FTL for 16 h, and then stimulated with LPS (60 EU/ml) and IFN-γ (2000 IU/ml) to determine (A) IL-12p70 and, (B) IP-10 production. Supernatants from the DC-tumor lysate cocultures were collected and evaluated by ELISA as described in Materials and Methods . The results are from 4 different normal healthy donors and are expressed as mean (pg/ml) ± standard errors.

    Journal: PLoS ONE

    Article Title: Day-4 Myeloid Dendritic Cells Pulsed with Whole Tumor Lysate Are Highly Immunogenic and Elicit Potent Anti-Tumor Responses

    doi: 10.1371/journal.pone.0028732

    Figure Lengend Snippet: Day-4 DCs produce the highest levels of IL-12p70 and IP-10. Day-2, Day-4, and Day-7 DCs were first pulsed with either UVBL or FTL for 16 h, and then stimulated with LPS (60 EU/ml) and IFN-γ (2000 IU/ml) to determine (A) IL-12p70 and, (B) IP-10 production. Supernatants from the DC-tumor lysate cocultures were collected and evaluated by ELISA as described in Materials and Methods . The results are from 4 different normal healthy donors and are expressed as mean (pg/ml) ± standard errors.

    Article Snippet: Briefly, NUNC MaxiSorp™ 96-well ELISA plates (Fisher Scientific, Rochester, NY, USA) were coated with capture IL-12p70 (BioLegend, San Diego, CA, USA) or IP-10 antibody (final concentration of 1.5 ng/ml; BD Pharmingen) for 2 h at 37°C, 5% CO2 .

    Techniques: Enzyme-linked Immunosorbent Assay

    COX-2 activity influences IFN-γ and IL-12p70 production in antigen stimulated cultures of DO11.10 CD4+ T cells. Peritoneal macrophages were isolated and cultured in the presence of medium alone (A) or viable Salmonella (B) . Following exposure for 45 minutes, cells were washed to remove any extracellular bacteria with gentamicin-containing medium. Magnetically separated splenic DO11.10 CD4 + T cells, B cells, and OVA peptide were added. To assess the role of COX-2, some cultures were supplemented with 1 uM celecoxib 20 minutes prior to infection. Levels of IFN-γ (A and B) and IL-12p70 (C) in 48-hour supernatants were measured by enzyme immunoassay, and are reported as mean values of triplicate determinations. These studies were performed three times with similar results.

    Journal: The Open Microbiology Journal

    Article Title: Cyclooxygenase-2 Inhibition Enhances Activation of T Helper Type 1 Responses During Salmonella Infection

    doi: 10.2174/1874285800903010023

    Figure Lengend Snippet: COX-2 activity influences IFN-γ and IL-12p70 production in antigen stimulated cultures of DO11.10 CD4+ T cells. Peritoneal macrophages were isolated and cultured in the presence of medium alone (A) or viable Salmonella (B) . Following exposure for 45 minutes, cells were washed to remove any extracellular bacteria with gentamicin-containing medium. Magnetically separated splenic DO11.10 CD4 + T cells, B cells, and OVA peptide were added. To assess the role of COX-2, some cultures were supplemented with 1 uM celecoxib 20 minutes prior to infection. Levels of IFN-γ (A and B) and IL-12p70 (C) in 48-hour supernatants were measured by enzyme immunoassay, and are reported as mean values of triplicate determinations. These studies were performed three times with similar results.

    Article Snippet: CD4+ T cells from BALB/c animals were stimulated with 50 pg/ml recombinant IL-12p70 (PharMingen).

    Techniques: Activity Assay, Isolation, Cell Culture, Infection, Enzyme-linked Immunosorbent Assay

    The impact of CD40 and CD40L deficiency on IL-12 and IFN-γ production. IL-12p70, IL-12p40, and IFN-γ production, measured by classical sandwich ELISA, in a mixture of purified monocytes and T cells, as indicated, upon stimulation with PHA from a healthy donor with (A) CD40L- and (B) CD40-deficient PBMCs. MDDCs obtained from two healthy controls (C2 and C3), P2 and P3, after 24 h of incubation alone or with LPS (1 μg/ml) plus IL-1β (10 ng/ml) as a control of CD40-independent activation, cocultured with L-cells transfected with human CD40L (L-cell-hCD40L) and nontransfected L-cells (L-cell): (C) IL-12p70, IL-12p40, TNF-α, and IL-6 production, measured by classical sandwich ELISA, and (D) FACS analysis of cell surface expression of CD40, CD80, and CD86 costimulatory molecules gating on CD1a-positive MDDCs. (E) Intracellular staining of NEMO protein in MDDCs from P2 and P3, and their respective controls (C2 and C3) in the experimental conditions of Fig. 3 . (F) IgE secretion by B cells in vitro, as measured by ELISA, after the activation of PBMCs from 20 healthy controls (C+, •), P2 (□), and a CD40-deficient patient as the negative control (C−,▵), with soluble CD40L in combination with IL-4. The results shown are representative of two independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: X-linked susceptibility to mycobacteria is caused by mutations in NEMO impairing CD40-dependent IL-12 production

    doi: 10.1084/jem.20060085

    Figure Lengend Snippet: The impact of CD40 and CD40L deficiency on IL-12 and IFN-γ production. IL-12p70, IL-12p40, and IFN-γ production, measured by classical sandwich ELISA, in a mixture of purified monocytes and T cells, as indicated, upon stimulation with PHA from a healthy donor with (A) CD40L- and (B) CD40-deficient PBMCs. MDDCs obtained from two healthy controls (C2 and C3), P2 and P3, after 24 h of incubation alone or with LPS (1 μg/ml) plus IL-1β (10 ng/ml) as a control of CD40-independent activation, cocultured with L-cells transfected with human CD40L (L-cell-hCD40L) and nontransfected L-cells (L-cell): (C) IL-12p70, IL-12p40, TNF-α, and IL-6 production, measured by classical sandwich ELISA, and (D) FACS analysis of cell surface expression of CD40, CD80, and CD86 costimulatory molecules gating on CD1a-positive MDDCs. (E) Intracellular staining of NEMO protein in MDDCs from P2 and P3, and their respective controls (C2 and C3) in the experimental conditions of Fig. 3 . (F) IgE secretion by B cells in vitro, as measured by ELISA, after the activation of PBMCs from 20 healthy controls (C+, •), P2 (□), and a CD40-deficient patient as the negative control (C−,▵), with soluble CD40L in combination with IL-4. The results shown are representative of two independent experiments.

    Article Snippet: Whole blood samples were diluted 1/2 in RPMI 1640 (GIBCO BRL) and infected by incubation with live M. bovis BCG (Pasteur substrain), at a multiplicity of infection of 20:1, alone or with recombinant IFN-γ (5,000 IU/ml; Imukin, Boehringer Ingelheim) or recombinant IL-12p70 (20 ng/ml; R & D Systems), LPS (from Salmonella minnesota , 1 μg/ml; Sigma-Aldrich), TNF-α (20 ng/ml; R & D Systems), IL-1β (10 ng/ml; R & D Systems), phorbol 12-myristate 13-acetate (PMA) (10−7 M; Sigma-Aldrich) with ionomycin (10−5 M; Sigma-Aldrich) and supernatants were recovered after 14 and 48 h. Cytokine production was normalized according to the number of PBMCs in the individual tested.

    Techniques: Sandwich ELISA, Purification, Incubation, Activation Assay, Transfection, FACS, Expressing, Staining, In Vitro, Enzyme-linked Immunosorbent Assay, Negative Control

    IL-12 and IFN-γ production by leukocytes. Cytokine production by PBMCs from 10 healthy donors, (C+, •), P2 (□), and P3 (◯) in response to PHA, alone or in combination with recombinant IL-12 (20 ng/ml), IL-23 (20 ng/ml), or IFNγ (5,000 UI/ml), anti-CD3 (10 ng/ml), LPS (1 μg/ml) and PMA/ionomycin (10 −7 /10 −5 M). (A) IFN-γ, (B) IL-12p70, (C) IL-12p40 production. (D) Cytokine production by whole blood cells from 50 healthy donors, P2, and P3 upon stimulation with live BCG alone or in combination with IL-12 (20 ng/ml) or IFN-γ (5,000 IU/ml): IFN-γ (top), IL-12p70 (middle), and IL-12p40 (bottom) secretion. For each patient, the results shown are representative of two independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: X-linked susceptibility to mycobacteria is caused by mutations in NEMO impairing CD40-dependent IL-12 production

    doi: 10.1084/jem.20060085

    Figure Lengend Snippet: IL-12 and IFN-γ production by leukocytes. Cytokine production by PBMCs from 10 healthy donors, (C+, •), P2 (□), and P3 (◯) in response to PHA, alone or in combination with recombinant IL-12 (20 ng/ml), IL-23 (20 ng/ml), or IFNγ (5,000 UI/ml), anti-CD3 (10 ng/ml), LPS (1 μg/ml) and PMA/ionomycin (10 −7 /10 −5 M). (A) IFN-γ, (B) IL-12p70, (C) IL-12p40 production. (D) Cytokine production by whole blood cells from 50 healthy donors, P2, and P3 upon stimulation with live BCG alone or in combination with IL-12 (20 ng/ml) or IFN-γ (5,000 IU/ml): IFN-γ (top), IL-12p70 (middle), and IL-12p40 (bottom) secretion. For each patient, the results shown are representative of two independent experiments.

    Article Snippet: Whole blood samples were diluted 1/2 in RPMI 1640 (GIBCO BRL) and infected by incubation with live M. bovis BCG (Pasteur substrain), at a multiplicity of infection of 20:1, alone or with recombinant IFN-γ (5,000 IU/ml; Imukin, Boehringer Ingelheim) or recombinant IL-12p70 (20 ng/ml; R & D Systems), LPS (from Salmonella minnesota , 1 μg/ml; Sigma-Aldrich), TNF-α (20 ng/ml; R & D Systems), IL-1β (10 ng/ml; R & D Systems), phorbol 12-myristate 13-acetate (PMA) (10−7 M; Sigma-Aldrich) with ionomycin (10−5 M; Sigma-Aldrich) and supernatants were recovered after 14 and 48 h. Cytokine production was normalized according to the number of PBMCs in the individual tested.

    Techniques: Recombinant

    IL-12 and IFN-γ production by cocultured monocytes and T cells from the patients studied and healthy controls. (A) IL-12p70, IL-12p40, and IFN-γ production, measured using classical sandwich ELISA, in a mixture of purified monocytes and T cells, as indicated upon stimulation with PHA. The results shown are representative of three independent experiments for P2 and one for P3. (B) The same coculture supernatants were analyzed for a multiplex of 16 cytokines, using the Bioplex array. Each column represents the data for one monocyte–T cell coculture system, and all four columns correspond to the same experiment. Each row corresponds to one cytokine. The gray-scale bar indicates the magnitude of cytokine expression, using the control/control (C/C) coculture system as a reference. For each data point, the amount of cytokine produced in the unstimulated system was subtracted from that produced in the PHA-activated system, and the result obtained was compared with the reference value (C/C). The production of *MCP-1 and *MIP-1β by monocytes was PHA-dependent but T cell–independent, as monocytes responded to PHA by producing large amounts of these cytokines, whereas the addition of T cells did not increase cytokine production. The defects in the production of IL-6, IL-12p70, G-CSF, IFN-γ, and MCP-1 were confirmed in three independent experiments on blood cells from P2 and one experiment on blood from P3.

    Journal: The Journal of Experimental Medicine

    Article Title: X-linked susceptibility to mycobacteria is caused by mutations in NEMO impairing CD40-dependent IL-12 production

    doi: 10.1084/jem.20060085

    Figure Lengend Snippet: IL-12 and IFN-γ production by cocultured monocytes and T cells from the patients studied and healthy controls. (A) IL-12p70, IL-12p40, and IFN-γ production, measured using classical sandwich ELISA, in a mixture of purified monocytes and T cells, as indicated upon stimulation with PHA. The results shown are representative of three independent experiments for P2 and one for P3. (B) The same coculture supernatants were analyzed for a multiplex of 16 cytokines, using the Bioplex array. Each column represents the data for one monocyte–T cell coculture system, and all four columns correspond to the same experiment. Each row corresponds to one cytokine. The gray-scale bar indicates the magnitude of cytokine expression, using the control/control (C/C) coculture system as a reference. For each data point, the amount of cytokine produced in the unstimulated system was subtracted from that produced in the PHA-activated system, and the result obtained was compared with the reference value (C/C). The production of *MCP-1 and *MIP-1β by monocytes was PHA-dependent but T cell–independent, as monocytes responded to PHA by producing large amounts of these cytokines, whereas the addition of T cells did not increase cytokine production. The defects in the production of IL-6, IL-12p70, G-CSF, IFN-γ, and MCP-1 were confirmed in three independent experiments on blood cells from P2 and one experiment on blood from P3.

    Article Snippet: Whole blood samples were diluted 1/2 in RPMI 1640 (GIBCO BRL) and infected by incubation with live M. bovis BCG (Pasteur substrain), at a multiplicity of infection of 20:1, alone or with recombinant IFN-γ (5,000 IU/ml; Imukin, Boehringer Ingelheim) or recombinant IL-12p70 (20 ng/ml; R & D Systems), LPS (from Salmonella minnesota , 1 μg/ml; Sigma-Aldrich), TNF-α (20 ng/ml; R & D Systems), IL-1β (10 ng/ml; R & D Systems), phorbol 12-myristate 13-acetate (PMA) (10−7 M; Sigma-Aldrich) with ionomycin (10−5 M; Sigma-Aldrich) and supernatants were recovered after 14 and 48 h. Cytokine production was normalized according to the number of PBMCs in the individual tested.

    Techniques: Sandwich ELISA, Purification, Multiplex Assay, Expressing, Produced

    Influence of ILT2/ILT5 on the cytokine secretion properties of dendritic cells. (A and B) Secretion of IL-12p70, IL-6, or TNF-α by mDC after stimulation with TLR7/8 ligands in the presence of ILT2 (A) or ILT5 (B) antibodies ( n = 10 study

    Journal: Journal of Virology

    Article Title: Leukocyte Immunoglobulin-Like Receptors Maintain Unique Antigen-Presenting Properties of Circulating Myeloid Dendritic Cells in HIV-1-Infected Elite Controllers ▿

    doi: 10.1128/JVI.01009-10

    Figure Lengend Snippet: Influence of ILT2/ILT5 on the cytokine secretion properties of dendritic cells. (A and B) Secretion of IL-12p70, IL-6, or TNF-α by mDC after stimulation with TLR7/8 ligands in the presence of ILT2 (A) or ILT5 (B) antibodies ( n = 10 study

    Article Snippet: For this purpose, PBMC were stimulated with TLR7/8 ligands in the presence or absence of ILT2/5-blocking monoclonal antibodies, and the secretion of TNF-α, IL-6, and IL-12p70 by mDC was assessed after 20 h using flow cytometry.

    Techniques: