Structured Review

Becton Dickinson il 12p40
Intracellular cytokine staining and infection status of individual cells. MDDCs were infected with IAV for 4 h before SP was added. After co-infection, brefeldin A or monensin was added to the cells to allow accumulation of intracellular cytokines. The cells were then fixed with paraformaldehyde, stained with specific antibodies and analysed for the presence of IAV nucleoprotein and TNFα, IL-6, <t>IL-12p40</t> and IL-12p70 by FACS. The graphs show one representative donor of three. Infection status of single cells in co-infected cultures. MDDCs were seeded on glass slides and sequentially infected as described. The cells were fixed 4 h after addition of SP and stained with specific antibodies for SP (green), IAV (red) and Hoechst DNA stain (blue). Four hundred cells per donor were examined. Numbers in the right column show the average frequency of the observed pattern ± SD from three independent experiments with different donors.
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1) Product Images from "Influenza A virus-mediated priming enhances cytokine secretion by human dendritic cells infected with Streptococcus pneumoniae"

Article Title: Influenza A virus-mediated priming enhances cytokine secretion by human dendritic cells infected with Streptococcus pneumoniae

Journal: Cellular Microbiology

doi: 10.1111/cmi.12122

Intracellular cytokine staining and infection status of individual cells. MDDCs were infected with IAV for 4 h before SP was added. After co-infection, brefeldin A or monensin was added to the cells to allow accumulation of intracellular cytokines. The cells were then fixed with paraformaldehyde, stained with specific antibodies and analysed for the presence of IAV nucleoprotein and TNFα, IL-6, IL-12p40 and IL-12p70 by FACS. The graphs show one representative donor of three. Infection status of single cells in co-infected cultures. MDDCs were seeded on glass slides and sequentially infected as described. The cells were fixed 4 h after addition of SP and stained with specific antibodies for SP (green), IAV (red) and Hoechst DNA stain (blue). Four hundred cells per donor were examined. Numbers in the right column show the average frequency of the observed pattern ± SD from three independent experiments with different donors.
Figure Legend Snippet: Intracellular cytokine staining and infection status of individual cells. MDDCs were infected with IAV for 4 h before SP was added. After co-infection, brefeldin A or monensin was added to the cells to allow accumulation of intracellular cytokines. The cells were then fixed with paraformaldehyde, stained with specific antibodies and analysed for the presence of IAV nucleoprotein and TNFα, IL-6, IL-12p40 and IL-12p70 by FACS. The graphs show one representative donor of three. Infection status of single cells in co-infected cultures. MDDCs were seeded on glass slides and sequentially infected as described. The cells were fixed 4 h after addition of SP and stained with specific antibodies for SP (green), IAV (red) and Hoechst DNA stain (blue). Four hundred cells per donor were examined. Numbers in the right column show the average frequency of the observed pattern ± SD from three independent experiments with different donors.

Techniques Used: Staining, Infection, FACS

Type I IFNs are responsible for the priming of DCs to produce IL-12p70. MDDCs were incubated with different concentrations of recombinant human IFN-α 4 h prior to infection with SP. The cells were further incubated for 18 h before the concentration of IL-12p70 in the supernatants was measured by ELISA. MDDCs were sequentially infected with IAV and SP in the presence of neutralizing antibodies against IFN-α, IFN-β and IFNAR. After 22 h incubation the cells were lysed and total RNA was isolated and assayed for the presence of IL-12p35 and IL-12p40 mRNA. Ct values were normalized against γ-actin and the relative induction of the genes was calculated using the ΔΔCt method. Values represent mean ± SEM three independent experiments with different donors (A) or one representative donor of 3 (B, C). Statistical analysis was performed using paired Student’s t -test. (* P
Figure Legend Snippet: Type I IFNs are responsible for the priming of DCs to produce IL-12p70. MDDCs were incubated with different concentrations of recombinant human IFN-α 4 h prior to infection with SP. The cells were further incubated for 18 h before the concentration of IL-12p70 in the supernatants was measured by ELISA. MDDCs were sequentially infected with IAV and SP in the presence of neutralizing antibodies against IFN-α, IFN-β and IFNAR. After 22 h incubation the cells were lysed and total RNA was isolated and assayed for the presence of IL-12p35 and IL-12p40 mRNA. Ct values were normalized against γ-actin and the relative induction of the genes was calculated using the ΔΔCt method. Values represent mean ± SEM three independent experiments with different donors (A) or one representative donor of 3 (B, C). Statistical analysis was performed using paired Student’s t -test. (* P

Techniques Used: Incubation, Recombinant, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Isolation

Enhanced IL-12p70 secretion is due to a specific induction of the IL-12p35 subunit. MDDCs were infected with IAV for 4 h before SP was added. The cells were then incubated for 18 h prior to monitoring the concentration of IL-12p40 (A) and IL-23 (B) in the supernatants by ELISA. Values represent mean ± SEM of five (A) or four (B) independent experiments with different donors. The transcription of the IL-12p40 (C) and IL-12p35 (D) subunits of IL-12p70 were monitored in MDDCs that were co-infected as described above. After 22 h incubation the cells were lysed and total RNA was isolated and assayed for the presence of IL-12p35 and IL-12p40 mRNA. Ct values were normalized against γ-actin and the relative induction of the genes was calculated using the ΔΔCt method. Values represent mean ± SEM of three independent experiments with different donors. Statistical analysis was performed using paired Student’s t -test. (* P
Figure Legend Snippet: Enhanced IL-12p70 secretion is due to a specific induction of the IL-12p35 subunit. MDDCs were infected with IAV for 4 h before SP was added. The cells were then incubated for 18 h prior to monitoring the concentration of IL-12p40 (A) and IL-23 (B) in the supernatants by ELISA. Values represent mean ± SEM of five (A) or four (B) independent experiments with different donors. The transcription of the IL-12p40 (C) and IL-12p35 (D) subunits of IL-12p70 were monitored in MDDCs that were co-infected as described above. After 22 h incubation the cells were lysed and total RNA was isolated and assayed for the presence of IL-12p35 and IL-12p40 mRNA. Ct values were normalized against γ-actin and the relative induction of the genes was calculated using the ΔΔCt method. Values represent mean ± SEM of three independent experiments with different donors. Statistical analysis was performed using paired Student’s t -test. (* P

Techniques Used: Infection, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Isolation

2) Product Images from "Lipoxin A4 and 15-Epi-Lipoxin A4 Protect against Experimental Cerebral Malaria by Inhibiting IL-12/IFN-? in the Brain"

Article Title: Lipoxin A4 and 15-Epi-Lipoxin A4 Protect against Experimental Cerebral Malaria by Inhibiting IL-12/IFN-? in the Brain

Journal: PLoS ONE

doi: 10.1371/journal.pone.0061882

Enhanced cytokine expression during P. berghei ANKA infection in 5-LO-deficient mice. C57Bl/6 WT and Alox5 −/− ( n = 4 mice/group) mice were infected i.p. with P. berghei ANKA strain. At 1 and 3 days after infection, animals were bled and serum levels of IL-12p40 ( A ) and IFN- γ ( B ) were determined by ELISA. Five days after infection, mice were sacrificed and brains, livers and spleens harvested. Tissues were homogenized, total RNA extracted and reverse transcripted for real-time RT-PCR determination of il12a ( C ), il12b ( D ) and ifng ( E ) expression. Data shown are representative of one out of three independent experiments performed. Statistical differences were determined using Mann Whitney test.
Figure Legend Snippet: Enhanced cytokine expression during P. berghei ANKA infection in 5-LO-deficient mice. C57Bl/6 WT and Alox5 −/− ( n = 4 mice/group) mice were infected i.p. with P. berghei ANKA strain. At 1 and 3 days after infection, animals were bled and serum levels of IL-12p40 ( A ) and IFN- γ ( B ) were determined by ELISA. Five days after infection, mice were sacrificed and brains, livers and spleens harvested. Tissues were homogenized, total RNA extracted and reverse transcripted for real-time RT-PCR determination of il12a ( C ), il12b ( D ) and ifng ( E ) expression. Data shown are representative of one out of three independent experiments performed. Statistical differences were determined using Mann Whitney test.

Techniques Used: Expressing, Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, MANN-WHITNEY

3) Product Images from "Vitamin D Regulates Cytokine Patterns Secreted by Dendritic Cells to Promote Differentiation of IL-22-Producing T Cells"

Article Title: Vitamin D Regulates Cytokine Patterns Secreted by Dendritic Cells to Promote Differentiation of IL-22-Producing T Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0130395

Effect of 25D on differentiation and/or stimulation of monocyte-derived DCs. Monocytes were differentiated for six days using rGM-CSF and rIL-4 in media with 10% FCS in the presence (25D diff -DCs or 25D diff/stim -DCs) or absence (serum-DCs or 25D stim -DCs) of additional 25D (10 −7 M). Subsequently, 25D-DCs and serum-DCs were stimulated with TLR2/1L (1 μg/ml) or left untreated in the presence (25D diff/stim -DCs or 25D stim -DCs) or absence (serum-DCs or 25D diff -DCs) of additional 25D (10 −7 M), and cultured in fresh media with 10% FCS for 18–24 hours. TNF-α, IL-6, IL-1β, IL-12p40, IL-12p70, IL-23 and IL-10, as well as total and active TGF-β levels in culture supernatants were measured by ELISA or CBA (mean cytokine levels in ng/ml ± SEM, n = 4). Results shown in Fig 1 and Fig. 2 were conducted using cells from the same cell preparations of identical donors. *p
Figure Legend Snippet: Effect of 25D on differentiation and/or stimulation of monocyte-derived DCs. Monocytes were differentiated for six days using rGM-CSF and rIL-4 in media with 10% FCS in the presence (25D diff -DCs or 25D diff/stim -DCs) or absence (serum-DCs or 25D stim -DCs) of additional 25D (10 −7 M). Subsequently, 25D-DCs and serum-DCs were stimulated with TLR2/1L (1 μg/ml) or left untreated in the presence (25D diff/stim -DCs or 25D stim -DCs) or absence (serum-DCs or 25D diff -DCs) of additional 25D (10 −7 M), and cultured in fresh media with 10% FCS for 18–24 hours. TNF-α, IL-6, IL-1β, IL-12p40, IL-12p70, IL-23 and IL-10, as well as total and active TGF-β levels in culture supernatants were measured by ELISA or CBA (mean cytokine levels in ng/ml ± SEM, n = 4). Results shown in Fig 1 and Fig. 2 were conducted using cells from the same cell preparations of identical donors. *p

Techniques Used: Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Crocin Bleaching Assay

Effect of 1,25D on differentiation and/or stimulation of monocyte-derived DCs. Monocytes were differentiated for six days with rGM-CSF and rIL-4 in media with 10% FCS in the presence (1,25D diff -DCs or 1,25D diff/stim -DCs) or absence (serum-DCs or 1,25D stim -DCs) of additional 1,25D (10 −8 M). Subsequently, 1,25D-DCs and serum-DCs were stimulated with TLR2/1L (1 μg/ml) or left untreated in the presence (1,25D diff/stim -DCs or 1,25D stim -DCs) or absence (serum-DCs or 1,25D diff -DCs) of additional 1,25D (10 −8 M) and cultured in fresh media with 10% FCS for 18–24 hours. TNF-α, IL-6, IL-1β, IL-12p40, IL-12p70, IL-23 and IL-10, as well as total and active TGF-β levels in culture supernatants were measured by ELISA or CBA (mean cytokine levels in ng/ml ± SEM, n = 4). Results shown in Fig. 1 and Fig 2 were conducted using cells from the same cell preparations of identical donors. *p
Figure Legend Snippet: Effect of 1,25D on differentiation and/or stimulation of monocyte-derived DCs. Monocytes were differentiated for six days with rGM-CSF and rIL-4 in media with 10% FCS in the presence (1,25D diff -DCs or 1,25D diff/stim -DCs) or absence (serum-DCs or 1,25D stim -DCs) of additional 1,25D (10 −8 M). Subsequently, 1,25D-DCs and serum-DCs were stimulated with TLR2/1L (1 μg/ml) or left untreated in the presence (1,25D diff/stim -DCs or 1,25D stim -DCs) or absence (serum-DCs or 1,25D diff -DCs) of additional 1,25D (10 −8 M) and cultured in fresh media with 10% FCS for 18–24 hours. TNF-α, IL-6, IL-1β, IL-12p40, IL-12p70, IL-23 and IL-10, as well as total and active TGF-β levels in culture supernatants were measured by ELISA or CBA (mean cytokine levels in ng/ml ± SEM, n = 4). Results shown in Fig. 1 and Fig 2 were conducted using cells from the same cell preparations of identical donors. *p

Techniques Used: Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Crocin Bleaching Assay

Cytokine profiles of 1,25D diff -DCs vs. serum-DCs stimulated with different ligands. Monocytes were differentiated for six days using rGM-CSF and rIL-4 in media with 10% FCS in the presence (1,25D diff -DCs, black bars) or absence (serum-DCs, white bars) of additional 1,25D (10 −8 M). Subsequently, 1,25D diff -DCs and serum-DCs were stimulated with TLR2/1L (1μg/ml), TLR4L (10 ng/ml) or CD40L (5 μg/ml) or left untreated, and cultured in fresh media with 10% FCS for 18–24 hours. TNF-α, IL-6, IL-1β, IL-12p40, IL-12p70, IL-23 and IL-10, as well as total and active TGF-β levels in culture supernatants were measured by ELISA or CBA (mean cytokine levels in ng/ml ± SEM, n of each condition indicated by numbers under the bars). *p
Figure Legend Snippet: Cytokine profiles of 1,25D diff -DCs vs. serum-DCs stimulated with different ligands. Monocytes were differentiated for six days using rGM-CSF and rIL-4 in media with 10% FCS in the presence (1,25D diff -DCs, black bars) or absence (serum-DCs, white bars) of additional 1,25D (10 −8 M). Subsequently, 1,25D diff -DCs and serum-DCs were stimulated with TLR2/1L (1μg/ml), TLR4L (10 ng/ml) or CD40L (5 μg/ml) or left untreated, and cultured in fresh media with 10% FCS for 18–24 hours. TNF-α, IL-6, IL-1β, IL-12p40, IL-12p70, IL-23 and IL-10, as well as total and active TGF-β levels in culture supernatants were measured by ELISA or CBA (mean cytokine levels in ng/ml ± SEM, n of each condition indicated by numbers under the bars). *p

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Crocin Bleaching Assay

4) Product Images from "Leishmania enriettii: biochemical characterisation of lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) and infectivity to Cavia porcellus"

Article Title: Leishmania enriettii: biochemical characterisation of lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) and infectivity to Cavia porcellus

Journal: Parasites & Vectors

doi: 10.1186/s13071-015-0633-8

IL-6 and IL12p40 production by IFN-γ-primed macrophages after stimulation with LPGs (10 μg/mL), GIPLs (10 μg/mL) and parasites. (A) IL-6 and (B) IL-12p40 concentrations were determined by flow cytometry. ANOVA test was performed and P
Figure Legend Snippet: IL-6 and IL12p40 production by IFN-γ-primed macrophages after stimulation with LPGs (10 μg/mL), GIPLs (10 μg/mL) and parasites. (A) IL-6 and (B) IL-12p40 concentrations were determined by flow cytometry. ANOVA test was performed and P

Techniques Used: Flow Cytometry, Cytometry

5) Product Images from "Cytokine patterns in a prospective cohort of HIV-infected patients with cryptococcal meningitis following initiation of antifungal and antiretroviral therapy"

Article Title: Cytokine patterns in a prospective cohort of HIV-infected patients with cryptococcal meningitis following initiation of antifungal and antiretroviral therapy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0176304

IL-12p40, TNF-α, IL-17A, IFN-γ, and CXCL-10 release. PBMCs (2 x 10 6 cells/mL) from HIV-infected patients with cryptococcal meningitis (CM + HIV + ) stimulated with 10 μg/mL of lipopolysaccharide (LPS) and 10 mg/mL of GXM for 48 h or treated with a control were evaluated at various times during treatment. Control groups: HIV patients without cryptococcosis (CM - HIV + ) and HIV-negative individuals (CM - HIV - ). Statistically significant differences are marked with * (p
Figure Legend Snippet: IL-12p40, TNF-α, IL-17A, IFN-γ, and CXCL-10 release. PBMCs (2 x 10 6 cells/mL) from HIV-infected patients with cryptococcal meningitis (CM + HIV + ) stimulated with 10 μg/mL of lipopolysaccharide (LPS) and 10 mg/mL of GXM for 48 h or treated with a control were evaluated at various times during treatment. Control groups: HIV patients without cryptococcosis (CM - HIV + ) and HIV-negative individuals (CM - HIV - ). Statistically significant differences are marked with * (p

Techniques Used: Infection

6) Product Images from "T-cell activation or tolerization: the Yin and Yang of bacterial superantigens"

Article Title: T-cell activation or tolerization: the Yin and Yang of bacterial superantigens

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2015.01153

Cyclosporine A (CsA) prevents secretion of cytokines . CD14 + monocytes were stimulated with SPEA (100 ng/ml) and CsA (1 μM) or Ethanol (equal volume to CsA) for 24 h. Supernatants were harvested and analyzed by ELISA for (A) IL-6, (B) TNF, (C) IL-12p40 and (D) IL-10. Shown is the mean concentration with standard deviation of 5 different donors. Significance codes: 0 ‘ ∗∗∗ ’; 0.001 ‘ ∗∗ ’; 0.01 ‘ ∗ ’ (Tukey Contrasts).
Figure Legend Snippet: Cyclosporine A (CsA) prevents secretion of cytokines . CD14 + monocytes were stimulated with SPEA (100 ng/ml) and CsA (1 μM) or Ethanol (equal volume to CsA) for 24 h. Supernatants were harvested and analyzed by ELISA for (A) IL-6, (B) TNF, (C) IL-12p40 and (D) IL-10. Shown is the mean concentration with standard deviation of 5 different donors. Significance codes: 0 ‘ ∗∗∗ ’; 0.001 ‘ ∗∗ ’; 0.01 ‘ ∗ ’ (Tukey Contrasts).

Techniques Used: Enzyme-linked Immunosorbent Assay, Concentration Assay, Standard Deviation

Signaling pathways involved in SPEA- mediated activation of APC. CD14+ monocytes were stimulated with SPEA (100ng/ml) in the presence or absence of (A) VIVIT (1 M), (B) Wortmannin (50 nM), (C) rapamycin (50nM) and (D) piceatannol (50 μM) or DMSO (equal volume to inhibitor) as solvent control. 24 h after stimulation supernatants were harvested and ELISAs were performed for IL-6, IL-10, IL-12p40 and TNF. Shown is the mean ±SD from 3 different donors, except VIVIT (n = 2). Statistical analysis was performed using a Multiple Comparison of Means (Tukey Contrasts). Significance codes: 0 ‘ ∗∗∗ ’; 0.001 ‘ ∗∗ ’; 0.01 ‘ ∗ ’.
Figure Legend Snippet: Signaling pathways involved in SPEA- mediated activation of APC. CD14+ monocytes were stimulated with SPEA (100ng/ml) in the presence or absence of (A) VIVIT (1 M), (B) Wortmannin (50 nM), (C) rapamycin (50nM) and (D) piceatannol (50 μM) or DMSO (equal volume to inhibitor) as solvent control. 24 h after stimulation supernatants were harvested and ELISAs were performed for IL-6, IL-10, IL-12p40 and TNF. Shown is the mean ±SD from 3 different donors, except VIVIT (n = 2). Statistical analysis was performed using a Multiple Comparison of Means (Tukey Contrasts). Significance codes: 0 ‘ ∗∗∗ ’; 0.001 ‘ ∗∗ ’; 0.01 ‘ ∗ ’.

Techniques Used: Activation Assay

Streptococcal pyrogenic exotoxin A (SPEA)- induced cytokines . CD14 + monocytes were stimulated with increasing concentrations of SPEA. 24 h after stimulation supernatants were analyzed by ELISA for (A) IL-6, (B) TNF, (C) IL-12p40 and (D) IL-10 (mean ± SD, n = 5). Statistical analysis was performed using a Multiple Comparison of Means (Tukey Contrasts). Significance code: 0 ‘ ∗∗∗ ’; 0.001 ‘ ∗∗ ’; 0.01 ‘ ∗ ’.
Figure Legend Snippet: Streptococcal pyrogenic exotoxin A (SPEA)- induced cytokines . CD14 + monocytes were stimulated with increasing concentrations of SPEA. 24 h after stimulation supernatants were analyzed by ELISA for (A) IL-6, (B) TNF, (C) IL-12p40 and (D) IL-10 (mean ± SD, n = 5). Statistical analysis was performed using a Multiple Comparison of Means (Tukey Contrasts). Significance code: 0 ‘ ∗∗∗ ’; 0.001 ‘ ∗∗ ’; 0.01 ‘ ∗ ’.

Techniques Used: Enzyme-linked Immunosorbent Assay

7) Product Images from "Virulent and Avirulent Strains of Toxoplasma gondii Which Differ in Their Glycosylphosphatidylinositol Content Induce Similar Biological Functions in Macrophages"

Article Title: Virulent and Avirulent Strains of Toxoplasma gondii Which Differ in Their Glycosylphosphatidylinositol Content Induce Similar Biological Functions in Macrophages

Journal: PLoS ONE

doi: 10.1371/journal.pone.0085386

GPIs of RH and PTG strains induce TNF-α and IL-12p40 secretion by macrophages. ( A ) Macrophages were incubated for 24 h with medium alone, or with individual GPIs of the PTG strain extracted from 1×10 8 parasites and assayed for TNF-α cytokine production. ( B ) Macrophages were incubated for 24 h with medium alone, or with individual GPIs of the RH (left panel) and the PTG strain (right panel) extracted from 2×10 8 parasites, respectively, and assayed for IL-12p40 cytokine production. ( C ) Macrophages were incubated for 24 h with medium alone, or with GPIa (3 mM), a chemically synthesized structure of RH strain GPI III core glycan and assayed for IL-12p40 cytokine production. *** P
Figure Legend Snippet: GPIs of RH and PTG strains induce TNF-α and IL-12p40 secretion by macrophages. ( A ) Macrophages were incubated for 24 h with medium alone, or with individual GPIs of the PTG strain extracted from 1×10 8 parasites and assayed for TNF-α cytokine production. ( B ) Macrophages were incubated for 24 h with medium alone, or with individual GPIs of the RH (left panel) and the PTG strain (right panel) extracted from 2×10 8 parasites, respectively, and assayed for IL-12p40 cytokine production. ( C ) Macrophages were incubated for 24 h with medium alone, or with GPIa (3 mM), a chemically synthesized structure of RH strain GPI III core glycan and assayed for IL-12p40 cytokine production. *** P

Techniques Used: Incubation, Synthesized

8) Product Images from "IL-12 Production Induced by Agaricus blazei Fraction H (ABH) Involves Toll-like Receptor (TLR)"

Article Title: IL-12 Production Induced by Agaricus blazei Fraction H (ABH) Involves Toll-like Receptor (TLR)

Journal: Evidence-based Complementary and Alternative Medicine

doi: 10.1093/ecam/neh043

ABH induces IL-12p40 and p70. ( A ) Ficoll-isolated human PBMC (1 × 10 6 /sample) were stimulated with ABH at various concentrations as indicated at the bottom of the panels. After incubation for 16 h, supernatants were collected and the amounts of IL-12 p40 were determined. ( B ) PBMCs were stimulated with 0.17 ng/ml ABH for 16 h. The supernatant was collected and the amounts of IL-12 p70 were determined. (A and B) IL-12 p40 and p70 in culture supernatants were measured by ELISA. Data of IL-12 p40 and p70 shown are from four and two different donors, respectively.
Figure Legend Snippet: ABH induces IL-12p40 and p70. ( A ) Ficoll-isolated human PBMC (1 × 10 6 /sample) were stimulated with ABH at various concentrations as indicated at the bottom of the panels. After incubation for 16 h, supernatants were collected and the amounts of IL-12 p40 were determined. ( B ) PBMCs were stimulated with 0.17 ng/ml ABH for 16 h. The supernatant was collected and the amounts of IL-12 p70 were determined. (A and B) IL-12 p40 and p70 in culture supernatants were measured by ELISA. Data of IL-12 p40 and p70 shown are from four and two different donors, respectively.

Techniques Used: Isolation, Incubation, Enzyme-linked Immunosorbent Assay

9) Product Images from "Altered Dendritic Cell Phenotype in Response to Leishmania amazonensis Amastigote Infection Is Mediated by MAP Kinase, ERK"

Article Title: Altered Dendritic Cell Phenotype in Response to Leishmania amazonensis Amastigote Infection Is Mediated by MAP Kinase, ERK

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2009.080905

ERK inhibition enhances DC maturation phenotype following L. amazonensis infection ( A–B ) BMDC were pretreated with the ERK inhibitor PD98059 (20 mmol/L) for 30 minutes and then infected with L. amazonensis amastigotes, and activated with LPS and IFN-γ. 24 hours post-infection cells were harvested and processed for ( A ) surface CD40 expression and analyzed via FACS scan and ( B ) supernatants were collected to determine IL-12p40 production via enzyme-linked immunosorbent assay. C–H: Mice were infected with L. amazonensis or L. major promastigotes and then treated with 100 mg/kg of CI-1040 via oral gavage started on day 0 post-infection and for the next 7 days, twice daily. On day 7 mice were sacrificed; draining lymph nodes were harvested to assess CD40 surface expression on DC populations. C: Representative histograms based on CD11c + CD11b + population, (gray line, isotype control; black CD40). Mean fluorescence intensity of CD40 surface expression on CD11c + cells in L. amazonensis- infected ( D ) or L. major -infected mice ( E ) treated with CI-1040 or DMSO-mock control. Number of IL-12p40-producing cells ( F and G ) as determined by Elispot analysis of total draining lymph node cells. Western blot of splenic lysates probed with anti-phosphorylated ERK1/2 and anti-total ERK1/2 ( H ). Data from at least three different experiments; error bars denote ±SEM; * P ≤ 0.05.
Figure Legend Snippet: ERK inhibition enhances DC maturation phenotype following L. amazonensis infection ( A–B ) BMDC were pretreated with the ERK inhibitor PD98059 (20 mmol/L) for 30 minutes and then infected with L. amazonensis amastigotes, and activated with LPS and IFN-γ. 24 hours post-infection cells were harvested and processed for ( A ) surface CD40 expression and analyzed via FACS scan and ( B ) supernatants were collected to determine IL-12p40 production via enzyme-linked immunosorbent assay. C–H: Mice were infected with L. amazonensis or L. major promastigotes and then treated with 100 mg/kg of CI-1040 via oral gavage started on day 0 post-infection and for the next 7 days, twice daily. On day 7 mice were sacrificed; draining lymph nodes were harvested to assess CD40 surface expression on DC populations. C: Representative histograms based on CD11c + CD11b + population, (gray line, isotype control; black CD40). Mean fluorescence intensity of CD40 surface expression on CD11c + cells in L. amazonensis- infected ( D ) or L. major -infected mice ( E ) treated with CI-1040 or DMSO-mock control. Number of IL-12p40-producing cells ( F and G ) as determined by Elispot analysis of total draining lymph node cells. Western blot of splenic lysates probed with anti-phosphorylated ERK1/2 and anti-total ERK1/2 ( H ). Data from at least three different experiments; error bars denote ±SEM; * P ≤ 0.05.

Techniques Used: Inhibition, Infection, Expressing, FACS, Enzyme-linked Immunosorbent Assay, Mouse Assay, Fluorescence, Enzyme-linked Immunospot, Western Blot

Decreased CD40 expression and IL-12p40-producing cells from DC subsets of total draining lymph node cells from L. amazonensis -infected mice at 7 days postinfection. Mice were infected with L. amazonensis or L. major promastigotes and sacrificed at 2 and 7 days post-infection. CD11c + DC subsets from total draining lymph node cells, collected on CD19 and CD3 double negative events ( A ) from L. amazonensis -infected, L. major -infected, and naïve mice. Cells were analyzed for CD40 surface expression via FACS scan. Histograms ( B and C ) indicate percent CD40 positive CD11c + CD11b + cells (gray, isotype control; black, CD40) and bar graphs ( D and E ) indicate CD40 mean fluorescence intensity of cells at 2dpi ( B and D ) and 7 dpi ( C and E ). F and G: Number of IL-12-producing cells from DLN of mice infected with L. amazonensis or L. major at 2dpi ( F ) or 7 dpi ( G ) measured via ELispot. All data are from three separate experiments; error bars indicate ±SEM; * P ≤ 0.05.
Figure Legend Snippet: Decreased CD40 expression and IL-12p40-producing cells from DC subsets of total draining lymph node cells from L. amazonensis -infected mice at 7 days postinfection. Mice were infected with L. amazonensis or L. major promastigotes and sacrificed at 2 and 7 days post-infection. CD11c + DC subsets from total draining lymph node cells, collected on CD19 and CD3 double negative events ( A ) from L. amazonensis -infected, L. major -infected, and naïve mice. Cells were analyzed for CD40 surface expression via FACS scan. Histograms ( B and C ) indicate percent CD40 positive CD11c + CD11b + cells (gray, isotype control; black, CD40) and bar graphs ( D and E ) indicate CD40 mean fluorescence intensity of cells at 2dpi ( B and D ) and 7 dpi ( C and E ). F and G: Number of IL-12-producing cells from DLN of mice infected with L. amazonensis or L. major at 2dpi ( F ) or 7 dpi ( G ) measured via ELispot. All data are from three separate experiments; error bars indicate ±SEM; * P ≤ 0.05.

Techniques Used: Expressing, Infection, Mouse Assay, FACS, Fluorescence, Enzyme-linked Immunospot

10) Product Images from "Role of Mitogen-Activated Protein Kinases and NF-?B in the Regulation of Proinflammatory and Anti-Inflammatory Cytokines by Porphyromonas gingivalis Hemagglutinin B "

Article Title: Role of Mitogen-Activated Protein Kinases and NF-?B in the Regulation of Proinflammatory and Anti-Inflammatory Cytokines by Porphyromonas gingivalis Hemagglutinin B

Journal: Infection and Immunity

doi: 10.1128/IAI.73.7.3990-3998.2005

Role of IL-10 in the enhancement of IL-12p40 production. C57BL/6 peritoneal macrophages (5 × 10 5 cells) were pretreated with a neutralizing MAb against IL-10 (10 μg/ml) or with its isotype control (IC) in the presence or absence of SB203580 (10 μM), U0126 (25 μM), or PD98059 (25 μM) for 2 h and then stimulated with rHagB (20 μg/ml) for 20 h. The levels of IL-10, IL-12p40, IFN-γ, and TNF-α in culture supernatants were determined by ELISA. Data are expressed as the mean of the increase in induction ( n -fold) over unstimulated control ± standard error of the mean of six separate experiments. Values were significantly different when P was
Figure Legend Snippet: Role of IL-10 in the enhancement of IL-12p40 production. C57BL/6 peritoneal macrophages (5 × 10 5 cells) were pretreated with a neutralizing MAb against IL-10 (10 μg/ml) or with its isotype control (IC) in the presence or absence of SB203580 (10 μM), U0126 (25 μM), or PD98059 (25 μM) for 2 h and then stimulated with rHagB (20 μg/ml) for 20 h. The levels of IL-10, IL-12p40, IFN-γ, and TNF-α in culture supernatants were determined by ELISA. Data are expressed as the mean of the increase in induction ( n -fold) over unstimulated control ± standard error of the mean of six separate experiments. Values were significantly different when P was

Techniques Used: Enzyme-linked Immunosorbent Assay

Dose-dependent effect of ERK and p38 inhibitors on rHagB-induced cytokine production. C57BL/6 peritoneal macrophages (5 × 10 5 cells) were pretreated with U0126, PD98059, or SB203580 (1 to 50 μM) for 2 h and then stimulated with rHagB (20 μg/ml) for 20 h. The levels of IL-10, IL-12p40, IFN-γ, and TNF-α in culture supernatants were determined by ELISA. Data are expressed as the mean percent increase over that seen in rHagB-stimulated culture with no inhibitor ± standard error of the means of six separate experiments. Significant increases (*, P
Figure Legend Snippet: Dose-dependent effect of ERK and p38 inhibitors on rHagB-induced cytokine production. C57BL/6 peritoneal macrophages (5 × 10 5 cells) were pretreated with U0126, PD98059, or SB203580 (1 to 50 μM) for 2 h and then stimulated with rHagB (20 μg/ml) for 20 h. The levels of IL-10, IL-12p40, IFN-γ, and TNF-α in culture supernatants were determined by ELISA. Data are expressed as the mean percent increase over that seen in rHagB-stimulated culture with no inhibitor ± standard error of the means of six separate experiments. Significant increases (*, P

Techniques Used: Enzyme-linked Immunosorbent Assay

rHagB induces cytokine mRNA expression by murine macrophages. C57BL/6 peritoneal macrophages (3 × 10 6 cells) were stimulated with rHagB (20 μg/ml) for 0 to 24 h. Total RNA was isolated and the presence of mRNA for IL-10, IL-12p35, IL-12p40, IFN-γ, TNF-α, and β-actin in each sample was assessed by real-time PCR using SYBR green. β-actin was used as the endogenous control. The results are representative of three independent experiments.
Figure Legend Snippet: rHagB induces cytokine mRNA expression by murine macrophages. C57BL/6 peritoneal macrophages (3 × 10 6 cells) were stimulated with rHagB (20 μg/ml) for 0 to 24 h. Total RNA was isolated and the presence of mRNA for IL-10, IL-12p35, IL-12p40, IFN-γ, TNF-α, and β-actin in each sample was assessed by real-time PCR using SYBR green. β-actin was used as the endogenous control. The results are representative of three independent experiments.

Techniques Used: Expressing, Isolation, Real-time Polymerase Chain Reaction, SYBR Green Assay

Effect of NF-κB inhibition on rHagB-induced cytokine production. C57BL/6 peritoneal macrophages (5 × 10 5 cells) were pretreated with SN50 (10 μM) or its inactive analog SN50 M (10 μM) for 2 h and then stimulated with rHagB (20 μg/ml) for 20 h. The levels of IL-10, IL-12p40, IFN-γ, and TNF-α in culture supernatants were determined by ELISA. Data are expressed as the mean of the increase in induction ( n -fold) over nonstimulated control ± standard error of the mean of six separate experiments. Significant differences (**, P
Figure Legend Snippet: Effect of NF-κB inhibition on rHagB-induced cytokine production. C57BL/6 peritoneal macrophages (5 × 10 5 cells) were pretreated with SN50 (10 μM) or its inactive analog SN50 M (10 μM) for 2 h and then stimulated with rHagB (20 μg/ml) for 20 h. The levels of IL-10, IL-12p40, IFN-γ, and TNF-α in culture supernatants were determined by ELISA. Data are expressed as the mean of the increase in induction ( n -fold) over nonstimulated control ± standard error of the mean of six separate experiments. Significant differences (**, P

Techniques Used: Inhibition, Enzyme-linked Immunosorbent Assay

rHagB induces cytokine production by murine macrophages. C57BL/6 peritoneal macrophages (5 × 10 5 cells) were stimulated with rHagB (0 to 20 μg/ml) for 20 h. The levels of IL-10, IL-12p40, IL-12p70, IFN-γ, and TNF-α in culture supernatants were determined by ELISA. Data represent the means ± standard deviations of triplicates. The results are representative of three separate experiments.
Figure Legend Snippet: rHagB induces cytokine production by murine macrophages. C57BL/6 peritoneal macrophages (5 × 10 5 cells) were stimulated with rHagB (0 to 20 μg/ml) for 20 h. The levels of IL-10, IL-12p40, IL-12p70, IFN-γ, and TNF-α in culture supernatants were determined by ELISA. Data represent the means ± standard deviations of triplicates. The results are representative of three separate experiments.

Techniques Used: Enzyme-linked Immunosorbent Assay

11) Product Images from "Systemic effects in naïve mice injected with immunomodulatory lectin ArtinM"

Article Title: Systemic effects in naïve mice injected with immunomodulatory lectin ArtinM

Journal: PLoS ONE

doi: 10.1371/journal.pone.0187151

Cytokine levels in tissues of naïve BALB/c mice after ArtinM administration. The levels of IL-12p40, IFN-γ, TNF-α, and IL-10 in the supernatant of organ homogenates obtained at the day 0 were assessed by ELISA. The organs were heart ( A – D ), lung ( E – H ), liver ( I – L ), kidney ( M – P ), and spleen ( Q – T ). Animals received ArtinM at the specified doses, PBS (negative control), or LPS (positive control). Results are expressed as mean ± SD, and the differences were considered significant when p
Figure Legend Snippet: Cytokine levels in tissues of naïve BALB/c mice after ArtinM administration. The levels of IL-12p40, IFN-γ, TNF-α, and IL-10 in the supernatant of organ homogenates obtained at the day 0 were assessed by ELISA. The organs were heart ( A – D ), lung ( E – H ), liver ( I – L ), kidney ( M – P ), and spleen ( Q – T ). Animals received ArtinM at the specified doses, PBS (negative control), or LPS (positive control). Results are expressed as mean ± SD, and the differences were considered significant when p

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Positive Control

12) Product Images from "A fundamental bimodal role for neuropeptide Y1 receptor in the immune system"

Article Title: A fundamental bimodal role for neuropeptide Y1 receptor in the immune system

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20051971

Impaired DC function in Y1 − / − mice. (A) OVA-FITC uptake by Y1 +/+ (white bar) and Y1 −/− (black bar) immature DCs. Flow cytometry was used to quantitated FITC-OVA uptake by DC, and results were plotted as mean fluorescence intensity (MFI). Four animals were analyzed per group. (B) Representative FACS plot showing OVA-FITC uptake by Y1 +/+ (thin line) and Y1 −/− (thick line) immature DCs. (C) Both Y1 −/− immature DCs (iDC) or DCs stimulated with LPS or anti-CD40 antibody as indicated (black bars) have impaired stimulatory functions in MLR compared with Y1 +/+ cells (white bars). Y1 +/+ T cells were used as responders. (D) IL-12p40 (top) and IL-12p70 (bottom) production by anti-CD40– (left) and LPS-stimulated (right) Y1 +/+ (white diamonds) and Y1 −/− (black circles) DCs measured by ELISA. The results are representative of two separate experiments. White and black squares represent control unstimulated Y1 +/+ and Y1 −/− cells, respectively. (E) Body weight of RAG1 −/− or RAG1 −/− × Y1 −/− mice days after transfer with Y1 −/− CD45RB hi CD4 + T cells as indicated. Four animals per group. (F) DTH response induced after injection of Y1 +/+ mice with mBSA-pulsed Y1 +/+ (white squares) or Y1 −/− (white diamonds) DCs. p-values as in Fig. 1 .
Figure Legend Snippet: Impaired DC function in Y1 − / − mice. (A) OVA-FITC uptake by Y1 +/+ (white bar) and Y1 −/− (black bar) immature DCs. Flow cytometry was used to quantitated FITC-OVA uptake by DC, and results were plotted as mean fluorescence intensity (MFI). Four animals were analyzed per group. (B) Representative FACS plot showing OVA-FITC uptake by Y1 +/+ (thin line) and Y1 −/− (thick line) immature DCs. (C) Both Y1 −/− immature DCs (iDC) or DCs stimulated with LPS or anti-CD40 antibody as indicated (black bars) have impaired stimulatory functions in MLR compared with Y1 +/+ cells (white bars). Y1 +/+ T cells were used as responders. (D) IL-12p40 (top) and IL-12p70 (bottom) production by anti-CD40– (left) and LPS-stimulated (right) Y1 +/+ (white diamonds) and Y1 −/− (black circles) DCs measured by ELISA. The results are representative of two separate experiments. White and black squares represent control unstimulated Y1 +/+ and Y1 −/− cells, respectively. (E) Body weight of RAG1 −/− or RAG1 −/− × Y1 −/− mice days after transfer with Y1 −/− CD45RB hi CD4 + T cells as indicated. Four animals per group. (F) DTH response induced after injection of Y1 +/+ mice with mBSA-pulsed Y1 +/+ (white squares) or Y1 −/− (white diamonds) DCs. p-values as in Fig. 1 .

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Fluorescence, FACS, Enzyme-linked Immunosorbent Assay, Injection

13) Product Images from "IL-1R signaling enables bystander cells to overcome bacterial blockade of host protein synthesis"

Article Title: IL-1R signaling enables bystander cells to overcome bacterial blockade of host protein synthesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1501289112

IL-12p40 and CD86 are produced by bystander cells during infection. ( A ) Bone marrow-derived macrophages (BMDMs) were infected with PBS or Lp02 Δ flaA or Δ dotA L. pneumophila at a multiplicity of infection, MOI = 5 for 4, 8, 12, or 16 h.
Figure Legend Snippet: IL-12p40 and CD86 are produced by bystander cells during infection. ( A ) Bone marrow-derived macrophages (BMDMs) were infected with PBS or Lp02 Δ flaA or Δ dotA L. pneumophila at a multiplicity of infection, MOI = 5 for 4, 8, 12, or 16 h.

Techniques Used: Produced, Infection, Derivative Assay

14) Product Images from "Murine Langerin+ dermal dendritic cells prime CD8+ T cells while Langerhans cells induce cross-tolerance"

Article Title: Murine Langerin+ dermal dendritic cells prime CD8+ T cells while Langerhans cells induce cross-tolerance

Journal: EMBO Molecular Medicine

doi: 10.15252/emmm.201303283

Langerin + dDCs, but not LCs, express CD70 upon pIC/40 stimulation in vivo Both ears of C57BL/6 mice were treated with imiquimod (imiq), poly(I:C) and anti-CD40 (pIC/40), or left untreated (nt). Two or 4 days later, a cell suspension was obtained from four auricular lymph nodes collected from two identically treated mice, and expression of CD70 and IL-12p40 was evaluated by flow cytometry. Measurements, each comprising cells from two mice, are pooled from two independent experiments (nt: n = 7; imiq—day 2: n = 4; imiq—day 4: n = 4; pIC/40—day 2: n = 4; pIC/40—day 4: n = 3) and compared using one-way ANOVA followed by Tukey's test (n.s., non-significant, P > 0.05). Representative stainings showing surface expression of CD70 in CD11c + Langerin + DCs. Percentages of Langerin + dDCs and LCs expressing CD70 (ANOVA: P
Figure Legend Snippet: Langerin + dDCs, but not LCs, express CD70 upon pIC/40 stimulation in vivo Both ears of C57BL/6 mice were treated with imiquimod (imiq), poly(I:C) and anti-CD40 (pIC/40), or left untreated (nt). Two or 4 days later, a cell suspension was obtained from four auricular lymph nodes collected from two identically treated mice, and expression of CD70 and IL-12p40 was evaluated by flow cytometry. Measurements, each comprising cells from two mice, are pooled from two independent experiments (nt: n = 7; imiq—day 2: n = 4; imiq—day 4: n = 4; pIC/40—day 2: n = 4; pIC/40—day 4: n = 3) and compared using one-way ANOVA followed by Tukey's test (n.s., non-significant, P > 0.05). Representative stainings showing surface expression of CD70 in CD11c + Langerin + DCs. Percentages of Langerin + dDCs and LCs expressing CD70 (ANOVA: P

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

15) Product Images from "Helminth products bypass the need for TSLP in Th2 immune responses by directly modulating dendritic cell function"

Article Title: Helminth products bypass the need for TSLP in Th2 immune responses by directly modulating dendritic cell function

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0906367106

H. polygyrus and N. brasiliensis products directly modulate IL-12p40 production by DC. ( A ) IL-12p40 production of C57BL/6 BM-DC after stimulation with ES products from H. polygyrus , N. brasiliensis , and T. muris . ( B ) Comparison of untreated or heat-inactivated
Figure Legend Snippet: H. polygyrus and N. brasiliensis products directly modulate IL-12p40 production by DC. ( A ) IL-12p40 production of C57BL/6 BM-DC after stimulation with ES products from H. polygyrus , N. brasiliensis , and T. muris . ( B ) Comparison of untreated or heat-inactivated

Techniques Used:

Susceptibility of TSLPR −/− mice to T. muris infection is reversed by IL-12p40 neutralization. ( A ) IL-12p40 serum levels in TSLPR −/− mice treated with anti-IL-12p40 (black bar, n = 4) or isotype control antibody (gray bar,
Figure Legend Snippet: Susceptibility of TSLPR −/− mice to T. muris infection is reversed by IL-12p40 neutralization. ( A ) IL-12p40 serum levels in TSLPR −/− mice treated with anti-IL-12p40 (black bar, n = 4) or isotype control antibody (gray bar,

Techniques Used: Mouse Assay, Infection, Neutralization

16) Product Images from "Production of interleukin-13 by human dendritic cells after stimulation with protein allergens is a key factor for induction of T helper 2 cytokines and is associated with activation of signal transducer and activator of transcription-6"

Article Title: Production of interleukin-13 by human dendritic cells after stimulation with protein allergens is a key factor for induction of T helper 2 cytokines and is associated with activation of signal transducer and activator of transcription-6

Journal: Immunology

doi: 10.1046/j.1365-2567.2003.01576.x

Neutralization of IL-13 has no effect on DC cytokine production, but reduces the potency to induce production of the Th2 cytokine IL-4 by T cells. (a) On day 6, immature DC were pulsed with different allergen doses as indicated and stimulated with IL-1β, TNF-α and PGE 2 with or without the addition of 5 µg/ml anti-IL-13 or 1 µg/ml anti-IL-4 mAbs. Supernatants were harvested 48 hr later and analysed for IL-6 and IL-12p40 content by ELISA. The results represent the means ± SD from four independent experiments. (b) 5 × 10 6 CD4 + T cells were cocultured with 5 × 10 5 autologous mature DC which were pulsed with 10 µg/ml allergen ± 1 µg/ml anti-IL-4, 5 µg/ml anti-IL-13, or 5 µg/ml mouse IgG 1 isotype control. DC were depleted after 5 hr of culture. Then, T cells were lysed and analysed for STAT6 expression by Western blot as described in Materials and Methods. (c) CD4 + T cells (5 × 10 5 ) were cocultured with 5 × 10 4 autologous mature DC which were pulsed with 0·1–10 µg/ml allergen ± 5 µg/ml anti-IL-13 mAbs. After 8 days of culture, which was additionally performed ± 5 µg/ml anti-IL-13 mAbs or 5 ng/ml recombinant IL-13, T cells were restimulated with 5 × 10 4 newly generated DC of the same donor which were pulsed with an optimal dose (5 µg/ml) of the respective allergen. Supernatants were harvested 24 hr later and analysed for IL-4, IL-5 and IFN-γ content by ELISA. Shown are the means ± SD of six (for IL-4) and 10 (for IL-5 and IFN-γ) independent experiments. *indicates statistically significant differences ( P ≤ 0·05) between absence or presence of anti-IL-13 or -IL-13.
Figure Legend Snippet: Neutralization of IL-13 has no effect on DC cytokine production, but reduces the potency to induce production of the Th2 cytokine IL-4 by T cells. (a) On day 6, immature DC were pulsed with different allergen doses as indicated and stimulated with IL-1β, TNF-α and PGE 2 with or without the addition of 5 µg/ml anti-IL-13 or 1 µg/ml anti-IL-4 mAbs. Supernatants were harvested 48 hr later and analysed for IL-6 and IL-12p40 content by ELISA. The results represent the means ± SD from four independent experiments. (b) 5 × 10 6 CD4 + T cells were cocultured with 5 × 10 5 autologous mature DC which were pulsed with 10 µg/ml allergen ± 1 µg/ml anti-IL-4, 5 µg/ml anti-IL-13, or 5 µg/ml mouse IgG 1 isotype control. DC were depleted after 5 hr of culture. Then, T cells were lysed and analysed for STAT6 expression by Western blot as described in Materials and Methods. (c) CD4 + T cells (5 × 10 5 ) were cocultured with 5 × 10 4 autologous mature DC which were pulsed with 0·1–10 µg/ml allergen ± 5 µg/ml anti-IL-13 mAbs. After 8 days of culture, which was additionally performed ± 5 µg/ml anti-IL-13 mAbs or 5 ng/ml recombinant IL-13, T cells were restimulated with 5 × 10 4 newly generated DC of the same donor which were pulsed with an optimal dose (5 µg/ml) of the respective allergen. Supernatants were harvested 24 hr later and analysed for IL-4, IL-5 and IFN-γ content by ELISA. Shown are the means ± SD of six (for IL-4) and 10 (for IL-5 and IFN-γ) independent experiments. *indicates statistically significant differences ( P ≤ 0·05) between absence or presence of anti-IL-13 or -IL-13.

Techniques Used: Neutralization, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Recombinant, Generated

Increased amounts of allergen induce increased levels of IL-6 and IL-12 production by DC. DC were generated from peripheral blood monocytes isolated from atopic donors. On day 6 of culture, immature DC were pulsed with different allergen doses as indicated and stimulated with IL-1β, TNF-α and PGE 2 to induce full maturation. At the same time point, 10 µg/ml PMB was added to neutralize endotoxin contamination in the allergen preparations. Supernatants were harvested 48 h later and analysed for IL-6 and IL-12p40 content by ELISA. The results represent the means ± SD from four independent experiments.
Figure Legend Snippet: Increased amounts of allergen induce increased levels of IL-6 and IL-12 production by DC. DC were generated from peripheral blood monocytes isolated from atopic donors. On day 6 of culture, immature DC were pulsed with different allergen doses as indicated and stimulated with IL-1β, TNF-α and PGE 2 to induce full maturation. At the same time point, 10 µg/ml PMB was added to neutralize endotoxin contamination in the allergen preparations. Supernatants were harvested 48 h later and analysed for IL-6 and IL-12p40 content by ELISA. The results represent the means ± SD from four independent experiments.

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

17) Product Images from "Role of Toll-Like Receptor 9 Signaling in Experimental Leishmania braziliensis Infection"

Article Title: Role of Toll-Like Receptor 9 Signaling in Experimental Leishmania braziliensis Infection

Journal: Infection and Immunity

doi: 10.1128/IAI.01401-12

IL-12p40 production and surface expression of costimulatory molecules by cDCs in response to L. braziliensis are dependent on TLR9. (A) GM-CSF-derived cDCs from WT and TLR9 −/− mice were stimulated or not with UV-treated L. braziliensis
Figure Legend Snippet: IL-12p40 production and surface expression of costimulatory molecules by cDCs in response to L. braziliensis are dependent on TLR9. (A) GM-CSF-derived cDCs from WT and TLR9 −/− mice were stimulated or not with UV-treated L. braziliensis

Techniques Used: Expressing, Derivative Assay, Mouse Assay

18) Product Images from "Myeloid differentiation factor 88 is required for resistance to Neospora caninum infection"

Article Title: Myeloid differentiation factor 88 is required for resistance to Neospora caninum infection

Journal: Veterinary Research

doi: 10.1051/vetres/2009015

MyD88 is responsible for the early induction of Th1-related cytokines after N. caninum infection. The presence of IL-12p40, IFN-γ, and IL-10 was evaluated by ELISA in the peritoneal exudate and serum samples of uninfected, and infected WT and MyD88 −/− mice at 3, 7, and 10 days p.i. ( n = 3 mice/group/date). Data are representative of two independent experiments. * Indicates significant statistical differences (mean ± SEM, p
Figure Legend Snippet: MyD88 is responsible for the early induction of Th1-related cytokines after N. caninum infection. The presence of IL-12p40, IFN-γ, and IL-10 was evaluated by ELISA in the peritoneal exudate and serum samples of uninfected, and infected WT and MyD88 −/− mice at 3, 7, and 10 days p.i. ( n = 3 mice/group/date). Data are representative of two independent experiments. * Indicates significant statistical differences (mean ± SEM, p

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

Replenishment of IL-12 and IFN-γ reverts susceptibility of MyD88 −/− mice to acute N. caninum infection. Survival curves of (A) WT, IL-12p40 −/− , MyD88 −/− and MyD88 −/− mice treated with rIL-12p70 or (B) WT, IFNγ −/− , MyD88 −/− and MyD88 −/− mice treated with rIFNγ ( n = 6 mice/group), infected at the same time with sub-lethal parasite doses. Survival was observed through a 30-day period. Data are representative of two independent experiments.
Figure Legend Snippet: Replenishment of IL-12 and IFN-γ reverts susceptibility of MyD88 −/− mice to acute N. caninum infection. Survival curves of (A) WT, IL-12p40 −/− , MyD88 −/− and MyD88 −/− mice treated with rIL-12p70 or (B) WT, IFNγ −/− , MyD88 −/− and MyD88 −/− mice treated with rIFNγ ( n = 6 mice/group), infected at the same time with sub-lethal parasite doses. Survival was observed through a 30-day period. Data are representative of two independent experiments.

Techniques Used: Mouse Assay, Infection

19) Product Images from "Francisella tularensis SchuS4 and SchuS4 lipids inhibit IL-12p40 in primary human dendritic cells by inhibition of IRF1 and IRF8 *"

Article Title: Francisella tularensis SchuS4 and SchuS4 lipids inhibit IL-12p40 in primary human dendritic cells by inhibition of IRF1 and IRF8 *

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

doi: 10.4049/jimmunol.1300867

Viable F. tularensis SchuS4 inhibits translocation of NF-κBp65 and IRF1 in hDC hDC were infected with a MOI = 50 viable SchuS4. As indicated, 24 hours after infection cells were treated with either Pam3CSK4 (5 μg/ml) or LPS (10 ng/ml). Eighteen hours later culture supernatants were assessed for IL-12p40 by ELISA (A) and 90 minutes after addition of Pam3CSK4 cells were assessed for translocation of NF-κBp65 by microscopy(B). Ninety minutes after addition of LPS cells were assessed for translocation of IRF1 and IRF3 by microscopy (C). Translocation of NF-κBp65, IRF1 and IRF3 was quantitated by scoring greater than 500 cells for presence of nuclear NF-κBp65, IRF1 or IRF3. ns = not significantly different. * = significantly less than uninfected cells (p
Figure Legend Snippet: Viable F. tularensis SchuS4 inhibits translocation of NF-κBp65 and IRF1 in hDC hDC were infected with a MOI = 50 viable SchuS4. As indicated, 24 hours after infection cells were treated with either Pam3CSK4 (5 μg/ml) or LPS (10 ng/ml). Eighteen hours later culture supernatants were assessed for IL-12p40 by ELISA (A) and 90 minutes after addition of Pam3CSK4 cells were assessed for translocation of NF-κBp65 by microscopy(B). Ninety minutes after addition of LPS cells were assessed for translocation of IRF1 and IRF3 by microscopy (C). Translocation of NF-κBp65, IRF1 and IRF3 was quantitated by scoring greater than 500 cells for presence of nuclear NF-κBp65, IRF1 or IRF3. ns = not significantly different. * = significantly less than uninfected cells (p

Techniques Used: Translocation Assay, Infection, Enzyme-linked Immunosorbent Assay, Microscopy

SchuS4 lipids inhibit binding of IRF1 and IRF8 to the human IL-12p40 promoter hDC were treated with SchuS4 lipids or EtOH (vehicle control) followed by LPS+IFN-γ. (A) Supernatants were assessed for IL-12p40 by ELISA. SchuS4 lipids significantly inhibited the ability of hDC to secrete IL-12p40 in response to LPS and IFN-γ. * = p
Figure Legend Snippet: SchuS4 lipids inhibit binding of IRF1 and IRF8 to the human IL-12p40 promoter hDC were treated with SchuS4 lipids or EtOH (vehicle control) followed by LPS+IFN-γ. (A) Supernatants were assessed for IL-12p40 by ELISA. SchuS4 lipids significantly inhibited the ability of hDC to secrete IL-12p40 in response to LPS and IFN-γ. * = p

Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay

SchuS4 lipids inhibit secretion of IL-12p40 by hDC hDC were treated with the indicated concentration of lipids isolated from SchuS4 or LVS for 18 hours followed by addition of LPS for an additional 20 hours. EtOH served as vehicle control. Culture supernatants were assessed for IL-12p40 by ELISA. ns= not significantly different. * = significantly less than EtOH+LPS treated controls (p
Figure Legend Snippet: SchuS4 lipids inhibit secretion of IL-12p40 by hDC hDC were treated with the indicated concentration of lipids isolated from SchuS4 or LVS for 18 hours followed by addition of LPS for an additional 20 hours. EtOH served as vehicle control. Culture supernatants were assessed for IL-12p40 by ELISA. ns= not significantly different. * = significantly less than EtOH+LPS treated controls (p

Techniques Used: Concentration Assay, Isolation, Enzyme-linked Immunosorbent Assay

Inhibition of IL-12p40 in hDC is mediated by components in SchuS4 membranes (A) SchuS4 hDC were incubated with the indicated concentration of total membrane fraction (MF) isolated from SchuS4 or LVS for 18 hours. Culture supernatants were assessed for IL-12p40 by ELISA. * = significantly greater than untreated or SchuS4 MF treated hDC (p
Figure Legend Snippet: Inhibition of IL-12p40 in hDC is mediated by components in SchuS4 membranes (A) SchuS4 hDC were incubated with the indicated concentration of total membrane fraction (MF) isolated from SchuS4 or LVS for 18 hours. Culture supernatants were assessed for IL-12p40 by ELISA. * = significantly greater than untreated or SchuS4 MF treated hDC (p

Techniques Used: Inhibition, Incubation, Concentration Assay, Isolation, Enzyme-linked Immunosorbent Assay

20) Product Images from "NOD2 Stimulation by Staphylococcus aureus-Derived Peptidoglycan Is Boosted by Toll-Like Receptor 2 Costimulation with Lipoproteins in Dendritic Cells"

Article Title: NOD2 Stimulation by Staphylococcus aureus-Derived Peptidoglycan Is Boosted by Toll-Like Receptor 2 Costimulation with Lipoproteins in Dendritic Cells

Journal: Infection and Immunity

doi: 10.1128/IAI.02043-14

Stimulation with PGNpol, but not PGNpolΔ lgt , leads to significant secretion of IL-6 and IL-12p40 in DCs. Incubation of DCs with PGNpol, but not PGNpolΔ lgt , stimulated IL-6 (A) and IL-12p40 (B) secretion, which was comparable to that induced
Figure Legend Snippet: Stimulation with PGNpol, but not PGNpolΔ lgt , leads to significant secretion of IL-6 and IL-12p40 in DCs. Incubation of DCs with PGNpol, but not PGNpolΔ lgt , stimulated IL-6 (A) and IL-12p40 (B) secretion, which was comparable to that induced

Techniques Used: Incubation

Combined stimulation of DCs with NOD2 and TLR2 ligands leads to stronger secretion of IL-12p40 and TNF than stimulation with each of the stimuli alone. DCs were stimulated with PGNpolΔ lgt (10 μg/ml), Pam3Cys (1 μg/ml), and LPS
Figure Legend Snippet: Combined stimulation of DCs with NOD2 and TLR2 ligands leads to stronger secretion of IL-12p40 and TNF than stimulation with each of the stimuli alone. DCs were stimulated with PGNpolΔ lgt (10 μg/ml), Pam3Cys (1 μg/ml), and LPS

Techniques Used:

Stimulation with PGNpol in DCs is dependent on a NOD2 costimulus. Stimulation with PGNpol led to significantly increased secretion of IL-6 (A) and IL-12p40 (B) in DCs from WT mice compared to unstimulated controls. In contrast, DCs derived from NOD2 −/−
Figure Legend Snippet: Stimulation with PGNpol in DCs is dependent on a NOD2 costimulus. Stimulation with PGNpol led to significantly increased secretion of IL-6 (A) and IL-12p40 (B) in DCs from WT mice compared to unstimulated controls. In contrast, DCs derived from NOD2 −/−

Techniques Used: Mouse Assay, Derivative Assay

21) Product Images from "Monocyte-Derived Dendritic Cells Can Revert In Vitro Antigen-Specific Cellular Anergy in Active Human Paracoccidioidomycosis"

Article Title: Monocyte-Derived Dendritic Cells Can Revert In Vitro Antigen-Specific Cellular Anergy in Active Human Paracoccidioidomycosis

Journal: Journal of Fungi

doi: 10.3390/jof7030201

Influence of gp43 and CFA of P. brasiliensis on secretion of cytokines by moDCs: Enzyme-linked immunosorbent assayed (ELISA) levels of ( a ) IL-12p40 (pg/mL), and ( b ) CCL18 (ng/mL) measured on moDCs of non-PCM control subjects (CO: white bars; n = 15) and patients with active PCM (AP: grey bars; n = 17) or treated PCM (TP: black bars; n = 22), after 48 h of stimulation with gp43 or CFA, with or without TNF-α, or left untreated (Medium). Results expressed as means with SEM of levels, and capped lines indicate statistically significant differences with respective p values: * p
Figure Legend Snippet: Influence of gp43 and CFA of P. brasiliensis on secretion of cytokines by moDCs: Enzyme-linked immunosorbent assayed (ELISA) levels of ( a ) IL-12p40 (pg/mL), and ( b ) CCL18 (ng/mL) measured on moDCs of non-PCM control subjects (CO: white bars; n = 15) and patients with active PCM (AP: grey bars; n = 17) or treated PCM (TP: black bars; n = 22), after 48 h of stimulation with gp43 or CFA, with or without TNF-α, or left untreated (Medium). Results expressed as means with SEM of levels, and capped lines indicate statistically significant differences with respective p values: * p

Techniques Used: Enzyme-linked Immunosorbent Assay

22) Product Images from "Beta 1-integrin ligation and TLR ligation enhance GM-CSF–induced ALDH1A2 expression in dendritic cells, but differentially regulate their anti-inflammatory properties"

Article Title: Beta 1-integrin ligation and TLR ligation enhance GM-CSF–induced ALDH1A2 expression in dendritic cells, but differentially regulate their anti-inflammatory properties

Journal: Scientific Reports

doi: 10.1038/srep37914

Delayed stimulation with TLR-L or E-cadherin of GM-CSF/Am80-treated BM-DCs enhances their ALDH1A2 expression without reducing CD103 expression. ( a ) Representative flow cytometric profiles of the ALDH activity and surface CD103 expression of BM-DCs cultured for 1 day with Pam3CSK4 or immobilized E-cadherin/Fc in the presence or absence of GM-CSF and Am80. ( b – c ) BM-DCs were cultured for 2 days with GM-CSF and Am80 and subsequently stimulated for 1 day with Pam3CSK4 or immobilized E-cadherin/Fc. ( b ) Representative flow cytometric profiles of ALDH activity and expression of the indicated surface molecules. Solid lines represent isotype controls. ( c ) Aldh1a2 expression was assessed by real-time PCR. Relative expression levels are presented as the mean + SD of triplicate samples relative to that of the cells cultured in medium alone. ( d ) Effects of simultaneous or sequential stimulation of GM-CSF/Am80-treated BM-DCs with E-cadherin/Fc and Pam3CSK4 on the production of IL-6, IL-12p40, and IL-23p19. GM-CSF/Am80-treated BM-DCs were stimulated for 1 day with Pam3CSK4 and immobilized E-cadherin/Fc or each one alone. Aliquots of the E-cadherin/Fc-treated BM-DCs were further stimulated for 1 day with Pam3CSK4 (E-cad → Pam3) or E-cadherin/Fc (E-cad → E-cad). Cytokine concentrations in the culture supernatants were assessed by ELISA. Results are presented as the mean + SD of triplicate samples. Statistical significance was determined by the one-way ANOVA with Tukey–Kramer multiple comparisons test. *** p
Figure Legend Snippet: Delayed stimulation with TLR-L or E-cadherin of GM-CSF/Am80-treated BM-DCs enhances their ALDH1A2 expression without reducing CD103 expression. ( a ) Representative flow cytometric profiles of the ALDH activity and surface CD103 expression of BM-DCs cultured for 1 day with Pam3CSK4 or immobilized E-cadherin/Fc in the presence or absence of GM-CSF and Am80. ( b – c ) BM-DCs were cultured for 2 days with GM-CSF and Am80 and subsequently stimulated for 1 day with Pam3CSK4 or immobilized E-cadherin/Fc. ( b ) Representative flow cytometric profiles of ALDH activity and expression of the indicated surface molecules. Solid lines represent isotype controls. ( c ) Aldh1a2 expression was assessed by real-time PCR. Relative expression levels are presented as the mean + SD of triplicate samples relative to that of the cells cultured in medium alone. ( d ) Effects of simultaneous or sequential stimulation of GM-CSF/Am80-treated BM-DCs with E-cadherin/Fc and Pam3CSK4 on the production of IL-6, IL-12p40, and IL-23p19. GM-CSF/Am80-treated BM-DCs were stimulated for 1 day with Pam3CSK4 and immobilized E-cadherin/Fc or each one alone. Aliquots of the E-cadherin/Fc-treated BM-DCs were further stimulated for 1 day with Pam3CSK4 (E-cad → Pam3) or E-cadherin/Fc (E-cad → E-cad). Cytokine concentrations in the culture supernatants were assessed by ELISA. Results are presented as the mean + SD of triplicate samples. Statistical significance was determined by the one-way ANOVA with Tukey–Kramer multiple comparisons test. *** p

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

23) Product Images from "Gallic Acid Is the Major Active Component of Cortex Moutan in Inhibiting Immune Maturation of Human Monocyte-Derived Dendritic Cells"

Article Title: Gallic Acid Is the Major Active Component of Cortex Moutan in Inhibiting Immune Maturation of Human Monocyte-Derived Dendritic Cells

Journal: Molecules

doi: 10.3390/molecules200916388

High-Speed Counter-Current Chromatography (HSCCC) of Cortex Moutan. ( a ) The percentage of total weight of 30 HSCCC fractions; ( b ) The effects of the 30 fractions and Cortex Moutan aqueous extract (CM) on IL-12p40 production in monocyte-derived dendritic cells. Values represent mean ± SEM ( n = 3). Fractions (200 µg/mL) were added to DC and cultured for 48 h.
Figure Legend Snippet: High-Speed Counter-Current Chromatography (HSCCC) of Cortex Moutan. ( a ) The percentage of total weight of 30 HSCCC fractions; ( b ) The effects of the 30 fractions and Cortex Moutan aqueous extract (CM) on IL-12p40 production in monocyte-derived dendritic cells. Values represent mean ± SEM ( n = 3). Fractions (200 µg/mL) were added to DC and cultured for 48 h.

Techniques Used: High Speed Counter-current Chromatography, Derivative Assay, Cell Culture

The effects of the active ingredients from Moutan Cortex on IL-12p40 production from human monocyte derived dendritic cells (moDC) ( n = 3). ( a ) The Effects of fraction 5 (F5) and gallic acid were compared to the crude water extracts of Cortex Moutan and Penta Herb formula (PHF) on IL-12p40 production from human moDC, and hyrodocotroisone (2 μM) was used as positive control; ( b ) The effects of three active ingredients from Cortex Moutan: paeoniflorin, paeonol and gallic acid on IL-12p40 production from human moDC. * and *** indicate p
Figure Legend Snippet: The effects of the active ingredients from Moutan Cortex on IL-12p40 production from human monocyte derived dendritic cells (moDC) ( n = 3). ( a ) The Effects of fraction 5 (F5) and gallic acid were compared to the crude water extracts of Cortex Moutan and Penta Herb formula (PHF) on IL-12p40 production from human moDC, and hyrodocotroisone (2 μM) was used as positive control; ( b ) The effects of three active ingredients from Cortex Moutan: paeoniflorin, paeonol and gallic acid on IL-12p40 production from human moDC. * and *** indicate p

Techniques Used: Derivative Assay, Positive Control

24) Product Images from "Serotonin drives the acquisition of a profibrotic and anti-inflammatory gene profile through the 5-HT7R-PKA signaling axis"

Article Title: Serotonin drives the acquisition of a profibrotic and anti-inflammatory gene profile through the 5-HT7R-PKA signaling axis

Journal: Scientific Reports

doi: 10.1038/s41598-017-15348-y

The inhibitory effect of 5-HT on the LPS-induced pro-inflammatory cytokine production of human macrophages is dependent on 5-HT7R and PKA. ( A ) Production of LPS-stimulated IL-12p40 and TNFα by M-MØ non-treated (-) or pretreated with 5-HT (6 h) in the presence or absence of the 5-HT7R antagonists SB269970 or SB258719 (n = 6; *p
Figure Legend Snippet: The inhibitory effect of 5-HT on the LPS-induced pro-inflammatory cytokine production of human macrophages is dependent on 5-HT7R and PKA. ( A ) Production of LPS-stimulated IL-12p40 and TNFα by M-MØ non-treated (-) or pretreated with 5-HT (6 h) in the presence or absence of the 5-HT7R antagonists SB269970 or SB258719 (n = 6; *p

Techniques Used:

25) Product Images from "IL-12 Production Induced by Agaricus blazei Fraction H (ABH) Involves Toll-like Receptor (TLR)"

Article Title: IL-12 Production Induced by Agaricus blazei Fraction H (ABH) Involves Toll-like Receptor (TLR)

Journal: Evidence-based Complementary and Alternative Medicine

doi: 10.1093/ecam/neh043

ABH induces IL-12p40 and p70. ( A ) Ficoll-isolated human PBMC (1 × 10 6 /sample) were stimulated with ABH at various concentrations as indicated at the bottom of the panels. After incubation for 16 h, supernatants were collected and the amounts of IL-12 p40 were determined. ( B ) PBMCs were stimulated with 0.17 ng/ml ABH for 16 h. The supernatant was collected and the amounts of IL-12 p70 were determined. (A and B) IL-12 p40 and p70 in culture supernatants were measured by ELISA. Data of IL-12 p40 and p70 shown are from four and two different donors, respectively.
Figure Legend Snippet: ABH induces IL-12p40 and p70. ( A ) Ficoll-isolated human PBMC (1 × 10 6 /sample) were stimulated with ABH at various concentrations as indicated at the bottom of the panels. After incubation for 16 h, supernatants were collected and the amounts of IL-12 p40 were determined. ( B ) PBMCs were stimulated with 0.17 ng/ml ABH for 16 h. The supernatant was collected and the amounts of IL-12 p70 were determined. (A and B) IL-12 p40 and p70 in culture supernatants were measured by ELISA. Data of IL-12 p40 and p70 shown are from four and two different donors, respectively.

Techniques Used: Isolation, Incubation, Enzyme-linked Immunosorbent Assay

26) Product Images from "2'-O-Methylation within Bacterial RNA Acts as Suppressor of TLR7/TLR8 Activation in Human Innate Immune Cells"

Article Title: 2'-O-Methylation within Bacterial RNA Acts as Suppressor of TLR7/TLR8 Activation in Human Innate Immune Cells

Journal: Journal of Innate Immunity

doi: 10.1159/000375460

2'-O-methylated RNA inhibits IFN-α, TNF and IL-12p40 production with similar efficiency. a-c PBMCs were cotransfected overnight with 1 µg/ml bRNA purified from S. aureus and different ratios of 2'-O-methylated inhibitory RNA (inh. RNA). Levels of IFN-α ( a ), TNF ( b ) and IL-12p40 ( c ) were analysed in cell-free supernatants by ELISA. Data were normalized to cytokine production induced by bRNA alone to account for donor variation. Curve fit and IC 50 ( d ) were calculated with R software using the 4-parameter Weibull function. Each data point represents the average value of 2–6 independent experiments with different donors.
Figure Legend Snippet: 2'-O-methylated RNA inhibits IFN-α, TNF and IL-12p40 production with similar efficiency. a-c PBMCs were cotransfected overnight with 1 µg/ml bRNA purified from S. aureus and different ratios of 2'-O-methylated inhibitory RNA (inh. RNA). Levels of IFN-α ( a ), TNF ( b ) and IL-12p40 ( c ) were analysed in cell-free supernatants by ELISA. Data were normalized to cytokine production induced by bRNA alone to account for donor variation. Curve fit and IC 50 ( d ) were calculated with R software using the 4-parameter Weibull function. Each data point represents the average value of 2–6 independent experiments with different donors.

Techniques Used: Methylation, Purification, Enzyme-linked Immunosorbent Assay, Software

Antagonistic effect of 2'-O-methylation on bRNA-induced cytokine production in human monocytes. a-d Human PBMCs were transfected overnight with 0.5 µg/ml total tRNA ( a,b ) or total bRNA ( c,d ) isolated from the indicated bacterial strains in the presence or absence of 2'-O-methylated RNA (inh. RNA, 1 µg/ml). e-g CD14+ sorted monocytes ( e,f ) or PBMCs ( g ) were transfected with total RNA purified from S. aureus (0.5 µg/ml) or were stimulated with R848 (0.5 µg/ml) or Pam3CSK4 (1 µg/ml) alone or with 1 µg/ml 2'-O-methylated RNA (inh. RNA). a-g Production of TNF, IL-12p40 and IL-6 was analysed in cell-free supernatants by ELISA. h HEK-Blue™ cells stably expressing human TLR8 together with an NF-κB-inducible SEAP reporter gene were transfected with ssRNA40 (1.5 µg/ml) in the presence or absence of 2'-O-methylated RNA (inh. RNA, 1.5 µg/ml) complexed with DOTAP. Stimulation was performed in HEK-Blue™ detection medium that allows measurement of SEAP activity by colour development upon substrate cleavage. The optical density at 620 nm was measured after overnight incubation. The data represent the mean values + SEM of 3 independent experiments ( a-g ) or 4 ( h ) independent experiments. n.d. = Not detectable; n.t. = non-treated.* p
Figure Legend Snippet: Antagonistic effect of 2'-O-methylation on bRNA-induced cytokine production in human monocytes. a-d Human PBMCs were transfected overnight with 0.5 µg/ml total tRNA ( a,b ) or total bRNA ( c,d ) isolated from the indicated bacterial strains in the presence or absence of 2'-O-methylated RNA (inh. RNA, 1 µg/ml). e-g CD14+ sorted monocytes ( e,f ) or PBMCs ( g ) were transfected with total RNA purified from S. aureus (0.5 µg/ml) or were stimulated with R848 (0.5 µg/ml) or Pam3CSK4 (1 µg/ml) alone or with 1 µg/ml 2'-O-methylated RNA (inh. RNA). a-g Production of TNF, IL-12p40 and IL-6 was analysed in cell-free supernatants by ELISA. h HEK-Blue™ cells stably expressing human TLR8 together with an NF-κB-inducible SEAP reporter gene were transfected with ssRNA40 (1.5 µg/ml) in the presence or absence of 2'-O-methylated RNA (inh. RNA, 1.5 µg/ml) complexed with DOTAP. Stimulation was performed in HEK-Blue™ detection medium that allows measurement of SEAP activity by colour development upon substrate cleavage. The optical density at 620 nm was measured after overnight incubation. The data represent the mean values + SEM of 3 independent experiments ( a-g ) or 4 ( h ) independent experiments. n.d. = Not detectable; n.t. = non-treated.* p

Techniques Used: Methylation, Transfection, Isolation, Purification, Enzyme-linked Immunosorbent Assay, Stable Transfection, Expressing, Activity Assay, Incubation

2'-O-methylated RNA impairs TLR activation at proximal levels. a,b,d Human PBMCs were transfected with bRNA (1 µg/ml) isolated from S. aureus in the presence or absence of 2'-O-methylated inhibitory (inh.) or unmethylated control (ctr.) RNA (2 µg/ml) or were stimulated with R848 (1 μg/ml) as positive control. Expression of TNF ( a ), IL-12p40 ( b ) and IRF7 ( d ) was detected by quantitative real-time PCR at the indicated time points. The data represent the mean values of 3 independent experiments normalized to the non-treated control. * p
Figure Legend Snippet: 2'-O-methylated RNA impairs TLR activation at proximal levels. a,b,d Human PBMCs were transfected with bRNA (1 µg/ml) isolated from S. aureus in the presence or absence of 2'-O-methylated inhibitory (inh.) or unmethylated control (ctr.) RNA (2 µg/ml) or were stimulated with R848 (1 μg/ml) as positive control. Expression of TNF ( a ), IL-12p40 ( b ) and IRF7 ( d ) was detected by quantitative real-time PCR at the indicated time points. The data represent the mean values of 3 independent experiments normalized to the non-treated control. * p

Techniques Used: Methylation, Activation Assay, Transfection, Isolation, Positive Control, Expressing, Real-time Polymerase Chain Reaction

27) Product Images from "2'-O-Methylation within Bacterial RNA Acts as Suppressor of TLR7/TLR8 Activation in Human Innate Immune Cells"

Article Title: 2'-O-Methylation within Bacterial RNA Acts as Suppressor of TLR7/TLR8 Activation in Human Innate Immune Cells

Journal: Journal of Innate Immunity

doi: 10.1159/000375460

2'-O-methylated RNA inhibits IFN-α, TNF and IL-12p40 production with similar efficiency. a-c PBMCs were cotransfected overnight with 1 µg/ml bRNA purified from S. aureus and different ratios of 2'-O-methylated inhibitory RNA (inh. RNA). Levels of IFN-α ( a ), TNF ( b ) and IL-12p40 ( c ) were analysed in cell-free supernatants by ELISA. Data were normalized to cytokine production induced by bRNA alone to account for donor variation. Curve fit and IC 50 ( d ) were calculated with R software using the 4-parameter Weibull function. Each data point represents the average value of 2–6 independent experiments with different donors.
Figure Legend Snippet: 2'-O-methylated RNA inhibits IFN-α, TNF and IL-12p40 production with similar efficiency. a-c PBMCs were cotransfected overnight with 1 µg/ml bRNA purified from S. aureus and different ratios of 2'-O-methylated inhibitory RNA (inh. RNA). Levels of IFN-α ( a ), TNF ( b ) and IL-12p40 ( c ) were analysed in cell-free supernatants by ELISA. Data were normalized to cytokine production induced by bRNA alone to account for donor variation. Curve fit and IC 50 ( d ) were calculated with R software using the 4-parameter Weibull function. Each data point represents the average value of 2–6 independent experiments with different donors.

Techniques Used: Methylation, Purification, Enzyme-linked Immunosorbent Assay, Software

Antagonistic effect of 2'-O-methylation on bRNA-induced cytokine production in human monocytes. a-d Human PBMCs were transfected overnight with 0.5 µg/ml total tRNA ( a,b ) or total bRNA ( c,d ) isolated from the indicated bacterial strains in the presence or absence of 2'-O-methylated RNA (inh. RNA, 1 µg/ml). e-g CD14+ sorted monocytes ( e,f ) or PBMCs ( g ) were transfected with total RNA purified from S. aureus (0.5 µg/ml) or were stimulated with R848 (0.5 µg/ml) or Pam3CSK4 (1 µg/ml) alone or with 1 µg/ml 2'-O-methylated RNA (inh. RNA). a-g Production of TNF, IL-12p40 and IL-6 was analysed in cell-free supernatants by ELISA. h HEK-Blue™ cells stably expressing human TLR8 together with an NF-κB-inducible SEAP reporter gene were transfected with ssRNA40 (1.5 µg/ml) in the presence or absence of 2'-O-methylated RNA (inh. RNA, 1.5 µg/ml) complexed with DOTAP. Stimulation was performed in HEK-Blue™ detection medium that allows measurement of SEAP activity by colour development upon substrate cleavage. The optical density at 620 nm was measured after overnight incubation. The data represent the mean values + SEM of 3 independent experiments ( a-g ) or 4 ( h ) independent experiments. n.d. = Not detectable; n.t. = non-treated.* p
Figure Legend Snippet: Antagonistic effect of 2'-O-methylation on bRNA-induced cytokine production in human monocytes. a-d Human PBMCs were transfected overnight with 0.5 µg/ml total tRNA ( a,b ) or total bRNA ( c,d ) isolated from the indicated bacterial strains in the presence or absence of 2'-O-methylated RNA (inh. RNA, 1 µg/ml). e-g CD14+ sorted monocytes ( e,f ) or PBMCs ( g ) were transfected with total RNA purified from S. aureus (0.5 µg/ml) or were stimulated with R848 (0.5 µg/ml) or Pam3CSK4 (1 µg/ml) alone or with 1 µg/ml 2'-O-methylated RNA (inh. RNA). a-g Production of TNF, IL-12p40 and IL-6 was analysed in cell-free supernatants by ELISA. h HEK-Blue™ cells stably expressing human TLR8 together with an NF-κB-inducible SEAP reporter gene were transfected with ssRNA40 (1.5 µg/ml) in the presence or absence of 2'-O-methylated RNA (inh. RNA, 1.5 µg/ml) complexed with DOTAP. Stimulation was performed in HEK-Blue™ detection medium that allows measurement of SEAP activity by colour development upon substrate cleavage. The optical density at 620 nm was measured after overnight incubation. The data represent the mean values + SEM of 3 independent experiments ( a-g ) or 4 ( h ) independent experiments. n.d. = Not detectable; n.t. = non-treated.* p

Techniques Used: Methylation, Transfection, Isolation, Purification, Enzyme-linked Immunosorbent Assay, Stable Transfection, Expressing, Activity Assay, Incubation

2'-O-methylated RNA impairs TLR activation at proximal levels. a,b,d Human PBMCs were transfected with bRNA (1 µg/ml) isolated from S. aureus in the presence or absence of 2'-O-methylated inhibitory (inh.) or unmethylated control (ctr.) RNA (2 µg/ml) or were stimulated with R848 (1 μg/ml) as positive control. Expression of TNF ( a ), IL-12p40 ( b ) and IRF7 ( d ) was detected by quantitative real-time PCR at the indicated time points. The data represent the mean values of 3 independent experiments normalized to the non-treated control. * p
Figure Legend Snippet: 2'-O-methylated RNA impairs TLR activation at proximal levels. a,b,d Human PBMCs were transfected with bRNA (1 µg/ml) isolated from S. aureus in the presence or absence of 2'-O-methylated inhibitory (inh.) or unmethylated control (ctr.) RNA (2 µg/ml) or were stimulated with R848 (1 μg/ml) as positive control. Expression of TNF ( a ), IL-12p40 ( b ) and IRF7 ( d ) was detected by quantitative real-time PCR at the indicated time points. The data represent the mean values of 3 independent experiments normalized to the non-treated control. * p

Techniques Used: Methylation, Activation Assay, Transfection, Isolation, Positive Control, Expressing, Real-time Polymerase Chain Reaction

28) Product Images from "Cinobufagin Modulates Human Innate Immune Responses and Triggers Antibacterial Activity"

Article Title: Cinobufagin Modulates Human Innate Immune Responses and Triggers Antibacterial Activity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0160734

Inhibited cytokine release of LPS-stimulated DCs. DCs were stimulated with LPS (100 ng/ml) in the presence of vehicle or CBG for 24 hours. Supernatants were collected and analyzed for IL-6, IL-8, IL-12p40, TNF-α and IL-10. Data shown represent cytokine production for each donor and mean values for 6–8 donors. *P
Figure Legend Snippet: Inhibited cytokine release of LPS-stimulated DCs. DCs were stimulated with LPS (100 ng/ml) in the presence of vehicle or CBG for 24 hours. Supernatants were collected and analyzed for IL-6, IL-8, IL-12p40, TNF-α and IL-10. Data shown represent cytokine production for each donor and mean values for 6–8 donors. *P

Techniques Used:

29) Product Images from "Myeloid differentiation primary response protein 88 (MyD88)-deficient dendritic cells exhibit a skewed cytokine response to BCG"

Article Title: Myeloid differentiation primary response protein 88 (MyD88)-deficient dendritic cells exhibit a skewed cytokine response to BCG

Journal: BMC Research Notes

doi: 10.1186/s13104-019-4086-6

Cytokine secretion by MyD88 −/− dendritic cells (DCs) in response to BCG. Bone marrow-derived DCs from wild-type (open bars) and MyD88 −/− mice (filled bars) were stimulated with BCG at MOI of 10. Culture supernatants were harvested after 24 h and analyzed for TNF-α, IL-12p40. Significant levels of TNF-α and IL-12p40 were produced by BCG-stimulated wild-type and MyD88 −/− DCs, compared with unstimulated DCs ( a , b ). Culture supernatants were also analyzed for IL-10 levels. Significant amount of IL-10 was secreted by BCG-stimulated wild-type, but not by MyD88 −/− DCs ( c ). Synthetic TLR2 ligand Pam 3 CSK 4 (100 ng/ml) was used as an experimental control. Data shown are mean ± SEM of three independent experiments. **p
Figure Legend Snippet: Cytokine secretion by MyD88 −/− dendritic cells (DCs) in response to BCG. Bone marrow-derived DCs from wild-type (open bars) and MyD88 −/− mice (filled bars) were stimulated with BCG at MOI of 10. Culture supernatants were harvested after 24 h and analyzed for TNF-α, IL-12p40. Significant levels of TNF-α and IL-12p40 were produced by BCG-stimulated wild-type and MyD88 −/− DCs, compared with unstimulated DCs ( a , b ). Culture supernatants were also analyzed for IL-10 levels. Significant amount of IL-10 was secreted by BCG-stimulated wild-type, but not by MyD88 −/− DCs ( c ). Synthetic TLR2 ligand Pam 3 CSK 4 (100 ng/ml) was used as an experimental control. Data shown are mean ± SEM of three independent experiments. **p

Techniques Used: Derivative Assay, Mouse Assay, Produced

30) Product Images from "Gold Nanorods Inhibit Respiratory Syncytial Virus by Stimulating the Innate Immune Response"

Article Title: Gold Nanorods Inhibit Respiratory Syncytial Virus by Stimulating the Innate Immune Response

Journal: Nanomedicine : nanotechnology, biology, and medicine

doi: 10.1016/j.nano.2016.06.006

Splenocytes (1 × 10 6 /mL) from each treated mice groups (PBS, RSV, GNR, GNR-RSV) were restimulated with LPS, RSV, GNR and GNR-RSV. Supernatants were collected and used to measure the levels of (A) IL-12p40, (B) IL-6, (C) IL-10, (D) TNF-α, and (E) IFN-γ cytokine production.
Figure Legend Snippet: Splenocytes (1 × 10 6 /mL) from each treated mice groups (PBS, RSV, GNR, GNR-RSV) were restimulated with LPS, RSV, GNR and GNR-RSV. Supernatants were collected and used to measure the levels of (A) IL-12p40, (B) IL-6, (C) IL-10, (D) TNF-α, and (E) IFN-γ cytokine production.

Techniques Used: Mouse Assay

31) Product Images from "Nuclear Localization of Suppressor of Cytokine Signaling-1 Regulates Local Immunity in the Lung"

Article Title: Nuclear Localization of Suppressor of Cytokine Signaling-1 Regulates Local Immunity in the Lung

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2016.00514

Generation of Socs1ΔNLS transgenic mice . (A) Schematic drawing of the bacterial artificial chromosome (BAC) consisting of a mutated Socs1 locus containing Socs1ΔNLS, eGFP , and LuciferaseCBG99 . 2A sequences result in cleavage of the polyprotein. (B) qPCR strategy to specifically detect Socs1 wildtype (wt) using the primer P1 and P2 in combination with a labeled probe within the NLS, Socs1ΔNLS using the reverse primer P3 in the NLS or total Socs1 using the primer P1 and P2. BMMs of Socs1 +/+ MGL tg mice of three different founders were stimulated with IFNγ (50 ng/ml) for 24 h and analyzed for (C) mRNA expression of Socs1 wt and Socs1ΔNLS , (D) luciferase activity, and (E) the percentage of GFP positive cells ( n = 4–5, mean + SD, two-way ANOVA including Bonferroni post-test). (F) CD11c + cells were isolated from lung homogenate and stimulated with CpG (1 μM), LPS (100 ng/ml), and pI:C (10 μg/ml) for 24 h. IL-12p40 protein levels were measured by ELISA ( n = 3–5, mean + SD, two-way ANOVA including Bonferroni post-test).
Figure Legend Snippet: Generation of Socs1ΔNLS transgenic mice . (A) Schematic drawing of the bacterial artificial chromosome (BAC) consisting of a mutated Socs1 locus containing Socs1ΔNLS, eGFP , and LuciferaseCBG99 . 2A sequences result in cleavage of the polyprotein. (B) qPCR strategy to specifically detect Socs1 wildtype (wt) using the primer P1 and P2 in combination with a labeled probe within the NLS, Socs1ΔNLS using the reverse primer P3 in the NLS or total Socs1 using the primer P1 and P2. BMMs of Socs1 +/+ MGL tg mice of three different founders were stimulated with IFNγ (50 ng/ml) for 24 h and analyzed for (C) mRNA expression of Socs1 wt and Socs1ΔNLS , (D) luciferase activity, and (E) the percentage of GFP positive cells ( n = 4–5, mean + SD, two-way ANOVA including Bonferroni post-test). (F) CD11c + cells were isolated from lung homogenate and stimulated with CpG (1 μM), LPS (100 ng/ml), and pI:C (10 μg/ml) for 24 h. IL-12p40 protein levels were measured by ELISA ( n = 3–5, mean + SD, two-way ANOVA including Bonferroni post-test).

Techniques Used: Transgenic Assay, Mouse Assay, BAC Assay, Real-time Polymerase Chain Reaction, Labeling, Expressing, Luciferase, Activity Assay, Isolation, Enzyme-linked Immunosorbent Assay

32) Product Images from "Intracellular Delivery of Lipopolysaccharide Induces Effective Th1-Immune Responses Independent of IL-12"

Article Title: Intracellular Delivery of Lipopolysaccharide Induces Effective Th1-Immune Responses Independent of IL-12

Journal: PLoS ONE

doi: 10.1371/journal.pone.0068671

Lipopolysaccharide (LPS)-liposomes induce interferon (IFN)-β production but not inflammatory cytokines in bone marrow-derived dendritic cells (BMDCs). (A) BMDCs from wild-type (WT) mice (1.5×10 5 ) were stimulated with LPS (100 ng/mL) or LPS-liposomes (100 ng/mL) for 2 h (TNF-α) or 24 h (IL-6, IL-12p40, and RANTES). Cytokine levels were determined by ELISA. (B) BMDCs from wild-type (WT) mice (1.5×10 5 ) were stimulated with LPS (100 ng/mL) or LPS-liposomes (100 ng/mL) for 9 h. IFN-β levels were determined by ELISA. (C) BMDCs (1×10 6 ) from WT mice were stimulated with LPS or LPS-liposomes (10 or 100 ng/mL) for 4 h and expression of IFN-β and β-actin mRNAs was determined by reverse transcription-polymerase chain reaction. PBS was control for LPS and liposomes was control for LPS-liposomes (Open columns). Data in A and B are average of three independent experiments. The values represent means ± S.E.M * P
Figure Legend Snippet: Lipopolysaccharide (LPS)-liposomes induce interferon (IFN)-β production but not inflammatory cytokines in bone marrow-derived dendritic cells (BMDCs). (A) BMDCs from wild-type (WT) mice (1.5×10 5 ) were stimulated with LPS (100 ng/mL) or LPS-liposomes (100 ng/mL) for 2 h (TNF-α) or 24 h (IL-6, IL-12p40, and RANTES). Cytokine levels were determined by ELISA. (B) BMDCs from wild-type (WT) mice (1.5×10 5 ) were stimulated with LPS (100 ng/mL) or LPS-liposomes (100 ng/mL) for 9 h. IFN-β levels were determined by ELISA. (C) BMDCs (1×10 6 ) from WT mice were stimulated with LPS or LPS-liposomes (10 or 100 ng/mL) for 4 h and expression of IFN-β and β-actin mRNAs was determined by reverse transcription-polymerase chain reaction. PBS was control for LPS and liposomes was control for LPS-liposomes (Open columns). Data in A and B are average of three independent experiments. The values represent means ± S.E.M * P

Techniques Used: Derivative Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

33) Product Images from "Evasion of Innate Immune Responses by the Highly Virulent Cryptococcus gattii by Altering Capsule Glucuronoxylomannan Structure"

Article Title: Evasion of Innate Immune Responses by the Highly Virulent Cryptococcus gattii by Altering Capsule Glucuronoxylomannan Structure

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2015.00101

Cytokine release from dendritic cells in response to native GXM purified from H99 and JP02 strains and the corresponding O-deacetylated products . IL-6 (A) and TNF-α (B) release from JAWSII cells (2 × 10 5 cells/mL) stimulated by GXMs (50 μg/mL). IL-12p40 (C) and TNF-α (D) release from BMDCs (1 × 10 6 cells/mL) stimulated by GXMs. The experiments were performed in triplicate (means ± SD). Data sets in (A) or (B) were compared by the unpaired t -test, except for vs. control in (A) , which were compared by the Mann–Whitney U-test. Treatment group means of (C) or (D) were compared to H99 GXM by the unpaired t -test, except for vs. JP02 at 10 μg/mL in (C) , which were compared by Welch's t -test. * P
Figure Legend Snippet: Cytokine release from dendritic cells in response to native GXM purified from H99 and JP02 strains and the corresponding O-deacetylated products . IL-6 (A) and TNF-α (B) release from JAWSII cells (2 × 10 5 cells/mL) stimulated by GXMs (50 μg/mL). IL-12p40 (C) and TNF-α (D) release from BMDCs (1 × 10 6 cells/mL) stimulated by GXMs. The experiments were performed in triplicate (means ± SD). Data sets in (A) or (B) were compared by the unpaired t -test, except for vs. control in (A) , which were compared by the Mann–Whitney U-test. Treatment group means of (C) or (D) were compared to H99 GXM by the unpaired t -test, except for vs. JP02 at 10 μg/mL in (C) , which were compared by Welch's t -test. * P

Techniques Used: Purification, MANN-WHITNEY

34) Product Images from "Immunomodulation of human monocytes following exposure to Lutzomyia intermedia saliva"

Article Title: Immunomodulation of human monocytes following exposure to Lutzomyia intermedia saliva

Journal: BMC Immunology

doi: 10.1186/1471-2172-9-12

Effect of L. intermedia SGS on LPS-stimulated human monocytes . Monocytes were pre-treated with SGS overnight and stimulated with LPS for 24 h (TNF-α and IL-6) or 48 h (IL-10 and IL-12p40). The presence of IL-10 (A), IL-6 (B), TNF-α (C) and IL-12p40 (D) was analyzed by ELISA. The data for individual donors are presented. (*p
Figure Legend Snippet: Effect of L. intermedia SGS on LPS-stimulated human monocytes . Monocytes were pre-treated with SGS overnight and stimulated with LPS for 24 h (TNF-α and IL-6) or 48 h (IL-10 and IL-12p40). The presence of IL-10 (A), IL-6 (B), TNF-α (C) and IL-12p40 (D) was analyzed by ELISA. The data for individual donors are presented. (*p

Techniques Used: Enzyme-linked Immunosorbent Assay

35) Product Images from "MyD88 Associated ROS Generation Is Crucial for Lactobacillus Induced IL-12 Production in Macrophage"

Article Title: MyD88 Associated ROS Generation Is Crucial for Lactobacillus Induced IL-12 Production in Macrophage

Journal: PLoS ONE

doi: 10.1371/journal.pone.0035880

Kinetic analysis of ROS production, IL-12 mRNA expression and IL-12 production from macrophage after LAB stimulation. Peritoneal macrophages were cultured with 1 µg/ml KW3110 and IL-12p35 and IL-12p40 mRNA expression (A), IL-12p40 and IL-12p70 production (B) and ROS production (C) were measured over time. Values are mean of duplicate culture (C) or mean ± SD of triplicate culture (A, B). Data are representative of three independent experiments yielding similar results.
Figure Legend Snippet: Kinetic analysis of ROS production, IL-12 mRNA expression and IL-12 production from macrophage after LAB stimulation. Peritoneal macrophages were cultured with 1 µg/ml KW3110 and IL-12p35 and IL-12p40 mRNA expression (A), IL-12p40 and IL-12p70 production (B) and ROS production (C) were measured over time. Values are mean of duplicate culture (C) or mean ± SD of triplicate culture (A, B). Data are representative of three independent experiments yielding similar results.

Techniques Used: Expressing, Cell Culture

ROS dependent IL-12 production from LAB stimulated macrophage. (A) Peritoneal macrophages were cultured with 1 µg/ml KW3110, ATCC53103 or 1 µM CpG for 24 hrs in the presence of 0, 0.1, 0.5 or 2 mM apocynin (Apo) or 0, 0.01, 0.05 or 0.2 mM propyl gallate (PG). IL-12p40 and IL-12p70 in the cell culture supernatant were measured by ELISA. (C) Peritoneal macrophages were cultured with FITC-labeled identical KW3110 (3 µg/ml) in the presence of Apocynin or propyl gallate. Phagocytosis index was calculated in the same manner as described in Fig. 1A . Data are representative of three independent experiments.
Figure Legend Snippet: ROS dependent IL-12 production from LAB stimulated macrophage. (A) Peritoneal macrophages were cultured with 1 µg/ml KW3110, ATCC53103 or 1 µM CpG for 24 hrs in the presence of 0, 0.1, 0.5 or 2 mM apocynin (Apo) or 0, 0.01, 0.05 or 0.2 mM propyl gallate (PG). IL-12p40 and IL-12p70 in the cell culture supernatant were measured by ELISA. (C) Peritoneal macrophages were cultured with FITC-labeled identical KW3110 (3 µg/ml) in the presence of Apocynin or propyl gallate. Phagocytosis index was calculated in the same manner as described in Fig. 1A . Data are representative of three independent experiments.

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Labeling

36) Product Images from "ISCOMATRIX Adjuvant Combines Immune Activation with Antigen Delivery to Dendritic Cells In Vivo Leading to Effective Cross-Priming of CD8+ T Cells"

Article Title: ISCOMATRIX Adjuvant Combines Immune Activation with Antigen Delivery to Dendritic Cells In Vivo Leading to Effective Cross-Priming of CD8+ T Cells

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

doi: 10.4049/jimmunol.1004114

Innate and adaptive immune cells in VDLNs upregulate activation marker expression and IFN-γ production. Mice were vaccinated s.c. with PBS, OVA, OVA/ISCOM, or ISCOMATRIX adjuvant alone and VDLNs were processed into single-cell suspensions after 24 h for flow cytometry. A , Expression of the activation marker CD69 by B cells, CD4 + T cells, and CD8 + T cells. B , Expression of CD69 and IFN-γ by CD3 − NK1.1 + NK cells and CD3 + NK1.1 + NKT cells. C , Expression of CD86 and IL-12p40 by CD8α + and CD8α − DC populations. Results from one representative experiment of two are shown as means ± SD comprising of five animals per group. * p
Figure Legend Snippet: Innate and adaptive immune cells in VDLNs upregulate activation marker expression and IFN-γ production. Mice were vaccinated s.c. with PBS, OVA, OVA/ISCOM, or ISCOMATRIX adjuvant alone and VDLNs were processed into single-cell suspensions after 24 h for flow cytometry. A , Expression of the activation marker CD69 by B cells, CD4 + T cells, and CD8 + T cells. B , Expression of CD69 and IFN-γ by CD3 − NK1.1 + NK cells and CD3 + NK1.1 + NKT cells. C , Expression of CD86 and IL-12p40 by CD8α + and CD8α − DC populations. Results from one representative experiment of two are shown as means ± SD comprising of five animals per group. * p

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

37) Product Images from "The Cell Surface Receptor SLAM Controls T Cell and Macrophage Functions"

Article Title: The Cell Surface Receptor SLAM Controls T Cell and Macrophage Functions

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20031835

Altered IL-12, TNF-α, IL-6, and NO production by peritoneal macrophages from SLAM −/− C57BL/6 mice. (A) IL-12 production by SLAM −/− C57BL/6 or WT SLAM +/+ C57BL/6 peritoneal macrophages. Resting peritoneal macrophages from 12 SLAM −/− C57BL/6 or WT SLAM +/+ C57BL/6 mice were stimulated with 200 U/ml IFN-γ, 2 ng/ml LPS, or with IFN-γ plus LPS for 24 h. IL-12p70 was determined by ELISA as described in Materials and Methods. M, cells incubated in medium only. (B) TNF-α production by SLAM −/− C57BL/6 or WT SLAM +/+ C57BL/6 peritoneal macrophages. Resting peritoneal macrophages from SLAM −/− C57BL/6 or WT SLAM +/+ C57BL/6 mice were stimulated as described in A. TNF-α was determined by ELISA in the same culture supernatants as described in Materials and Methods. M, cells incubated in medium only. (C) IL-6 production by SLAM −/− C57BL/6 or WT SLAM +/+ C57BL/6 peritoneal macrophages. Resting peritoneal macrophages from SLAM −/− C57BL/6 or WT SLAM +/+ C57BL/6 mice were stimulated as described in A. IL-6 was determined by ELISA in the same culture supernatants as described in Materials and Methods. M, cells incubated in medium only. (D) NO production by SLAM −/− C57BL/6 or WT SLAM +/+ C57BL/6 peritoneal macrophages. Resting peritoneal macrophages from SLAM −/− C57BL/6 or WT SLAM +/+ C57BL/6 mice were stimulated as described in A. NO was determined in the same culture supernatants as described in Materials and Methods. M, cells incubated in medium only. (E) IL-12p40 production is impaired in thioglycollate-induced peritoneal macrophages from SLAM −/− C57BL/6 mice, but not from SAP −/− C57BL/6 mice. Thioglycollate-elicited macrophages were isolated from the peritoneal cavity of SLAM −/− , SAP −/− , or WT SLAM +/+ C57BL/6 mice. 2 × 10 6 macrophages were activated with increasing amounts of LPS (at 1, 10, or 100 ng/ml). Supernatants were collected after 24 h and IL-12p40 was determined by ELISA. (F) IL-12p40 production by macrophages in response to CpG and PGN. Thioglycollate-elicited peritoneal SLAM −/− or SLAM +/+ BALB/c macrophages (10 6 per well) were treated with 10 μM CpG or 25 μg/ml peptidoglycan for 24 h. Supernatants were removed and IL-12p40 levels were determined by ELISA. (G) TNF-α production by macrophages in response to CpG and PGN. Thioglycollate-elicited peritoneal SLAM −/− or SLAM +/+ BALB/c macrophages (10 6 per well) were treated with 10 μM CpG or 25 μg/ml peptidoglycan for 24 h. Supernatants were used to determine IL-12p40 levels by ELISA. (H) Antigen presentation by macrophages. 2 × 10 5 thioglycollate-elicited peritoneal SLAM −/− or SLAM +/+ BALB/c macrophages were pulsed with 0.01–1 μg/ml of the OVA peptide OVA 323–339. Next, antigen-pulsed macrophages were incubated for 3 d with 10 5 KJ126 + CD4 + T cells isolated from DO11.10 TCR transgenic mice. To determine DNA synthesis, cultures were pulsed with [ 3 H]thymidine in the final 18 h. (I) Phagocytosis of F18 E. coli by macrophages. Thioglycollate-elicited peritoneal SLAM −/− or SLAM +/+ BALB/c macrophages were incubated with E. coli F18 bacteria. After 1 h, cells were lysed and phagocytosis rates were obtained by counting colonies after 24 h of incubation. CFU, colony-forming units.
Figure Legend Snippet: Altered IL-12, TNF-α, IL-6, and NO production by peritoneal macrophages from SLAM −/− C57BL/6 mice. (A) IL-12 production by SLAM −/− C57BL/6 or WT SLAM +/+ C57BL/6 peritoneal macrophages. Resting peritoneal macrophages from 12 SLAM −/− C57BL/6 or WT SLAM +/+ C57BL/6 mice were stimulated with 200 U/ml IFN-γ, 2 ng/ml LPS, or with IFN-γ plus LPS for 24 h. IL-12p70 was determined by ELISA as described in Materials and Methods. M, cells incubated in medium only. (B) TNF-α production by SLAM −/− C57BL/6 or WT SLAM +/+ C57BL/6 peritoneal macrophages. Resting peritoneal macrophages from SLAM −/− C57BL/6 or WT SLAM +/+ C57BL/6 mice were stimulated as described in A. TNF-α was determined by ELISA in the same culture supernatants as described in Materials and Methods. M, cells incubated in medium only. (C) IL-6 production by SLAM −/− C57BL/6 or WT SLAM +/+ C57BL/6 peritoneal macrophages. Resting peritoneal macrophages from SLAM −/− C57BL/6 or WT SLAM +/+ C57BL/6 mice were stimulated as described in A. IL-6 was determined by ELISA in the same culture supernatants as described in Materials and Methods. M, cells incubated in medium only. (D) NO production by SLAM −/− C57BL/6 or WT SLAM +/+ C57BL/6 peritoneal macrophages. Resting peritoneal macrophages from SLAM −/− C57BL/6 or WT SLAM +/+ C57BL/6 mice were stimulated as described in A. NO was determined in the same culture supernatants as described in Materials and Methods. M, cells incubated in medium only. (E) IL-12p40 production is impaired in thioglycollate-induced peritoneal macrophages from SLAM −/− C57BL/6 mice, but not from SAP −/− C57BL/6 mice. Thioglycollate-elicited macrophages were isolated from the peritoneal cavity of SLAM −/− , SAP −/− , or WT SLAM +/+ C57BL/6 mice. 2 × 10 6 macrophages were activated with increasing amounts of LPS (at 1, 10, or 100 ng/ml). Supernatants were collected after 24 h and IL-12p40 was determined by ELISA. (F) IL-12p40 production by macrophages in response to CpG and PGN. Thioglycollate-elicited peritoneal SLAM −/− or SLAM +/+ BALB/c macrophages (10 6 per well) were treated with 10 μM CpG or 25 μg/ml peptidoglycan for 24 h. Supernatants were removed and IL-12p40 levels were determined by ELISA. (G) TNF-α production by macrophages in response to CpG and PGN. Thioglycollate-elicited peritoneal SLAM −/− or SLAM +/+ BALB/c macrophages (10 6 per well) were treated with 10 μM CpG or 25 μg/ml peptidoglycan for 24 h. Supernatants were used to determine IL-12p40 levels by ELISA. (H) Antigen presentation by macrophages. 2 × 10 5 thioglycollate-elicited peritoneal SLAM −/− or SLAM +/+ BALB/c macrophages were pulsed with 0.01–1 μg/ml of the OVA peptide OVA 323–339. Next, antigen-pulsed macrophages were incubated for 3 d with 10 5 KJ126 + CD4 + T cells isolated from DO11.10 TCR transgenic mice. To determine DNA synthesis, cultures were pulsed with [ 3 H]thymidine in the final 18 h. (I) Phagocytosis of F18 E. coli by macrophages. Thioglycollate-elicited peritoneal SLAM −/− or SLAM +/+ BALB/c macrophages were incubated with E. coli F18 bacteria. After 1 h, cells were lysed and phagocytosis rates were obtained by counting colonies after 24 h of incubation. CFU, colony-forming units.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Incubation, Isolation, Transgenic Assay, DNA Synthesis

38) Product Images from "RIPK1-dependent apoptosis bypasses pathogen blockade of innate signaling to promote immune defense"

Article Title: RIPK1-dependent apoptosis bypasses pathogen blockade of innate signaling to promote immune defense

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20170347

RIPK1 kinase activity is required for Yersinia -induced apoptosis. WT and Ripk1 kd BMDMs were treated with 60 µM of the RIPK1 inhibitor Nec-1 or control media and infected with YopJ-deficient (ΔYopJ) or WT ( Yp ) Y. pseudotuberculosis. (A) Cell death was measured by LDH release assay at 4 h postinfection. (B and C) Apoptotic caspase cleavage was assessed by flow cytometry staining for CC3 (B) and Western blotting of lysates (C) 2 h postinfection. Molecular mass is indicated in kilodaltons. (D and E) Supernatants of BMDMs were measured for release of TNF at 4 h postinfection (D) or for TNF, IL-12p40, and IL-6 at 6 h after treatment with TLR ligands (E). Data are representative of more than three independent experiments in A and two independent experiments in B–E. Graphs show mean and SD of triplicate experiments. (F) Splenocytes were harvested 4 h after intravenous infection with Yp and stained for the indicated cell types and CC3. (G) Percentage of WT and Ripk1 kd CC3 + spleen cells infected with Yp relative to WT mice infected with the virulence plasmid–deficient strain ( YpP − ). Flow cytometry numbers and bar graph show mean and SD ( n = 4–5 mice per group) values representative of two independent experiments. #, P
Figure Legend Snippet: RIPK1 kinase activity is required for Yersinia -induced apoptosis. WT and Ripk1 kd BMDMs were treated with 60 µM of the RIPK1 inhibitor Nec-1 or control media and infected with YopJ-deficient (ΔYopJ) or WT ( Yp ) Y. pseudotuberculosis. (A) Cell death was measured by LDH release assay at 4 h postinfection. (B and C) Apoptotic caspase cleavage was assessed by flow cytometry staining for CC3 (B) and Western blotting of lysates (C) 2 h postinfection. Molecular mass is indicated in kilodaltons. (D and E) Supernatants of BMDMs were measured for release of TNF at 4 h postinfection (D) or for TNF, IL-12p40, and IL-6 at 6 h after treatment with TLR ligands (E). Data are representative of more than three independent experiments in A and two independent experiments in B–E. Graphs show mean and SD of triplicate experiments. (F) Splenocytes were harvested 4 h after intravenous infection with Yp and stained for the indicated cell types and CC3. (G) Percentage of WT and Ripk1 kd CC3 + spleen cells infected with Yp relative to WT mice infected with the virulence plasmid–deficient strain ( YpP − ). Flow cytometry numbers and bar graph show mean and SD ( n = 4–5 mice per group) values representative of two independent experiments. #, P

Techniques Used: Activity Assay, Infection, Lactate Dehydrogenase Assay, Flow Cytometry, Staining, Western Blot, Mouse Assay, Plasmid Preparation

39) Product Images from "Innate PI3K p110δ regulates Th1/Th17 development and microbiota-dependent colitis"

Article Title: Innate PI3K p110δ regulates Th1/Th17 development and microbiota-dependent colitis

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

doi: 10.4049/jimmunol.1301533

Defective p110δ activity alters macrophage production of IL-10 and IL-12p40 in response to bacterial products
Figure Legend Snippet: Defective p110δ activity alters macrophage production of IL-10 and IL-12p40 in response to bacterial products

Techniques Used: Activity Assay

A p110δ-specific inhibitor decreases IL-10 and augments Il-12p40 production in WT macrophages stimulated with bacterial products
Figure Legend Snippet: A p110δ-specific inhibitor decreases IL-10 and augments Il-12p40 production in WT macrophages stimulated with bacterial products

Techniques Used:

40) Product Images from "Cutting Edge: Synchronization of IRF1, JunB, and C/EBPβ Activities during TLR3–TLR7 Cross-Talk Orchestrates Timely Cytokine Synergy in the Proinflammatory Response"

Article Title: Cutting Edge: Synchronization of IRF1, JunB, and C/EBPβ Activities during TLR3–TLR7 Cross-Talk Orchestrates Timely Cytokine Synergy in the Proinflammatory Response

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1402358

IRF1 and JunB collaborate to synergize IL-6 and IL-12p40 production. J774 cells were transfected with sequence-specific siRNA targeting Irf1 or Junb singly or doubly or were transfected with a nontargeting scramble siRNA control for 24 h. Cells were then
Figure Legend Snippet: IRF1 and JunB collaborate to synergize IL-6 and IL-12p40 production. J774 cells were transfected with sequence-specific siRNA targeting Irf1 or Junb singly or doubly or were transfected with a nontargeting scramble siRNA control for 24 h. Cells were then

Techniques Used: Transfection, Sequencing

IRF1 and C/EBPβ collaborate to synergize IL-6 and IL-12p40 production. J774 cells were transfected with sequence-specific siRNA targeting Irf1 or Cebpb , singly or doubly, or were transfected with a nontargeting scramble siRNA control for 24 h.
Figure Legend Snippet: IRF1 and C/EBPβ collaborate to synergize IL-6 and IL-12p40 production. J774 cells were transfected with sequence-specific siRNA targeting Irf1 or Cebpb , singly or doubly, or were transfected with a nontargeting scramble siRNA control for 24 h.

Techniques Used: Transfection, Sequencing

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Enzyme-linked Immunosorbent Assay:

Article Title: Triptolide Inhibits IL-12/IL-23 Expression in APCs via CCAAT/Enhancer-Binding Protein ?
Article Snippet: Cytokine secretion was measured by ELISA, using appropriately diluted culture supernatants. .. Human IL-12p40 and p70, and mouse IL-12p40, p70 were measured by the respective ELISA kits from BD Pharmingen (San Diego, CA), mouse IL-23 was measured by an ELISA kit from Biolegend (San Diego, CA), in triplicates. .. Total RNAs were isolated using the RNeasy mini kit (Qiagen, Hilden, Germany) and reverse-transcribed into cDNA.

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    Becton Dickinson murine il 12p40
    IL-10 is not sufficient to suppress IL12B induction in MDMs. MDMs were differentiated in R5 medium for 6–7 days and stimulated with LPS and IFNγ, with recombinant IL-10 from human cells, or both for 24 h. (A) IL12B expression was measured by RT-qPCR ( N = 11). (B) <t>IL-12p40</t> in culture supernatants ( N = 9) was measured by ELISA. Each square denotes a biological replicate (individual healthy donor). Colors encode individual healthy donors, and colors are consistent between panels within this figure. Median values are indicated by horizontal bars. Statistical significances were calculated with paired t -tests. ** P
    Murine Il 12p40, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of 25D on differentiation and/or stimulation of monocyte-derived DCs. Monocytes were differentiated for six days using rGM-CSF and rIL-4 in media with 10% FCS in the presence (25D diff -DCs or 25D diff/stim -DCs) or absence (serum-DCs or 25D stim -DCs) of additional 25D (10 −7 M). Subsequently, 25D-DCs and serum-DCs were stimulated with TLR2/1L (1 μg/ml) or left untreated in the presence (25D diff/stim -DCs or 25D stim -DCs) or absence (serum-DCs or 25D diff -DCs) of additional 25D (10 −7 M), and cultured in fresh media with 10% FCS for 18–24 hours. TNF-α, IL-6, IL-1β, <t>IL-12p40,</t> IL-12p70, IL-23 and IL-10, as well as total and active TGF-β levels in culture supernatants were measured by ELISA or CBA (mean cytokine levels in ng/ml ± SEM, n = 4). Results shown in Fig 1 and Fig. 2 were conducted using cells from the same cell preparations of identical donors. *p
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    Becton Dickinson monoclonal antibodies against il 12p40
    MyD88 is responsible for the early induction of Th1-related cytokines after N. caninum infection. The presence of <t>IL-12p40,</t> IFN-γ, and IL-10 was evaluated by ELISA in the peritoneal exudate and serum samples of uninfected, and infected WT and MyD88 −/− mice at 3, 7, and 10 days p.i. ( n = 3 mice/group/date). Data are representative of two independent experiments. * Indicates significant statistical differences (mean ± SEM, p
    Monoclonal Antibodies Against Il 12p40, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    IL-10 is not sufficient to suppress IL12B induction in MDMs. MDMs were differentiated in R5 medium for 6–7 days and stimulated with LPS and IFNγ, with recombinant IL-10 from human cells, or both for 24 h. (A) IL12B expression was measured by RT-qPCR ( N = 11). (B) IL-12p40 in culture supernatants ( N = 9) was measured by ELISA. Each square denotes a biological replicate (individual healthy donor). Colors encode individual healthy donors, and colors are consistent between panels within this figure. Median values are indicated by horizontal bars. Statistical significances were calculated with paired t -tests. ** P

    Journal: Frontiers in Immunology

    Article Title: Chromatin Binding of c-REL and p65 Is Not Limiting for Macrophage IL12B Transcription During Immediate Suppression by Ovarian Carcinoma Ascites

    doi: 10.3389/fimmu.2018.01425

    Figure Lengend Snippet: IL-10 is not sufficient to suppress IL12B induction in MDMs. MDMs were differentiated in R5 medium for 6–7 days and stimulated with LPS and IFNγ, with recombinant IL-10 from human cells, or both for 24 h. (A) IL12B expression was measured by RT-qPCR ( N = 11). (B) IL-12p40 in culture supernatants ( N = 9) was measured by ELISA. Each square denotes a biological replicate (individual healthy donor). Colors encode individual healthy donors, and colors are consistent between panels within this figure. Median values are indicated by horizontal bars. Statistical significances were calculated with paired t -tests. ** P

    Article Snippet: Murine Il-12p40 was measured with an ELISA kit (BD Biosciences, no. 555165) according to the manufacturer’s protocol.

    Techniques: Recombinant, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    The role of Rel in the induction of Il12b expression. Murine Rel knockout BMDMs or wild-type BMDMs ( N = 2 each) were differentiated in vitro and stimulated with LPS or LPS and IFNγ for 24 h. Cells were used for RT-qPCR analysis of Il12b (A) , and Il-12p40 was measured in supernatants by ELISA (B) ; n.d., not detected.

    Journal: Frontiers in Immunology

    Article Title: Chromatin Binding of c-REL and p65 Is Not Limiting for Macrophage IL12B Transcription During Immediate Suppression by Ovarian Carcinoma Ascites

    doi: 10.3389/fimmu.2018.01425

    Figure Lengend Snippet: The role of Rel in the induction of Il12b expression. Murine Rel knockout BMDMs or wild-type BMDMs ( N = 2 each) were differentiated in vitro and stimulated with LPS or LPS and IFNγ for 24 h. Cells were used for RT-qPCR analysis of Il12b (A) , and Il-12p40 was measured in supernatants by ELISA (B) ; n.d., not detected.

    Article Snippet: Murine Il-12p40 was measured with an ELISA kit (BD Biosciences, no. 555165) according to the manufacturer’s protocol.

    Techniques: Expressing, Knock-Out, In Vitro, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    IL-12 production by tumor-associated macrophages from ovarian carcinoma patients is suppressed by ascites in vitro . (A) Ex vivo TAMs ( N = 8) were cultivated in autologous ascites in the presence or in the absence of IFNγ or in normal medium (R5) as indicated, and non-cultivated ex vivo TAMs served as an additional control. LPS and IFNγ or their respective solvents (vehicle) were added to the culture supernatants for 24 h prior to harvesting, and IL12B expression was measured by RT-qPCR. (B) Secreted IL-12p40 from these TAMs was measured by ELISA ( N ≥ 6). Cell-free autologous ascites was used as additional control. Each dot denotes a biological replicate. Median values are indicated by horizontal bars. Colors encode individual patients, and colors are consistent between panels within this figure. Statistical significances were calculated with paired t -tests. * P

    Journal: Frontiers in Immunology

    Article Title: Chromatin Binding of c-REL and p65 Is Not Limiting for Macrophage IL12B Transcription During Immediate Suppression by Ovarian Carcinoma Ascites

    doi: 10.3389/fimmu.2018.01425

    Figure Lengend Snippet: IL-12 production by tumor-associated macrophages from ovarian carcinoma patients is suppressed by ascites in vitro . (A) Ex vivo TAMs ( N = 8) were cultivated in autologous ascites in the presence or in the absence of IFNγ or in normal medium (R5) as indicated, and non-cultivated ex vivo TAMs served as an additional control. LPS and IFNγ or their respective solvents (vehicle) were added to the culture supernatants for 24 h prior to harvesting, and IL12B expression was measured by RT-qPCR. (B) Secreted IL-12p40 from these TAMs was measured by ELISA ( N ≥ 6). Cell-free autologous ascites was used as additional control. Each dot denotes a biological replicate. Median values are indicated by horizontal bars. Colors encode individual patients, and colors are consistent between panels within this figure. Statistical significances were calculated with paired t -tests. * P

    Article Snippet: Murine Il-12p40 was measured with an ELISA kit (BD Biosciences, no. 555165) according to the manufacturer’s protocol.

    Techniques: In Vitro, Ex Vivo, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Effect of 25D on differentiation and/or stimulation of monocyte-derived DCs. Monocytes were differentiated for six days using rGM-CSF and rIL-4 in media with 10% FCS in the presence (25D diff -DCs or 25D diff/stim -DCs) or absence (serum-DCs or 25D stim -DCs) of additional 25D (10 −7 M). Subsequently, 25D-DCs and serum-DCs were stimulated with TLR2/1L (1 μg/ml) or left untreated in the presence (25D diff/stim -DCs or 25D stim -DCs) or absence (serum-DCs or 25D diff -DCs) of additional 25D (10 −7 M), and cultured in fresh media with 10% FCS for 18–24 hours. TNF-α, IL-6, IL-1β, IL-12p40, IL-12p70, IL-23 and IL-10, as well as total and active TGF-β levels in culture supernatants were measured by ELISA or CBA (mean cytokine levels in ng/ml ± SEM, n = 4). Results shown in Fig 1 and Fig. 2 were conducted using cells from the same cell preparations of identical donors. *p

    Journal: PLoS ONE

    Article Title: Vitamin D Regulates Cytokine Patterns Secreted by Dendritic Cells to Promote Differentiation of IL-22-Producing T Cells

    doi: 10.1371/journal.pone.0130395

    Figure Lengend Snippet: Effect of 25D on differentiation and/or stimulation of monocyte-derived DCs. Monocytes were differentiated for six days using rGM-CSF and rIL-4 in media with 10% FCS in the presence (25D diff -DCs or 25D diff/stim -DCs) or absence (serum-DCs or 25D stim -DCs) of additional 25D (10 −7 M). Subsequently, 25D-DCs and serum-DCs were stimulated with TLR2/1L (1 μg/ml) or left untreated in the presence (25D diff/stim -DCs or 25D stim -DCs) or absence (serum-DCs or 25D diff -DCs) of additional 25D (10 −7 M), and cultured in fresh media with 10% FCS for 18–24 hours. TNF-α, IL-6, IL-1β, IL-12p40, IL-12p70, IL-23 and IL-10, as well as total and active TGF-β levels in culture supernatants were measured by ELISA or CBA (mean cytokine levels in ng/ml ± SEM, n = 4). Results shown in Fig 1 and Fig. 2 were conducted using cells from the same cell preparations of identical donors. *p

    Article Snippet: TNF-α, IL-6, IL-1β, IL-12p40, IL-12p70, IL-23 and IL-10, as well as total and active TGF-β levels in the supernatants were determined by ELISA or CBA (BD Biosciences).

    Techniques: Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Crocin Bleaching Assay

    Effect of 1,25D on differentiation and/or stimulation of monocyte-derived DCs. Monocytes were differentiated for six days with rGM-CSF and rIL-4 in media with 10% FCS in the presence (1,25D diff -DCs or 1,25D diff/stim -DCs) or absence (serum-DCs or 1,25D stim -DCs) of additional 1,25D (10 −8 M). Subsequently, 1,25D-DCs and serum-DCs were stimulated with TLR2/1L (1 μg/ml) or left untreated in the presence (1,25D diff/stim -DCs or 1,25D stim -DCs) or absence (serum-DCs or 1,25D diff -DCs) of additional 1,25D (10 −8 M) and cultured in fresh media with 10% FCS for 18–24 hours. TNF-α, IL-6, IL-1β, IL-12p40, IL-12p70, IL-23 and IL-10, as well as total and active TGF-β levels in culture supernatants were measured by ELISA or CBA (mean cytokine levels in ng/ml ± SEM, n = 4). Results shown in Fig. 1 and Fig 2 were conducted using cells from the same cell preparations of identical donors. *p

    Journal: PLoS ONE

    Article Title: Vitamin D Regulates Cytokine Patterns Secreted by Dendritic Cells to Promote Differentiation of IL-22-Producing T Cells

    doi: 10.1371/journal.pone.0130395

    Figure Lengend Snippet: Effect of 1,25D on differentiation and/or stimulation of monocyte-derived DCs. Monocytes were differentiated for six days with rGM-CSF and rIL-4 in media with 10% FCS in the presence (1,25D diff -DCs or 1,25D diff/stim -DCs) or absence (serum-DCs or 1,25D stim -DCs) of additional 1,25D (10 −8 M). Subsequently, 1,25D-DCs and serum-DCs were stimulated with TLR2/1L (1 μg/ml) or left untreated in the presence (1,25D diff/stim -DCs or 1,25D stim -DCs) or absence (serum-DCs or 1,25D diff -DCs) of additional 1,25D (10 −8 M) and cultured in fresh media with 10% FCS for 18–24 hours. TNF-α, IL-6, IL-1β, IL-12p40, IL-12p70, IL-23 and IL-10, as well as total and active TGF-β levels in culture supernatants were measured by ELISA or CBA (mean cytokine levels in ng/ml ± SEM, n = 4). Results shown in Fig. 1 and Fig 2 were conducted using cells from the same cell preparations of identical donors. *p

    Article Snippet: TNF-α, IL-6, IL-1β, IL-12p40, IL-12p70, IL-23 and IL-10, as well as total and active TGF-β levels in the supernatants were determined by ELISA or CBA (BD Biosciences).

    Techniques: Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Crocin Bleaching Assay

    Cytokine profiles of 1,25D diff -DCs vs. serum-DCs stimulated with different ligands. Monocytes were differentiated for six days using rGM-CSF and rIL-4 in media with 10% FCS in the presence (1,25D diff -DCs, black bars) or absence (serum-DCs, white bars) of additional 1,25D (10 −8 M). Subsequently, 1,25D diff -DCs and serum-DCs were stimulated with TLR2/1L (1μg/ml), TLR4L (10 ng/ml) or CD40L (5 μg/ml) or left untreated, and cultured in fresh media with 10% FCS for 18–24 hours. TNF-α, IL-6, IL-1β, IL-12p40, IL-12p70, IL-23 and IL-10, as well as total and active TGF-β levels in culture supernatants were measured by ELISA or CBA (mean cytokine levels in ng/ml ± SEM, n of each condition indicated by numbers under the bars). *p

    Journal: PLoS ONE

    Article Title: Vitamin D Regulates Cytokine Patterns Secreted by Dendritic Cells to Promote Differentiation of IL-22-Producing T Cells

    doi: 10.1371/journal.pone.0130395

    Figure Lengend Snippet: Cytokine profiles of 1,25D diff -DCs vs. serum-DCs stimulated with different ligands. Monocytes were differentiated for six days using rGM-CSF and rIL-4 in media with 10% FCS in the presence (1,25D diff -DCs, black bars) or absence (serum-DCs, white bars) of additional 1,25D (10 −8 M). Subsequently, 1,25D diff -DCs and serum-DCs were stimulated with TLR2/1L (1μg/ml), TLR4L (10 ng/ml) or CD40L (5 μg/ml) or left untreated, and cultured in fresh media with 10% FCS for 18–24 hours. TNF-α, IL-6, IL-1β, IL-12p40, IL-12p70, IL-23 and IL-10, as well as total and active TGF-β levels in culture supernatants were measured by ELISA or CBA (mean cytokine levels in ng/ml ± SEM, n of each condition indicated by numbers under the bars). *p

    Article Snippet: TNF-α, IL-6, IL-1β, IL-12p40, IL-12p70, IL-23 and IL-10, as well as total and active TGF-β levels in the supernatants were determined by ELISA or CBA (BD Biosciences).

    Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Crocin Bleaching Assay

    IL-6 and IL12p40 production by IFN-γ-primed macrophages after stimulation with LPGs (10 μg/mL), GIPLs (10 μg/mL) and parasites. (A) IL-6 and (B) IL-12p40 concentrations were determined by flow cytometry. ANOVA test was performed and P

    Journal: Parasites & Vectors

    Article Title: Leishmania enriettii: biochemical characterisation of lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) and infectivity to Cavia porcellus

    doi: 10.1186/s13071-015-0633-8

    Figure Lengend Snippet: IL-6 and IL12p40 production by IFN-γ-primed macrophages after stimulation with LPGs (10 μg/mL), GIPLs (10 μg/mL) and parasites. (A) IL-6 and (B) IL-12p40 concentrations were determined by flow cytometry. ANOVA test was performed and P

    Article Snippet: Supernatants were collected and IL1-β, IL-6, IL-10, IL-12p40 and TNF-α were determined using the BD CBA Mouse Cytokine assay kits according to the manufacturer’s specifications (BD Biosciences, CA, USA).

    Techniques: Flow Cytometry, Cytometry

    MyD88 is responsible for the early induction of Th1-related cytokines after N. caninum infection. The presence of IL-12p40, IFN-γ, and IL-10 was evaluated by ELISA in the peritoneal exudate and serum samples of uninfected, and infected WT and MyD88 −/− mice at 3, 7, and 10 days p.i. ( n = 3 mice/group/date). Data are representative of two independent experiments. * Indicates significant statistical differences (mean ± SEM, p

    Journal: Veterinary Research

    Article Title: Myeloid differentiation factor 88 is required for resistance to Neospora caninum infection

    doi: 10.1051/vetres/2009015

    Figure Lengend Snippet: MyD88 is responsible for the early induction of Th1-related cytokines after N. caninum infection. The presence of IL-12p40, IFN-γ, and IL-10 was evaluated by ELISA in the peritoneal exudate and serum samples of uninfected, and infected WT and MyD88 −/− mice at 3, 7, and 10 days p.i. ( n = 3 mice/group/date). Data are representative of two independent experiments. * Indicates significant statistical differences (mean ± SEM, p

    Article Snippet: Monoclonal antibodies against IL-12p40 and IFN-γ conjugated to biotin and streptotavidin-PE-Cy5, were used to detect intracellular cytokine production, together with a commercially available permeabilization kit, which was used according to the manufacturer’s instructions (Becton Dickinson, Franklin Lakes, NJ, USA).

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, Mouse Assay

    Replenishment of IL-12 and IFN-γ reverts susceptibility of MyD88 −/− mice to acute N. caninum infection. Survival curves of (A) WT, IL-12p40 −/− , MyD88 −/− and MyD88 −/− mice treated with rIL-12p70 or (B) WT, IFNγ −/− , MyD88 −/− and MyD88 −/− mice treated with rIFNγ ( n = 6 mice/group), infected at the same time with sub-lethal parasite doses. Survival was observed through a 30-day period. Data are representative of two independent experiments.

    Journal: Veterinary Research

    Article Title: Myeloid differentiation factor 88 is required for resistance to Neospora caninum infection

    doi: 10.1051/vetres/2009015

    Figure Lengend Snippet: Replenishment of IL-12 and IFN-γ reverts susceptibility of MyD88 −/− mice to acute N. caninum infection. Survival curves of (A) WT, IL-12p40 −/− , MyD88 −/− and MyD88 −/− mice treated with rIL-12p70 or (B) WT, IFNγ −/− , MyD88 −/− and MyD88 −/− mice treated with rIFNγ ( n = 6 mice/group), infected at the same time with sub-lethal parasite doses. Survival was observed through a 30-day period. Data are representative of two independent experiments.

    Article Snippet: Monoclonal antibodies against IL-12p40 and IFN-γ conjugated to biotin and streptotavidin-PE-Cy5, were used to detect intracellular cytokine production, together with a commercially available permeabilization kit, which was used according to the manufacturer’s instructions (Becton Dickinson, Franklin Lakes, NJ, USA).

    Techniques: Mouse Assay, Infection