mouse il‐10 elisa set Search Results


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  • 95
    Thermo Fisher mouse il 10 elisa ready set go
    Expression data of human and mouse <t>IL-10</t> in transiently transformed Nicotiana benthamiana leaves. Use of the thk-tag gives an increasing boost in yield for both human and mouse IL-10 from 2 days post infiltration (dpi). Strikingly, mouse IL-10 yield was significantly higher compared to human IL-10, regardless of ER-retention. Differences in yield could not be explained by differences in mRNA transcript levels. (A) Schematic representation of expression cassettes and vector used. Expressed genes include the native coding sequence of the human (h) or mouse (m) IL-10 gene including signal peptide for secretion (SP) with or without a 3′ tag coding for a thrombin cleavage site, a 6xHis-tag and the ER retention sequence KDEL (thk). All expression cassettes include the 35S promoter of the Cauliflower mosaic virus with duplicated enhancer (d35S), 5′ leader sequence of the Alfalfa mosaic virus RNA 4 (AlMV) and Agrobacterium tumefaciens nopaline synthase transcription terminator (Tnos). (B) Relative transcript levels of IL-10 versus actin as determined by Q-PCR on 2 and 3 dpi ( n = 3, error bars indicate standard error). (C/D) Human and mouse IL-10 yield in crude extracts (1 to 6 dpi) in µg per mg total soluble protein (TSP) as determined by <t>ELISA</t> ( n = 3, error bars indicate standard error).
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    Becton Dickinson mouse il 10 elisa set
    Wnts suppress TLR induced proinflammatory cytokine production a) Effect of Wnts on LPS responses of DC from normal mouse lymph nodes. CD11c+ DC were stimulated in vitro with LPS (5μg/ml) in combination with Wnt3A (3μg/ml) or Wnt5A (3μg/ml) for 20 hours. Cytokine secretion was determined by multiplex luminex analysis of culture supernatants. Data represent cytokine concentrations from one of two experiments performed with similar results. b) Wnts inhibit CpG as well as LPS stimulation of IL-6 in DC. CD11c+ DC isolated from pooled lymph nodes from normal mice were stimulated in vitro with LPS (5μg/ml) or CpGA (5μg/ml) in combination with Wnt3a or Wnt5a (3μg/ml) for 20 hours. Cytokine secretion was determined by <t>ELISA.</t> Data show cytokine concentrations from 3–5 experiments. Mean values ± standard error of the mean (SEM). Symbols indicate individual experiments. c) Dose dependent Wnt effects on TLR responses. Total FACS sorted cDC (CD11c high , B220−) or CD103+ cDC (inserted graphs) isolated from mesenteric lymph nodes (IL-6, IL-12p40 and <t>IL-10</t> graphs) or total CD11c+ DCs isolated from pooled lymph nodes (VEGF-A and IFN-α graphs) of FLT3L-treated mice were stimulated in vitro with LPS (5μg/ml), CpGA (5μg/ml) or Pam3CSK4 (5μg/ml) in the presence of varying doses of Wnt3A or Wnt5A for 20 hours. Cytokine secretion was determined by ELISA of culture supernatants. For IL-6, IL-12p40 and IFN-α data are presented as % of cytokine concentration in the presence of TLR alone, without Wnt. [In the absence of Wnts values were as follows in pg/ml: IL-6 (for the Wnt3A graph) LPS 820, CpGA 3400, Pam3CSK4 730; IL-6 (for the Wnt5a graph) LPS 670, CpGA 4150, Pam3CSK4 660; IL-12p40 (for the Wnt3A graph) LPS 18310, CpGA 25220, Pam3CSK4 18900; IL-12p40 (for the Wnt5A graph) LPS 16550, CpGA 32140, Pam3CSK4 13530; and IFN-α in units/μl (for both Wnt3A and Wnt5A graphs) 1650.] For IL-10 and VEGF-A induction, data are % of maximum cytokine concentration induced by each TLR ligand or control. [Maximum values in pg/ml were as follows: IL-10 medium 80, LPS 120, CpGA 110, Pam3CSK4 240; VEGF-A medium 22580, LPS 20940, CpGA 17100.] Mean ± SEM are presented, n=3, with the exception of IL-12p40 and IFN-α in response to CpGA, and VEGF-A in response to all stimulations, mean ± StDev n=2. CD103+ DC responses (graph inserts) are from one of two experiments with similar results.
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    Becton Dickinson opteia mouse il 10 elisa set
    PMT stimulated release of TNF-α or IL-6 is independent of the mTOR pathway. RAW264.7 cells were stimulated with PMT (5 nM) w or w/o pre-incubation with rapamycin (10 ng/ml) for 1 h. As control, cells were untreated, treated with the solvent control DMSO, with rapamycin or LPS (100 ng/ml) as positive control. After 24 h, supernatants were removed and subjected to <t>ELISA</t> for the measurement of ( a ) IL-6-, ( b ) TNF-α-, ( c ) IL12p40- or ( d ) <t>IL-10</t> release. Shown are the results of three independent experiments (mean ± SD; n = 3)
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    Becton Dickinson opteiatm mouse il 10 elisa set bd biosciences
    PMT stimulated release of TNF-α or IL-6 is independent of the mTOR pathway. RAW264.7 cells were stimulated with PMT (5 nM) w or w/o pre-incubation with rapamycin (10 ng/ml) for 1 h. As control, cells were untreated, treated with the solvent control DMSO, with rapamycin or LPS (100 ng/ml) as positive control. After 24 h, supernatants were removed and subjected to <t>ELISA</t> for the measurement of ( a ) IL-6-, ( b ) TNF-α-, ( c ) IL12p40- or ( d ) <t>IL-10</t> release. Shown are the results of three independent experiments (mean ± SD; n = 3)
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    94
    Thermo Fisher mouse interleukin 10 enzyme linked immunosorbent assay kit
    PMT stimulated release of TNF-α or IL-6 is independent of the mTOR pathway. RAW264.7 cells were stimulated with PMT (5 nM) w or w/o pre-incubation with rapamycin (10 ng/ml) for 1 h. As control, cells were untreated, treated with the solvent control DMSO, with rapamycin or LPS (100 ng/ml) as positive control. After 24 h, supernatants were removed and subjected to <t>ELISA</t> for the measurement of ( a ) IL-6-, ( b ) TNF-α-, ( c ) IL12p40- or ( d ) <t>IL-10</t> release. Shown are the results of three independent experiments (mean ± SD; n = 3)
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    BioLegend mouse il 10 elisa kit
    An expanded pool of <t>IL-10</t> + B cells are present in the uterus after implantation. (A) Known regulatory B cell subsets were analyzed from B cells collected from non-pregnant and pregnant uteri ( n = 6–7, unpaired t -test). (B) IL-10 production by stimulated populations of purified B cells obtained from the spleen, virgin uterus, day 5.5 pc uterus, or in vitro induced Bregs was assessed by <t>ELISA.</t> Data represent pooled supernatants of duplicate wells from three independent experiments. Data is presented as mean ± SEM and compared by one-way ANOVA, followed by Dunnett's post-hoc multiple comparisons test against unstimulated control. * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001. Additional Dunnett's analysis assessed against non-pregnant virgin uterus reveal a significant increase in IL-10 produced by day 5.5 pc uterine B cells and iBregs ( # p ≤ 0.05, #### p ≤ 0.0001).
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    Becton Dickinson mouse il 10 opt eia set
    An expanded pool of <t>IL-10</t> + B cells are present in the uterus after implantation. (A) Known regulatory B cell subsets were analyzed from B cells collected from non-pregnant and pregnant uteri ( n = 6–7, unpaired t -test). (B) IL-10 production by stimulated populations of purified B cells obtained from the spleen, virgin uterus, day 5.5 pc uterus, or in vitro induced Bregs was assessed by <t>ELISA.</t> Data represent pooled supernatants of duplicate wells from three independent experiments. Data is presented as mean ± SEM and compared by one-way ANOVA, followed by Dunnett's post-hoc multiple comparisons test against unstimulated control. * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001. Additional Dunnett's analysis assessed against non-pregnant virgin uterus reveal a significant increase in IL-10 produced by day 5.5 pc uterine B cells and iBregs ( # p ≤ 0.05, #### p ≤ 0.0001).
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    Thermo Fisher human il 10 elisa ready set go
    Pre-fixed platelets but not plastic microbead particles nor lymphocytes mediate regulatory response. (A) Pre-fixed platelets can mediate IgG-bound platelet-induced regulatory response. Paraformaldehyde (PFA)-fixed human platelets and sort-purified monocytes were incubated with an anti-CD61 mAb following LPS stimulation for 24 h. The <t>IL-10</t> levels in culture supernatants were determined by <t>ELISA.</t> Data are shown as means for triplicate samples ± SEM. The results are representative of more than three independent experiments with similar results. ** P
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    Becton Dickinson mouse elisa set
    Pre-fixed platelets but not plastic microbead particles nor lymphocytes mediate regulatory response. (A) Pre-fixed platelets can mediate IgG-bound platelet-induced regulatory response. Paraformaldehyde (PFA)-fixed human platelets and sort-purified monocytes were incubated with an anti-CD61 mAb following LPS stimulation for 24 h. The <t>IL-10</t> levels in culture supernatants were determined by <t>ELISA.</t> Data are shown as means for triplicate samples ± SEM. The results are representative of more than three independent experiments with similar results. ** P
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    Thermo Fisher il 22 enzyme linked immunosorbent assay elisa ready set go kit
    Pre-fixed platelets but not plastic microbead particles nor lymphocytes mediate regulatory response. (A) Pre-fixed platelets can mediate IgG-bound platelet-induced regulatory response. Paraformaldehyde (PFA)-fixed human platelets and sort-purified monocytes were incubated with an anti-CD61 mAb following LPS stimulation for 24 h. The <t>IL-10</t> levels in culture supernatants were determined by <t>ELISA.</t> Data are shown as means for triplicate samples ± SEM. The results are representative of more than three independent experiments with similar results. ** P
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    Becton Dickinson opteia mouse il 10 set
    Pre-fixed platelets but not plastic microbead particles nor lymphocytes mediate regulatory response. (A) Pre-fixed platelets can mediate IgG-bound platelet-induced regulatory response. Paraformaldehyde (PFA)-fixed human platelets and sort-purified monocytes were incubated with an anti-CD61 mAb following LPS stimulation for 24 h. The <t>IL-10</t> levels in culture supernatants were determined by <t>ELISA.</t> Data are shown as means for triplicate samples ± SEM. The results are representative of more than three independent experiments with similar results. ** P
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    BioLegend human il 10 elisa max standard
    Effect of ERK1/2 inhibition on Sp1 and <t>IL-10</t> levels Eµ- TCL1 CLL cells were cultured with the ERK1/2 inhibitor (SCH772984) (2µM). A) IL-10 levels in 24-hour supernatants were measured by <t>ELISA.</t> Values represent mean±SD of triplicates. B) Levels of p-ERK1/2, total ERK1, p-STAT3, total STAT3 and Sp1 were quantified by immunoblot analysis. Results are representative of three experiments. C) Sp1 mRNA levels were quantified by qRT-PCR. Fold change was normalized to the no-treatment group. Values represent mean±SD of triplicates ***p
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    Pharmingen opt eia mouse il 10 set
    Effect of ERK1/2 inhibition on Sp1 and <t>IL-10</t> levels Eµ- TCL1 CLL cells were cultured with the ERK1/2 inhibitor (SCH772984) (2µM). A) IL-10 levels in 24-hour supernatants were measured by <t>ELISA.</t> Values represent mean±SD of triplicates. B) Levels of p-ERK1/2, total ERK1, p-STAT3, total STAT3 and Sp1 were quantified by immunoblot analysis. Results are representative of three experiments. C) Sp1 mRNA levels were quantified by qRT-PCR. Fold change was normalized to the no-treatment group. Values represent mean±SD of triplicates ***p
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    BioLegend mouse il 10 elisa max deluxe
    Effect of ERK1/2 inhibition on Sp1 and <t>IL-10</t> levels Eµ- TCL1 CLL cells were cultured with the ERK1/2 inhibitor (SCH772984) (2µM). A) IL-10 levels in 24-hour supernatants were measured by <t>ELISA.</t> Values represent mean±SD of triplicates. B) Levels of p-ERK1/2, total ERK1, p-STAT3, total STAT3 and Sp1 were quantified by immunoblot analysis. Results are representative of three experiments. C) Sp1 mRNA levels were quantified by qRT-PCR. Fold change was normalized to the no-treatment group. Values represent mean±SD of triplicates ***p
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    Thermo Fisher mouse tnfα elisa ready set go
    Effect of ERK1/2 inhibition on Sp1 and <t>IL-10</t> levels Eµ- TCL1 CLL cells were cultured with the ERK1/2 inhibitor (SCH772984) (2µM). A) IL-10 levels in 24-hour supernatants were measured by <t>ELISA.</t> Values represent mean±SD of triplicates. B) Levels of p-ERK1/2, total ERK1, p-STAT3, total STAT3 and Sp1 were quantified by immunoblot analysis. Results are representative of three experiments. C) Sp1 mRNA levels were quantified by qRT-PCR. Fold change was normalized to the no-treatment group. Values represent mean±SD of triplicates ***p
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    Expression data of human and mouse IL-10 in transiently transformed Nicotiana benthamiana leaves. Use of the thk-tag gives an increasing boost in yield for both human and mouse IL-10 from 2 days post infiltration (dpi). Strikingly, mouse IL-10 yield was significantly higher compared to human IL-10, regardless of ER-retention. Differences in yield could not be explained by differences in mRNA transcript levels. (A) Schematic representation of expression cassettes and vector used. Expressed genes include the native coding sequence of the human (h) or mouse (m) IL-10 gene including signal peptide for secretion (SP) with or without a 3′ tag coding for a thrombin cleavage site, a 6xHis-tag and the ER retention sequence KDEL (thk). All expression cassettes include the 35S promoter of the Cauliflower mosaic virus with duplicated enhancer (d35S), 5′ leader sequence of the Alfalfa mosaic virus RNA 4 (AlMV) and Agrobacterium tumefaciens nopaline synthase transcription terminator (Tnos). (B) Relative transcript levels of IL-10 versus actin as determined by Q-PCR on 2 and 3 dpi ( n = 3, error bars indicate standard error). (C/D) Human and mouse IL-10 yield in crude extracts (1 to 6 dpi) in µg per mg total soluble protein (TSP) as determined by ELISA ( n = 3, error bars indicate standard error).

    Journal: PLoS ONE

    Article Title: 3D Domain Swapping Causes Extensive Multimerisation of Human Interleukin-10 When Expressed In Planta

    doi: 10.1371/journal.pone.0046460

    Figure Lengend Snippet: Expression data of human and mouse IL-10 in transiently transformed Nicotiana benthamiana leaves. Use of the thk-tag gives an increasing boost in yield for both human and mouse IL-10 from 2 days post infiltration (dpi). Strikingly, mouse IL-10 yield was significantly higher compared to human IL-10, regardless of ER-retention. Differences in yield could not be explained by differences in mRNA transcript levels. (A) Schematic representation of expression cassettes and vector used. Expressed genes include the native coding sequence of the human (h) or mouse (m) IL-10 gene including signal peptide for secretion (SP) with or without a 3′ tag coding for a thrombin cleavage site, a 6xHis-tag and the ER retention sequence KDEL (thk). All expression cassettes include the 35S promoter of the Cauliflower mosaic virus with duplicated enhancer (d35S), 5′ leader sequence of the Alfalfa mosaic virus RNA 4 (AlMV) and Agrobacterium tumefaciens nopaline synthase transcription terminator (Tnos). (B) Relative transcript levels of IL-10 versus actin as determined by Q-PCR on 2 and 3 dpi ( n = 3, error bars indicate standard error). (C/D) Human and mouse IL-10 yield in crude extracts (1 to 6 dpi) in µg per mg total soluble protein (TSP) as determined by ELISA ( n = 3, error bars indicate standard error).

    Article Snippet: Human and mouse IL-10 ELISA Ready-SET-Go!® kits (eBioscience) were used according to suppliers protocol using the model 680 plate reader (BioRad) to measure the OD at 450 nm with correction filter of 690 nm.

    Techniques: Expressing, Transformation Assay, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Analysis of the effect of N -glycosylation at Asn29 on granulation. Glycosylation of IL-10 plays a role in preventing granulation. (A/B) Whole mount confocal microscopy output of leaves expressing GFP fused C-terminally to human (h) and mouse (m) IL-10 including native signal peptide (SP) and with introduced (S29N) or removed (N29S) glycosylation site, respectively. (C/D) Western blot analysis under reducing conditions of plant produced (p) hIL-10 and mIL-10 with and without glycosylation site. As controls, empty vector (EV) and 50 ng recombinant (r) E. coli produced hL-10 and mIL-10 were used. A molecular weight marker is indicated in kDa. (E/F) Yield of hIL-10 and mIL-10 with and without glycosylation site in crude extracts 2 to 5 days post infiltration (dpi) as determined by ELISA ( n = 4, error bars indicate standard error).

    Journal: PLoS ONE

    Article Title: 3D Domain Swapping Causes Extensive Multimerisation of Human Interleukin-10 When Expressed In Planta

    doi: 10.1371/journal.pone.0046460

    Figure Lengend Snippet: Analysis of the effect of N -glycosylation at Asn29 on granulation. Glycosylation of IL-10 plays a role in preventing granulation. (A/B) Whole mount confocal microscopy output of leaves expressing GFP fused C-terminally to human (h) and mouse (m) IL-10 including native signal peptide (SP) and with introduced (S29N) or removed (N29S) glycosylation site, respectively. (C/D) Western blot analysis under reducing conditions of plant produced (p) hIL-10 and mIL-10 with and without glycosylation site. As controls, empty vector (EV) and 50 ng recombinant (r) E. coli produced hL-10 and mIL-10 were used. A molecular weight marker is indicated in kDa. (E/F) Yield of hIL-10 and mIL-10 with and without glycosylation site in crude extracts 2 to 5 days post infiltration (dpi) as determined by ELISA ( n = 4, error bars indicate standard error).

    Article Snippet: Human and mouse IL-10 ELISA Ready-SET-Go!® kits (eBioscience) were used according to suppliers protocol using the model 680 plate reader (BioRad) to measure the OD at 450 nm with correction filter of 690 nm.

    Techniques: Confocal Microscopy, Expressing, Western Blot, Produced, Plasmid Preparation, Recombinant, Molecular Weight, Marker, Enzyme-linked Immunosorbent Assay

    Analysis of expression of a stable monomeric form of human IL-10. A stable monomeric form of human IL-10 (hIL-10 mono ) does not granulate and yield increases 30-fold. (A) Three cartoons illustrating the human IL-10 (I) dimer, (II) monomer and (III) stable monomer structure, as well as a schematic representation of the human (h) IL-10 alpha helices A–F. Helices are represented by ovals, whereby a fragment of the amino acid sequence and the location of insertion of the small GS-linker is indicated. (B) Whole mount confocal microscopy output of GFP fused C-terminally to hIL-10 mono including native signal peptide (SP). (C) Western blot analysis under non-reducing conditions of plant produced hIL-10 and hIL-10 mono . As controls, empty vector (EV) and 50 ng recombinant (r) E. coli produced hL-10 were used. A molecular weight marker is indicated in kDa. (D) Yield of hIL-10 and hIL-10 mono in crude extracts 2 to 5 days post infiltration as determined by ELISA ( n = 3, error bars indicate standard error). Average yield of hIL-10 mono was significantly higher compared to hIL-10.

    Journal: PLoS ONE

    Article Title: 3D Domain Swapping Causes Extensive Multimerisation of Human Interleukin-10 When Expressed In Planta

    doi: 10.1371/journal.pone.0046460

    Figure Lengend Snippet: Analysis of expression of a stable monomeric form of human IL-10. A stable monomeric form of human IL-10 (hIL-10 mono ) does not granulate and yield increases 30-fold. (A) Three cartoons illustrating the human IL-10 (I) dimer, (II) monomer and (III) stable monomer structure, as well as a schematic representation of the human (h) IL-10 alpha helices A–F. Helices are represented by ovals, whereby a fragment of the amino acid sequence and the location of insertion of the small GS-linker is indicated. (B) Whole mount confocal microscopy output of GFP fused C-terminally to hIL-10 mono including native signal peptide (SP). (C) Western blot analysis under non-reducing conditions of plant produced hIL-10 and hIL-10 mono . As controls, empty vector (EV) and 50 ng recombinant (r) E. coli produced hL-10 were used. A molecular weight marker is indicated in kDa. (D) Yield of hIL-10 and hIL-10 mono in crude extracts 2 to 5 days post infiltration as determined by ELISA ( n = 3, error bars indicate standard error). Average yield of hIL-10 mono was significantly higher compared to hIL-10.

    Article Snippet: Human and mouse IL-10 ELISA Ready-SET-Go!® kits (eBioscience) were used according to suppliers protocol using the model 680 plate reader (BioRad) to measure the OD at 450 nm with correction filter of 690 nm.

    Techniques: Expressing, Sequencing, Confocal Microscopy, Western Blot, Produced, Plasmid Preparation, Recombinant, Molecular Weight, Marker, Enzyme-linked Immunosorbent Assay

    Biological activity of human and mouse IL-10 variants on human and mouse macrophages. Plant produced (p) and recombinant (r) E. coli produced human (h) or mouse (m) IL-10 were calibrated to contain the same amount of IL-10 as well as total soluble protein by using the empty vector control. Human (THP-1) and mouse (RAW264.7) macrophages were then pretreated with 10 ng/ml hIL-10 or mIL-10 for 20 min and subsequently stimulated with 1 µg/ml E. coli lipopolysaccharide. Tumor Necrosis Factor-alpha (TNF-α) expression was determined by ELISA and IL-10 activity is indicated as the percentage of inhibition of TNF-α expression as compared to the empty vector control ( n = 3, error bars indicate standard error).

    Journal: PLoS ONE

    Article Title: 3D Domain Swapping Causes Extensive Multimerisation of Human Interleukin-10 When Expressed In Planta

    doi: 10.1371/journal.pone.0046460

    Figure Lengend Snippet: Biological activity of human and mouse IL-10 variants on human and mouse macrophages. Plant produced (p) and recombinant (r) E. coli produced human (h) or mouse (m) IL-10 were calibrated to contain the same amount of IL-10 as well as total soluble protein by using the empty vector control. Human (THP-1) and mouse (RAW264.7) macrophages were then pretreated with 10 ng/ml hIL-10 or mIL-10 for 20 min and subsequently stimulated with 1 µg/ml E. coli lipopolysaccharide. Tumor Necrosis Factor-alpha (TNF-α) expression was determined by ELISA and IL-10 activity is indicated as the percentage of inhibition of TNF-α expression as compared to the empty vector control ( n = 3, error bars indicate standard error).

    Article Snippet: Human and mouse IL-10 ELISA Ready-SET-Go!® kits (eBioscience) were used according to suppliers protocol using the model 680 plate reader (BioRad) to measure the OD at 450 nm with correction filter of 690 nm.

    Techniques: Activity Assay, Produced, Recombinant, Plasmid Preparation, Expressing, Enzyme-linked Immunosorbent Assay, Inhibition

    Analysis of biological activity and expression of human IL-10 and human IL-10 mono fused to Fcα. Forced dimerization of human IL-10 mono restores biological activity. (A) Bioactivity assay of hIL-10 mono and Fcα-hIL-10 fusion proteins on mouse macrophages (RAW267.4). Plant produced (p) and recombinant (r) E. coli produced hIL-10 were calibrated to contain the same amount of IL-10 as well as total soluble protein by using the empty vector control. Cells were then pretreated with 50 ng/ml hIL-10 for 20 min and subsequently stimulated with 1 µg/ml E. coli lipopolysaccharide. Tumor Necrosis Factor-alpha (TNF-α) expression was determined by ELISA and IL-10 activity is indicated as the percentage of inhibition of TNF-α expression as compared to the empty vector control ( n = 4, error bars indicate standard error). Significant difference ( P

    Journal: PLoS ONE

    Article Title: 3D Domain Swapping Causes Extensive Multimerisation of Human Interleukin-10 When Expressed In Planta

    doi: 10.1371/journal.pone.0046460

    Figure Lengend Snippet: Analysis of biological activity and expression of human IL-10 and human IL-10 mono fused to Fcα. Forced dimerization of human IL-10 mono restores biological activity. (A) Bioactivity assay of hIL-10 mono and Fcα-hIL-10 fusion proteins on mouse macrophages (RAW267.4). Plant produced (p) and recombinant (r) E. coli produced hIL-10 were calibrated to contain the same amount of IL-10 as well as total soluble protein by using the empty vector control. Cells were then pretreated with 50 ng/ml hIL-10 for 20 min and subsequently stimulated with 1 µg/ml E. coli lipopolysaccharide. Tumor Necrosis Factor-alpha (TNF-α) expression was determined by ELISA and IL-10 activity is indicated as the percentage of inhibition of TNF-α expression as compared to the empty vector control ( n = 4, error bars indicate standard error). Significant difference ( P

    Article Snippet: Human and mouse IL-10 ELISA Ready-SET-Go!® kits (eBioscience) were used according to suppliers protocol using the model 680 plate reader (BioRad) to measure the OD at 450 nm with correction filter of 690 nm.

    Techniques: Activity Assay, Expressing, Produced, Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Inhibition

    hASC and hASC-CM maintain a correct pro- and anti-inflammatory cytokine balance in sciatic nerves, DRG and spinal cord of STZ mice. IL-1β, IL-6, TNFα and IL-10 protein content in nervous tissues was evaluated by ELISA and reported as pg cytokine/mg total protein. ( a – d ) IL-1β ( a ) IL-6 ( b ), TNF-α ( c ) and IL-10 ( d ) in sciatic nerve, DRG and spinal cord of STZ mice treated 2 weeks after STZ with hASC or hASC-CM; cytokines were evaluated after 1 week from treatments. ( e , f ) IL-1β ( e ) and IL-10 ( f ) levels in spinal cord, measured 14 weeks after STZ in animals treated with hASC or hASC-CM either 2 weeks (W2) or 6 weeks (W6) after STZ. Data represent mean ± SEM of 6 mice per group. One-way ANOVA was used for statistical evaluation, followed by Bonferroni’s post hoc test for multiple comparisons. *p

    Journal: Scientific Reports

    Article Title: Therapeutic effect of human adipose-derived stem cells and their secretome in experimental diabetic pain

    doi: 10.1038/s41598-017-09487-5

    Figure Lengend Snippet: hASC and hASC-CM maintain a correct pro- and anti-inflammatory cytokine balance in sciatic nerves, DRG and spinal cord of STZ mice. IL-1β, IL-6, TNFα and IL-10 protein content in nervous tissues was evaluated by ELISA and reported as pg cytokine/mg total protein. ( a – d ) IL-1β ( a ) IL-6 ( b ), TNF-α ( c ) and IL-10 ( d ) in sciatic nerve, DRG and spinal cord of STZ mice treated 2 weeks after STZ with hASC or hASC-CM; cytokines were evaluated after 1 week from treatments. ( e , f ) IL-1β ( e ) and IL-10 ( f ) levels in spinal cord, measured 14 weeks after STZ in animals treated with hASC or hASC-CM either 2 weeks (W2) or 6 weeks (W6) after STZ. Data represent mean ± SEM of 6 mice per group. One-way ANOVA was used for statistical evaluation, followed by Bonferroni’s post hoc test for multiple comparisons. *p

    Article Snippet: DuoSet ELISA development systems for mouse IL-2, IFN-γ and IL-4 were from R & D Systems (Minneapolis, USA) while mouse IL-1β, IL-6, TNFα and IL-10 ELISA Ready-SET-Go from eBioscience (San Diego, CA).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    hASC and hASC-CM treatments modulate cytokine release from splenocytes. IFN-γ ( a and b ), IL-2 ( c and d ), IL-4 ( e and f ) and IL-10 ( g and h ) were evaluated by ELISA, and reported as protein concentrations in culture media. ( a , c , e and g ) depict the levels of cytokine evaluated 3 weeks after STZ and 1 week after hASC or hASC-CM treatments. ( b , d , f and h ) Report cytokines levels measured 14 weeks after STZ in animals treated with hASC or hASC-CM either 2 weeks (W2) or 6 weeks (W6) after STZ. Data represent mean ± SEM of 6 mice per group, and have been statistically analyzed with One-way ANOVA, followed by Bonferroni’s test for multiple comparisons. *p

    Journal: Scientific Reports

    Article Title: Therapeutic effect of human adipose-derived stem cells and their secretome in experimental diabetic pain

    doi: 10.1038/s41598-017-09487-5

    Figure Lengend Snippet: hASC and hASC-CM treatments modulate cytokine release from splenocytes. IFN-γ ( a and b ), IL-2 ( c and d ), IL-4 ( e and f ) and IL-10 ( g and h ) were evaluated by ELISA, and reported as protein concentrations in culture media. ( a , c , e and g ) depict the levels of cytokine evaluated 3 weeks after STZ and 1 week after hASC or hASC-CM treatments. ( b , d , f and h ) Report cytokines levels measured 14 weeks after STZ in animals treated with hASC or hASC-CM either 2 weeks (W2) or 6 weeks (W6) after STZ. Data represent mean ± SEM of 6 mice per group, and have been statistically analyzed with One-way ANOVA, followed by Bonferroni’s test for multiple comparisons. *p

    Article Snippet: DuoSet ELISA development systems for mouse IL-2, IFN-γ and IL-4 were from R & D Systems (Minneapolis, USA) while mouse IL-1β, IL-6, TNFα and IL-10 ELISA Ready-SET-Go from eBioscience (San Diego, CA).

    Techniques: Enzyme-linked Immunosorbent Assay, Mouse Assay

    Muscle macrophage phenotype Macrophages were isolated by magnetic separation from uninjured (0d) or lacerated gastrocnemius muscles at the indicated time points. Muscle macrophages were obtained as the CD11b-positive, Ly6G/CD3/CD19-negative cell fraction. As positive and negative controls, bone marrow derived macrophages (right side of vertical bar in panels A–J) were activated with IFNγ and TNFα (M1), IL-4 (M2a), or IL-10 (M2c). Total RNA was isolated and reverse transcribed, and expression of IL-1β (A), TNFα (B), iNOS (C), IDO1 (D), CXCL10 (E), CD206 (F), CD36 (G), TGFβ (H), Ym1 (I), and IL-10 (J) was analyzed by real-time PCR. Expression of each gene was determined by the 2 −ΔΔCT method using GAPDH as endogenous control. M1-associated genes (A–E) were normalized to in vitro activated M1 macrophages, and M2-associated genes (F–J) were normalized to in vitro activated M2a macrophages. (K) Macrophages were isolated by magnetic separation from uninjured or injured muscles, equal numbers of macrophages were incubated for 20 hours, and IL-10 secretion was measured by ELISA on the conditioned medium. Data did not pass tests of normality and equal variance, and are presented with center line as median, boxes representing the 25 th and 75 th percentiles, whiskers representing the 10 th and 90 th percentiles, and outliers as dots. * p

    Journal: The Journal of pathology

    Article Title: Macrophage Activation and Skeletal Muscle Healing Following Traumatic Injury

    doi: 10.1002/path.4301

    Figure Lengend Snippet: Muscle macrophage phenotype Macrophages were isolated by magnetic separation from uninjured (0d) or lacerated gastrocnemius muscles at the indicated time points. Muscle macrophages were obtained as the CD11b-positive, Ly6G/CD3/CD19-negative cell fraction. As positive and negative controls, bone marrow derived macrophages (right side of vertical bar in panels A–J) were activated with IFNγ and TNFα (M1), IL-4 (M2a), or IL-10 (M2c). Total RNA was isolated and reverse transcribed, and expression of IL-1β (A), TNFα (B), iNOS (C), IDO1 (D), CXCL10 (E), CD206 (F), CD36 (G), TGFβ (H), Ym1 (I), and IL-10 (J) was analyzed by real-time PCR. Expression of each gene was determined by the 2 −ΔΔCT method using GAPDH as endogenous control. M1-associated genes (A–E) were normalized to in vitro activated M1 macrophages, and M2-associated genes (F–J) were normalized to in vitro activated M2a macrophages. (K) Macrophages were isolated by magnetic separation from uninjured or injured muscles, equal numbers of macrophages were incubated for 20 hours, and IL-10 secretion was measured by ELISA on the conditioned medium. Data did not pass tests of normality and equal variance, and are presented with center line as median, boxes representing the 25 th and 75 th percentiles, whiskers representing the 10 th and 90 th percentiles, and outliers as dots. * p

    Article Snippet: Muscle-derived macrophages were cultured for 20 hours, conditioned media were collected and centrifuged, and the supernatant was analyzed for IL-10 using the Mouse IL-10 ELISA Ready-SET-Go kit (eBioscience).

    Techniques: Isolation, Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, In Vitro, Incubation, Enzyme-linked Immunosorbent Assay

    Macrophages at 3 days after muscle laceration are not separable into M1 and M2a subsets Cells were isolated from gastrocnemius muscles at 3 days post-injury and labeled for flow cytometry. Macrophages were defined as FITC-F4/80+ cells (A) or PEF4/80+ cells (C), with lower threshold set based on background FL1 (B) or FL2 (D) fluorescence in unlabeled cells. Density plots display macrophage expression of TGFβ versus TNFα (E), IL-10 versus TNFα (F), CD36 versus IL-10 (G), CD36 versus IL-1b (H) and CD36 versus TNFα (I). In panel G, cells in the upper left quadrant (arrow) likely represent non-specific labeling, as this population was also seen in IgG control plots (not shown). Data are representative of 5 independent experiments of n=1–2 per experiment.

    Journal: The Journal of pathology

    Article Title: Macrophage Activation and Skeletal Muscle Healing Following Traumatic Injury

    doi: 10.1002/path.4301

    Figure Lengend Snippet: Macrophages at 3 days after muscle laceration are not separable into M1 and M2a subsets Cells were isolated from gastrocnemius muscles at 3 days post-injury and labeled for flow cytometry. Macrophages were defined as FITC-F4/80+ cells (A) or PEF4/80+ cells (C), with lower threshold set based on background FL1 (B) or FL2 (D) fluorescence in unlabeled cells. Density plots display macrophage expression of TGFβ versus TNFα (E), IL-10 versus TNFα (F), CD36 versus IL-10 (G), CD36 versus IL-1b (H) and CD36 versus TNFα (I). In panel G, cells in the upper left quadrant (arrow) likely represent non-specific labeling, as this population was also seen in IgG control plots (not shown). Data are representative of 5 independent experiments of n=1–2 per experiment.

    Article Snippet: Muscle-derived macrophages were cultured for 20 hours, conditioned media were collected and centrifuged, and the supernatant was analyzed for IL-10 using the Mouse IL-10 ELISA Ready-SET-Go kit (eBioscience).

    Techniques: Isolation, Labeling, Flow Cytometry, Cytometry, Fluorescence, Expressing

    Wnts suppress TLR induced proinflammatory cytokine production a) Effect of Wnts on LPS responses of DC from normal mouse lymph nodes. CD11c+ DC were stimulated in vitro with LPS (5μg/ml) in combination with Wnt3A (3μg/ml) or Wnt5A (3μg/ml) for 20 hours. Cytokine secretion was determined by multiplex luminex analysis of culture supernatants. Data represent cytokine concentrations from one of two experiments performed with similar results. b) Wnts inhibit CpG as well as LPS stimulation of IL-6 in DC. CD11c+ DC isolated from pooled lymph nodes from normal mice were stimulated in vitro with LPS (5μg/ml) or CpGA (5μg/ml) in combination with Wnt3a or Wnt5a (3μg/ml) for 20 hours. Cytokine secretion was determined by ELISA. Data show cytokine concentrations from 3–5 experiments. Mean values ± standard error of the mean (SEM). Symbols indicate individual experiments. c) Dose dependent Wnt effects on TLR responses. Total FACS sorted cDC (CD11c high , B220−) or CD103+ cDC (inserted graphs) isolated from mesenteric lymph nodes (IL-6, IL-12p40 and IL-10 graphs) or total CD11c+ DCs isolated from pooled lymph nodes (VEGF-A and IFN-α graphs) of FLT3L-treated mice were stimulated in vitro with LPS (5μg/ml), CpGA (5μg/ml) or Pam3CSK4 (5μg/ml) in the presence of varying doses of Wnt3A or Wnt5A for 20 hours. Cytokine secretion was determined by ELISA of culture supernatants. For IL-6, IL-12p40 and IFN-α data are presented as % of cytokine concentration in the presence of TLR alone, without Wnt. [In the absence of Wnts values were as follows in pg/ml: IL-6 (for the Wnt3A graph) LPS 820, CpGA 3400, Pam3CSK4 730; IL-6 (for the Wnt5a graph) LPS 670, CpGA 4150, Pam3CSK4 660; IL-12p40 (for the Wnt3A graph) LPS 18310, CpGA 25220, Pam3CSK4 18900; IL-12p40 (for the Wnt5A graph) LPS 16550, CpGA 32140, Pam3CSK4 13530; and IFN-α in units/μl (for both Wnt3A and Wnt5A graphs) 1650.] For IL-10 and VEGF-A induction, data are % of maximum cytokine concentration induced by each TLR ligand or control. [Maximum values in pg/ml were as follows: IL-10 medium 80, LPS 120, CpGA 110, Pam3CSK4 240; VEGF-A medium 22580, LPS 20940, CpGA 17100.] Mean ± SEM are presented, n=3, with the exception of IL-12p40 and IFN-α in response to CpGA, and VEGF-A in response to all stimulations, mean ± StDev n=2. CD103+ DC responses (graph inserts) are from one of two experiments with similar results.

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

    Article Title: Canonical and non-canonical Wnt proteins program dendritic cell responses for tolerance

    doi: 10.4049/jimmunol.1203002

    Figure Lengend Snippet: Wnts suppress TLR induced proinflammatory cytokine production a) Effect of Wnts on LPS responses of DC from normal mouse lymph nodes. CD11c+ DC were stimulated in vitro with LPS (5μg/ml) in combination with Wnt3A (3μg/ml) or Wnt5A (3μg/ml) for 20 hours. Cytokine secretion was determined by multiplex luminex analysis of culture supernatants. Data represent cytokine concentrations from one of two experiments performed with similar results. b) Wnts inhibit CpG as well as LPS stimulation of IL-6 in DC. CD11c+ DC isolated from pooled lymph nodes from normal mice were stimulated in vitro with LPS (5μg/ml) or CpGA (5μg/ml) in combination with Wnt3a or Wnt5a (3μg/ml) for 20 hours. Cytokine secretion was determined by ELISA. Data show cytokine concentrations from 3–5 experiments. Mean values ± standard error of the mean (SEM). Symbols indicate individual experiments. c) Dose dependent Wnt effects on TLR responses. Total FACS sorted cDC (CD11c high , B220−) or CD103+ cDC (inserted graphs) isolated from mesenteric lymph nodes (IL-6, IL-12p40 and IL-10 graphs) or total CD11c+ DCs isolated from pooled lymph nodes (VEGF-A and IFN-α graphs) of FLT3L-treated mice were stimulated in vitro with LPS (5μg/ml), CpGA (5μg/ml) or Pam3CSK4 (5μg/ml) in the presence of varying doses of Wnt3A or Wnt5A for 20 hours. Cytokine secretion was determined by ELISA of culture supernatants. For IL-6, IL-12p40 and IFN-α data are presented as % of cytokine concentration in the presence of TLR alone, without Wnt. [In the absence of Wnts values were as follows in pg/ml: IL-6 (for the Wnt3A graph) LPS 820, CpGA 3400, Pam3CSK4 730; IL-6 (for the Wnt5a graph) LPS 670, CpGA 4150, Pam3CSK4 660; IL-12p40 (for the Wnt3A graph) LPS 18310, CpGA 25220, Pam3CSK4 18900; IL-12p40 (for the Wnt5A graph) LPS 16550, CpGA 32140, Pam3CSK4 13530; and IFN-α in units/μl (for both Wnt3A and Wnt5A graphs) 1650.] For IL-10 and VEGF-A induction, data are % of maximum cytokine concentration induced by each TLR ligand or control. [Maximum values in pg/ml were as follows: IL-10 medium 80, LPS 120, CpGA 110, Pam3CSK4 240; VEGF-A medium 22580, LPS 20940, CpGA 17100.] Mean ± SEM are presented, n=3, with the exception of IL-12p40 and IFN-α in response to CpGA, and VEGF-A in response to all stimulations, mean ± StDev n=2. CD103+ DC responses (graph inserts) are from one of two experiments with similar results.

    Article Snippet: The following reagents were used for ELISA: mouse IL-6 ELISA set, mouse IL-12p40 ELISA set, mouse TNFα ELISA set, mouse IL-10 ELISA set (BD Biosciences), mouse VEGF ELISA set (R & D Systems) and IFNα ELISA was performed using anti Mu-IFNα (Rmma-1) as capture antibody and Rabbit-Pab against Mu-IFNα for detection (PBL InterferonSource, Piscataway, NJ).

    Techniques: In Vitro, Multiplex Assay, Luminex, Isolation, Mouse Assay, Enzyme-linked Immunosorbent Assay, FACS, Concentration Assay

    Wnt3A and Wnt5A differentially stimulate tolerogenic cytokine production in resting DC Isolated CD11c+ DC from pooled peripheral and mesenteric lymph nodes were stimulated in vitro with Wnt3A (3μg/ml) or Wnt5A (3μg/ml) for 20 hours. Cytokine secretion was determined by multiplex luminex analysis (VEGF and TGFβ) or ELISA (IL-10) of culture supernatants. Results are presented as mean ± standard error of the mean (SEM), n=3. DC were from normal mice (n=2, TGFβ and VEGF) or Flt3L-treated mice (n=1, TGFβ and VEGF; n=3, IL-10). Wnt5A but not Wnt3A also induced an increase in IL-10 from normal mouse lymph node DC in an independent experiment (not shown). P

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

    Article Title: Canonical and non-canonical Wnt proteins program dendritic cell responses for tolerance

    doi: 10.4049/jimmunol.1203002

    Figure Lengend Snippet: Wnt3A and Wnt5A differentially stimulate tolerogenic cytokine production in resting DC Isolated CD11c+ DC from pooled peripheral and mesenteric lymph nodes were stimulated in vitro with Wnt3A (3μg/ml) or Wnt5A (3μg/ml) for 20 hours. Cytokine secretion was determined by multiplex luminex analysis (VEGF and TGFβ) or ELISA (IL-10) of culture supernatants. Results are presented as mean ± standard error of the mean (SEM), n=3. DC were from normal mice (n=2, TGFβ and VEGF) or Flt3L-treated mice (n=1, TGFβ and VEGF; n=3, IL-10). Wnt5A but not Wnt3A also induced an increase in IL-10 from normal mouse lymph node DC in an independent experiment (not shown). P

    Article Snippet: The following reagents were used for ELISA: mouse IL-6 ELISA set, mouse IL-12p40 ELISA set, mouse TNFα ELISA set, mouse IL-10 ELISA set (BD Biosciences), mouse VEGF ELISA set (R & D Systems) and IFNα ELISA was performed using anti Mu-IFNα (Rmma-1) as capture antibody and Rabbit-Pab against Mu-IFNα for detection (PBL InterferonSource, Piscataway, NJ).

    Techniques: Isolation, In Vitro, Multiplex Assay, Luminex, Enzyme-linked Immunosorbent Assay, Mouse Assay

    Wnt5A does not counteract Wnt3A induced β-catenin signaling or VEGF secretion nor does Wnt3A affect Wnt5A induced IL-10 secretion CD11c+ DC from pooled lymph nodes of FLT3L treated mice were treated with Wnt3A or Wnt5A or Wnt3A in combination with Wnt5A at the indicated concentrations. a) DC were cultured 2 hours and nuclear extracts were analyzed by western blot for β-catenin and for lamin B as a loading control. Data shown is representative of 3 independent experiments with similar results. b) DC were cultured for 20 hours and supernatants were analyzed by ELISA to determine concentration of secreted cytokines. Results are presented as % of maximum cytokine concentration induced, Maximum values were 120 pg/ml for IL-10, 4200 pg/ml for VEGF-A. Data shown is representative of 2 independent experiments with similar results.

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

    Article Title: Canonical and non-canonical Wnt proteins program dendritic cell responses for tolerance

    doi: 10.4049/jimmunol.1203002

    Figure Lengend Snippet: Wnt5A does not counteract Wnt3A induced β-catenin signaling or VEGF secretion nor does Wnt3A affect Wnt5A induced IL-10 secretion CD11c+ DC from pooled lymph nodes of FLT3L treated mice were treated with Wnt3A or Wnt5A or Wnt3A in combination with Wnt5A at the indicated concentrations. a) DC were cultured 2 hours and nuclear extracts were analyzed by western blot for β-catenin and for lamin B as a loading control. Data shown is representative of 3 independent experiments with similar results. b) DC were cultured for 20 hours and supernatants were analyzed by ELISA to determine concentration of secreted cytokines. Results are presented as % of maximum cytokine concentration induced, Maximum values were 120 pg/ml for IL-10, 4200 pg/ml for VEGF-A. Data shown is representative of 2 independent experiments with similar results.

    Article Snippet: The following reagents were used for ELISA: mouse IL-6 ELISA set, mouse IL-12p40 ELISA set, mouse TNFα ELISA set, mouse IL-10 ELISA set (BD Biosciences), mouse VEGF ELISA set (R & D Systems) and IFNα ELISA was performed using anti Mu-IFNα (Rmma-1) as capture antibody and Rabbit-Pab against Mu-IFNα for detection (PBL InterferonSource, Piscataway, NJ).

    Techniques: Mouse Assay, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay

    PMT stimulated release of TNF-α or IL-6 is independent of the mTOR pathway. RAW264.7 cells were stimulated with PMT (5 nM) w or w/o pre-incubation with rapamycin (10 ng/ml) for 1 h. As control, cells were untreated, treated with the solvent control DMSO, with rapamycin or LPS (100 ng/ml) as positive control. After 24 h, supernatants were removed and subjected to ELISA for the measurement of ( a ) IL-6-, ( b ) TNF-α-, ( c ) IL12p40- or ( d ) IL-10 release. Shown are the results of three independent experiments (mean ± SD; n = 3)

    Journal: Cell Communication and Signaling : CCS

    Article Title: Pasteurella multocida toxin- induced osteoclastogenesis requires mTOR activation

    doi: 10.1186/s12964-015-0117-7

    Figure Lengend Snippet: PMT stimulated release of TNF-α or IL-6 is independent of the mTOR pathway. RAW264.7 cells were stimulated with PMT (5 nM) w or w/o pre-incubation with rapamycin (10 ng/ml) for 1 h. As control, cells were untreated, treated with the solvent control DMSO, with rapamycin or LPS (100 ng/ml) as positive control. After 24 h, supernatants were removed and subjected to ELISA for the measurement of ( a ) IL-6-, ( b ) TNF-α-, ( c ) IL12p40- or ( d ) IL-10 release. Shown are the results of three independent experiments (mean ± SD; n = 3)

    Article Snippet: ELISA The production of IL-6, TNF-α, IL-12(p40) and IL-10 was measured by commercial assays: mouse IL-6 ELISA MAX™ Standard Set (BioLegend), mouse TNF-α ELISA MAX™ (BioLegend) Standard Set, the BD OptEIA™ Mouse IL-12(p40) ELISA Set (BD Biosciences) or BD OptEIA™ Mouse IL-10 ELISA Set (BD Biosciences).

    Techniques: Incubation, Positive Control, Enzyme-linked Immunosorbent Assay

    An expanded pool of IL-10 + B cells are present in the uterus after implantation. (A) Known regulatory B cell subsets were analyzed from B cells collected from non-pregnant and pregnant uteri ( n = 6–7, unpaired t -test). (B) IL-10 production by stimulated populations of purified B cells obtained from the spleen, virgin uterus, day 5.5 pc uterus, or in vitro induced Bregs was assessed by ELISA. Data represent pooled supernatants of duplicate wells from three independent experiments. Data is presented as mean ± SEM and compared by one-way ANOVA, followed by Dunnett's post-hoc multiple comparisons test against unstimulated control. * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001. Additional Dunnett's analysis assessed against non-pregnant virgin uterus reveal a significant increase in IL-10 produced by day 5.5 pc uterine B cells and iBregs ( # p ≤ 0.05, #### p ≤ 0.0001).

    Journal: Frontiers in Immunology

    Article Title: Uterine B Cells Exhibit Regulatory Properties During the Peri-Implantation Stage of Murine Pregnancy

    doi: 10.3389/fimmu.2019.02899

    Figure Lengend Snippet: An expanded pool of IL-10 + B cells are present in the uterus after implantation. (A) Known regulatory B cell subsets were analyzed from B cells collected from non-pregnant and pregnant uteri ( n = 6–7, unpaired t -test). (B) IL-10 production by stimulated populations of purified B cells obtained from the spleen, virgin uterus, day 5.5 pc uterus, or in vitro induced Bregs was assessed by ELISA. Data represent pooled supernatants of duplicate wells from three independent experiments. Data is presented as mean ± SEM and compared by one-way ANOVA, followed by Dunnett's post-hoc multiple comparisons test against unstimulated control. * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001. Additional Dunnett's analysis assessed against non-pregnant virgin uterus reveal a significant increase in IL-10 produced by day 5.5 pc uterine B cells and iBregs ( # p ≤ 0.05, #### p ≤ 0.0001).

    Article Snippet: IL-10 Detection by ELISA IL-10 quantities in supernatants were measured using the ELISA MaxTM Standard Set Mouse IL-10 Kit (Biolegend) following the manufacturer's instructions.

    Techniques: Purification, In Vitro, Enzyme-linked Immunosorbent Assay, Produced

    Heparin treatment affects the mRNA expression of CCR2, IL1 β , and TLR7 in kidneys of B/W mice. The mRNA expression of CCR2 (a), IL1 β (b), IL10 (c), TLR2 (d), and TLR7 (e) was increased in Group 3 mice compared to Group 1 mice. The mRNA expression of CCR2 (a), IL1 β (b), and TLR7 (e) was significantly reduced in Heparin-treated mice compared to Group 3 mice. Data is given as Log 2 of mean ± SEM of fold change values normalized against 4-week-old mice ( n = 3). P values are calculated using one-way ANOVA followed by Bonferroni posttest. * P

    Journal: Clinical and Developmental Immunology

    Article Title: LMW Heparin Prevents Increased Kidney Expression of Proinflammatory Mediators in (NZBxNZW)F1 Mice

    doi: 10.1155/2013/791262

    Figure Lengend Snippet: Heparin treatment affects the mRNA expression of CCR2, IL1 β , and TLR7 in kidneys of B/W mice. The mRNA expression of CCR2 (a), IL1 β (b), IL10 (c), TLR2 (d), and TLR7 (e) was increased in Group 3 mice compared to Group 1 mice. The mRNA expression of CCR2 (a), IL1 β (b), and TLR7 (e) was significantly reduced in Heparin-treated mice compared to Group 3 mice. Data is given as Log 2 of mean ± SEM of fold change values normalized against 4-week-old mice ( n = 3). P values are calculated using one-way ANOVA followed by Bonferroni posttest. * P

    Article Snippet: Measurements of Cytokines in Cell Supernatants Cytokine analyses were performed with ELISA MAX Standard Sets for mouse IL10 (BioLegend, San Diego, CA, USA) or mouse TNFα ELISA kit (Thermo scientific, Rockford, IL, USA).

    Techniques: Expressing, Mouse Assay

    Pre-fixed platelets but not plastic microbead particles nor lymphocytes mediate regulatory response. (A) Pre-fixed platelets can mediate IgG-bound platelet-induced regulatory response. Paraformaldehyde (PFA)-fixed human platelets and sort-purified monocytes were incubated with an anti-CD61 mAb following LPS stimulation for 24 h. The IL-10 levels in culture supernatants were determined by ELISA. Data are shown as means for triplicate samples ± SEM. The results are representative of more than three independent experiments with similar results. ** P

    Journal: BMC Immunology

    Article Title: Platelets convert peripheral blood circulating monocytes to regulatory cells via immunoglobulin G and activating-type Fcγ receptors

    doi: 10.1186/s12865-015-0086-z

    Figure Lengend Snippet: Pre-fixed platelets but not plastic microbead particles nor lymphocytes mediate regulatory response. (A) Pre-fixed platelets can mediate IgG-bound platelet-induced regulatory response. Paraformaldehyde (PFA)-fixed human platelets and sort-purified monocytes were incubated with an anti-CD61 mAb following LPS stimulation for 24 h. The IL-10 levels in culture supernatants were determined by ELISA. Data are shown as means for triplicate samples ± SEM. The results are representative of more than three independent experiments with similar results. ** P

    Article Snippet: ELISA ELISAs were performed to measure human IL-1β, human IL-10, human IL-12, mouse IL-6, mouse IL-10, and mouse IL-12 using Human IL-1β ELISA Ready-SET-Go!, Human IL-10 ELISA Ready-SET-Go!, Human IL-12 ELISA Ready-SET-Go! (eBioscience, San Diego, CA), Mouse IL-6 ELISA MAX standard, Mouse IL-10 ELISA MAX standard, and Mouse IL-12 ELISA MAX standard, respectively (BioLegend).

    Techniques: Purification, Incubation, Enzyme-linked Immunosorbent Assay

    Augmented serum IL-10 in a murine in vivo setting. (A, B) Wild-type B6 mice were administered intravenously an anti-human/mouse CD61 or isotype control mAb and subsequently with LPS intraperitoneally. The IL-10 and IL-6 levels in serum samples collected at 3 h after LPS stimulation were measured by ELISA (A) . IL-10 upregulation in sera was observed also in FcγRIIB-deficient but not FcRγ-deficient mice ( n = 4 mice per group) (B) . The results are representative of more than three independent experiments with similar results. * P

    Journal: BMC Immunology

    Article Title: Platelets convert peripheral blood circulating monocytes to regulatory cells via immunoglobulin G and activating-type Fcγ receptors

    doi: 10.1186/s12865-015-0086-z

    Figure Lengend Snippet: Augmented serum IL-10 in a murine in vivo setting. (A, B) Wild-type B6 mice were administered intravenously an anti-human/mouse CD61 or isotype control mAb and subsequently with LPS intraperitoneally. The IL-10 and IL-6 levels in serum samples collected at 3 h after LPS stimulation were measured by ELISA (A) . IL-10 upregulation in sera was observed also in FcγRIIB-deficient but not FcRγ-deficient mice ( n = 4 mice per group) (B) . The results are representative of more than three independent experiments with similar results. * P

    Article Snippet: ELISA ELISAs were performed to measure human IL-1β, human IL-10, human IL-12, mouse IL-6, mouse IL-10, and mouse IL-12 using Human IL-1β ELISA Ready-SET-Go!, Human IL-10 ELISA Ready-SET-Go!, Human IL-12 ELISA Ready-SET-Go! (eBioscience, San Diego, CA), Mouse IL-6 ELISA MAX standard, Mouse IL-10 ELISA MAX standard, and Mouse IL-12 ELISA MAX standard, respectively (BioLegend).

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

    IgG-opsonized platelets augment IL-10 release but decrease proinflammatory cytokine release from monocytes via their direct contact. (A) A crude PBMC preparation was stimulated with LPS or poly(I:C) in the presence of anti-human CD61 mAb or isotype-matched control (cIg). The IL-10, IL-1β, and IL-12 p40 levels in the culture supernatants collected at different time points were determined by ELISA. (B) A crude PBMC preparation was stimulated with LPS in the presence of an anti-CD61 or anti-CD41 mAb or isotype control (cIg). The IL-10 and IL-12 levels in the culture supernatant after 24 h were determined by ELISA. Blank (−) indicates cells with no antibody or stimulator for monitoring spontaneous production of cytokines. (C) Transwell assaying of cytokine production. Sort-purified monocytes were stimulated with LPS in the presence or absence of platelets and an anti-CD61 mAb. The bottom chamber contained monocytes or was left blank, and the upper chamber contained platelets. Both chambers contained LPS and the anti-CD61 mAb. The IL-10 ( left ) and IL-1β ( right ) levels in the culture supernatant were measured after 24 h. * P

    Journal: BMC Immunology

    Article Title: Platelets convert peripheral blood circulating monocytes to regulatory cells via immunoglobulin G and activating-type Fcγ receptors

    doi: 10.1186/s12865-015-0086-z

    Figure Lengend Snippet: IgG-opsonized platelets augment IL-10 release but decrease proinflammatory cytokine release from monocytes via their direct contact. (A) A crude PBMC preparation was stimulated with LPS or poly(I:C) in the presence of anti-human CD61 mAb or isotype-matched control (cIg). The IL-10, IL-1β, and IL-12 p40 levels in the culture supernatants collected at different time points were determined by ELISA. (B) A crude PBMC preparation was stimulated with LPS in the presence of an anti-CD61 or anti-CD41 mAb or isotype control (cIg). The IL-10 and IL-12 levels in the culture supernatant after 24 h were determined by ELISA. Blank (−) indicates cells with no antibody or stimulator for monitoring spontaneous production of cytokines. (C) Transwell assaying of cytokine production. Sort-purified monocytes were stimulated with LPS in the presence or absence of platelets and an anti-CD61 mAb. The bottom chamber contained monocytes or was left blank, and the upper chamber contained platelets. Both chambers contained LPS and the anti-CD61 mAb. The IL-10 ( left ) and IL-1β ( right ) levels in the culture supernatant were measured after 24 h. * P

    Article Snippet: ELISA ELISAs were performed to measure human IL-1β, human IL-10, human IL-12, mouse IL-6, mouse IL-10, and mouse IL-12 using Human IL-1β ELISA Ready-SET-Go!, Human IL-10 ELISA Ready-SET-Go!, Human IL-12 ELISA Ready-SET-Go! (eBioscience, San Diego, CA), Mouse IL-6 ELISA MAX standard, Mouse IL-10 ELISA MAX standard, and Mouse IL-12 ELISA MAX standard, respectively (BioLegend).

    Techniques: Enzyme-linked Immunosorbent Assay, Purification

    Effect of ERK1/2 inhibition on Sp1 and IL-10 levels Eµ- TCL1 CLL cells were cultured with the ERK1/2 inhibitor (SCH772984) (2µM). A) IL-10 levels in 24-hour supernatants were measured by ELISA. Values represent mean±SD of triplicates. B) Levels of p-ERK1/2, total ERK1, p-STAT3, total STAT3 and Sp1 were quantified by immunoblot analysis. Results are representative of three experiments. C) Sp1 mRNA levels were quantified by qRT-PCR. Fold change was normalized to the no-treatment group. Values represent mean±SD of triplicates ***p

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

    Article Title: Chronic lymphocytic leukemia derived interleukin-10 suppresses anti-tumor immunity

    doi: 10.4049/jimmunol.1800241

    Figure Lengend Snippet: Effect of ERK1/2 inhibition on Sp1 and IL-10 levels Eµ- TCL1 CLL cells were cultured with the ERK1/2 inhibitor (SCH772984) (2µM). A) IL-10 levels in 24-hour supernatants were measured by ELISA. Values represent mean±SD of triplicates. B) Levels of p-ERK1/2, total ERK1, p-STAT3, total STAT3 and Sp1 were quantified by immunoblot analysis. Results are representative of three experiments. C) Sp1 mRNA levels were quantified by qRT-PCR. Fold change was normalized to the no-treatment group. Values represent mean±SD of triplicates ***p

    Article Snippet: Human plasma or secreted IL-10 levels were quantified using IL-10 ELISA MAX set (BioLegend #430601).

    Techniques: Inhibition, Cell Culture, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

    IL-10 production by Eµ TCL1 CLL cells is dependent on ERK1/2 MAPK and the transcription factor Sp1 but not on p38MAPK or STAT3 A) Eµ- TCL1 cells were treated with Syk inhibitor-IV (5µM) for indicated time periods. Levels of key molecules downstream of BCR signaling were quantified by Immunoblotting. Band densitometry analysis was performed using the NIH ImageJ program. Phospho-protein levels were normalized to total protein. Results are representative of three experiments. B) Sp1 mRNA levels were quantified by qRT-PCR after treatment of Eµ- TCL1 cells with αIgM ±Syk inhibitor-IV. Fold change was normalized to the no-treatment group. Values represent mean±SD of triplicate determinations. C) Eµ- TCL1 cells were treated with various doses of mithramycin A for 24 hours and IL-10 in the supernatant was quantified by ELISA. D) Immunoblot analysis of IL-10 protein levels in CLL cells after treatment with mithramycin A (5µM). E) ChIP with anti-Sp1 or control IgG was carried out as described in the Methods. qRT-PCR was performed on the ChIP DNA product using primers specific for the consensus Sp1 binding site sequence in the IL-10 promoter. Results are calculated using the Fold Enrichment Method. *p

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

    Article Title: Chronic lymphocytic leukemia derived interleukin-10 suppresses anti-tumor immunity

    doi: 10.4049/jimmunol.1800241

    Figure Lengend Snippet: IL-10 production by Eµ TCL1 CLL cells is dependent on ERK1/2 MAPK and the transcription factor Sp1 but not on p38MAPK or STAT3 A) Eµ- TCL1 cells were treated with Syk inhibitor-IV (5µM) for indicated time periods. Levels of key molecules downstream of BCR signaling were quantified by Immunoblotting. Band densitometry analysis was performed using the NIH ImageJ program. Phospho-protein levels were normalized to total protein. Results are representative of three experiments. B) Sp1 mRNA levels were quantified by qRT-PCR after treatment of Eµ- TCL1 cells with αIgM ±Syk inhibitor-IV. Fold change was normalized to the no-treatment group. Values represent mean±SD of triplicate determinations. C) Eµ- TCL1 cells were treated with various doses of mithramycin A for 24 hours and IL-10 in the supernatant was quantified by ELISA. D) Immunoblot analysis of IL-10 protein levels in CLL cells after treatment with mithramycin A (5µM). E) ChIP with anti-Sp1 or control IgG was carried out as described in the Methods. qRT-PCR was performed on the ChIP DNA product using primers specific for the consensus Sp1 binding site sequence in the IL-10 promoter. Results are calculated using the Fold Enrichment Method. *p

    Article Snippet: Human plasma or secreted IL-10 levels were quantified using IL-10 ELISA MAX set (BioLegend #430601).

    Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Chromatin Immunoprecipitation, Binding Assay, Sequencing

    Human CLL cells utilize BCR signaling for IL-10 production A–B) IL-10 levels in 24-hour supernatants of human CLL cells ±αIgM (A) or in the plasma of human CLL patients (n=15) and normal donors (n=28) (B). C) Human CLL cells were cultured with αIL-10 or αIL-10R antibodies ±αIgM for 48 hours. Survival of CLL cells was measured by MTT. D) Human total CLL cells or purified CLL B-cells were stimulated with αIgM±inhibitors of Btk, Syk, SFK or ERK1/2 for 24 hours (2µM). IL-10 levels in the supernatants were measured by ELISA. Human B-cell CLL cells were purified by negative selection (MojoSort Human B cells (CD43 − ) Isolation Kit from Biolegend. E) Human CLL cells were treated with Syk inhibitor-IV (2µM). Protein levels indicated were quantified by immunoblot analysis. For (A, C and D) , values represent mean±SD of triplicate cultures. *p

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

    Article Title: Chronic lymphocytic leukemia derived interleukin-10 suppresses anti-tumor immunity

    doi: 10.4049/jimmunol.1800241

    Figure Lengend Snippet: Human CLL cells utilize BCR signaling for IL-10 production A–B) IL-10 levels in 24-hour supernatants of human CLL cells ±αIgM (A) or in the plasma of human CLL patients (n=15) and normal donors (n=28) (B). C) Human CLL cells were cultured with αIL-10 or αIL-10R antibodies ±αIgM for 48 hours. Survival of CLL cells was measured by MTT. D) Human total CLL cells or purified CLL B-cells were stimulated with αIgM±inhibitors of Btk, Syk, SFK or ERK1/2 for 24 hours (2µM). IL-10 levels in the supernatants were measured by ELISA. Human B-cell CLL cells were purified by negative selection (MojoSort Human B cells (CD43 − ) Isolation Kit from Biolegend. E) Human CLL cells were treated with Syk inhibitor-IV (2µM). Protein levels indicated were quantified by immunoblot analysis. For (A, C and D) , values represent mean±SD of triplicate cultures. *p

    Article Snippet: Human plasma or secreted IL-10 levels were quantified using IL-10 ELISA MAX set (BioLegend #430601).

    Techniques: Cell Culture, MTT Assay, Purification, Enzyme-linked Immunosorbent Assay, Selection, Isolation

    Constitutive IL-10 production by CLL cells and its role in CLL cell survival A) CLL cells were harvested from spleens of Eµ- TCL1 (n=23) or adoptive transfer mice (n=9). B-1a cells were isolated from the peritoneal cavity (PC) (n=3) and normal CD19+ cells were isolated from spleens of C57BL/6J mice (n=5). Purified CD19+ Eµ- TCL1 cells were cultured for 24 hours and IL-10 was measured in the supernatants by ELISA. Bars represent mean±SE. B) Splenic Eµ- TCL1 cells were cultured with αIL-10 or αIL-10R antibodies ±LPS (5µg/ml) for 48 hours. Survival was measured by MTT. Values represent mean±SD of triplicate cultures. C) Flow cytometric analysis of IL-10R expression by Eµ- TCL1 cells (left) and protein lysates of Eµ- TCL1 CLL cells stimulated with exogenous IL-10 were analyzed for STAT3 activation by immunoblot (right) . β-actin is used for loading control. *p

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

    Article Title: Chronic lymphocytic leukemia derived interleukin-10 suppresses anti-tumor immunity

    doi: 10.4049/jimmunol.1800241

    Figure Lengend Snippet: Constitutive IL-10 production by CLL cells and its role in CLL cell survival A) CLL cells were harvested from spleens of Eµ- TCL1 (n=23) or adoptive transfer mice (n=9). B-1a cells were isolated from the peritoneal cavity (PC) (n=3) and normal CD19+ cells were isolated from spleens of C57BL/6J mice (n=5). Purified CD19+ Eµ- TCL1 cells were cultured for 24 hours and IL-10 was measured in the supernatants by ELISA. Bars represent mean±SE. B) Splenic Eµ- TCL1 cells were cultured with αIL-10 or αIL-10R antibodies ±LPS (5µg/ml) for 48 hours. Survival was measured by MTT. Values represent mean±SD of triplicate cultures. C) Flow cytometric analysis of IL-10R expression by Eµ- TCL1 cells (left) and protein lysates of Eµ- TCL1 CLL cells stimulated with exogenous IL-10 were analyzed for STAT3 activation by immunoblot (right) . β-actin is used for loading control. *p

    Article Snippet: Human plasma or secreted IL-10 levels were quantified using IL-10 ELISA MAX set (BioLegend #430601).

    Techniques: Adoptive Transfer Assay, Mouse Assay, Isolation, Purification, Cell Culture, Enzyme-linked Immunosorbent Assay, MTT Assay, Flow Cytometry, Expressing, Activation Assay