cytokine production analysis cells Search Results


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  • 93
    Thermo Fisher elisa
    Elisa, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 13759 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore ionomycin
    Ionomycin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 46801 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore brefeldin a
    Brefeldin A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 33643 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher tnf α
    Regulatory effect of tribulusamide D on the production of IL-6, IL-10 and <t>TNF-α</t> in LPS-stimulated RAW 264.7 cells. Cells were treated with tribulusamide D (25–100 µM) for 1 h prior to LPS (0.5 µg/ml) stimulation for 24
    Tnf α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 21729 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher brefeldin a
    Regulatory effect of tribulusamide D on the production of IL-6, IL-10 and <t>TNF-α</t> in LPS-stimulated RAW 264.7 cells. Cells were treated with tribulusamide D (25–100 µM) for 1 h prior to LPS (0.5 µg/ml) stimulation for 24
    Brefeldin A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8963 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson cytokine production
    CD4 + T cells from sarcoidosis progressors exhibit characteristics of exhaustion. (a, b, and c) Representative functional data for a single healthy control, sarcoidosis progressor, and sarcoidosis resolver. (a) <t>Cytokine</t> secretion; data have been normalized to the mean value for the healthy control cohort (hashed line). (b and c) Proliferation analysis. (a) Apoptosis analysis. (b and c) Percent PD-1 + CD4 + T cells; overlay of naïve (CCR7 + CD45RO − ; shaded) CD4 + cells and total CD4 + T cells (unshaded). (d) Immune activity of sarcoidosis progressors ( n = 12) and resolvers ( n = 5) relative to healthy controls. High = activity greater than one standard deviation above the healthy control mean (yellow). Low = activity greater than one standard deviation below the healthy control mean (magenta). Normal = within one standard deviation of the healthy control mean (grey). White spaces indicate data not present due to lack of available cells.
    Cytokine Production, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 3241 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher elisa kits
    CD4 + T cells from sarcoidosis progressors exhibit characteristics of exhaustion. (a, b, and c) Representative functional data for a single healthy control, sarcoidosis progressor, and sarcoidosis resolver. (a) <t>Cytokine</t> secretion; data have been normalized to the mean value for the healthy control cohort (hashed line). (b and c) Proliferation analysis. (a) Apoptosis analysis. (b and c) Percent PD-1 + CD4 + T cells; overlay of naïve (CCR7 + CD45RO − ; shaded) CD4 + cells and total CD4 + T cells (unshaded). (d) Immune activity of sarcoidosis progressors ( n = 12) and resolvers ( n = 5) relative to healthy controls. High = activity greater than one standard deviation above the healthy control mean (yellow). Low = activity greater than one standard deviation below the healthy control mean (magenta). Normal = within one standard deviation of the healthy control mean (grey). White spaces indicate data not present due to lack of available cells.
    Elisa Kits, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8254 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher elisa kit
    CD4 + T cells from sarcoidosis progressors exhibit characteristics of exhaustion. (a, b, and c) Representative functional data for a single healthy control, sarcoidosis progressor, and sarcoidosis resolver. (a) <t>Cytokine</t> secretion; data have been normalized to the mean value for the healthy control cohort (hashed line). (b and c) Proliferation analysis. (a) Apoptosis analysis. (b and c) Percent PD-1 + CD4 + T cells; overlay of naïve (CCR7 + CD45RO − ; shaded) CD4 + cells and total CD4 + T cells (unshaded). (d) Immune activity of sarcoidosis progressors ( n = 12) and resolvers ( n = 5) relative to healthy controls. High = activity greater than one standard deviation above the healthy control mean (yellow). Low = activity greater than one standard deviation below the healthy control mean (magenta). Normal = within one standard deviation of the healthy control mean (grey). White spaces indicate data not present due to lack of available cells.
    Elisa Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6578 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore phorbol 12 myristate 13 acetate
    CD4 + T cells from sarcoidosis progressors exhibit characteristics of exhaustion. (a, b, and c) Representative functional data for a single healthy control, sarcoidosis progressor, and sarcoidosis resolver. (a) <t>Cytokine</t> secretion; data have been normalized to the mean value for the healthy control cohort (hashed line). (b and c) Proliferation analysis. (a) Apoptosis analysis. (b and c) Percent PD-1 + CD4 + T cells; overlay of naïve (CCR7 + CD45RO − ; shaded) CD4 + cells and total CD4 + T cells (unshaded). (d) Immune activity of sarcoidosis progressors ( n = 12) and resolvers ( n = 5) relative to healthy controls. High = activity greater than one standard deviation above the healthy control mean (yellow). Low = activity greater than one standard deviation below the healthy control mean (magenta). Normal = within one standard deviation of the healthy control mean (grey). White spaces indicate data not present due to lack of available cells.
    Phorbol 12 Myristate 13 Acetate, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 17372 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore pma
    Perinatal HI brain injury shifts the peripheral T cell immune response towards pro-inflammatory. Mononuclear cells were isolated from the spleen of pups 7 days after HI injury and the phenotype of T helper cells was assessed by flow cytometry. a Proportion of CD4+ lymphocytes. b Proportion of CD4+IL4+ T cells. c Proportion of CD4+IFN-γ+ T cells. d Proportion of CD4+IL17+ T cells. e Ratio of Th1 cells (IFN-γ+) to Th2 cells (IL4+). f Ratio of Th17 cells (IL-17+) to Th2 cells (IL4+). g Concentration of IL4 protein produced by <t>ionomycin</t> and <t>PMA</t> stimulated spleen cells. h Proportion of regulatory T cells in the spleen. ( n = 7–14 pups per group, * P
    Pma, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 21647 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson flow cytometry
    Perinatal HI brain injury shifts the peripheral T cell immune response towards pro-inflammatory. Mononuclear cells were isolated from the spleen of pups 7 days after HI injury and the phenotype of T helper cells was assessed by flow cytometry. a Proportion of CD4+ lymphocytes. b Proportion of CD4+IL4+ T cells. c Proportion of CD4+IFN-γ+ T cells. d Proportion of CD4+IL17+ T cells. e Ratio of Th1 cells (IFN-γ+) to Th2 cells (IL4+). f Ratio of Th17 cells (IL-17+) to Th2 cells (IL4+). g Concentration of IL4 protein produced by <t>ionomycin</t> and <t>PMA</t> stimulated spleen cells. h Proportion of regulatory T cells in the spleen. ( n = 7–14 pups per group, * P
    Flow Cytometry, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 188544 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson golgistop
    Distribution of T naïve , T CM , T EM subpopulations and IL-17 + /T naïve , IL-17 + /T EM and IL-17 + /T CM , subpopulations of CD4 + T lymphocytes at 1 and 3 month after transplantation compared to before transplantation. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of <t>GolgiStop.CD4</t> + lymphocytes were stained with mAbs to CD45RA and CCR7, which identified three subsets. In addition, analysis of IL-17 in CD4 + T cell subsets by intracellular flow cytometry was done. After surface staining with CD45 and CCR7 mAbs, cells were fixated and permeabilized and intracellular accumulated cytokines were detected with IL-17 mAbs. (A) T naïve /CD4 + T (CD45RA + CCR7 + /CD4 + Tcells), (B) IL-17 + /T naïve , (C) T CM /CD4 + T (CD45RA − CCR7 + /CD4 + Tcells), (D) IL-17 + /T CM + , (E) T EM /CD4 + T (CD45RA − CCR7 − /CD4 + Tcells), (F) IL-17 + /T EM + . Bars show the means. * P
    Golgistop, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 16164 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson brefeldin a
    An analysis of the development of regulatory T cells from spleen and Peyer’s patch (PP) cells in response to various bacteria. Spleen and PP cells prepared from ovalbumin-specific T-cell receptor transgenic (OVA-TCR-Tg) mice were primarily cultured with or without (OVA only) various bacteria (10 μg/ml) in the presence of OVA for 6 days in order to induce the development of regulatory T cells. The cultured cells were harvested, re-stimulated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin in the presence of <t>brefeldin</t> A for 4 hr and stained with fluorescein isothiocyanate (FITC)-labelled anti-CD4 and phycoerythrin (PE)-labelled anti-IL-10 or biotinylated anti-transforming growth factor (TGF)-β1 antibodies. Biotinylated antibody was detected using PE-labelled streptavidin. For the analysis of Foxp3-positive cells, the cells after the primary culture were stained with FITC-labelled anti-CD4 and PE-labelled anti-Foxp3 antibodies without re-stimulation. The stained cells were analysed by flow cytometry. The values represent the mean ± standard deviation of the percentages of cytokine-producing or Foxp3-expressing cells in the CD4 + T cells in three independent experiments. Lc, Lactobacillus casei ; Bb, Bifidobacterium bifidum ; Sa, Staphylococcus aureus ; Ec, Escherichia coli .
    Brefeldin A, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 15653 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cell stimulation cocktail
    An analysis of the development of regulatory T cells from spleen and Peyer’s patch (PP) cells in response to various bacteria. Spleen and PP cells prepared from ovalbumin-specific T-cell receptor transgenic (OVA-TCR-Tg) mice were primarily cultured with or without (OVA only) various bacteria (10 μg/ml) in the presence of OVA for 6 days in order to induce the development of regulatory T cells. The cultured cells were harvested, re-stimulated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin in the presence of <t>brefeldin</t> A for 4 hr and stained with fluorescein isothiocyanate (FITC)-labelled anti-CD4 and phycoerythrin (PE)-labelled anti-IL-10 or biotinylated anti-transforming growth factor (TGF)-β1 antibodies. Biotinylated antibody was detected using PE-labelled streptavidin. For the analysis of Foxp3-positive cells, the cells after the primary culture were stained with FITC-labelled anti-CD4 and PE-labelled anti-Foxp3 antibodies without re-stimulation. The stained cells were analysed by flow cytometry. The values represent the mean ± standard deviation of the percentages of cytokine-producing or Foxp3-expressing cells in the CD4 + T cells in three independent experiments. Lc, Lactobacillus casei ; Bb, Bifidobacterium bifidum ; Sa, Staphylococcus aureus ; Ec, Escherichia coli .
    Cell Stimulation Cocktail, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1996 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher bca protein assay kit
    An analysis of the development of regulatory T cells from spleen and Peyer’s patch (PP) cells in response to various bacteria. Spleen and PP cells prepared from ovalbumin-specific T-cell receptor transgenic (OVA-TCR-Tg) mice were primarily cultured with or without (OVA only) various bacteria (10 μg/ml) in the presence of OVA for 6 days in order to induce the development of regulatory T cells. The cultured cells were harvested, re-stimulated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin in the presence of <t>brefeldin</t> A for 4 hr and stained with fluorescein isothiocyanate (FITC)-labelled anti-CD4 and phycoerythrin (PE)-labelled anti-IL-10 or biotinylated anti-transforming growth factor (TGF)-β1 antibodies. Biotinylated antibody was detected using PE-labelled streptavidin. For the analysis of Foxp3-positive cells, the cells after the primary culture were stained with FITC-labelled anti-CD4 and PE-labelled anti-Foxp3 antibodies without re-stimulation. The stained cells were analysed by flow cytometry. The values represent the mean ± standard deviation of the percentages of cytokine-producing or Foxp3-expressing cells in the CD4 + T cells in three independent experiments. Lc, Lactobacillus casei ; Bb, Bifidobacterium bifidum ; Sa, Staphylococcus aureus ; Ec, Escherichia coli .
    Bca Protein Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 160639 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc stat3
    <t>STAT3</t> signaling is not disrupted in JNK KO glands. a Immunohistochemistry (left, scale bar = 100 µm) and immunofluorescence (right, scale bar = 30 µm) were performed on sections prepared from #4 mammary glands of single parous female mice on involution day 3 ( n = 5 JNK WT mice and n = 4 JNK KO mice) using an antibody to phospho-STAT3 (p-STAT3) and counter-stained with hematoxylin or DAPI, respectively. An antibody to keratin 8 (K8) was used to label epithelial cells during immunofluorescence staining. Representative images are presented. b K8, p-STAT3, and DAPI fluorescence intensities of involution day 3 glands from JNK WT ( n = 5 mice) and JNK KO ( n = 4 mice) were quantitated and p-STAT3 intensity was normalized to K8 and DAPI fluorescence intensity. No significant differences (unpaired, two-tailed t -test) between JNK WT and JNK KO glands were detected. c Protein extracts prepared from involution day 3 mammary glands were examined by immunoblot analysis by probing with antibodies to p-STAT3, STAT3, and Tubulin. Two representative mice are presented. The quantitative data presented are the mean ± SEM ( n = 9 JNK WT mice and n = 5 JNK KO mice). No significant differences (unpaired, two-tailed t -test) between JNK WT and JNK KO glands were detected. d The mRNA expression of Socs3 measured by RNA-seq analysis is presented as the mean FPKM ± SEM; n = 3 JNK WT mice and n = 3 JNK KO mice per condition. No significant differences between JNK WT and JNK KO glands were detected (calculated by applying the Benjamini–Hochberg method to the p -value)
    Stat3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 16391 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson monensin
    Ligands for CXCR3 and CCR5 are overexpressed in the livers of hepatosplenic subjects. The highest proportions of Tregs among PBMCs were associated with the lowest levels of IFNγ production by PBMCs. A-B) Expression of the ligands for CXCR3 , CCR5 and CCR7 in the liver and spleen of eight hepatosplenic patients (Group 2). Messenger RNA levels are expressed relative to the arithmetic mean values obtained for 11 healthy controls. C ) The proportion of IFNγ + cells among blood CD4 + T cells is negatively correlated ( r = -0.73, p = 0.002) with the proportion of eTregs in the blood in Group 1. The proportion of IFNγ + CD4 + T cells was determined after 6h of stimulation with PMA, ionomycin and <t>monensin.</t> Nonparametric statistical tests were used * p
    Monensin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 8728 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rna
    Ligands for CXCR3 and CCR5 are overexpressed in the livers of hepatosplenic subjects. The highest proportions of Tregs among PBMCs were associated with the lowest levels of IFNγ production by PBMCs. A-B) Expression of the ligands for CXCR3 , CCR5 and CCR7 in the liver and spleen of eight hepatosplenic patients (Group 2). Messenger RNA levels are expressed relative to the arithmetic mean values obtained for 11 healthy controls. C ) The proportion of IFNγ + cells among blood CD4 + T cells is negatively correlated ( r = -0.73, p = 0.002) with the proportion of eTregs in the blood in Group 1. The proportion of IFNγ + CD4 + T cells was determined after 6h of stimulation with PMA, ionomycin and <t>monensin.</t> Nonparametric statistical tests were used * p
    Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 177224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher monensin
    Ligands for CXCR3 and CCR5 are overexpressed in the livers of hepatosplenic subjects. The highest proportions of Tregs among PBMCs were associated with the lowest levels of IFNγ production by PBMCs. A-B) Expression of the ligands for CXCR3 , CCR5 and CCR7 in the liver and spleen of eight hepatosplenic patients (Group 2). Messenger RNA levels are expressed relative to the arithmetic mean values obtained for 11 healthy controls. C ) The proportion of IFNγ + cells among blood CD4 + T cells is negatively correlated ( r = -0.73, p = 0.002) with the proportion of eTregs in the blood in Group 1. The proportion of IFNγ + CD4 + T cells was determined after 6h of stimulation with PMA, ionomycin and <t>monensin.</t> Nonparametric statistical tests were used * p
    Monensin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3983 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson cytometric bead array
    Production of Th2 cytokines in the duodenal mucosa of hookworm infected individuals. Duodenal biopsies from Trial 1, taken from either the duodenum at week 20 post-infection or from directly adjacent to an adult hookworm attachment site (HW site – determined by endoscopy) at week 21 in the hookworm group only, were cultured for 24 h in tissue culture medium at 37°C with 95% O 2 /5% CO 2 . Cell supernatants were removed and levels of IL-4 (A), IL-5 (B) and IL-13 (C) were determined using a <t>Cytometric</t> Bead Array. Data were analysed by Mann-Whitney U test.
    Cytometric Bead Array, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 4672 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monensin
    Production of Th2 cytokines in the duodenal mucosa of hookworm infected individuals. Duodenal biopsies from Trial 1, taken from either the duodenum at week 20 post-infection or from directly adjacent to an adult hookworm attachment site (HW site – determined by endoscopy) at week 21 in the hookworm group only, were cultured for 24 h in tissue culture medium at 37°C with 95% O 2 /5% CO 2 . Cell supernatants were removed and levels of IL-4 (A), IL-5 (B) and IL-13 (C) were determined using a <t>Cytometric</t> Bead Array. Data were analysed by Mann-Whitney U test.
    Monensin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6165 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Regulatory effect of tribulusamide D on the production of IL-6, IL-10 and TNF-α in LPS-stimulated RAW 264.7 cells. Cells were treated with tribulusamide D (25–100 µM) for 1 h prior to LPS (0.5 µg/ml) stimulation for 24

    Journal: Molecular Medicine Reports

    Article Title: Anti-inflammatory effect of tribulusamide D isolated from Tribulus terrestris in lipopolysaccharide-stimulated RAW264.7 macrophages

    doi: 10.3892/mmr.2017.7208

    Figure Lengend Snippet: Regulatory effect of tribulusamide D on the production of IL-6, IL-10 and TNF-α in LPS-stimulated RAW 264.7 cells. Cells were treated with tribulusamide D (25–100 µM) for 1 h prior to LPS (0.5 µg/ml) stimulation for 24

    Article Snippet: RAW264.7 cells were plated at a density of 4×104 cells/well in 96-well plates and treated with tribulusamide D (25–100 µM) for 1 h prior to LPS (0.5 µg/ml) stimulation for 24 h. Levels of IL-6, IL-10 and TNF-α in culture medium were quantified using platinum IL-6 (cat. no. BMS603/2), IL-10 (cat. no. BMS614/2), TNF-α (cat. no. BMS607/3) ELISA kits (eBioscience, Inc., San Diego, CA, USA), and the PGE2 (cat. no. KGE004B) concentration in culture medium was quantified using a competitive enzyme ELISA kit (R & D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer's instructions.

    Techniques:

    CD4 + T cells from sarcoidosis progressors exhibit characteristics of exhaustion. (a, b, and c) Representative functional data for a single healthy control, sarcoidosis progressor, and sarcoidosis resolver. (a) Cytokine secretion; data have been normalized to the mean value for the healthy control cohort (hashed line). (b and c) Proliferation analysis. (a) Apoptosis analysis. (b and c) Percent PD-1 + CD4 + T cells; overlay of naïve (CCR7 + CD45RO − ; shaded) CD4 + cells and total CD4 + T cells (unshaded). (d) Immune activity of sarcoidosis progressors ( n = 12) and resolvers ( n = 5) relative to healthy controls. High = activity greater than one standard deviation above the healthy control mean (yellow). Low = activity greater than one standard deviation below the healthy control mean (magenta). Normal = within one standard deviation of the healthy control mean (grey). White spaces indicate data not present due to lack of available cells.

    Journal: Journal of Immunology Research

    Article Title: Local and Systemic CD4+ T Cell Exhaustion Reverses with Clinical Resolution of Pulmonary Sarcoidosis

    doi: 10.1155/2017/3642832

    Figure Lengend Snippet: CD4 + T cells from sarcoidosis progressors exhibit characteristics of exhaustion. (a, b, and c) Representative functional data for a single healthy control, sarcoidosis progressor, and sarcoidosis resolver. (a) Cytokine secretion; data have been normalized to the mean value for the healthy control cohort (hashed line). (b and c) Proliferation analysis. (a) Apoptosis analysis. (b and c) Percent PD-1 + CD4 + T cells; overlay of naïve (CCR7 + CD45RO − ; shaded) CD4 + cells and total CD4 + T cells (unshaded). (d) Immune activity of sarcoidosis progressors ( n = 12) and resolvers ( n = 5) relative to healthy controls. High = activity greater than one standard deviation above the healthy control mean (yellow). Low = activity greater than one standard deviation below the healthy control mean (magenta). Normal = within one standard deviation of the healthy control mean (grey). White spaces indicate data not present due to lack of available cells.

    Article Snippet: Cytokine Production and Proliferation Assays Cytokine bead array (BD Biosciences, San Jose, CA) was conducted to assay spontaneous and TCR-stimulated cytokine secretion as previously described [ ].

    Techniques: Functional Assay, Activity Assay, Standard Deviation

    Spontaneous and TCR-stimulated cytokine secretion in CD4 + T cells. CD4 + T cells were isolated from the PBMC of healthy control (HC) or sarcoidosis (S) subjects and cultured for 24 hours in the absence (a and b) or presence (c and d) of polyclonal TCR stimulation. Cell culture supernatants were collected 24 hours post TCR stimulation and analyzed for cytokine secretion by cytokine bead array. (a) Spontaneous IL-2 secretion in pg/mL. (b) Spontaneous IFN- γ secretion in pg/mL. (c) TCR-stimulated IL-2 secretion in pg/mL. (d) TCR-stimulated IFN- γ secretion in pg/mL. (e) Spontaneous and TCR-stimulated IL-2 secretion by sarcoidosis CD4 + T cells in pg/mL. (f) Spontaneous and TCR-stimulated IFN- γ secretion by sarcoidosis CD4 + T cells in pg/mL. Horizontal lines represent mean values of cytokine secretion for each cohort. Statistical analysis was performed using the Mann–Whitney U test. ∗ p

    Journal: Journal of Immunology Research

    Article Title: Local and Systemic CD4+ T Cell Exhaustion Reverses with Clinical Resolution of Pulmonary Sarcoidosis

    doi: 10.1155/2017/3642832

    Figure Lengend Snippet: Spontaneous and TCR-stimulated cytokine secretion in CD4 + T cells. CD4 + T cells were isolated from the PBMC of healthy control (HC) or sarcoidosis (S) subjects and cultured for 24 hours in the absence (a and b) or presence (c and d) of polyclonal TCR stimulation. Cell culture supernatants were collected 24 hours post TCR stimulation and analyzed for cytokine secretion by cytokine bead array. (a) Spontaneous IL-2 secretion in pg/mL. (b) Spontaneous IFN- γ secretion in pg/mL. (c) TCR-stimulated IL-2 secretion in pg/mL. (d) TCR-stimulated IFN- γ secretion in pg/mL. (e) Spontaneous and TCR-stimulated IL-2 secretion by sarcoidosis CD4 + T cells in pg/mL. (f) Spontaneous and TCR-stimulated IFN- γ secretion by sarcoidosis CD4 + T cells in pg/mL. Horizontal lines represent mean values of cytokine secretion for each cohort. Statistical analysis was performed using the Mann–Whitney U test. ∗ p

    Article Snippet: Cytokine Production and Proliferation Assays Cytokine bead array (BD Biosciences, San Jose, CA) was conducted to assay spontaneous and TCR-stimulated cytokine secretion as previously described [ ].

    Techniques: Isolation, Cell Culture, MANN-WHITNEY

    Immune dysfunction in sarcoidosis BAL versus PBMC. BAL cells and matching PBMC isolated from sarcoidosis subjects were analyzed according to immune parameters associated with T cell exhaustion. (a, b, c, and d) Intracellular staining for cytokine production: cells were incubated in the absence (a and b) or presence (c and d) of TCR stimulation for 6 hours with Golgi Stop. Cells were then stained with anti-CD3, anti-CD4, and anti-CD8 before permeabilization and staining with PE-conjugated anti-IL-2. Flow cytometry was conducted and live, singlet cells were gated to assess the percentage of CD4 + CD3 + cells that had produced IL-2 or IFN- γ . Unstimulated cells (baseline) were used to help determine the IL-2 production in TCR-stimulated cells. (a and b) Baseline IL-2 and IFN- γ production. (c and d) TCR-stimulated IL-2 and IFN- γ production. (e) The percentage of apoptosing CD4 + T cells in matching BAL and PBMC was assessed using 7AAD and Annexin V staining. (f) PD-1 expression in the population of CD4 + T cells was determined in matching BAL and PBMC. Statistical analysis was performed using the Wilcoxon matched-pairs signed-rank test. ∗ p

    Journal: Journal of Immunology Research

    Article Title: Local and Systemic CD4+ T Cell Exhaustion Reverses with Clinical Resolution of Pulmonary Sarcoidosis

    doi: 10.1155/2017/3642832

    Figure Lengend Snippet: Immune dysfunction in sarcoidosis BAL versus PBMC. BAL cells and matching PBMC isolated from sarcoidosis subjects were analyzed according to immune parameters associated with T cell exhaustion. (a, b, c, and d) Intracellular staining for cytokine production: cells were incubated in the absence (a and b) or presence (c and d) of TCR stimulation for 6 hours with Golgi Stop. Cells were then stained with anti-CD3, anti-CD4, and anti-CD8 before permeabilization and staining with PE-conjugated anti-IL-2. Flow cytometry was conducted and live, singlet cells were gated to assess the percentage of CD4 + CD3 + cells that had produced IL-2 or IFN- γ . Unstimulated cells (baseline) were used to help determine the IL-2 production in TCR-stimulated cells. (a and b) Baseline IL-2 and IFN- γ production. (c and d) TCR-stimulated IL-2 and IFN- γ production. (e) The percentage of apoptosing CD4 + T cells in matching BAL and PBMC was assessed using 7AAD and Annexin V staining. (f) PD-1 expression in the population of CD4 + T cells was determined in matching BAL and PBMC. Statistical analysis was performed using the Wilcoxon matched-pairs signed-rank test. ∗ p

    Article Snippet: Cytokine Production and Proliferation Assays Cytokine bead array (BD Biosciences, San Jose, CA) was conducted to assay spontaneous and TCR-stimulated cytokine secretion as previously described [ ].

    Techniques: Isolation, Staining, Incubation, Flow Cytometry, Cytometry, Produced, Expressing

    Perinatal HI brain injury shifts the peripheral T cell immune response towards pro-inflammatory. Mononuclear cells were isolated from the spleen of pups 7 days after HI injury and the phenotype of T helper cells was assessed by flow cytometry. a Proportion of CD4+ lymphocytes. b Proportion of CD4+IL4+ T cells. c Proportion of CD4+IFN-γ+ T cells. d Proportion of CD4+IL17+ T cells. e Ratio of Th1 cells (IFN-γ+) to Th2 cells (IL4+). f Ratio of Th17 cells (IL-17+) to Th2 cells (IL4+). g Concentration of IL4 protein produced by ionomycin and PMA stimulated spleen cells. h Proportion of regulatory T cells in the spleen. ( n = 7–14 pups per group, * P

    Journal: Journal of Neuroinflammation

    Article Title: Effects of umbilical cord blood cells, and subtypes, to reduce neuroinflammation following perinatal hypoxic-ischemic brain injury

    doi: 10.1186/s12974-018-1089-5

    Figure Lengend Snippet: Perinatal HI brain injury shifts the peripheral T cell immune response towards pro-inflammatory. Mononuclear cells were isolated from the spleen of pups 7 days after HI injury and the phenotype of T helper cells was assessed by flow cytometry. a Proportion of CD4+ lymphocytes. b Proportion of CD4+IL4+ T cells. c Proportion of CD4+IFN-γ+ T cells. d Proportion of CD4+IL17+ T cells. e Ratio of Th1 cells (IFN-γ+) to Th2 cells (IL4+). f Ratio of Th17 cells (IL-17+) to Th2 cells (IL4+). g Concentration of IL4 protein produced by ionomycin and PMA stimulated spleen cells. h Proportion of regulatory T cells in the spleen. ( n = 7–14 pups per group, * P

    Article Snippet: Briefly, using a 24-well plate, 2.5 × 106 splenocytes were cultured for 48 h in complete RPMI medium alone, or media supplemented with 800 ng/ml ionomycin and 20 pg/ml PMA (Sigma-Aldrich), following manufacturer’s instructions.

    Techniques: Isolation, Flow Cytometry, Cytometry, Concentration Assay, Produced

    Immunosuppressive potential of different cell types found in hUCB. a Proliferative response of human PBMCs stimulated with PMA and ionomycin in the presence or absence of hUCB mononuclear cells at different hUCB to PBMC ratios. b Proliferative response of human PBMCs stimulated with PMA and ionomycin in the presence or absence of different cell types found in hUCB added at a ratio of 1:10 of hUCB cells to PBMCs. ( n = 3 PBMC donors with a minimum three pooled hUCB cell donors, all performed in triplicate, ** P

    Journal: Journal of Neuroinflammation

    Article Title: Effects of umbilical cord blood cells, and subtypes, to reduce neuroinflammation following perinatal hypoxic-ischemic brain injury

    doi: 10.1186/s12974-018-1089-5

    Figure Lengend Snippet: Immunosuppressive potential of different cell types found in hUCB. a Proliferative response of human PBMCs stimulated with PMA and ionomycin in the presence or absence of hUCB mononuclear cells at different hUCB to PBMC ratios. b Proliferative response of human PBMCs stimulated with PMA and ionomycin in the presence or absence of different cell types found in hUCB added at a ratio of 1:10 of hUCB cells to PBMCs. ( n = 3 PBMC donors with a minimum three pooled hUCB cell donors, all performed in triplicate, ** P

    Article Snippet: Briefly, using a 24-well plate, 2.5 × 106 splenocytes were cultured for 48 h in complete RPMI medium alone, or media supplemented with 800 ng/ml ionomycin and 20 pg/ml PMA (Sigma-Aldrich), following manufacturer’s instructions.

    Techniques:

    Distribution of T naïve , T CM , T EM subpopulations and IL-17 + /T naïve , IL-17 + /T EM and IL-17 + /T CM , subpopulations of CD4 + T lymphocytes at 1 and 3 month after transplantation compared to before transplantation. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of GolgiStop.CD4 + lymphocytes were stained with mAbs to CD45RA and CCR7, which identified three subsets. In addition, analysis of IL-17 in CD4 + T cell subsets by intracellular flow cytometry was done. After surface staining with CD45 and CCR7 mAbs, cells were fixated and permeabilized and intracellular accumulated cytokines were detected with IL-17 mAbs. (A) T naïve /CD4 + T (CD45RA + CCR7 + /CD4 + Tcells), (B) IL-17 + /T naïve , (C) T CM /CD4 + T (CD45RA − CCR7 + /CD4 + Tcells), (D) IL-17 + /T CM + , (E) T EM /CD4 + T (CD45RA − CCR7 − /CD4 + Tcells), (F) IL-17 + /T EM + . Bars show the means. * P

    Journal: PLoS ONE

    Article Title: Dysregulation of Th17 Cells during the Early Post-Transplant Period in Patients under Calcineurin Inhibitor Based Immunosuppression

    doi: 10.1371/journal.pone.0042011

    Figure Lengend Snippet: Distribution of T naïve , T CM , T EM subpopulations and IL-17 + /T naïve , IL-17 + /T EM and IL-17 + /T CM , subpopulations of CD4 + T lymphocytes at 1 and 3 month after transplantation compared to before transplantation. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of GolgiStop.CD4 + lymphocytes were stained with mAbs to CD45RA and CCR7, which identified three subsets. In addition, analysis of IL-17 in CD4 + T cell subsets by intracellular flow cytometry was done. After surface staining with CD45 and CCR7 mAbs, cells were fixated and permeabilized and intracellular accumulated cytokines were detected with IL-17 mAbs. (A) T naïve /CD4 + T (CD45RA + CCR7 + /CD4 + Tcells), (B) IL-17 + /T naïve , (C) T CM /CD4 + T (CD45RA − CCR7 + /CD4 + Tcells), (D) IL-17 + /T CM + , (E) T EM /CD4 + T (CD45RA − CCR7 − /CD4 + Tcells), (F) IL-17 + /T EM + . Bars show the means. * P

    Article Snippet: For analysis of human intracellular cytokine production, PBMC were stimulated with PMA and ionomycin in the presence of GolgiStop (BD Biosciences, San Diego, CA) for 4 hours.

    Techniques: Transplantation Assay, Ex Vivo, Staining, Flow Cytometry, Cytometry

    Expression of IL-1beta, and HMGB1 associated with Th17 cell at 1 and 3 month after transplantation compared to before transplantation. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of GolgiStop. PBMC from all groups were treated as described in Figure 1 and Materials and Methods . The expression of IL-1beta (A), HMGB1(B) mRNA was measured using real-time PCR. Bars show the means.

    Journal: PLoS ONE

    Article Title: Dysregulation of Th17 Cells during the Early Post-Transplant Period in Patients under Calcineurin Inhibitor Based Immunosuppression

    doi: 10.1371/journal.pone.0042011

    Figure Lengend Snippet: Expression of IL-1beta, and HMGB1 associated with Th17 cell at 1 and 3 month after transplantation compared to before transplantation. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of GolgiStop. PBMC from all groups were treated as described in Figure 1 and Materials and Methods . The expression of IL-1beta (A), HMGB1(B) mRNA was measured using real-time PCR. Bars show the means.

    Article Snippet: For analysis of human intracellular cytokine production, PBMC were stimulated with PMA and ionomycin in the presence of GolgiStop (BD Biosciences, San Diego, CA) for 4 hours.

    Techniques: Expressing, Transplantation Assay, Ex Vivo, Real-time Polymerase Chain Reaction

    Effects of Tac in Th1, Th2, Th17 and Treg subpopulations of CD4+T lymphocytes from the peripheral blood of healthy donors. PBMC were preincubated for 1 h in the presence of Tac and stimulated with 1 µg/ml anti-CD3 and anti-CD28. Flow cytometry of intracellular IFN-r (A), IL-4 (B), Th17 (C) and Treg (D) in CD4+ T cells stimulated in the presence of plate-bound anti-CD3 plus anti-CD28, assessed after 48 h and then stimulated for 4 h with PMA and ionomycin in the presence of GolgiStop. The data are representative of three independent experiments. The values are expressed as the mean ± SEM. * P

    Journal: PLoS ONE

    Article Title: Dysregulation of Th17 Cells during the Early Post-Transplant Period in Patients under Calcineurin Inhibitor Based Immunosuppression

    doi: 10.1371/journal.pone.0042011

    Figure Lengend Snippet: Effects of Tac in Th1, Th2, Th17 and Treg subpopulations of CD4+T lymphocytes from the peripheral blood of healthy donors. PBMC were preincubated for 1 h in the presence of Tac and stimulated with 1 µg/ml anti-CD3 and anti-CD28. Flow cytometry of intracellular IFN-r (A), IL-4 (B), Th17 (C) and Treg (D) in CD4+ T cells stimulated in the presence of plate-bound anti-CD3 plus anti-CD28, assessed after 48 h and then stimulated for 4 h with PMA and ionomycin in the presence of GolgiStop. The data are representative of three independent experiments. The values are expressed as the mean ± SEM. * P

    Article Snippet: For analysis of human intracellular cytokine production, PBMC were stimulated with PMA and ionomycin in the presence of GolgiStop (BD Biosciences, San Diego, CA) for 4 hours.

    Techniques: Flow Cytometry, Cytometry

    Effect of Tac in Th17 subpopulations of CD4+T lymphocytes from the peripheral blood of early-post transplant recipients. We used flow cytometry to examine how Tacrolimus regulates in vitro Th17 subpopulations of CD4+ T lymphocytes in Th17-polarizing condition. PBMC from renal transplant recipients were preincubated for 1 h in the presence of Tacrolimus and stimulated with Th17-polarizing condition for 48 h. Anti-CD3 (1 µg/ml), anti-CD28 (1 µg/ml), IL-1b (20 ng/ml), IL-6 (20 ng/ml) and IL-23 (20 ng/ml) were added to stimulate the differentiation of Th17 cells. Neutralizing antibodies to IFN-gamma (2 µg/ml) and IL-4 (2 µg/ml) were added in some experiments (R D Systems). Flow cytometry of intracellular Th17 in CD4+ T cells stimulated in the presence of Th17-polarizing condition, assessed after 48 h and then stimulated for 4 h with PMA and ionomycin in the presence of GolgiStop. The data are representative of three independent experiments. The values are expressed as the mean ± SEM.

    Journal: PLoS ONE

    Article Title: Dysregulation of Th17 Cells during the Early Post-Transplant Period in Patients under Calcineurin Inhibitor Based Immunosuppression

    doi: 10.1371/journal.pone.0042011

    Figure Lengend Snippet: Effect of Tac in Th17 subpopulations of CD4+T lymphocytes from the peripheral blood of early-post transplant recipients. We used flow cytometry to examine how Tacrolimus regulates in vitro Th17 subpopulations of CD4+ T lymphocytes in Th17-polarizing condition. PBMC from renal transplant recipients were preincubated for 1 h in the presence of Tacrolimus and stimulated with Th17-polarizing condition for 48 h. Anti-CD3 (1 µg/ml), anti-CD28 (1 µg/ml), IL-1b (20 ng/ml), IL-6 (20 ng/ml) and IL-23 (20 ng/ml) were added to stimulate the differentiation of Th17 cells. Neutralizing antibodies to IFN-gamma (2 µg/ml) and IL-4 (2 µg/ml) were added in some experiments (R D Systems). Flow cytometry of intracellular Th17 in CD4+ T cells stimulated in the presence of Th17-polarizing condition, assessed after 48 h and then stimulated for 4 h with PMA and ionomycin in the presence of GolgiStop. The data are representative of three independent experiments. The values are expressed as the mean ± SEM.

    Article Snippet: For analysis of human intracellular cytokine production, PBMC were stimulated with PMA and ionomycin in the presence of GolgiStop (BD Biosciences, San Diego, CA) for 4 hours.

    Techniques: Flow Cytometry, Cytometry, In Vitro

    TCR γδ + T cells were the major IL‐17A‐producing cells in the lungs of M. tuberculosis ‐infected mice. Wild‐type C57BL/6 mice were inoculated i.t. with M. tuberculosis H37Rv or left untreated (A and B). The PIF cells (5 × 10 5 cells) were prepared on day 60, and were cultured with PPD (5 μg/ml) in the presence of naive spleen antigen‐presenting cells (1 × 10 5 cells) for 18 h at 37°C, and with GolgiPlug for the last 6 h. The cells were also stimulated with PMA and ionomycin. After the culture, the cells were surface stained with FITC‐CD3e, PerCP‐Cy5.5, or APC‐conjugated anti‐TCR Cβ and APC‐conjugated TCR Cδ mAbs. Surface‐stained cells were subjected to intercellular cytokine staining with a PE‐conjugated anti‐IL‐17A mAb. The samples were analyzed by FCM (A, naïve; B, infected). Wild‐type C57BL/6, IL‐17A KO, and TCR Cδ KO mice were inoculated i.t. with 1 × 10 3 CFU of M. tuberculosis H37Rv (C), and the CFU in the lungs was determined on day 60 after the infection. The statistical analysis was performed with ANOVA. Asterisks (*) indicate significant difference between two groups.

    Journal: Immunity, Inflammation and Disease

    Article Title: Involvement of IL‐17A‐producing TCR γδ T cells in late protective immunity against pulmonary Mycobacterium tuberculosis infection

    doi: 10.1002/iid3.121

    Figure Lengend Snippet: TCR γδ + T cells were the major IL‐17A‐producing cells in the lungs of M. tuberculosis ‐infected mice. Wild‐type C57BL/6 mice were inoculated i.t. with M. tuberculosis H37Rv or left untreated (A and B). The PIF cells (5 × 10 5 cells) were prepared on day 60, and were cultured with PPD (5 μg/ml) in the presence of naive spleen antigen‐presenting cells (1 × 10 5 cells) for 18 h at 37°C, and with GolgiPlug for the last 6 h. The cells were also stimulated with PMA and ionomycin. After the culture, the cells were surface stained with FITC‐CD3e, PerCP‐Cy5.5, or APC‐conjugated anti‐TCR Cβ and APC‐conjugated TCR Cδ mAbs. Surface‐stained cells were subjected to intercellular cytokine staining with a PE‐conjugated anti‐IL‐17A mAb. The samples were analyzed by FCM (A, naïve; B, infected). Wild‐type C57BL/6, IL‐17A KO, and TCR Cδ KO mice were inoculated i.t. with 1 × 10 3 CFU of M. tuberculosis H37Rv (C), and the CFU in the lungs was determined on day 60 after the infection. The statistical analysis was performed with ANOVA. Asterisks (*) indicate significant difference between two groups.

    Article Snippet: Flow cytometric analysis of intracellular cytokine To analyze the IL‐17A expression of the cells of the in vivo infection system, PIF cells or pulmonary lymphocytes from M. tuberculosis ‐infected mice were incubated with or without 1 μg/ml calcium ionophore A‐23187 (Calbiochem, San Diego, CA) and 25 ng/ml phorbol 12‐myristate 13‐acetate (PMA, Sigma) for 6 h at 37°C and 5% CO2 in the presence of GolgiPlug (BD).

    Techniques: Infection, Mouse Assay, Cell Culture, Staining

    BCG vaccination did not induce Th17 cells in the lungs after M. tuberculosis challenge. Wild‐type C57BL/6 mice were vaccinated with M. bovis BCG 30 or 60 days before M. tuberculosis H37Rv infection. The PIF cells (5 × 10 5 cells) were prepared on the 4th week after the M. tuberculosis infection, and were cultured with PPD (5 μg/ml) in the presence of naive spleen antigen presenting cells (1 × 10 5 cells) for 18 h at 37°C, and with GolgiPlug for the last 6 h. After the culture, the cells were surface stained with FITC‐CD3 and APC‐conjugated anti‐CD4. Surface‐stained cells were subjected to intercellular cytokine staining with a PE‐conjugated anti‐IL‐17A mAb. The samples were analyzed by FCM.

    Journal: Immunity, Inflammation and Disease

    Article Title: Involvement of IL‐17A‐producing TCR γδ T cells in late protective immunity against pulmonary Mycobacterium tuberculosis infection

    doi: 10.1002/iid3.121

    Figure Lengend Snippet: BCG vaccination did not induce Th17 cells in the lungs after M. tuberculosis challenge. Wild‐type C57BL/6 mice were vaccinated with M. bovis BCG 30 or 60 days before M. tuberculosis H37Rv infection. The PIF cells (5 × 10 5 cells) were prepared on the 4th week after the M. tuberculosis infection, and were cultured with PPD (5 μg/ml) in the presence of naive spleen antigen presenting cells (1 × 10 5 cells) for 18 h at 37°C, and with GolgiPlug for the last 6 h. After the culture, the cells were surface stained with FITC‐CD3 and APC‐conjugated anti‐CD4. Surface‐stained cells were subjected to intercellular cytokine staining with a PE‐conjugated anti‐IL‐17A mAb. The samples were analyzed by FCM.

    Article Snippet: Flow cytometric analysis of intracellular cytokine To analyze the IL‐17A expression of the cells of the in vivo infection system, PIF cells or pulmonary lymphocytes from M. tuberculosis ‐infected mice were incubated with or without 1 μg/ml calcium ionophore A‐23187 (Calbiochem, San Diego, CA) and 25 ng/ml phorbol 12‐myristate 13‐acetate (PMA, Sigma) for 6 h at 37°C and 5% CO2 in the presence of GolgiPlug (BD).

    Techniques: Mouse Assay, Infection, Cell Culture, Staining

    Mucosal iNKT secrete pro-inflammatory cytokines. (A) Comparison between pro-inflammatory cytokines (TNF, IFNγ, IL17A, and IL13) produced upon brief polyclonal stimulation by CD161+ and CD161− expressing cells among CD4 + iNKT (left panels) and conventional T cells (right panels) isolated from the LP of HDs (white bars, n = 27), UC patients (blue bars, n = 16), and CD patients (red bars, n = 24). Statistical significance was calculated using the Kruskal–Wallis nonparametric test for multiple comparisons. P

    Journal: Life Science Alliance

    Article Title: Mucosa-associated microbiota drives pathogenic functions in IBD-derived intestinal iNKT cells

    doi: 10.26508/lsa.201800229

    Figure Lengend Snippet: Mucosal iNKT secrete pro-inflammatory cytokines. (A) Comparison between pro-inflammatory cytokines (TNF, IFNγ, IL17A, and IL13) produced upon brief polyclonal stimulation by CD161+ and CD161− expressing cells among CD4 + iNKT (left panels) and conventional T cells (right panels) isolated from the LP of HDs (white bars, n = 27), UC patients (blue bars, n = 16), and CD patients (red bars, n = 24). Statistical significance was calculated using the Kruskal–Wallis nonparametric test for multiple comparisons. P

    Article Snippet: Multiplexing analysis of cytokines in supernatants collected after in vitro stimulation was performed with a cytometric bead array assay, according to the manufacturer’s protocol (BD).

    Techniques: Produced, Expressing, Isolation

    Gating strategy to identify iNKT cells amongst LPMCs and PMBCs. (A) From left, forward and side scatter to identify lymphocytes, CD3/hCD1d:PBS57 tetramer expression. Among CD3+Tet+ or CD3+Tet-cells, expression of CD4 and CD161. On the different subsets, intracellular cytokines (IL-17A, IFNγ, TNF, and IL-13). (B) Dot plots indicate iNKT cells staining with unloaded (left panels) or PBS57-loaded hCD1d tetramer.

    Journal: Life Science Alliance

    Article Title: Mucosa-associated microbiota drives pathogenic functions in IBD-derived intestinal iNKT cells

    doi: 10.26508/lsa.201800229

    Figure Lengend Snippet: Gating strategy to identify iNKT cells amongst LPMCs and PMBCs. (A) From left, forward and side scatter to identify lymphocytes, CD3/hCD1d:PBS57 tetramer expression. Among CD3+Tet+ or CD3+Tet-cells, expression of CD4 and CD161. On the different subsets, intracellular cytokines (IL-17A, IFNγ, TNF, and IL-13). (B) Dot plots indicate iNKT cells staining with unloaded (left panels) or PBS57-loaded hCD1d tetramer.

    Article Snippet: Multiplexing analysis of cytokines in supernatants collected after in vitro stimulation was performed with a cytometric bead array assay, according to the manufacturer’s protocol (BD).

    Techniques: Expressing, Staining

    LP iNKT cells express CD4 and CD161 and secrete pro-inflammatory cytokines. (A, B) Representative dot plots (A) and summary of frequencies (B) of circulating iNKT (hCD1d:PBS57 tetramer + , plain bars) and of conventional T cells (hCD1d:PBS57 tetramer − , dotted bars) among total CD3 + lineage cells in the peripheral blood (PB) of HDs (n = 15) or IBD patients (n = 5). (C–E) Representative dot plots (C), summary of frequencies (D and E, left panels), and absolute numbers (D and E, right panels) of colonic iNKT (D) and of conventional T cells (E) in the LP of uniflamed donors (HDs, white bars, n = 27), UC patients (blue bars, n = 16), and CD patients (red bars, n = 24). (F, G) Representative dot plots (right panels) and cumulative frequencies of CD4/CD161–expressing cells among iNKT (tetramer + , F) and conventional T cells (tetramer − , G) isolated from the LP of HDs (left panels), UC patients (middle panels), and CD patients (right panels). (H, I) Comparison between CD161+ and CD161− expressing cells among CD4 + iNKT (H, tetramer + ) and conventional T cells (I, tetramer − ) isolated from the LP of HDs (white bars, n = 27), UC patients (blue bars, n = 16), and CD patients (red bars, n = 24). Left panels, frequencies; right panels, absolute numbers. Statistical significance was calculated using the Kruskal–Wallis nonparametric test for multiple comparisons. P

    Journal: Life Science Alliance

    Article Title: Mucosa-associated microbiota drives pathogenic functions in IBD-derived intestinal iNKT cells

    doi: 10.26508/lsa.201800229

    Figure Lengend Snippet: LP iNKT cells express CD4 and CD161 and secrete pro-inflammatory cytokines. (A, B) Representative dot plots (A) and summary of frequencies (B) of circulating iNKT (hCD1d:PBS57 tetramer + , plain bars) and of conventional T cells (hCD1d:PBS57 tetramer − , dotted bars) among total CD3 + lineage cells in the peripheral blood (PB) of HDs (n = 15) or IBD patients (n = 5). (C–E) Representative dot plots (C), summary of frequencies (D and E, left panels), and absolute numbers (D and E, right panels) of colonic iNKT (D) and of conventional T cells (E) in the LP of uniflamed donors (HDs, white bars, n = 27), UC patients (blue bars, n = 16), and CD patients (red bars, n = 24). (F, G) Representative dot plots (right panels) and cumulative frequencies of CD4/CD161–expressing cells among iNKT (tetramer + , F) and conventional T cells (tetramer − , G) isolated from the LP of HDs (left panels), UC patients (middle panels), and CD patients (right panels). (H, I) Comparison between CD161+ and CD161− expressing cells among CD4 + iNKT (H, tetramer + ) and conventional T cells (I, tetramer − ) isolated from the LP of HDs (white bars, n = 27), UC patients (blue bars, n = 16), and CD patients (red bars, n = 24). Left panels, frequencies; right panels, absolute numbers. Statistical significance was calculated using the Kruskal–Wallis nonparametric test for multiple comparisons. P

    Article Snippet: Multiplexing analysis of cytokines in supernatants collected after in vitro stimulation was performed with a cytometric bead array assay, according to the manufacturer’s protocol (BD).

    Techniques: Expressing, Isolation

    Pro-inflammatory cytokine secretion by CD4 − Tetramer + and Tetramer − T cells. Frequency of pro-inflammatory cytokines (TNF, IFNg, IL17A, and IL13) produced upon brief polyclonal stimulation by CD4 − CD161+ (left graphs) or CD4 − CD161 − (right graphs) intestinal ex vivo–isolated iNKT cells (tetramer+, upper panels) or conventional T cells (tetramer−, lower panels) from HDs (n = 27), UC patients (n = 16), and CD patients (n = 24).

    Journal: Life Science Alliance

    Article Title: Mucosa-associated microbiota drives pathogenic functions in IBD-derived intestinal iNKT cells

    doi: 10.26508/lsa.201800229

    Figure Lengend Snippet: Pro-inflammatory cytokine secretion by CD4 − Tetramer + and Tetramer − T cells. Frequency of pro-inflammatory cytokines (TNF, IFNg, IL17A, and IL13) produced upon brief polyclonal stimulation by CD4 − CD161+ (left graphs) or CD4 − CD161 − (right graphs) intestinal ex vivo–isolated iNKT cells (tetramer+, upper panels) or conventional T cells (tetramer−, lower panels) from HDs (n = 27), UC patients (n = 16), and CD patients (n = 24).

    Article Snippet: Multiplexing analysis of cytokines in supernatants collected after in vitro stimulation was performed with a cytometric bead array assay, according to the manufacturer’s protocol (BD).

    Techniques: Produced, Ex Vivo, Isolation

    An analysis of the development of regulatory T cells from spleen and Peyer’s patch (PP) cells in response to various bacteria. Spleen and PP cells prepared from ovalbumin-specific T-cell receptor transgenic (OVA-TCR-Tg) mice were primarily cultured with or without (OVA only) various bacteria (10 μg/ml) in the presence of OVA for 6 days in order to induce the development of regulatory T cells. The cultured cells were harvested, re-stimulated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin in the presence of brefeldin A for 4 hr and stained with fluorescein isothiocyanate (FITC)-labelled anti-CD4 and phycoerythrin (PE)-labelled anti-IL-10 or biotinylated anti-transforming growth factor (TGF)-β1 antibodies. Biotinylated antibody was detected using PE-labelled streptavidin. For the analysis of Foxp3-positive cells, the cells after the primary culture were stained with FITC-labelled anti-CD4 and PE-labelled anti-Foxp3 antibodies without re-stimulation. The stained cells were analysed by flow cytometry. The values represent the mean ± standard deviation of the percentages of cytokine-producing or Foxp3-expressing cells in the CD4 + T cells in three independent experiments. Lc, Lactobacillus casei ; Bb, Bifidobacterium bifidum ; Sa, Staphylococcus aureus ; Ec, Escherichia coli .

    Journal: Immunology

    Article Title: Well-controlled proinflammatory cytokine responses of Peyer's patch cells to probiotic Lactobacillus casei

    doi: 10.1111/j.1365-2567.2009.03204.x

    Figure Lengend Snippet: An analysis of the development of regulatory T cells from spleen and Peyer’s patch (PP) cells in response to various bacteria. Spleen and PP cells prepared from ovalbumin-specific T-cell receptor transgenic (OVA-TCR-Tg) mice were primarily cultured with or without (OVA only) various bacteria (10 μg/ml) in the presence of OVA for 6 days in order to induce the development of regulatory T cells. The cultured cells were harvested, re-stimulated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin in the presence of brefeldin A for 4 hr and stained with fluorescein isothiocyanate (FITC)-labelled anti-CD4 and phycoerythrin (PE)-labelled anti-IL-10 or biotinylated anti-transforming growth factor (TGF)-β1 antibodies. Biotinylated antibody was detected using PE-labelled streptavidin. For the analysis of Foxp3-positive cells, the cells after the primary culture were stained with FITC-labelled anti-CD4 and PE-labelled anti-Foxp3 antibodies without re-stimulation. The stained cells were analysed by flow cytometry. The values represent the mean ± standard deviation of the percentages of cytokine-producing or Foxp3-expressing cells in the CD4 + T cells in three independent experiments. Lc, Lactobacillus casei ; Bb, Bifidobacterium bifidum ; Sa, Staphylococcus aureus ; Ec, Escherichia coli .

    Article Snippet: To analyse cytokine-producing effector T-cell development, cultured cells were harvested, washed with RPMI-1640 medium, and stimulated with phorbol 12-myristate 13-acetate and ionomycin in the presence of brefeldin A (Leukocyte Activation Cocktail; BD Pharmingen) for 4 hr.

    Techniques: Transgenic Assay, Mouse Assay, Cell Culture, Staining, Flow Cytometry, Cytometry, Standard Deviation, Expressing

    The importance of interleukin (IL)-12 for the development of T helper type 1 (Th1) cells. (a) Spleen and Peyer’s patch (PP) cells prepared from ovalbumin-specific T-cell receptor transgenic (OVA-TCR-Tg) mice were cultured either with or without (open circle) Lactobacillus casei (closed circle) or Bifidobacterium bifidum (closed triangle, 10 μg/ml) in the presence of OVA. The levels of IL-12 in culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA) on days 1, 3 and 5. All experiments were repeated twice with similar results. (b) Spleen and PP cells prepared from OVA-TCR-Tg mice were primarily cultured with or without (OVA only) L. casei (Lc) or B. bifidum (Bb, 10 μg/ml) in the presence of OVA for 6 days in order to induce the development of Th1 cells. Control rat IgG2a (ContAb) or rat anti-mouse IL-12 (αIL-12) antibody was also added to the primary cultures. The cultured cells were harvested, re-stimulated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin in the presence of brefeldin A for 4 hr, stained with fluorescein isothiocyanate (FITC)-labelled anti-CD4 and phycoerythrin (PE)-labelled anti-interferon (IFN)-γ antibodies, and then analysed by flow cytometry. The values represent the percentages of IFN-γ-producing cells in CD4 + T cells. All experiments were repeated twice with similar results.

    Journal: Immunology

    Article Title: Well-controlled proinflammatory cytokine responses of Peyer's patch cells to probiotic Lactobacillus casei

    doi: 10.1111/j.1365-2567.2009.03204.x

    Figure Lengend Snippet: The importance of interleukin (IL)-12 for the development of T helper type 1 (Th1) cells. (a) Spleen and Peyer’s patch (PP) cells prepared from ovalbumin-specific T-cell receptor transgenic (OVA-TCR-Tg) mice were cultured either with or without (open circle) Lactobacillus casei (closed circle) or Bifidobacterium bifidum (closed triangle, 10 μg/ml) in the presence of OVA. The levels of IL-12 in culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA) on days 1, 3 and 5. All experiments were repeated twice with similar results. (b) Spleen and PP cells prepared from OVA-TCR-Tg mice were primarily cultured with or without (OVA only) L. casei (Lc) or B. bifidum (Bb, 10 μg/ml) in the presence of OVA for 6 days in order to induce the development of Th1 cells. Control rat IgG2a (ContAb) or rat anti-mouse IL-12 (αIL-12) antibody was also added to the primary cultures. The cultured cells were harvested, re-stimulated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin in the presence of brefeldin A for 4 hr, stained with fluorescein isothiocyanate (FITC)-labelled anti-CD4 and phycoerythrin (PE)-labelled anti-interferon (IFN)-γ antibodies, and then analysed by flow cytometry. The values represent the percentages of IFN-γ-producing cells in CD4 + T cells. All experiments were repeated twice with similar results.

    Article Snippet: To analyse cytokine-producing effector T-cell development, cultured cells were harvested, washed with RPMI-1640 medium, and stimulated with phorbol 12-myristate 13-acetate and ionomycin in the presence of brefeldin A (Leukocyte Activation Cocktail; BD Pharmingen) for 4 hr.

    Techniques: Transgenic Assay, Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Cytometry

    An analysis of cytokine-producing T cells developed from spleen and Peyer’s patch (PP) cells in response to various bacteria. Spleen and PP cells prepared from ovalbumin-specific T-cell receptor transgenic (OVA-TCR-Tg) mice were primarily cultured with or without (OVA only) various bacteria (10 μg/ml) in the presence of OVA for 6 days in order to induce the development of effector T helper (Th) cells. The cultured cells and freshly isolated spleen and PP cells were stimulated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin in the presence of brefeldin A for 4 hr, stained with fluorescein isothiocyanate (FITC)-labelled anti-CD4 and phycoerythrin (PE)-labelled anti-interferon (IFN)-γ, interleukin (IL)-4 or IL-17 antibodies, and then analysed by flow cytometry. The values represent the mean ± standard deviation of percentages of cytokine-producing cells in CD4 + T cells in three independent experiments. # P

    Journal: Immunology

    Article Title: Well-controlled proinflammatory cytokine responses of Peyer's patch cells to probiotic Lactobacillus casei

    doi: 10.1111/j.1365-2567.2009.03204.x

    Figure Lengend Snippet: An analysis of cytokine-producing T cells developed from spleen and Peyer’s patch (PP) cells in response to various bacteria. Spleen and PP cells prepared from ovalbumin-specific T-cell receptor transgenic (OVA-TCR-Tg) mice were primarily cultured with or without (OVA only) various bacteria (10 μg/ml) in the presence of OVA for 6 days in order to induce the development of effector T helper (Th) cells. The cultured cells and freshly isolated spleen and PP cells were stimulated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin in the presence of brefeldin A for 4 hr, stained with fluorescein isothiocyanate (FITC)-labelled anti-CD4 and phycoerythrin (PE)-labelled anti-interferon (IFN)-γ, interleukin (IL)-4 or IL-17 antibodies, and then analysed by flow cytometry. The values represent the mean ± standard deviation of percentages of cytokine-producing cells in CD4 + T cells in three independent experiments. # P

    Article Snippet: To analyse cytokine-producing effector T-cell development, cultured cells were harvested, washed with RPMI-1640 medium, and stimulated with phorbol 12-myristate 13-acetate and ionomycin in the presence of brefeldin A (Leukocyte Activation Cocktail; BD Pharmingen) for 4 hr.

    Techniques: Transgenic Assay, Mouse Assay, Cell Culture, Isolation, Staining, Flow Cytometry, Cytometry, Standard Deviation

    STAT3 signaling is not disrupted in JNK KO glands. a Immunohistochemistry (left, scale bar = 100 µm) and immunofluorescence (right, scale bar = 30 µm) were performed on sections prepared from #4 mammary glands of single parous female mice on involution day 3 ( n = 5 JNK WT mice and n = 4 JNK KO mice) using an antibody to phospho-STAT3 (p-STAT3) and counter-stained with hematoxylin or DAPI, respectively. An antibody to keratin 8 (K8) was used to label epithelial cells during immunofluorescence staining. Representative images are presented. b K8, p-STAT3, and DAPI fluorescence intensities of involution day 3 glands from JNK WT ( n = 5 mice) and JNK KO ( n = 4 mice) were quantitated and p-STAT3 intensity was normalized to K8 and DAPI fluorescence intensity. No significant differences (unpaired, two-tailed t -test) between JNK WT and JNK KO glands were detected. c Protein extracts prepared from involution day 3 mammary glands were examined by immunoblot analysis by probing with antibodies to p-STAT3, STAT3, and Tubulin. Two representative mice are presented. The quantitative data presented are the mean ± SEM ( n = 9 JNK WT mice and n = 5 JNK KO mice). No significant differences (unpaired, two-tailed t -test) between JNK WT and JNK KO glands were detected. d The mRNA expression of Socs3 measured by RNA-seq analysis is presented as the mean FPKM ± SEM; n = 3 JNK WT mice and n = 3 JNK KO mice per condition. No significant differences between JNK WT and JNK KO glands were detected (calculated by applying the Benjamini–Hochberg method to the p -value)

    Journal: Cell Death and Differentiation

    Article Title: The cJUN NH2-terminal kinase (JNK) pathway contributes to mouse mammary gland remodeling during involution

    doi: 10.1038/s41418-018-0081-z

    Figure Lengend Snippet: STAT3 signaling is not disrupted in JNK KO glands. a Immunohistochemistry (left, scale bar = 100 µm) and immunofluorescence (right, scale bar = 30 µm) were performed on sections prepared from #4 mammary glands of single parous female mice on involution day 3 ( n = 5 JNK WT mice and n = 4 JNK KO mice) using an antibody to phospho-STAT3 (p-STAT3) and counter-stained with hematoxylin or DAPI, respectively. An antibody to keratin 8 (K8) was used to label epithelial cells during immunofluorescence staining. Representative images are presented. b K8, p-STAT3, and DAPI fluorescence intensities of involution day 3 glands from JNK WT ( n = 5 mice) and JNK KO ( n = 4 mice) were quantitated and p-STAT3 intensity was normalized to K8 and DAPI fluorescence intensity. No significant differences (unpaired, two-tailed t -test) between JNK WT and JNK KO glands were detected. c Protein extracts prepared from involution day 3 mammary glands were examined by immunoblot analysis by probing with antibodies to p-STAT3, STAT3, and Tubulin. Two representative mice are presented. The quantitative data presented are the mean ± SEM ( n = 9 JNK WT mice and n = 5 JNK KO mice). No significant differences (unpaired, two-tailed t -test) between JNK WT and JNK KO glands were detected. d The mRNA expression of Socs3 measured by RNA-seq analysis is presented as the mean FPKM ± SEM; n = 3 JNK WT mice and n = 3 JNK KO mice per condition. No significant differences between JNK WT and JNK KO glands were detected (calculated by applying the Benjamini–Hochberg method to the p -value)

    Article Snippet: Extracts (30 µg) were subjected to immunoblot analysis with antibodies to STAT3 (Cell Signaling Technology Cat# 9139 RRID:AB_331757; dilution 1:1000), phospho-STAT3 (Cell Signaling Technology, Cat# 9145 RRID:AB_2491009; dilution 1:2000), and αTubulin (Sigma-Aldrich Cat# T5168; RRID:AB_477579).

    Techniques: Immunohistochemistry, Immunofluorescence, Mouse Assay, Staining, Fluorescence, Two Tailed Test, Expressing, RNA Sequencing Assay

    Ligands for CXCR3 and CCR5 are overexpressed in the livers of hepatosplenic subjects. The highest proportions of Tregs among PBMCs were associated with the lowest levels of IFNγ production by PBMCs. A-B) Expression of the ligands for CXCR3 , CCR5 and CCR7 in the liver and spleen of eight hepatosplenic patients (Group 2). Messenger RNA levels are expressed relative to the arithmetic mean values obtained for 11 healthy controls. C ) The proportion of IFNγ + cells among blood CD4 + T cells is negatively correlated ( r = -0.73, p = 0.002) with the proportion of eTregs in the blood in Group 1. The proportion of IFNγ + CD4 + T cells was determined after 6h of stimulation with PMA, ionomycin and monensin. Nonparametric statistical tests were used * p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: FOXP3+ Regulatory T Cells in Hepatic Fibrosis and Splenomegaly Caused by Schistosoma japonicum: The Spleen May Be a Major Source of Tregs in Subjects with Splenomegaly

    doi: 10.1371/journal.pntd.0004306

    Figure Lengend Snippet: Ligands for CXCR3 and CCR5 are overexpressed in the livers of hepatosplenic subjects. The highest proportions of Tregs among PBMCs were associated with the lowest levels of IFNγ production by PBMCs. A-B) Expression of the ligands for CXCR3 , CCR5 and CCR7 in the liver and spleen of eight hepatosplenic patients (Group 2). Messenger RNA levels are expressed relative to the arithmetic mean values obtained for 11 healthy controls. C ) The proportion of IFNγ + cells among blood CD4 + T cells is negatively correlated ( r = -0.73, p = 0.002) with the proportion of eTregs in the blood in Group 1. The proportion of IFNγ + CD4 + T cells was determined after 6h of stimulation with PMA, ionomycin and monensin. Nonparametric statistical tests were used * p

    Article Snippet: They were incubated with 100 ng/ml PMA, 1 μg/ml ionomycin and monensin (BD GolgiStop) for 6 hours at 37°C before intracellular cytokine labeling.

    Techniques: Expressing

    Production of Th2 cytokines in the duodenal mucosa of hookworm infected individuals. Duodenal biopsies from Trial 1, taken from either the duodenum at week 20 post-infection or from directly adjacent to an adult hookworm attachment site (HW site – determined by endoscopy) at week 21 in the hookworm group only, were cultured for 24 h in tissue culture medium at 37°C with 95% O 2 /5% CO 2 . Cell supernatants were removed and levels of IL-4 (A), IL-5 (B) and IL-13 (C) were determined using a Cytometric Bead Array. Data were analysed by Mann-Whitney U test.

    Journal: PLoS Pathogens

    Article Title: Characterising the Mucosal and Systemic Immune Responses to Experimental Human Hookworm Infection

    doi: 10.1371/journal.ppat.1002520

    Figure Lengend Snippet: Production of Th2 cytokines in the duodenal mucosa of hookworm infected individuals. Duodenal biopsies from Trial 1, taken from either the duodenum at week 20 post-infection or from directly adjacent to an adult hookworm attachment site (HW site – determined by endoscopy) at week 21 in the hookworm group only, were cultured for 24 h in tissue culture medium at 37°C with 95% O 2 /5% CO 2 . Cell supernatants were removed and levels of IL-4 (A), IL-5 (B) and IL-13 (C) were determined using a Cytometric Bead Array. Data were analysed by Mann-Whitney U test.

    Article Snippet: Cell-free supernatants were collected and analysed using a Cytometric Bead Array (CBA; BD Biosciences).

    Techniques: Infection, Cell Culture, MANN-WHITNEY

    Systemic production of hookworm-specific cytokines. Peripheral blood mononuclear cells were harvested from subjects in Trial 1 and cultured for 120 h at 37°C in either tissue culture medium (MED) or MED containing 10 µg/ml Necator americanus ES products (NaES). Cell supernatants were removed and levels of IL-4 (A), IL-5 (B) and IL-13 (C) determined using a Cytometric Bead Array. Cytokine levels from PBMCs stimulated with just MED alone were subtracted from those stimulated with NaES. Data were analysed by Kruskal-Wallis non-parametric ANOVA, comparing time points at weeks 4, 12 and 20 to week 0 within each group.

    Journal: PLoS Pathogens

    Article Title: Characterising the Mucosal and Systemic Immune Responses to Experimental Human Hookworm Infection

    doi: 10.1371/journal.ppat.1002520

    Figure Lengend Snippet: Systemic production of hookworm-specific cytokines. Peripheral blood mononuclear cells were harvested from subjects in Trial 1 and cultured for 120 h at 37°C in either tissue culture medium (MED) or MED containing 10 µg/ml Necator americanus ES products (NaES). Cell supernatants were removed and levels of IL-4 (A), IL-5 (B) and IL-13 (C) determined using a Cytometric Bead Array. Cytokine levels from PBMCs stimulated with just MED alone were subtracted from those stimulated with NaES. Data were analysed by Kruskal-Wallis non-parametric ANOVA, comparing time points at weeks 4, 12 and 20 to week 0 within each group.

    Article Snippet: Cell-free supernatants were collected and analysed using a Cytometric Bead Array (CBA; BD Biosciences).

    Techniques: Cell Culture

    Systemic and mucosal hookworm specific immune responses. Peripheral blood mononuclear cells (PBMCs) from Trial 2 were cultured for 120 h with either tissue culture medium (Med) or Med containing 10 µg/ml Necator americanus ES products (NaES). Duodenal biopsies were also taken at week 20 post-infection, and cultured for 24 h at 37°C, 95% O2/5% CO 2 , in either Med alone or 10 µg/ml NaES in Med. Cell-free supernatants were taken from both PBMCs and biopsy cultures and levels of soluble cytokines were determined by cytometric bead array. RNA was also prepared from biopsies after stimulation and quantitative real time RT-PCR was used to determine levels of cytokine gene transcripts. Soluble cytokines released from PBMCs are shown in panels A, D, G, J and M. Biopsy-derived soluble cytokines are shown in panels B, E, H, K and N. Biopsy cytokine transcripts are shown in panels C, F, I, L and O. Cytokine levels determined were IL-4 (A–C), IL-5 (D–F), IL-13 (G–I), IL-9 (J–L) and IL-10 (M–O).

    Journal: PLoS Pathogens

    Article Title: Characterising the Mucosal and Systemic Immune Responses to Experimental Human Hookworm Infection

    doi: 10.1371/journal.ppat.1002520

    Figure Lengend Snippet: Systemic and mucosal hookworm specific immune responses. Peripheral blood mononuclear cells (PBMCs) from Trial 2 were cultured for 120 h with either tissue culture medium (Med) or Med containing 10 µg/ml Necator americanus ES products (NaES). Duodenal biopsies were also taken at week 20 post-infection, and cultured for 24 h at 37°C, 95% O2/5% CO 2 , in either Med alone or 10 µg/ml NaES in Med. Cell-free supernatants were taken from both PBMCs and biopsy cultures and levels of soluble cytokines were determined by cytometric bead array. RNA was also prepared from biopsies after stimulation and quantitative real time RT-PCR was used to determine levels of cytokine gene transcripts. Soluble cytokines released from PBMCs are shown in panels A, D, G, J and M. Biopsy-derived soluble cytokines are shown in panels B, E, H, K and N. Biopsy cytokine transcripts are shown in panels C, F, I, L and O. Cytokine levels determined were IL-4 (A–C), IL-5 (D–F), IL-13 (G–I), IL-9 (J–L) and IL-10 (M–O).

    Article Snippet: Cell-free supernatants were collected and analysed using a Cytometric Bead Array (CBA; BD Biosciences).

    Techniques: Cell Culture, Infection, Quantitative RT-PCR, Derivative Assay

    Systemic and mucosal hookworm specific immune responses. Peripheral blood mononuclear cells (PBMCs) from Trial 2 were cultured for 120 h with either tissue culture medium (Med) or Med containing 10 µg/ml Necator americanus ES products (NaES). Duodenal biopsies were also taken at week 20 post-infection, and cultured for 24 h at 37°C, 95% O2/5% CO2, in either Med alone or 10 µg/ml NaES in Med. Cell-free supernatants were taken from both PBMCs and biopsy cultures and levels of soluble cytokines determined by Cytometric Bead Array. RNA was also prepared from biopsies after stimulation and quantitative real time RT-PCR was used to determine levels of cytokine gene transcripts. Soluble cytokines released from PBMCs are shown in panels A, D and G. Biopsy-derived soluble cytokines are shown in panels B, E and H. Biopsy cytokine transcripts are shown in panels C, F, I, J and K. Cytokine levels determined were IL-2 (A–C), IFN-γ (D–F), IL-17A (G–I), IL-15 (J), TGF-β (K) and IL-22 (L).

    Journal: PLoS Pathogens

    Article Title: Characterising the Mucosal and Systemic Immune Responses to Experimental Human Hookworm Infection

    doi: 10.1371/journal.ppat.1002520

    Figure Lengend Snippet: Systemic and mucosal hookworm specific immune responses. Peripheral blood mononuclear cells (PBMCs) from Trial 2 were cultured for 120 h with either tissue culture medium (Med) or Med containing 10 µg/ml Necator americanus ES products (NaES). Duodenal biopsies were also taken at week 20 post-infection, and cultured for 24 h at 37°C, 95% O2/5% CO2, in either Med alone or 10 µg/ml NaES in Med. Cell-free supernatants were taken from both PBMCs and biopsy cultures and levels of soluble cytokines determined by Cytometric Bead Array. RNA was also prepared from biopsies after stimulation and quantitative real time RT-PCR was used to determine levels of cytokine gene transcripts. Soluble cytokines released from PBMCs are shown in panels A, D and G. Biopsy-derived soluble cytokines are shown in panels B, E and H. Biopsy cytokine transcripts are shown in panels C, F, I, J and K. Cytokine levels determined were IL-2 (A–C), IFN-γ (D–F), IL-17A (G–I), IL-15 (J), TGF-β (K) and IL-22 (L).

    Article Snippet: Cell-free supernatants were collected and analysed using a Cytometric Bead Array (CBA; BD Biosciences).

    Techniques: Cell Culture, Infection, Quantitative RT-PCR, Derivative Assay