human il-4 Search Results


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  • 90
    RayBiotech raybio human il 4
    Raybio Human Il 4, supplied by RayBiotech, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher human il 4
    Mouse CCL8–responsive T H 2-R2A cells are enriched for IL-5, IL-25R, TNF and OX40. ( a ) Flow cytometry of <t>IL-4</t> and IL-5 by ICS in T H 2-R1, T H 2-R2, and T H 2-R2A cells; representative of six experiments. Below each plot is the chemotactic index of the
    Human Il 4, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cellgenix human il 4
    Dendritic cell (DC) manufacturing. (A) Flow chart of the standard operating procedure (SOP) for the manufacturing of a cancer vaccine. From patients undergoing tumour surgery a piece of tumour tissue is delivered to the manufacturing facility. The tumour tissue is disrupted mechanically, and the tumour cells are lysed to enrich soluble protein containing tumour antigens. After recovery from surgery, leucocyte apheresis is performed to collect peripheral blood mononuclear cells (PBMCs) from the patients, the monocytes are enriched and cultivated for 6 days in the presence of interleukin <t>(IL)-4</t> and granulocyte-macrophage colony-stimulation factor (GM-CSF) in order to obtain iDCs. The iDCs are charged with tumour antigens, exposed to LPS/IFN-γ to trigger maturation and cryopreserved until treatment. An aliquot of the DC cancer vaccine is subjected to quality control, a potency assay and sterility control. If all criteria are met the DC cancer vaccine is released for treatment. ( B ) Flow chart of DC manufacturing using different monocyte enrichment protocols. Monocytes are isolated from PBMCs of a healthy donor or cancer patient using leucocyte apheresis. The PBMCs are further subjected to monocyte enrichment using adherence, or semi-automated elutriation, CD14 selection, or CD2/19 depletion. The differentiation into DCs is done in the same way independently of the enrichment procedure used (see Fig. 1A ).
    Human Il 4, supplied by Cellgenix, used in various techniques. Bioz Stars score: 92/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Schering-Plough human il 4
    Receptor dimerization-independent activation of Stat6 in PV-treated L929 cells. L929 cells (lacking the γc chain) and NIH 3T3 cells (positive control) were treated with murine <t>IL-4</t> (20 ng/ml) or PV (200 μM) for 30 min or left untreated. WCE derived from these cells were subjected to EMSA using the N 6 -GAS probe.
    Human Il 4, supplied by Schering-Plough, used in various techniques. Bioz Stars score: 90/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Miltenyi Biotec human il 4
    Effect of HspX and ESAT-6 on BCG-induced cytokine secretion by DCs. Monocytes were treated (5 days) with 50 ng/ml GM-CSF and 20 ng/ml <t>IL-4</t> to obtain immature DCs, that were subsequently cultured (24 hrs) in the absence (CTRL) or presence of 50 µg/ml BCG, alone or combined with 10 µg/ml HspX and/or 10 µg/ml ESAT-6. DCs were also cultured with 50 µg/ml Mtb as a positive control. Release of the indicated cytokines in culture supernatants was evaluated by ELISA. Results are expressed as the mean value+SD of seven independent experiments. Statistical analysis: DCs treated with BCG alone vs BCG plus HspX and ESAT-6 added alone or in combination; ns P > 0.05, *P
    Human Il 4, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 92/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pharmingen human il 4
    TARC-responsive cells are Th2. ( A ) A polyclonal Th2-polarized cell line was loaded with Indo-1 and challenged with 100 ng/ml TARC. Fluxing and nonfluxing cells were sorted over a 1-min period. Unsorted ( B ), and sorted fluxing ( C ) and nonfluxing ( D ) T cells were stimulated with PMA + ionomycin for 4 h and IFN-γ and <t>IL-4</t> were measured by intracellular staining.
    Human Il 4, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GENTAUR human il 4
    TARC-responsive cells are Th2. ( A ) A polyclonal Th2-polarized cell line was loaded with Indo-1 and challenged with 100 ng/ml TARC. Fluxing and nonfluxing cells were sorted over a 1-min period. Unsorted ( B ), and sorted fluxing ( C ) and nonfluxing ( D ) T cells were stimulated with PMA + ionomycin for 4 h and IFN-γ and <t>IL-4</t> were measured by intracellular staining.
    Human Il 4, supplied by GENTAUR, used in various techniques. Bioz Stars score: 90/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bender MedSystems human il 4
    TARC-responsive cells are Th2. ( A ) A polyclonal Th2-polarized cell line was loaded with Indo-1 and challenged with 100 ng/ml TARC. Fluxing and nonfluxing cells were sorted over a 1-min period. Unsorted ( B ), and sorted fluxing ( C ) and nonfluxing ( D ) T cells were stimulated with PMA + ionomycin for 4 h and IFN-γ and <t>IL-4</t> were measured by intracellular staining.
    Human Il 4, supplied by Bender MedSystems, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Shenandoah Biotechnology human il 4
    TARC-responsive cells are Th2. ( A ) A polyclonal Th2-polarized cell line was loaded with Indo-1 and challenged with 100 ng/ml TARC. Fluxing and nonfluxing cells were sorted over a 1-min period. Unsorted ( B ), and sorted fluxing ( C ) and nonfluxing ( D ) T cells were stimulated with PMA + ionomycin for 4 h and IFN-γ and <t>IL-4</t> were measured by intracellular staining.
    Human Il 4, supplied by Shenandoah Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson human il 4
    The novel glycolipid RK is recognized by human natural killer T (NKT) cells. (A) Staining of an NKT-iPSC line with RK- or galactosylceramide (GC)-loaded human CD1d dimers. Flow cytometry plots are representative from triplicate samples per group. Numbers indicate the percentage of human CD1d dimer + CD3 + cells within the 7-AAD − viable lymphocyte gates. (B) The mean fluorescence intensity (MFI) levels of human CD1d dimers loaded with RK- or GC, which were gated as shown in panel (A) . Data are mean ± SEM from triplicate samples per group. Experiments were repeated three times with similar results. (C) Human NKT cell expansion upon culturing with RK. Umbilical cord blood mononuclear cells were cultured in the presence or absence of the indicated glycolipids (100 ng/mL) for 19 days in complete media supplemented with 100 U/mL hIL-2. The cultures were re-stimulated with fresh glycolipid-pulsed antigen-presenting cells on culture day 9. Numbers on flow cytometry plots show frequencies (mean ± SEM, n = 3 samples per group) of Vα24 + CD3 + human NKT cells within gated viable lymphocytes. (D) Absolute NKT cell numbers (mean ± SEM, n = 3 samples per group) of Vα24 + CD3 + human NKT cells as shown in panel (C) . Experiments were repeated with two different donors with similar results. (E) IFN-γ and <t>IL-4</t> release from human NKT cells activated with RK-pulsed dendritic cells (DCs). Human NKT cells (5 × 10 4 per well) were co-cultured for 48 h with the same numbers of peripheral blood monocyte-derived DCs that were pulsed overnight with RK or GC at 100 ng/mL. IFN-γ and IL-4 levels in culture supernatants were measured with ELISA or cytometric bead array, respectively. Data are mean ± SEM from triplicate wells. Experiments were repeated three times with similar results. ** P
    Human Il 4, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ImmunoTools human il 4
    Coculture with breast cancer cells increases miR-375 in human MΦ. a – l Human PBMC-derived MΦ were used. a Heatmap of differentially expressed miRs from control and MΦ cocultured with MCF-7 cells ( n = 4). b Representative differentially expressed miRs in control MΦ vs. cocultured MΦ and c MA -plot. d MiR-375 abundance was measured in control, 48 h cocultured, polarized (LPS + IFNγ for 24 h, <t>IL-4</t> for 48 h) and resolution-phase (resolvin D1 for 6 h) MΦ via qPCR, and normalized to untreated MΦ ( n ≥ 3). e Primary human MΦ were treated for 3 h with actinomycin D (Act D) or a DMSO control. Cells were washed and cocultured with MCF-7 cells for 24 h. MiR-375 abundance was quantified via qPCR and normalized to MΦ control ( n = 8). f PPARgamma mRNA expression was measured as a positive control ( n ≥ 5). g – j MΦ were transfected with nonspecific control (ns siRNA) or DICER siRNA for 24 h and cocultured with MCF-7 cells for another 48 h ( n = 5–6). g DICER mRNA expression in MΦ. h Endogenous miR-21-5p and miR-142-3p were measured by qPCR as a control. i MiR-375 abundance and j pre-miR-375 expression were measured by qPCR in control and cocultured MΦ. k MΦ were cocultured with indicated cell lines for 24 h. MiR-375 levels were measured by qPCR and normalized to MΦ control ( n ≥ 5). l MΦ were cocultured with MCF-7 control (empty vector transfected) or decoy (miR-375 decoy transfected) cells for 24 h. MiR-375 expression was measured by qPCR and normalized to MΦ control ( n = 27) using different MCF-7 cell passages. Data of b and d – I are mean ± SEM and p -values were calculated using two-tailed Student’s t -test ( d , f , g – k ) and one-sample t -test ( e , l ). * p
    Human Il 4, supplied by ImmunoTools, used in various techniques. Bioz Stars score: 92/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems human anti il4
    Coculture with breast cancer cells increases miR-375 in human MΦ. a – l Human PBMC-derived MΦ were used. a Heatmap of differentially expressed miRs from control and MΦ cocultured with MCF-7 cells ( n = 4). b Representative differentially expressed miRs in control MΦ vs. cocultured MΦ and c MA -plot. d MiR-375 abundance was measured in control, 48 h cocultured, polarized (LPS + IFNγ for 24 h, <t>IL-4</t> for 48 h) and resolution-phase (resolvin D1 for 6 h) MΦ via qPCR, and normalized to untreated MΦ ( n ≥ 3). e Primary human MΦ were treated for 3 h with actinomycin D (Act D) or a DMSO control. Cells were washed and cocultured with MCF-7 cells for 24 h. MiR-375 abundance was quantified via qPCR and normalized to MΦ control ( n = 8). f PPARgamma mRNA expression was measured as a positive control ( n ≥ 5). g – j MΦ were transfected with nonspecific control (ns siRNA) or DICER siRNA for 24 h and cocultured with MCF-7 cells for another 48 h ( n = 5–6). g DICER mRNA expression in MΦ. h Endogenous miR-21-5p and miR-142-3p were measured by qPCR as a control. i MiR-375 abundance and j pre-miR-375 expression were measured by qPCR in control and cocultured MΦ. k MΦ were cocultured with indicated cell lines for 24 h. MiR-375 levels were measured by qPCR and normalized to MΦ control ( n ≥ 5). l MΦ were cocultured with MCF-7 control (empty vector transfected) or decoy (miR-375 decoy transfected) cells for 24 h. MiR-375 expression was measured by qPCR and normalized to MΦ control ( n = 27) using different MCF-7 cell passages. Data of b and d – I are mean ± SEM and p -values were calculated using two-tailed Student’s t -test ( d , f , g – k ) and one-sample t -test ( e , l ). * p
    Human Anti Il4, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems human il 4
    The relative activity of complete DC-SIGN promoters and those without AP-1 or ETS-1 transcription factor binding factors. The activities of luciferase reporter vectors of different DC-SIGN promoters were detected in 293T cells (part a), Hacat cells (part b) and untreated, PMA or <t>PMA+IL-4-treated</t> THP-1 cells (part c). The abbreviation in the figures: AP-B, pGL-3/Basic/DC-SIGN without AP-1 transcription factor binding factor; AP-E, pGL-3/Enhancer/DC-SIGN without AP-1 transcription factor binding factor; ETS-B, pGL-3/Basic/DC-SIGN without ETS-1 transcription factor binding factor; ETS-E, pGL-3/Enhancer/DC-SIGN without ETS-1 transcription factor binding factor; B, pGL-3/Basic/DC-SIG; E, pGL-3/Enhancer/DC-SIGN; Negative, the internal control plasmid pRL-TK. *means P
    Human Il 4, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 342 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam human il 4 elisa kit
    The proinflammatory cytokines were elevated in ALI patients. The concentrations of cytokines including IL-1β (A) , IL-6 (B) , IL-15 (C) , TNF-α (D) , <t>IL-4</t> (E) , and IL-13 (F) were measured using <t>ELISA</t> kits in serum samples obtained from 24 NSCLC patients (Control) under T0 stage and 24 ALI patients. *** P
    Human Il 4 Elisa Kit, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PeproTech human interleukin 4
    Changes in monocyte function after surgery. Functional properties of the circulating MO were tested using, phagocytosis assays ( a ), ability to stimulate allogeneic T cells ( b ), functional plasticity by incubate cells in vitro with <t>IL-4</t> and GM-CSF and measuring an emergence of DC specific marker ( c ) or ability to induce proliferative T cells response. a There was a significant increase (t 0 : Mean = 10.7 ± 0.05 vs t 3m : Mean = 1.64 ± 0.49 CI 95% : Δx = [0.23, 0.90]; p = 0.003; d = 1.36) in the phagocytic capabilities of peripheral blood monocytes taken from patients at 3 months after the surgery, as shown by uptake of zymosan. b The ability of peripheral blood monocytes to stimulate allogeneic T cells was significantly (t 0 : Mean = 0.65 ± 0.13 vs t 3m : Mean = 0.46 ± 0.31; CI 95% Δx [− 0.33, − 0.04]; p = 0.016; d = − 0.68) diminished at 3 months. c There was a significant decline in the emergence of CD1a (+) (immature DC marker) (t 0 : Me = 1964.58; IQR[745.82, 7586.04] vs t 3m : Me = 519.19; IQR[428.24, 785.52]; CI 95% : ΔpMe[761.49, 5459.51]; p =
    Human Interleukin 4, supplied by PeproTech, used in various techniques. Bioz Stars score: 99/100, based on 160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend human il 4
    α-LacCer suppresses α-GalCer-stimulated cytokine production by competing for CD1d binding. (A,B) Sorted mouse iNKT cells were cultured in the CD1-coated plate pre-loaded with 100 ng/ml α-GalCer or 1 μg/ml α-LacCer with or without the second loading of α-GalCer or α-LacCer for 48 h. The levels of IFN-γ (A) and <t>IL-4</t> (B) in the culture supernatant were assessed ( n = 3). (C,D) Sorted human iNKT cells were cultured in the CD1d-coated plate pre-loaded with α-LacCer (1 or 10 μg/ml) with the second loading of α-GalCer (100 ng/ml) for 48 h. The levels of IFN-γ (C) and IL-4 (D) in the supernatant were assessed ( n = 3–6). Data are representative of three independent experiments and are presented as means ± s.e.m. [ * P
    Human Il 4, supplied by BioLegend, used in various techniques. Bioz Stars score: 94/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher human il 4 us elisa kit
    α-LacCer suppresses α-GalCer-stimulated cytokine production by competing for CD1d binding. (A,B) Sorted mouse iNKT cells were cultured in the CD1-coated plate pre-loaded with 100 ng/ml α-GalCer or 1 μg/ml α-LacCer with or without the second loading of α-GalCer or α-LacCer for 48 h. The levels of IFN-γ (A) and <t>IL-4</t> (B) in the culture supernatant were assessed ( n = 3). (C,D) Sorted human iNKT cells were cultured in the CD1d-coated plate pre-loaded with α-LacCer (1 or 10 μg/ml) with the second loading of α-GalCer (100 ng/ml) for 48 h. The levels of IFN-γ (C) and IL-4 (D) in the supernatant were assessed ( n = 3–6). Data are representative of three independent experiments and are presented as means ± s.e.m. [ * P
    Human Il 4 Us Elisa Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boster Bio human il 4
    Correlation between vitamin D and <t>IL-4</t> in the study group
    Human Il 4, supplied by Boster Bio, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore human il 4
    Na + –K + –ATPase activity in Caco-2 cells treated for 24 h with vehicle, interleukin-12 (IL-12, 500 U ml −1 ), interferon- γ (IFN- γ , 500 U ml −1 ), interleukin-10 (IL-10, 500 U ml −1 ), <t>interleukin-4</t> <t>(IL-4,</t> 500 U ml −1 ), transforming growth factor β (TGF- β , 500 U ml −1 ) or insulin growth factor I (IGF-I, 500 U ml −1 ). Enzyme activity is expressed in nanomoles of Pi per mg of protein per min. Columns represent the mean of five experiments per group; vertical lines show s.e.m. Values are percent of control for Na + –K + –ATPase activity (absolute levels in nmol Pi per mg protein per min were 44.7±1.2, n =5). * Significantly different from control values ( P
    Human Il 4, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend human il 4 elisa max deluxe
    Serum concentrations of IL-2, <t>IL-4,</t> IL-10 and IL-21 were determined by <t>ELISA.</t> Normalized values were presented as the mean ± standard deviation.*P
    Human Il 4 Elisa Max Deluxe, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend human il 4 elisa max standard
    Serum concentrations of IL-2, <t>IL-4,</t> IL-10 and IL-21 were determined by <t>ELISA.</t> Normalized values were presented as the mean ± standard deviation.*P
    Human Il 4 Elisa Max Standard, supplied by BioLegend, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems human il 4 biotinylated antibody
    Serum concentrations of IL-2, <t>IL-4,</t> IL-10 and IL-21 were determined by <t>ELISA.</t> Normalized values were presented as the mean ± standard deviation.*P
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    BioLegend recombinant human il4
    Serum concentrations of IL-2, <t>IL-4,</t> IL-10 and IL-21 were determined by <t>ELISA.</t> Normalized values were presented as the mean ± standard deviation.*P
    Recombinant Human Il4, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Mouse CCL8–responsive T H 2-R2A cells are enriched for IL-5, IL-25R, TNF and OX40. ( a ) Flow cytometry of IL-4 and IL-5 by ICS in T H 2-R1, T H 2-R2, and T H 2-R2A cells; representative of six experiments. Below each plot is the chemotactic index of the

    Journal: Nature immunology

    Article Title: Mouse CCL8, a CCR8 agonist, promotes atopic dermatitis by recruiting IL-5+ TH2 cells

    doi: 10.1038/ni.1984

    Figure Lengend Snippet: Mouse CCL8–responsive T H 2-R2A cells are enriched for IL-5, IL-25R, TNF and OX40. ( a ) Flow cytometry of IL-4 and IL-5 by ICS in T H 2-R1, T H 2-R2, and T H 2-R2A cells; representative of six experiments. Below each plot is the chemotactic index of the

    Article Snippet: Antibodies to CCR4 (205410), CXCR5 (51505), CCR7 (150503) and IL-25R (170220) were from R & D Systems; antibody to T1/ST2 (DJ8) was from MD Biosciences; antibody to CRTh2 (BM16) was from Miltenyi; and FITC-conjugated antibody to human IL-4 (MP4-25D2) and APC-conjugated antibody to IL-5 (TRFK5) were from eBioscience.

    Techniques: Flow Cytometry, Cytometry

    Dendritic cell (DC) manufacturing. (A) Flow chart of the standard operating procedure (SOP) for the manufacturing of a cancer vaccine. From patients undergoing tumour surgery a piece of tumour tissue is delivered to the manufacturing facility. The tumour tissue is disrupted mechanically, and the tumour cells are lysed to enrich soluble protein containing tumour antigens. After recovery from surgery, leucocyte apheresis is performed to collect peripheral blood mononuclear cells (PBMCs) from the patients, the monocytes are enriched and cultivated for 6 days in the presence of interleukin (IL)-4 and granulocyte-macrophage colony-stimulation factor (GM-CSF) in order to obtain iDCs. The iDCs are charged with tumour antigens, exposed to LPS/IFN-γ to trigger maturation and cryopreserved until treatment. An aliquot of the DC cancer vaccine is subjected to quality control, a potency assay and sterility control. If all criteria are met the DC cancer vaccine is released for treatment. ( B ) Flow chart of DC manufacturing using different monocyte enrichment protocols. Monocytes are isolated from PBMCs of a healthy donor or cancer patient using leucocyte apheresis. The PBMCs are further subjected to monocyte enrichment using adherence, or semi-automated elutriation, CD14 selection, or CD2/19 depletion. The differentiation into DCs is done in the same way independently of the enrichment procedure used (see Fig. 1A ).

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Comparative evaluation of techniques for the manufacturing of dendritic cell-based cancer vaccines

    doi: 10.1111/j.1582-4934.2008.00304.x

    Figure Lengend Snippet: Dendritic cell (DC) manufacturing. (A) Flow chart of the standard operating procedure (SOP) for the manufacturing of a cancer vaccine. From patients undergoing tumour surgery a piece of tumour tissue is delivered to the manufacturing facility. The tumour tissue is disrupted mechanically, and the tumour cells are lysed to enrich soluble protein containing tumour antigens. After recovery from surgery, leucocyte apheresis is performed to collect peripheral blood mononuclear cells (PBMCs) from the patients, the monocytes are enriched and cultivated for 6 days in the presence of interleukin (IL)-4 and granulocyte-macrophage colony-stimulation factor (GM-CSF) in order to obtain iDCs. The iDCs are charged with tumour antigens, exposed to LPS/IFN-γ to trigger maturation and cryopreserved until treatment. An aliquot of the DC cancer vaccine is subjected to quality control, a potency assay and sterility control. If all criteria are met the DC cancer vaccine is released for treatment. ( B ) Flow chart of DC manufacturing using different monocyte enrichment protocols. Monocytes are isolated from PBMCs of a healthy donor or cancer patient using leucocyte apheresis. The PBMCs are further subjected to monocyte enrichment using adherence, or semi-automated elutriation, CD14 selection, or CD2/19 depletion. The differentiation into DCs is done in the same way independently of the enrichment procedure used (see Fig. 1A ).

    Article Snippet: Based on optimization and validation experiments, the culture media AIM-V/2%OP or CellGro were supplemented with 1000 U/ml human GM-CSF and 300 U/ml human IL-4 (both from CellGenix Freiburg, Germany) and replaced with the same volume and units of medium plus GM-CSF and IL-4 on day 3.

    Techniques: Flow Cytometry, Potency Assay, Sterility, Isolation, Selection

    Receptor dimerization-independent activation of Stat6 in PV-treated L929 cells. L929 cells (lacking the γc chain) and NIH 3T3 cells (positive control) were treated with murine IL-4 (20 ng/ml) or PV (200 μM) for 30 min or left untreated. WCE derived from these cells were subjected to EMSA using the N 6 -GAS probe.

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

    Article Title: Receptor-associated constitutive protein tyrosine phosphatase activity controls the kinase function of JAK1

    doi:

    Figure Lengend Snippet: Receptor dimerization-independent activation of Stat6 in PV-treated L929 cells. L929 cells (lacking the γc chain) and NIH 3T3 cells (positive control) were treated with murine IL-4 (20 ng/ml) or PV (200 μM) for 30 min or left untreated. WCE derived from these cells were subjected to EMSA using the N 6 -GAS probe.

    Article Snippet: Human IL-4 was obtained from Schering-Plough.

    Techniques: Activation Assay, Positive Control, Derivative Assay

    ( A ) Activation of Stat6 in PV-treated Daudi cells. The cells were treated with 200 μM of PV for 30 min, 20 ng/ml of human IL-4 for 30 min, or were left untreated. WCE was prepared. An N 6 -GAS oligonucleotide, end-labeled with 32 P, was used for EMSA and an unlabeled N 6 -GAS probe was used at a 50-fold molar excess in the competition assay (lane 4), showing the specificity of the DNA-protein complex. A supershift was performed with anti-Stat6 antibody (lane 3) to verify the presence of Stat6 in the DNA-protein complex. ( B ) Stat6 activation by PV requires Jak1 activity. Wild-type and Jak1 mutant Daudi-100K cells were treated with 200 μM of PV for 30 min or left untreated; WCEs prepared were used in EMSA with the N 6 -GAS probe. ( C ) Restoration of Stat6 activation by PV or IL-4 in complemented Jak1 mutant cells. U4A cells lacking functional Jak1 activity and U4A cells complemented with either a functional Jak1 or a kinase-inactive Jak1 were treated with 200 μM of PV or 20 ng/ml of human IL-4 for 30 min or left untreated. WCE and the N 6 -GAS probe were used for EMSA.

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

    Article Title: Receptor-associated constitutive protein tyrosine phosphatase activity controls the kinase function of JAK1

    doi:

    Figure Lengend Snippet: ( A ) Activation of Stat6 in PV-treated Daudi cells. The cells were treated with 200 μM of PV for 30 min, 20 ng/ml of human IL-4 for 30 min, or were left untreated. WCE was prepared. An N 6 -GAS oligonucleotide, end-labeled with 32 P, was used for EMSA and an unlabeled N 6 -GAS probe was used at a 50-fold molar excess in the competition assay (lane 4), showing the specificity of the DNA-protein complex. A supershift was performed with anti-Stat6 antibody (lane 3) to verify the presence of Stat6 in the DNA-protein complex. ( B ) Stat6 activation by PV requires Jak1 activity. Wild-type and Jak1 mutant Daudi-100K cells were treated with 200 μM of PV for 30 min or left untreated; WCEs prepared were used in EMSA with the N 6 -GAS probe. ( C ) Restoration of Stat6 activation by PV or IL-4 in complemented Jak1 mutant cells. U4A cells lacking functional Jak1 activity and U4A cells complemented with either a functional Jak1 or a kinase-inactive Jak1 were treated with 200 μM of PV or 20 ng/ml of human IL-4 for 30 min or left untreated. WCE and the N 6 -GAS probe were used for EMSA.

    Article Snippet: Human IL-4 was obtained from Schering-Plough.

    Techniques: Activation Assay, Labeling, Competitive Binding Assay, Activity Assay, Mutagenesis, Functional Assay

    ( A ) Lack of Stat6 activation in human glioblastoma cell lines. T98G, GRE, and M007 cells were treated with human IL-4 (50 ng/ml) or PV (500 μM) for 30 min or left untreated. WCE prepared from these cells were subjected to EMSA with the N 6 -GAS probe. WCE from PV-treated Daudi cells was used as positive control for activated Stat6. ( B ) Stat6 activation by PV or IL-4 in T98G cells expressing full-length human IL-4Rα protein. Two representative clones of T98G cells that stably express the full-length IL-4Rα (T98G/F1 and T98G/F2) and one stable clone (T98G/T1) expressing a truncated IL-4Rα (the N-terminal 404 amino acids lacking the Stat6 docking sites), all under the simian virus 40 promoter, were treated with human IL-4 (20 ng/ml) or PV (200 μM) for 30 min or left untreated. WCE prepared from these cells were used in EMSA with an N 6 -GAS probe.

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

    Article Title: Receptor-associated constitutive protein tyrosine phosphatase activity controls the kinase function of JAK1

    doi:

    Figure Lengend Snippet: ( A ) Lack of Stat6 activation in human glioblastoma cell lines. T98G, GRE, and M007 cells were treated with human IL-4 (50 ng/ml) or PV (500 μM) for 30 min or left untreated. WCE prepared from these cells were subjected to EMSA with the N 6 -GAS probe. WCE from PV-treated Daudi cells was used as positive control for activated Stat6. ( B ) Stat6 activation by PV or IL-4 in T98G cells expressing full-length human IL-4Rα protein. Two representative clones of T98G cells that stably express the full-length IL-4Rα (T98G/F1 and T98G/F2) and one stable clone (T98G/T1) expressing a truncated IL-4Rα (the N-terminal 404 amino acids lacking the Stat6 docking sites), all under the simian virus 40 promoter, were treated with human IL-4 (20 ng/ml) or PV (200 μM) for 30 min or left untreated. WCE prepared from these cells were used in EMSA with an N 6 -GAS probe.

    Article Snippet: Human IL-4 was obtained from Schering-Plough.

    Techniques: Activation Assay, Positive Control, Expressing, Clone Assay, Stable Transfection

    Effect of HspX and ESAT-6 on BCG-induced cytokine secretion by DCs. Monocytes were treated (5 days) with 50 ng/ml GM-CSF and 20 ng/ml IL-4 to obtain immature DCs, that were subsequently cultured (24 hrs) in the absence (CTRL) or presence of 50 µg/ml BCG, alone or combined with 10 µg/ml HspX and/or 10 µg/ml ESAT-6. DCs were also cultured with 50 µg/ml Mtb as a positive control. Release of the indicated cytokines in culture supernatants was evaluated by ELISA. Results are expressed as the mean value+SD of seven independent experiments. Statistical analysis: DCs treated with BCG alone vs BCG plus HspX and ESAT-6 added alone or in combination; ns P > 0.05, *P

    Journal: PLoS ONE

    Article Title: ESAT-6 and HspX Improve the Effectiveness of BCG to Induce Human Dendritic Cells-Dependent Th1 and NK Cells Activation

    doi: 10.1371/journal.pone.0075684

    Figure Lengend Snippet: Effect of HspX and ESAT-6 on BCG-induced cytokine secretion by DCs. Monocytes were treated (5 days) with 50 ng/ml GM-CSF and 20 ng/ml IL-4 to obtain immature DCs, that were subsequently cultured (24 hrs) in the absence (CTRL) or presence of 50 µg/ml BCG, alone or combined with 10 µg/ml HspX and/or 10 µg/ml ESAT-6. DCs were also cultured with 50 µg/ml Mtb as a positive control. Release of the indicated cytokines in culture supernatants was evaluated by ELISA. Results are expressed as the mean value+SD of seven independent experiments. Statistical analysis: DCs treated with BCG alone vs BCG plus HspX and ESAT-6 added alone or in combination; ns P > 0.05, *P

    Article Snippet: Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and human IL-4 were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

    Techniques: Cell Culture, Positive Control, Enzyme-linked Immunosorbent Assay

    TARC-responsive cells are Th2. ( A ) A polyclonal Th2-polarized cell line was loaded with Indo-1 and challenged with 100 ng/ml TARC. Fluxing and nonfluxing cells were sorted over a 1-min period. Unsorted ( B ), and sorted fluxing ( C ) and nonfluxing ( D ) T cells were stimulated with PMA + ionomycin for 4 h and IFN-γ and IL-4 were measured by intracellular staining.

    Journal: The Journal of Experimental Medicine

    Article Title: Flexible Programs of Chemokine Receptor Expression on Human Polarized T Helper 1 and 2 Lymphocytes

    doi:

    Figure Lengend Snippet: TARC-responsive cells are Th2. ( A ) A polyclonal Th2-polarized cell line was loaded with Indo-1 and challenged with 100 ng/ml TARC. Fluxing and nonfluxing cells were sorted over a 1-min period. Unsorted ( B ), and sorted fluxing ( C ) and nonfluxing ( D ) T cells were stimulated with PMA + ionomycin for 4 h and IFN-γ and IL-4 were measured by intracellular staining.

    Article Snippet: Cord blood T cells were stimulated with PHA (1 μg/ml; Murex Ltd., Dartford, UK) and rIL-2 (40 U/ml) under Th1-polarizing conditions (human rIL-12 [2 ng/ml; Hoffmann–La Roche, Nutley, NJ] plus neutralizing mAb to human IL-4 [200 ng/ml; PharMingen , San Diego, CA]) or Th2-polarizing conditions (human rIL-4 [200 U/ml] plus neutralizing mAb to human IL-12 [2 μg/ml; R & D Sys., Inc., Minneapolis, MN]).

    Techniques: Staining

    Chemokine receptor expression on T cell clones. ( A–C ) IFN-γ and IL-4 production and chemokine receptor expression (both on four-decade logarithmic scale) on three representative Th0, Th1, and Th2 clones; ( D ) CXCR3 expression level and ( E ) percent of CCR5 + cells in Th0, Th1, and Th2 clones. The mean and standard deviation for each group is also shown. No direct correlation was found in the expression of CXCR3 and CCR5. T cell clones were defined according to cytokine profile ( Th0 , IFN-γ and IL-4; Th1 , IFN-γ and no IL-4; Th2 , IL-4 and no IFN-γ).

    Journal: The Journal of Experimental Medicine

    Article Title: Flexible Programs of Chemokine Receptor Expression on Human Polarized T Helper 1 and 2 Lymphocytes

    doi:

    Figure Lengend Snippet: Chemokine receptor expression on T cell clones. ( A–C ) IFN-γ and IL-4 production and chemokine receptor expression (both on four-decade logarithmic scale) on three representative Th0, Th1, and Th2 clones; ( D ) CXCR3 expression level and ( E ) percent of CCR5 + cells in Th0, Th1, and Th2 clones. The mean and standard deviation for each group is also shown. No direct correlation was found in the expression of CXCR3 and CCR5. T cell clones were defined according to cytokine profile ( Th0 , IFN-γ and IL-4; Th1 , IFN-γ and no IL-4; Th2 , IL-4 and no IFN-γ).

    Article Snippet: Cord blood T cells were stimulated with PHA (1 μg/ml; Murex Ltd., Dartford, UK) and rIL-2 (40 U/ml) under Th1-polarizing conditions (human rIL-12 [2 ng/ml; Hoffmann–La Roche, Nutley, NJ] plus neutralizing mAb to human IL-4 [200 ng/ml; PharMingen , San Diego, CA]) or Th2-polarizing conditions (human rIL-4 [200 U/ml] plus neutralizing mAb to human IL-12 [2 μg/ml; R & D Sys., Inc., Minneapolis, MN]).

    Techniques: Expressing, Clone Assay, Standard Deviation

    Acquisition of CXCR3, CCR5, and CCR3 after T cell polarization in vitro. Cord blood T cells were activated for two consecutive cycles under Th1- (IL-12 + α–IL-4) or Th2- (IL-4 + α–IL-12) polarizing conditions. The cells were analyzed for IFN-γ and IL-4 production by intracellular staining (dot plots in a four-decade logarithmic scale) and for expression of chemokine receptors (histograms in a four-decade logarithmic scale) 10 d after the first polarization ( A ) and 10 d after the second polarization ( B ). Comparable results were obtained in three additional experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Flexible Programs of Chemokine Receptor Expression on Human Polarized T Helper 1 and 2 Lymphocytes

    doi:

    Figure Lengend Snippet: Acquisition of CXCR3, CCR5, and CCR3 after T cell polarization in vitro. Cord blood T cells were activated for two consecutive cycles under Th1- (IL-12 + α–IL-4) or Th2- (IL-4 + α–IL-12) polarizing conditions. The cells were analyzed for IFN-γ and IL-4 production by intracellular staining (dot plots in a four-decade logarithmic scale) and for expression of chemokine receptors (histograms in a four-decade logarithmic scale) 10 d after the first polarization ( A ) and 10 d after the second polarization ( B ). Comparable results were obtained in three additional experiments.

    Article Snippet: Cord blood T cells were stimulated with PHA (1 μg/ml; Murex Ltd., Dartford, UK) and rIL-2 (40 U/ml) under Th1-polarizing conditions (human rIL-12 [2 ng/ml; Hoffmann–La Roche, Nutley, NJ] plus neutralizing mAb to human IL-4 [200 ng/ml; PharMingen , San Diego, CA]) or Th2-polarizing conditions (human rIL-4 [200 U/ml] plus neutralizing mAb to human IL-12 [2 μg/ml; R & D Sys., Inc., Minneapolis, MN]).

    Techniques: In Vitro, Staining, Expressing

    Modulation of IFN-γ and IL-4 production and CCR3 and CXCR3 expression in polarized T cell lines by TGF-β and IFN-α. Cord blood T cells were polarized by two consecutive cycles of stimulation in the presence of IL-12 + α-IL-4 (Th1) or IL-4 + α-IL-12 (Th2) alone or together with TGF-β or IFN-α. ( A ) IFN-γ and IL-4 production at the single cell level measured by intracellular staining (four-decade logarithmic scale); ( B ) CCR3 and CXCR3 expression (four-decade logarithmic scale); ( C ) IL-12Rβ2 mRNA expression as determined by Northern blot on total RNA using as probe the full-length human IL-12Rβ2 subunit cDNA. Exposure time was 7 d using an intensifying screen at −70°C. Two major messages were found as described ( 42 ). As a loading control, the blot was stripped and rehybridized for β-actin.

    Journal: The Journal of Experimental Medicine

    Article Title: Flexible Programs of Chemokine Receptor Expression on Human Polarized T Helper 1 and 2 Lymphocytes

    doi:

    Figure Lengend Snippet: Modulation of IFN-γ and IL-4 production and CCR3 and CXCR3 expression in polarized T cell lines by TGF-β and IFN-α. Cord blood T cells were polarized by two consecutive cycles of stimulation in the presence of IL-12 + α-IL-4 (Th1) or IL-4 + α-IL-12 (Th2) alone or together with TGF-β or IFN-α. ( A ) IFN-γ and IL-4 production at the single cell level measured by intracellular staining (four-decade logarithmic scale); ( B ) CCR3 and CXCR3 expression (four-decade logarithmic scale); ( C ) IL-12Rβ2 mRNA expression as determined by Northern blot on total RNA using as probe the full-length human IL-12Rβ2 subunit cDNA. Exposure time was 7 d using an intensifying screen at −70°C. Two major messages were found as described ( 42 ). As a loading control, the blot was stripped and rehybridized for β-actin.

    Article Snippet: Cord blood T cells were stimulated with PHA (1 μg/ml; Murex Ltd., Dartford, UK) and rIL-2 (40 U/ml) under Th1-polarizing conditions (human rIL-12 [2 ng/ml; Hoffmann–La Roche, Nutley, NJ] plus neutralizing mAb to human IL-4 [200 ng/ml; PharMingen , San Diego, CA]) or Th2-polarizing conditions (human rIL-4 [200 U/ml] plus neutralizing mAb to human IL-12 [2 μg/ml; R & D Sys., Inc., Minneapolis, MN]).

    Techniques: Expressing, Staining, Northern Blot

    The novel glycolipid RK is recognized by human natural killer T (NKT) cells. (A) Staining of an NKT-iPSC line with RK- or galactosylceramide (GC)-loaded human CD1d dimers. Flow cytometry plots are representative from triplicate samples per group. Numbers indicate the percentage of human CD1d dimer + CD3 + cells within the 7-AAD − viable lymphocyte gates. (B) The mean fluorescence intensity (MFI) levels of human CD1d dimers loaded with RK- or GC, which were gated as shown in panel (A) . Data are mean ± SEM from triplicate samples per group. Experiments were repeated three times with similar results. (C) Human NKT cell expansion upon culturing with RK. Umbilical cord blood mononuclear cells were cultured in the presence or absence of the indicated glycolipids (100 ng/mL) for 19 days in complete media supplemented with 100 U/mL hIL-2. The cultures were re-stimulated with fresh glycolipid-pulsed antigen-presenting cells on culture day 9. Numbers on flow cytometry plots show frequencies (mean ± SEM, n = 3 samples per group) of Vα24 + CD3 + human NKT cells within gated viable lymphocytes. (D) Absolute NKT cell numbers (mean ± SEM, n = 3 samples per group) of Vα24 + CD3 + human NKT cells as shown in panel (C) . Experiments were repeated with two different donors with similar results. (E) IFN-γ and IL-4 release from human NKT cells activated with RK-pulsed dendritic cells (DCs). Human NKT cells (5 × 10 4 per well) were co-cultured for 48 h with the same numbers of peripheral blood monocyte-derived DCs that were pulsed overnight with RK or GC at 100 ng/mL. IFN-γ and IL-4 levels in culture supernatants were measured with ELISA or cytometric bead array, respectively. Data are mean ± SEM from triplicate wells. Experiments were repeated three times with similar results. ** P

    Journal: Frontiers in Immunology

    Article Title: Natural Killer T Cell-Targeted Immunotherapy Mediating Long-term Memory Responses and Strong Antitumor Activity

    doi: 10.3389/fimmu.2017.01206

    Figure Lengend Snippet: The novel glycolipid RK is recognized by human natural killer T (NKT) cells. (A) Staining of an NKT-iPSC line with RK- or galactosylceramide (GC)-loaded human CD1d dimers. Flow cytometry plots are representative from triplicate samples per group. Numbers indicate the percentage of human CD1d dimer + CD3 + cells within the 7-AAD − viable lymphocyte gates. (B) The mean fluorescence intensity (MFI) levels of human CD1d dimers loaded with RK- or GC, which were gated as shown in panel (A) . Data are mean ± SEM from triplicate samples per group. Experiments were repeated three times with similar results. (C) Human NKT cell expansion upon culturing with RK. Umbilical cord blood mononuclear cells were cultured in the presence or absence of the indicated glycolipids (100 ng/mL) for 19 days in complete media supplemented with 100 U/mL hIL-2. The cultures were re-stimulated with fresh glycolipid-pulsed antigen-presenting cells on culture day 9. Numbers on flow cytometry plots show frequencies (mean ± SEM, n = 3 samples per group) of Vα24 + CD3 + human NKT cells within gated viable lymphocytes. (D) Absolute NKT cell numbers (mean ± SEM, n = 3 samples per group) of Vα24 + CD3 + human NKT cells as shown in panel (C) . Experiments were repeated with two different donors with similar results. (E) IFN-γ and IL-4 release from human NKT cells activated with RK-pulsed dendritic cells (DCs). Human NKT cells (5 × 10 4 per well) were co-cultured for 48 h with the same numbers of peripheral blood monocyte-derived DCs that were pulsed overnight with RK or GC at 100 ng/mL. IFN-γ and IL-4 levels in culture supernatants were measured with ELISA or cytometric bead array, respectively. Data are mean ± SEM from triplicate wells. Experiments were repeated three times with similar results. ** P

    Article Snippet: The levels of mouse IL-12p70, mouse IL-4, and human IL-4 were measured with a cytometric bead array (CBA) (BD Biosciences).

    Techniques: Staining, Flow Cytometry, Cytometry, Fluorescence, Cell Culture, Derivative Assay, Enzyme-linked Immunosorbent Assay

    IFN-γ release from mouse Vα14 + natural killer T (NKT) cells activated with RK or galactosylceramide (GC) glycolipids. (A) NKT cell hybridoma E210 cells were plated 1 × 10 5 cells per well into 96-well culture plates, which were previously coated with soluble CD1d and incubated with RK or GC at the indicated concentrations overnight. Vehicle was used as a control in the NKT cell hybridoma activation assay. The culture supernatants were collected after 16 h and IFN-γ levels were measured by ELISA. Data are mean ± SEM from triplicate wells. The data are representative from two independent experiments with similar results. (B) In vivo antigen presentation assay with RK- or GC-pulsed dendritic cells (DCs). B6 mice were injected intravenously with 5 × 10 5 RK- or GC-pulsed DCs per mouse, and levels of IFN-γ and IL-4 in the sera collected at the indicated time points were measured by ELISA or cytometric bead array, respectively. Data are mean ± SEM from three mice and repeated three times with similar results. ** P

    Journal: Frontiers in Immunology

    Article Title: Natural Killer T Cell-Targeted Immunotherapy Mediating Long-term Memory Responses and Strong Antitumor Activity

    doi: 10.3389/fimmu.2017.01206

    Figure Lengend Snippet: IFN-γ release from mouse Vα14 + natural killer T (NKT) cells activated with RK or galactosylceramide (GC) glycolipids. (A) NKT cell hybridoma E210 cells were plated 1 × 10 5 cells per well into 96-well culture plates, which were previously coated with soluble CD1d and incubated with RK or GC at the indicated concentrations overnight. Vehicle was used as a control in the NKT cell hybridoma activation assay. The culture supernatants were collected after 16 h and IFN-γ levels were measured by ELISA. Data are mean ± SEM from triplicate wells. The data are representative from two independent experiments with similar results. (B) In vivo antigen presentation assay with RK- or GC-pulsed dendritic cells (DCs). B6 mice were injected intravenously with 5 × 10 5 RK- or GC-pulsed DCs per mouse, and levels of IFN-γ and IL-4 in the sera collected at the indicated time points were measured by ELISA or cytometric bead array, respectively. Data are mean ± SEM from three mice and repeated three times with similar results. ** P

    Article Snippet: The levels of mouse IL-12p70, mouse IL-4, and human IL-4 were measured with a cytometric bead array (CBA) (BD Biosciences).

    Techniques: Incubation, Activation Assay, Enzyme-linked Immunosorbent Assay, In Vivo, Mouse Assay, Injection

    Coculture with breast cancer cells increases miR-375 in human MΦ. a – l Human PBMC-derived MΦ were used. a Heatmap of differentially expressed miRs from control and MΦ cocultured with MCF-7 cells ( n = 4). b Representative differentially expressed miRs in control MΦ vs. cocultured MΦ and c MA -plot. d MiR-375 abundance was measured in control, 48 h cocultured, polarized (LPS + IFNγ for 24 h, IL-4 for 48 h) and resolution-phase (resolvin D1 for 6 h) MΦ via qPCR, and normalized to untreated MΦ ( n ≥ 3). e Primary human MΦ were treated for 3 h with actinomycin D (Act D) or a DMSO control. Cells were washed and cocultured with MCF-7 cells for 24 h. MiR-375 abundance was quantified via qPCR and normalized to MΦ control ( n = 8). f PPARgamma mRNA expression was measured as a positive control ( n ≥ 5). g – j MΦ were transfected with nonspecific control (ns siRNA) or DICER siRNA for 24 h and cocultured with MCF-7 cells for another 48 h ( n = 5–6). g DICER mRNA expression in MΦ. h Endogenous miR-21-5p and miR-142-3p were measured by qPCR as a control. i MiR-375 abundance and j pre-miR-375 expression were measured by qPCR in control and cocultured MΦ. k MΦ were cocultured with indicated cell lines for 24 h. MiR-375 levels were measured by qPCR and normalized to MΦ control ( n ≥ 5). l MΦ were cocultured with MCF-7 control (empty vector transfected) or decoy (miR-375 decoy transfected) cells for 24 h. MiR-375 expression was measured by qPCR and normalized to MΦ control ( n = 27) using different MCF-7 cell passages. Data of b and d – I are mean ± SEM and p -values were calculated using two-tailed Student’s t -test ( d , f , g – k ) and one-sample t -test ( e , l ). * p

    Journal: Nature Communications

    Article Title: Apoptotic tumor cell-derived microRNA-375 uses CD36 to alter the tumor-associated macrophage phenotype

    doi: 10.1038/s41467-019-08989-2

    Figure Lengend Snippet: Coculture with breast cancer cells increases miR-375 in human MΦ. a – l Human PBMC-derived MΦ were used. a Heatmap of differentially expressed miRs from control and MΦ cocultured with MCF-7 cells ( n = 4). b Representative differentially expressed miRs in control MΦ vs. cocultured MΦ and c MA -plot. d MiR-375 abundance was measured in control, 48 h cocultured, polarized (LPS + IFNγ for 24 h, IL-4 for 48 h) and resolution-phase (resolvin D1 for 6 h) MΦ via qPCR, and normalized to untreated MΦ ( n ≥ 3). e Primary human MΦ were treated for 3 h with actinomycin D (Act D) or a DMSO control. Cells were washed and cocultured with MCF-7 cells for 24 h. MiR-375 abundance was quantified via qPCR and normalized to MΦ control ( n = 8). f PPARgamma mRNA expression was measured as a positive control ( n ≥ 5). g – j MΦ were transfected with nonspecific control (ns siRNA) or DICER siRNA for 24 h and cocultured with MCF-7 cells for another 48 h ( n = 5–6). g DICER mRNA expression in MΦ. h Endogenous miR-21-5p and miR-142-3p were measured by qPCR as a control. i MiR-375 abundance and j pre-miR-375 expression were measured by qPCR in control and cocultured MΦ. k MΦ were cocultured with indicated cell lines for 24 h. MiR-375 levels were measured by qPCR and normalized to MΦ control ( n ≥ 5). l MΦ were cocultured with MCF-7 control (empty vector transfected) or decoy (miR-375 decoy transfected) cells for 24 h. MiR-375 expression was measured by qPCR and normalized to MΦ control ( n = 27) using different MCF-7 cell passages. Data of b and d – I are mean ± SEM and p -values were calculated using two-tailed Student’s t -test ( d , f , g – k ) and one-sample t -test ( e , l ). * p

    Article Snippet: Human IL-4, IFNγ, the monoclonal CD36-blocking antibody (#21270361; 2 µg/mL), and corresponding IgG control (#21915011; 2 µg/mL) were ordered from ImmunoTools (Friesoythe, Germany).

    Techniques: Derivative Assay, Real-time Polymerase Chain Reaction, Activated Clotting Time Assay, Expressing, Positive Control, Transfection, Plasmid Preparation, Two Tailed Test

    The relative activity of complete DC-SIGN promoters and those without AP-1 or ETS-1 transcription factor binding factors. The activities of luciferase reporter vectors of different DC-SIGN promoters were detected in 293T cells (part a), Hacat cells (part b) and untreated, PMA or PMA+IL-4-treated THP-1 cells (part c). The abbreviation in the figures: AP-B, pGL-3/Basic/DC-SIGN without AP-1 transcription factor binding factor; AP-E, pGL-3/Enhancer/DC-SIGN without AP-1 transcription factor binding factor; ETS-B, pGL-3/Basic/DC-SIGN without ETS-1 transcription factor binding factor; ETS-E, pGL-3/Enhancer/DC-SIGN without ETS-1 transcription factor binding factor; B, pGL-3/Basic/DC-SIG; E, pGL-3/Enhancer/DC-SIGN; Negative, the internal control plasmid pRL-TK. *means P

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Multiple Signaling Pathways Are Involved in the Interleukine-4 Regulated Expression of DC-SIGN in THP-1 Cell Line

    doi: 10.1155/2012/357060

    Figure Lengend Snippet: The relative activity of complete DC-SIGN promoters and those without AP-1 or ETS-1 transcription factor binding factors. The activities of luciferase reporter vectors of different DC-SIGN promoters were detected in 293T cells (part a), Hacat cells (part b) and untreated, PMA or PMA+IL-4-treated THP-1 cells (part c). The abbreviation in the figures: AP-B, pGL-3/Basic/DC-SIGN without AP-1 transcription factor binding factor; AP-E, pGL-3/Enhancer/DC-SIGN without AP-1 transcription factor binding factor; ETS-B, pGL-3/Basic/DC-SIGN without ETS-1 transcription factor binding factor; ETS-E, pGL-3/Enhancer/DC-SIGN without ETS-1 transcription factor binding factor; B, pGL-3/Basic/DC-SIG; E, pGL-3/Enhancer/DC-SIGN; Negative, the internal control plasmid pRL-TK. *means P

    Article Snippet: Cytokines and Antibodies Recombined human IL-4 was obtained from R & D System (Minneapolis, USA) and used at 1000 units/mL.

    Techniques: Activity Assay, Binding Assay, Luciferase, Plasmid Preparation

    Inhibition of DC-SIGN expression by specific inhibitors of signaling pathways. DC-SIGN expression was detected on THP-1 cells differentiated by PMA, PMA plus IL-4, or PMA plus IL-4 treated with specific inhibitors (Helenalin, AG490, SB202190, and PD98059). (a), The relative levels of DC-SIGN mRNA detected by real-time PCR. The levels of DC-SIGN mRNA in PMA plus IL-4 treated THP-1 cells were valued as 100%, and used as a reference. *means P

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Multiple Signaling Pathways Are Involved in the Interleukine-4 Regulated Expression of DC-SIGN in THP-1 Cell Line

    doi: 10.1155/2012/357060

    Figure Lengend Snippet: Inhibition of DC-SIGN expression by specific inhibitors of signaling pathways. DC-SIGN expression was detected on THP-1 cells differentiated by PMA, PMA plus IL-4, or PMA plus IL-4 treated with specific inhibitors (Helenalin, AG490, SB202190, and PD98059). (a), The relative levels of DC-SIGN mRNA detected by real-time PCR. The levels of DC-SIGN mRNA in PMA plus IL-4 treated THP-1 cells were valued as 100%, and used as a reference. *means P

    Article Snippet: Cytokines and Antibodies Recombined human IL-4 was obtained from R & D System (Minneapolis, USA) and used at 1000 units/mL.

    Techniques: Inhibition, Expressing, Real-time Polymerase Chain Reaction

    Phosphorylation of kinase and factors over time in the signaling pathways within 120 min in IL-4-induced THP-1 cells. (a), the cytoplasmic levels of protein kinase and phosphorylated kinase in the signaling pathways. (b), the concentration of phosphorylated protein kinase in the nucleus over time. Protein kinase with “p” means phosphorylated protein kinase.

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Multiple Signaling Pathways Are Involved in the Interleukine-4 Regulated Expression of DC-SIGN in THP-1 Cell Line

    doi: 10.1155/2012/357060

    Figure Lengend Snippet: Phosphorylation of kinase and factors over time in the signaling pathways within 120 min in IL-4-induced THP-1 cells. (a), the cytoplasmic levels of protein kinase and phosphorylated kinase in the signaling pathways. (b), the concentration of phosphorylated protein kinase in the nucleus over time. Protein kinase with “p” means phosphorylated protein kinase.

    Article Snippet: Cytokines and Antibodies Recombined human IL-4 was obtained from R & D System (Minneapolis, USA) and used at 1000 units/mL.

    Techniques: Concentration Assay

    IL-4-induced high expression of DC-SIGN on THP-1 cells over time. THP-1 cells were treated with or without PMA for 24 hours, or with PMA for 24 hours and IL-4 for up to 72 hours. (a), the quantitative analysis of the level of DC-SIGN mRNA in differentiated THP-1 cells by PMA and IL-4 by SYBR Green real-time PCR. (b), analysis of induced DC-SIGN expression on surface of differentiated THP-1 cells by flow cytometry. The percentage of positive cells (top number) and mean fluorescence intensity (figure below) are shown. *means P

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Multiple Signaling Pathways Are Involved in the Interleukine-4 Regulated Expression of DC-SIGN in THP-1 Cell Line

    doi: 10.1155/2012/357060

    Figure Lengend Snippet: IL-4-induced high expression of DC-SIGN on THP-1 cells over time. THP-1 cells were treated with or without PMA for 24 hours, or with PMA for 24 hours and IL-4 for up to 72 hours. (a), the quantitative analysis of the level of DC-SIGN mRNA in differentiated THP-1 cells by PMA and IL-4 by SYBR Green real-time PCR. (b), analysis of induced DC-SIGN expression on surface of differentiated THP-1 cells by flow cytometry. The percentage of positive cells (top number) and mean fluorescence intensity (figure below) are shown. *means P

    Article Snippet: Cytokines and Antibodies Recombined human IL-4 was obtained from R & D System (Minneapolis, USA) and used at 1000 units/mL.

    Techniques: Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Fluorescence

    Signaling pathways involved in IL-4-induced DC-SIGN expression on THP-1 cells. ERK signaling pathway plays a main role by transcription factor of Ets-1 to active DC-SIGN promoter. JAK-STAT and NF- κ B signaling pathways are also involved, which may be induced by IL-4/IL-4R directly or by the interactions with ERK pathway indirectly.

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Multiple Signaling Pathways Are Involved in the Interleukine-4 Regulated Expression of DC-SIGN in THP-1 Cell Line

    doi: 10.1155/2012/357060

    Figure Lengend Snippet: Signaling pathways involved in IL-4-induced DC-SIGN expression on THP-1 cells. ERK signaling pathway plays a main role by transcription factor of Ets-1 to active DC-SIGN promoter. JAK-STAT and NF- κ B signaling pathways are also involved, which may be induced by IL-4/IL-4R directly or by the interactions with ERK pathway indirectly.

    Article Snippet: Cytokines and Antibodies Recombined human IL-4 was obtained from R & D System (Minneapolis, USA) and used at 1000 units/mL.

    Techniques: Expressing

    Vitamin D is required for concomitant production of IFNγ and cathelicidin in DC–T cell cultures. Relative expression (A) and production (B) of IFNγ and relative CXCL9 (C) , CXCL10 (D) , and cathelicidin (E) expression in DC–T cell co-cultures incubated in the presence or absence of 100 nM 25(OH)D 3 and Th1-polarizing medium (IL-12 + anti-IL-4). Data are normalized to the values obtained from co-cultures incubated in the absence of 25(OH)D 3 and Th1-polarizing medium (mean + SEM, n = 4).

    Journal: Frontiers in Immunology

    Article Title: Vitamin D Counteracts Mycobacterium tuberculosis-Induced Cathelicidin Downregulation in Dendritic Cells and Allows Th1 Differentiation and IFNγ Secretion

    doi: 10.3389/fimmu.2017.00656

    Figure Lengend Snippet: Vitamin D is required for concomitant production of IFNγ and cathelicidin in DC–T cell cultures. Relative expression (A) and production (B) of IFNγ and relative CXCL9 (C) , CXCL10 (D) , and cathelicidin (E) expression in DC–T cell co-cultures incubated in the presence or absence of 100 nM 25(OH)D 3 and Th1-polarizing medium (IL-12 + anti-IL-4). Data are normalized to the values obtained from co-cultures incubated in the absence of 25(OH)D 3 and Th1-polarizing medium (mean + SEM, n = 4).

    Article Snippet: In Th1 polarization studies, purified naïve CD4+ T cells were cultured and stimulated as described above in the presence of recombinant human IL-12 (10 ng/ml) plus human IL-4 antibody (4 µg/ml, MAB204, R & D Systems).

    Techniques: Expressing, Incubation

    IL-12-mediated signaling is initiated before vitamin D signaling. (A) Representative Western blots (lower panel) and quantification (upper panel) of VDR with GAPDH as loading control from T cells activated for 0, 12, 24, and 48 h in the presence or absence of 100 nM 25(OH)D 3 and Th1-polarizing medium (IL-12 + anti-IL-4) (mean + SEM, n = 2). IFNγ concentration in the supernatants of T cells activated for 12 h (B) , 24 h (C) , or 48 h (D) in the presence or absence of 100 nM 25(OH)D 3 and Th1-polarizing medium (mean + SEM, n = 4). Western blots including protein ladder are shown in the Figure S3 in Supplementary Material.

    Journal: Frontiers in Immunology

    Article Title: Vitamin D Counteracts Mycobacterium tuberculosis-Induced Cathelicidin Downregulation in Dendritic Cells and Allows Th1 Differentiation and IFNγ Secretion

    doi: 10.3389/fimmu.2017.00656

    Figure Lengend Snippet: IL-12-mediated signaling is initiated before vitamin D signaling. (A) Representative Western blots (lower panel) and quantification (upper panel) of VDR with GAPDH as loading control from T cells activated for 0, 12, 24, and 48 h in the presence or absence of 100 nM 25(OH)D 3 and Th1-polarizing medium (IL-12 + anti-IL-4) (mean + SEM, n = 2). IFNγ concentration in the supernatants of T cells activated for 12 h (B) , 24 h (C) , or 48 h (D) in the presence or absence of 100 nM 25(OH)D 3 and Th1-polarizing medium (mean + SEM, n = 4). Western blots including protein ladder are shown in the Figure S3 in Supplementary Material.

    Article Snippet: In Th1 polarization studies, purified naïve CD4+ T cells were cultured and stimulated as described above in the presence of recombinant human IL-12 (10 ng/ml) plus human IL-4 antibody (4 µg/ml, MAB204, R & D Systems).

    Techniques: Western Blot, Concentration Assay

    Allergen-specific IL-9 production by PBMC from nickel-allergic patients PBMC were isolated from 7 different patients with ACD to nickel ( Table 1 ), and cultured with nickel chloride (1–10 μg/ml of culture medium) for ninety six hours. Supernatants were harvested, and the following cytokines were assayed in cell-free culture supernatants: IL-9, IFN-γ, IL-4, and IL-2 (data depicted represent mean +/− SD of samples assayed in triplicate). (A) Dose response curve to Nickel titration from two nickel allergic patients and two nickel tolerant patients. (B) IL-9 production from the same two nickel allergic and tolerant patients, demonstrating that both groups can produce IL-9 in an equivalent manner after PHA stimulation. (C) Scatter plot represents the peak cytokine production from the seven allergic patients. The scatter plot of peak cytokine production (horizontal bar represents mean value) for both IFN-γ and IL-9 were significantly higher than of IL-4 (P

    Journal: The Journal of investigative dermatology

    Article Title: IL-9 Regulates Allergen-Specific Th1 Responses in Allergic Contact Dermatitis

    doi: 10.1038/jid.2014.61

    Figure Lengend Snippet: Allergen-specific IL-9 production by PBMC from nickel-allergic patients PBMC were isolated from 7 different patients with ACD to nickel ( Table 1 ), and cultured with nickel chloride (1–10 μg/ml of culture medium) for ninety six hours. Supernatants were harvested, and the following cytokines were assayed in cell-free culture supernatants: IL-9, IFN-γ, IL-4, and IL-2 (data depicted represent mean +/− SD of samples assayed in triplicate). (A) Dose response curve to Nickel titration from two nickel allergic patients and two nickel tolerant patients. (B) IL-9 production from the same two nickel allergic and tolerant patients, demonstrating that both groups can produce IL-9 in an equivalent manner after PHA stimulation. (C) Scatter plot represents the peak cytokine production from the seven allergic patients. The scatter plot of peak cytokine production (horizontal bar represents mean value) for both IFN-γ and IL-9 were significantly higher than of IL-4 (P

    Article Snippet: Blocking IL-4 or IL-9 in vitro PBMCs were incubated with anti-human IL-9 (polyclonal goat IgG, R & D systems, Minneapolis, MN) at a concentration of 0.5μg/ml or 1.0 μg/ml; anti-human IL-4 (polyclonal goat IgG, R & D systems, Minneapolis, MN) at a concentration of 0.5 μg/ml; or normal goat IgG (polyclonal, R & D systems, Minneapolis, MN) at a concentration of 0.5μg/ml during the stimulation with metal nickel chloride (10μg/ml) or medium and then incubated at 37°C for 96 hours.

    Techniques: Isolation, Cell Culture, Titration

    Increased expression of IL-9 and associated genes in AC and the detection of Th9 cells in allergic contact dermatitis (A) Mean relative-gene expression of IL-9, was elevated on average 5 (range 2–18) fold higher than paired control skin. IL-9 increase is similar to increases in IFN-γ, IL-4, IL-17A, CCL11 and CD3ε. (B) Mean relative gene expression of IL-9/Th9-associated transcription factors PU.1, IRF4, ETS1 and GcN5 was 2–3 (range 1.5–8) fold greater than paired control skin. Sections of skin biopsy specimens from a representative (C) Stained slides from an ACD patient were stained with anti-PU.1 (red) and anti-CD4 (green); nuclei are counter-stained with DAPI (blue). Stained T lymphocytes were identified as (D) PU.1 + /CD3 + or (E) PU.1 + /CD4 + . No PU.1 + /CD8 + cells were identified. To quantify cell populations, twelve fields from each double-stained section were counted with a mean of 40 infiltrating cells per field. Mean +/− SEM is depicted. For gene expression studies, skin biopsy samples were taken from positive patch tests to nickel or cobalt from seven different patients (ACD 6–12 in Table 1 ). For the immunochemistry, skin biopsy specimens were taken from five different positive patch tests in five different patients (ACD1–5 in Table 1 ).

    Journal: The Journal of investigative dermatology

    Article Title: IL-9 Regulates Allergen-Specific Th1 Responses in Allergic Contact Dermatitis

    doi: 10.1038/jid.2014.61

    Figure Lengend Snippet: Increased expression of IL-9 and associated genes in AC and the detection of Th9 cells in allergic contact dermatitis (A) Mean relative-gene expression of IL-9, was elevated on average 5 (range 2–18) fold higher than paired control skin. IL-9 increase is similar to increases in IFN-γ, IL-4, IL-17A, CCL11 and CD3ε. (B) Mean relative gene expression of IL-9/Th9-associated transcription factors PU.1, IRF4, ETS1 and GcN5 was 2–3 (range 1.5–8) fold greater than paired control skin. Sections of skin biopsy specimens from a representative (C) Stained slides from an ACD patient were stained with anti-PU.1 (red) and anti-CD4 (green); nuclei are counter-stained with DAPI (blue). Stained T lymphocytes were identified as (D) PU.1 + /CD3 + or (E) PU.1 + /CD4 + . No PU.1 + /CD8 + cells were identified. To quantify cell populations, twelve fields from each double-stained section were counted with a mean of 40 infiltrating cells per field. Mean +/− SEM is depicted. For gene expression studies, skin biopsy samples were taken from positive patch tests to nickel or cobalt from seven different patients (ACD 6–12 in Table 1 ). For the immunochemistry, skin biopsy specimens were taken from five different positive patch tests in five different patients (ACD1–5 in Table 1 ).

    Article Snippet: Blocking IL-4 or IL-9 in vitro PBMCs were incubated with anti-human IL-9 (polyclonal goat IgG, R & D systems, Minneapolis, MN) at a concentration of 0.5μg/ml or 1.0 μg/ml; anti-human IL-4 (polyclonal goat IgG, R & D systems, Minneapolis, MN) at a concentration of 0.5 μg/ml; or normal goat IgG (polyclonal, R & D systems, Minneapolis, MN) at a concentration of 0.5μg/ml during the stimulation with metal nickel chloride (10μg/ml) or medium and then incubated at 37°C for 96 hours.

    Techniques: Expressing, Staining

    IL-4 but not IL-9 directly regulates IFN-γ during allergen-specific Th1 activation in vitro (A) PBMC from a nickel allergic patient were cultured in medium alone or in medium containing nickel-chloride, the indicated doses of IL-9 alone, IL-4 alone, or IL-9 and IL-4 together were added to the nickel-chloride stimulated PBMC. Supernatants were collected 96 h after nickel-stimulation. (B) IL-4 secretion was also measured after addition of IL-9. (C) IL-9 was measured in those culture conditions which had added IL-4. (D) Specific blocking anti-sera were added to the nickel culture, and IFN-γ was assayed using multiplex ELISA. Data depicted represent mean +/− SD of samples assayed in triplicate. This experiment was repeated with PBMC three times, and found to be reproducible. The data in ( A–D ) represent PBMC from distinct nickel-allergic patients.

    Journal: The Journal of investigative dermatology

    Article Title: IL-9 Regulates Allergen-Specific Th1 Responses in Allergic Contact Dermatitis

    doi: 10.1038/jid.2014.61

    Figure Lengend Snippet: IL-4 but not IL-9 directly regulates IFN-γ during allergen-specific Th1 activation in vitro (A) PBMC from a nickel allergic patient were cultured in medium alone or in medium containing nickel-chloride, the indicated doses of IL-9 alone, IL-4 alone, or IL-9 and IL-4 together were added to the nickel-chloride stimulated PBMC. Supernatants were collected 96 h after nickel-stimulation. (B) IL-4 secretion was also measured after addition of IL-9. (C) IL-9 was measured in those culture conditions which had added IL-4. (D) Specific blocking anti-sera were added to the nickel culture, and IFN-γ was assayed using multiplex ELISA. Data depicted represent mean +/− SD of samples assayed in triplicate. This experiment was repeated with PBMC three times, and found to be reproducible. The data in ( A–D ) represent PBMC from distinct nickel-allergic patients.

    Article Snippet: Blocking IL-4 or IL-9 in vitro PBMCs were incubated with anti-human IL-9 (polyclonal goat IgG, R & D systems, Minneapolis, MN) at a concentration of 0.5μg/ml or 1.0 μg/ml; anti-human IL-4 (polyclonal goat IgG, R & D systems, Minneapolis, MN) at a concentration of 0.5 μg/ml; or normal goat IgG (polyclonal, R & D systems, Minneapolis, MN) at a concentration of 0.5μg/ml during the stimulation with metal nickel chloride (10μg/ml) or medium and then incubated at 37°C for 96 hours.

    Techniques: Activation Assay, In Vitro, Cell Culture, Blocking Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay

    The proinflammatory cytokines were elevated in ALI patients. The concentrations of cytokines including IL-1β (A) , IL-6 (B) , IL-15 (C) , TNF-α (D) , IL-4 (E) , and IL-13 (F) were measured using ELISA kits in serum samples obtained from 24 NSCLC patients (Control) under T0 stage and 24 ALI patients. *** P

    Journal: International Journal of Biological Sciences

    Article Title: Downregulation of miR-199a-3p mediated by the CtBP2-HDAC1-FOXP3 transcriptional complex contributes to acute lung injury by targeting NLRP1

    doi: 10.7150/ijbs.37133

    Figure Lengend Snippet: The proinflammatory cytokines were elevated in ALI patients. The concentrations of cytokines including IL-1β (A) , IL-6 (B) , IL-15 (C) , TNF-α (D) , IL-4 (E) , and IL-13 (F) were measured using ELISA kits in serum samples obtained from 24 NSCLC patients (Control) under T0 stage and 24 ALI patients. *** P

    Article Snippet: Blood samples were immediately centrifuged at 800 × g for 10 min to obtain serum, which was applied to measure the levels of cytokines including IL-1β (#ab214025), IL-4 (#ab215089), IL-6 (#ab100573), IL-13 (#ab46038), IL-15 (#ab100554), and TNF-α (ab181421) using ELISA kits purchased from Abcam (Cambridge, MA, USA).

    Techniques: Enzyme-linked Immunosorbent Assay

    Changes in monocyte function after surgery. Functional properties of the circulating MO were tested using, phagocytosis assays ( a ), ability to stimulate allogeneic T cells ( b ), functional plasticity by incubate cells in vitro with IL-4 and GM-CSF and measuring an emergence of DC specific marker ( c ) or ability to induce proliferative T cells response. a There was a significant increase (t 0 : Mean = 10.7 ± 0.05 vs t 3m : Mean = 1.64 ± 0.49 CI 95% : Δx = [0.23, 0.90]; p = 0.003; d = 1.36) in the phagocytic capabilities of peripheral blood monocytes taken from patients at 3 months after the surgery, as shown by uptake of zymosan. b The ability of peripheral blood monocytes to stimulate allogeneic T cells was significantly (t 0 : Mean = 0.65 ± 0.13 vs t 3m : Mean = 0.46 ± 0.31; CI 95% Δx [− 0.33, − 0.04]; p = 0.016; d = − 0.68) diminished at 3 months. c There was a significant decline in the emergence of CD1a (+) (immature DC marker) (t 0 : Me = 1964.58; IQR[745.82, 7586.04] vs t 3m : Me = 519.19; IQR[428.24, 785.52]; CI 95% : ΔpMe[761.49, 5459.51]; p =

    Journal: Journal of Translational Medicine

    Article Title: Acquired immunological imbalance after surgery with cardiopulmonary bypass due to epigenetic over-activation of PU.1/M-CSF

    doi: 10.1186/s12967-018-1518-3

    Figure Lengend Snippet: Changes in monocyte function after surgery. Functional properties of the circulating MO were tested using, phagocytosis assays ( a ), ability to stimulate allogeneic T cells ( b ), functional plasticity by incubate cells in vitro with IL-4 and GM-CSF and measuring an emergence of DC specific marker ( c ) or ability to induce proliferative T cells response. a There was a significant increase (t 0 : Mean = 10.7 ± 0.05 vs t 3m : Mean = 1.64 ± 0.49 CI 95% : Δx = [0.23, 0.90]; p = 0.003; d = 1.36) in the phagocytic capabilities of peripheral blood monocytes taken from patients at 3 months after the surgery, as shown by uptake of zymosan. b The ability of peripheral blood monocytes to stimulate allogeneic T cells was significantly (t 0 : Mean = 0.65 ± 0.13 vs t 3m : Mean = 0.46 ± 0.31; CI 95% Δx [− 0.33, − 0.04]; p = 0.016; d = − 0.68) diminished at 3 months. c There was a significant decline in the emergence of CD1a (+) (immature DC marker) (t 0 : Me = 1964.58; IQR[745.82, 7586.04] vs t 3m : Me = 519.19; IQR[428.24, 785.52]; CI 95% : ΔpMe[761.49, 5459.51]; p =

    Article Snippet: Fresh monocytes were incubated in X-VIVO 15™ Media with Gentamycin and Phenol Red (Lonza, Cohasset, MN) supplemented with human interleukin 4 (hIL-4; Peprotech, Rocky Hill, NJ) at 500 IU/ml and human granulocyte monocyte colony stimulation factor (hGM-CSF; Peprotech, Rocky Hill, NJ) 1000 IU/ml 1 at 37 °C 5% CO2 in the dark.

    Techniques: Functional Assay, In Vitro, Marker

    α-LacCer suppresses α-GalCer-stimulated cytokine production by competing for CD1d binding. (A,B) Sorted mouse iNKT cells were cultured in the CD1-coated plate pre-loaded with 100 ng/ml α-GalCer or 1 μg/ml α-LacCer with or without the second loading of α-GalCer or α-LacCer for 48 h. The levels of IFN-γ (A) and IL-4 (B) in the culture supernatant were assessed ( n = 3). (C,D) Sorted human iNKT cells were cultured in the CD1d-coated plate pre-loaded with α-LacCer (1 or 10 μg/ml) with the second loading of α-GalCer (100 ng/ml) for 48 h. The levels of IFN-γ (C) and IL-4 (D) in the supernatant were assessed ( n = 3–6). Data are representative of three independent experiments and are presented as means ± s.e.m. [ * P

    Journal: Frontiers in Chemistry

    Article Title: α-Lactosylceramide Protects Against iNKT-Mediated Murine Airway Hyperreactivity and Liver Injury Through Competitive Inhibition of Cd1d Binding

    doi: 10.3389/fchem.2019.00811

    Figure Lengend Snippet: α-LacCer suppresses α-GalCer-stimulated cytokine production by competing for CD1d binding. (A,B) Sorted mouse iNKT cells were cultured in the CD1-coated plate pre-loaded with 100 ng/ml α-GalCer or 1 μg/ml α-LacCer with or without the second loading of α-GalCer or α-LacCer for 48 h. The levels of IFN-γ (A) and IL-4 (B) in the culture supernatant were assessed ( n = 3). (C,D) Sorted human iNKT cells were cultured in the CD1d-coated plate pre-loaded with α-LacCer (1 or 10 μg/ml) with the second loading of α-GalCer (100 ng/ml) for 48 h. The levels of IFN-γ (C) and IL-4 (D) in the supernatant were assessed ( n = 3–6). Data are representative of three independent experiments and are presented as means ± s.e.m. [ * P

    Article Snippet: For intracellular staining, single cell suspensions were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich), 1 μg/mL ionomycin (Sigma-Aldrich) and 1 μg/mL Golgi stop A (BD Biosciences) for 4 h. After surface staining, cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD biosciences) and further stained intracellularly with human IFNγ (B27, BioLegend) and human IL-4 (MP4-25D2, BioLegend).

    Techniques: Binding Assay, Cell Culture

    α-LacCer suppresses α-GalCer-induced cytokine production in vitro . (A,B) Splenocytes from BALB/c mice were stimulated with 100 ng/ml α-GalCer in the presence or absence of α-LacCer (5 or 20 μg/ml) for 48 h. The levels of IFN-γ (A) and IL-4 (B) in the supernatant were measured ( n = 3). (C,D) Sorted mouse iNKT cells were co-cultured with irradiated BMDCs and stimulated with α-GalCer (100 ng/ml) in the presence or absence of α-LacCer (5 or 20 μg/ml) for 48 h. The levels of IFN-γ (C) and IL-4 (D) in the supernatant were measured ( n = 3). (E) NK1.2 cells in co-culture with A20-CD1d cells were stimulated with 100 ng/ml α-GalCer in the presence or absence of α-LacCer (100, 1,000, or 10,000 ng/ml) for 48 h. The level of IL-2 in supernatants was measured ( n = 3). (F) NK1.2 cells in co-culture with A20-CD1d cells were stimulated with 100 ng/ml α-GalCer in the presence of incremental levels of α-LacCer (0.1–100 μg/ml) for 48 h. The level of IL-2 in supernatants was measured ( n = 4), and GraphPad Prism was utilized for calculating the IC50. Data are representative of three independent experiments and presented as means ± s.e.m. [n.s., not significant; ** P

    Journal: Frontiers in Chemistry

    Article Title: α-Lactosylceramide Protects Against iNKT-Mediated Murine Airway Hyperreactivity and Liver Injury Through Competitive Inhibition of Cd1d Binding

    doi: 10.3389/fchem.2019.00811

    Figure Lengend Snippet: α-LacCer suppresses α-GalCer-induced cytokine production in vitro . (A,B) Splenocytes from BALB/c mice were stimulated with 100 ng/ml α-GalCer in the presence or absence of α-LacCer (5 or 20 μg/ml) for 48 h. The levels of IFN-γ (A) and IL-4 (B) in the supernatant were measured ( n = 3). (C,D) Sorted mouse iNKT cells were co-cultured with irradiated BMDCs and stimulated with α-GalCer (100 ng/ml) in the presence or absence of α-LacCer (5 or 20 μg/ml) for 48 h. The levels of IFN-γ (C) and IL-4 (D) in the supernatant were measured ( n = 3). (E) NK1.2 cells in co-culture with A20-CD1d cells were stimulated with 100 ng/ml α-GalCer in the presence or absence of α-LacCer (100, 1,000, or 10,000 ng/ml) for 48 h. The level of IL-2 in supernatants was measured ( n = 3). (F) NK1.2 cells in co-culture with A20-CD1d cells were stimulated with 100 ng/ml α-GalCer in the presence of incremental levels of α-LacCer (0.1–100 μg/ml) for 48 h. The level of IL-2 in supernatants was measured ( n = 4), and GraphPad Prism was utilized for calculating the IC50. Data are representative of three independent experiments and presented as means ± s.e.m. [n.s., not significant; ** P

    Article Snippet: For intracellular staining, single cell suspensions were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich), 1 μg/mL ionomycin (Sigma-Aldrich) and 1 μg/mL Golgi stop A (BD Biosciences) for 4 h. After surface staining, cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD biosciences) and further stained intracellularly with human IFNγ (B27, BioLegend) and human IL-4 (MP4-25D2, BioLegend).

    Techniques: In Vitro, Mouse Assay, Cell Culture, Irradiation, Co-Culture Assay

    α-GalCer-induced iNKT cell activation and proliferation are suppressed by α-LacCer in vivo . BALB/c mice were injected intraperitoneally with 1 μg of α-GalCer and α-LacCer, and the lungs were harvested 3 days post-injection. (A) Flow cytometry analysis of CD69 and Ki67 expressions in iNKT cells (CD45 + TCRβ + CD1d tetramer + cells). (B) Absolute numbers of total, CD69 + , and Ki67 + iNKT cells calculated from the flow cytometry data ( n = 3). (C) IFN-γ and IL-4 levels in the lung lysates were measured by ELISA ( n = 3). Data are representative of three independent experiments and presented as means ± s.e.m. [ ** P

    Journal: Frontiers in Chemistry

    Article Title: α-Lactosylceramide Protects Against iNKT-Mediated Murine Airway Hyperreactivity and Liver Injury Through Competitive Inhibition of Cd1d Binding

    doi: 10.3389/fchem.2019.00811

    Figure Lengend Snippet: α-GalCer-induced iNKT cell activation and proliferation are suppressed by α-LacCer in vivo . BALB/c mice were injected intraperitoneally with 1 μg of α-GalCer and α-LacCer, and the lungs were harvested 3 days post-injection. (A) Flow cytometry analysis of CD69 and Ki67 expressions in iNKT cells (CD45 + TCRβ + CD1d tetramer + cells). (B) Absolute numbers of total, CD69 + , and Ki67 + iNKT cells calculated from the flow cytometry data ( n = 3). (C) IFN-γ and IL-4 levels in the lung lysates were measured by ELISA ( n = 3). Data are representative of three independent experiments and presented as means ± s.e.m. [ ** P

    Article Snippet: For intracellular staining, single cell suspensions were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich), 1 μg/mL ionomycin (Sigma-Aldrich) and 1 μg/mL Golgi stop A (BD Biosciences) for 4 h. After surface staining, cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD biosciences) and further stained intracellularly with human IFNγ (B27, BioLegend) and human IL-4 (MP4-25D2, BioLegend).

    Techniques: Activation Assay, In Vivo, Mouse Assay, Injection, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    α-LacCer suppresses α-GalCer and GSL-1-induced airway hyperreactivity. BALB/c mice were treated intranasally with 1 μg α-GalCer (A–C) or 10 μg GSL-1 (D) in the presence or absence of 1 μg α-LacCer for 24 h. (A,D) Changes in lung resistance (R L ) in response to increasing doses of methacholine [ n = 6–8/group in (A) ; n = 3–5/group in (D) ]. (B) The numbers of macrophage (Mac), neutrophil (Neu), eosinophil (Eos), and lymphocyte (Lym) in the BALF ( n = 7–8). (C) The levels of IL-4 and IL-13 in the lungs assessed using ELISA ( n = 4). Data are representative of three independent experiments and presented as means ± s.e.m. [ * P

    Journal: Frontiers in Chemistry

    Article Title: α-Lactosylceramide Protects Against iNKT-Mediated Murine Airway Hyperreactivity and Liver Injury Through Competitive Inhibition of Cd1d Binding

    doi: 10.3389/fchem.2019.00811

    Figure Lengend Snippet: α-LacCer suppresses α-GalCer and GSL-1-induced airway hyperreactivity. BALB/c mice were treated intranasally with 1 μg α-GalCer (A–C) or 10 μg GSL-1 (D) in the presence or absence of 1 μg α-LacCer for 24 h. (A,D) Changes in lung resistance (R L ) in response to increasing doses of methacholine [ n = 6–8/group in (A) ; n = 3–5/group in (D) ]. (B) The numbers of macrophage (Mac), neutrophil (Neu), eosinophil (Eos), and lymphocyte (Lym) in the BALF ( n = 7–8). (C) The levels of IL-4 and IL-13 in the lungs assessed using ELISA ( n = 4). Data are representative of three independent experiments and presented as means ± s.e.m. [ * P

    Article Snippet: For intracellular staining, single cell suspensions were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich), 1 μg/mL ionomycin (Sigma-Aldrich) and 1 μg/mL Golgi stop A (BD Biosciences) for 4 h. After surface staining, cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD biosciences) and further stained intracellularly with human IFNγ (B27, BioLegend) and human IL-4 (MP4-25D2, BioLegend).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    α-LacCer suppresses α-GalCer-induced cytokine production by human iNKT cells in vitro . (A) Sorted human iNKT cells were stimulated with α-GalCer and α-LacCer (1 μg/ml) in the absence or presence of anti-CD1b or anti-CD1d antibody for 72 h. The levels of IFN-γ and IL-4 in the supernatant were analyzed ( n = 3–6). (B,C) Human PBMCs were cultured with 1 μg/ml α-GalCer and α-LacCer for 7 days. (B) FACS analysis of CD69, IFN-γ, and IL-4 expressions in human iNKT cells (CD45 + tetra-CD1d + TCRβ + ). (C) Absolute numbers of total and CD69 + iNKT cells. (D) Human PBMCs were stimulated with α-GalCer (100 ng/ml) in the presence or absence of α-LacCer (1 μg/ml) for 3 days. The level of IFN-γ was analyzed ( n = 3). (E) Human iNKT cells were cultured with irradiated DCs pre-treated with 100 ng/ml α-GalCer in the absence or presence of 1 μg/ml α-LacCer for 3 days. The levels of IFN-γ and IL-4 were analyzed ( n = 3–5). Data are representative of three independent experiments and presented as means ± s.e.m. [ n.s ., not significant; * P

    Journal: Frontiers in Chemistry

    Article Title: α-Lactosylceramide Protects Against iNKT-Mediated Murine Airway Hyperreactivity and Liver Injury Through Competitive Inhibition of Cd1d Binding

    doi: 10.3389/fchem.2019.00811

    Figure Lengend Snippet: α-LacCer suppresses α-GalCer-induced cytokine production by human iNKT cells in vitro . (A) Sorted human iNKT cells were stimulated with α-GalCer and α-LacCer (1 μg/ml) in the absence or presence of anti-CD1b or anti-CD1d antibody for 72 h. The levels of IFN-γ and IL-4 in the supernatant were analyzed ( n = 3–6). (B,C) Human PBMCs were cultured with 1 μg/ml α-GalCer and α-LacCer for 7 days. (B) FACS analysis of CD69, IFN-γ, and IL-4 expressions in human iNKT cells (CD45 + tetra-CD1d + TCRβ + ). (C) Absolute numbers of total and CD69 + iNKT cells. (D) Human PBMCs were stimulated with α-GalCer (100 ng/ml) in the presence or absence of α-LacCer (1 μg/ml) for 3 days. The level of IFN-γ was analyzed ( n = 3). (E) Human iNKT cells were cultured with irradiated DCs pre-treated with 100 ng/ml α-GalCer in the absence or presence of 1 μg/ml α-LacCer for 3 days. The levels of IFN-γ and IL-4 were analyzed ( n = 3–5). Data are representative of three independent experiments and presented as means ± s.e.m. [ n.s ., not significant; * P

    Article Snippet: For intracellular staining, single cell suspensions were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich), 1 μg/mL ionomycin (Sigma-Aldrich) and 1 μg/mL Golgi stop A (BD Biosciences) for 4 h. After surface staining, cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD biosciences) and further stained intracellularly with human IFNγ (B27, BioLegend) and human IL-4 (MP4-25D2, BioLegend).

    Techniques: In Vitro, Cell Culture, FACS, Irradiation

    α-LacCer activates NK1.2 hybridoma and mouse iNKT cells in a CD1d-dependent manner. (A) The structures of α-galactosyl ceramide (α-GalCer) and its analogs, α-lactosyl ceramide (α-LacCer). (B) Vα14 + T hybridoma cells (NK1.2) were co-cultured with A20-CD1d cells in the presence of α-GalCer (100, 1,000 ng/ml) or α-LacCer (10, 100, 1,000 ng/ml) for 48 h. The level of IL-2 in the supernatant was measured ( n = 3–4). (C,D) Sorted mouse iNKT cells in co-culture with irradiated BMDCs were stimulated with α-GalCer (100 ng/ml) or α-LacCer (10, 100, 1,000 ng/ml) for 48 h, and the levels of IFN-γ (C) and IL-4 (D) in the supernatant were measured ( n = 3). (E,F) Sorted mouse iNKT cells in co-culture with irradiated CD1d −/− or WT BMDCs were stimulated with α-GalCer (100 ng/ml) or α-LacCer (100 ng/ml) for 48 h. The levels of IFN-γ (E) and IL-4 (F) in the supernatant were measured ( n = 3). Data are representative of three independent experiments and presented as means ± s.e.m. [ * P

    Journal: Frontiers in Chemistry

    Article Title: α-Lactosylceramide Protects Against iNKT-Mediated Murine Airway Hyperreactivity and Liver Injury Through Competitive Inhibition of Cd1d Binding

    doi: 10.3389/fchem.2019.00811

    Figure Lengend Snippet: α-LacCer activates NK1.2 hybridoma and mouse iNKT cells in a CD1d-dependent manner. (A) The structures of α-galactosyl ceramide (α-GalCer) and its analogs, α-lactosyl ceramide (α-LacCer). (B) Vα14 + T hybridoma cells (NK1.2) were co-cultured with A20-CD1d cells in the presence of α-GalCer (100, 1,000 ng/ml) or α-LacCer (10, 100, 1,000 ng/ml) for 48 h. The level of IL-2 in the supernatant was measured ( n = 3–4). (C,D) Sorted mouse iNKT cells in co-culture with irradiated BMDCs were stimulated with α-GalCer (100 ng/ml) or α-LacCer (10, 100, 1,000 ng/ml) for 48 h, and the levels of IFN-γ (C) and IL-4 (D) in the supernatant were measured ( n = 3). (E,F) Sorted mouse iNKT cells in co-culture with irradiated CD1d −/− or WT BMDCs were stimulated with α-GalCer (100 ng/ml) or α-LacCer (100 ng/ml) for 48 h. The levels of IFN-γ (E) and IL-4 (F) in the supernatant were measured ( n = 3). Data are representative of three independent experiments and presented as means ± s.e.m. [ * P

    Article Snippet: For intracellular staining, single cell suspensions were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich), 1 μg/mL ionomycin (Sigma-Aldrich) and 1 μg/mL Golgi stop A (BD Biosciences) for 4 h. After surface staining, cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD biosciences) and further stained intracellularly with human IFNγ (B27, BioLegend) and human IL-4 (MP4-25D2, BioLegend).

    Techniques: Cell Culture, Co-Culture Assay, Irradiation

    Correlation between vitamin D and IL-4 in the study group

    Journal: Turkish Archives of Otorhinolaryngology

    Article Title: The Relationship between Th1/Th2 Balance and 1α, 25-Dihydroxyvitamin D3 in Patients with Allergic Rhinitis

    doi: 10.5152/tao.2015.1187

    Figure Lengend Snippet: Correlation between vitamin D and IL-4 in the study group

    Article Snippet: The IL-4, IL-10, and IFN-γ levels were measured using enzyme- linked immunosorbent assay (ELISA), using commercial human IL-4, IL-10 and IFN-γ kits (Boster Immunoleader; Wuhan, China).

    Techniques:

    Na + –K + –ATPase activity in Caco-2 cells treated for 24 h with vehicle, interleukin-12 (IL-12, 500 U ml −1 ), interferon- γ (IFN- γ , 500 U ml −1 ), interleukin-10 (IL-10, 500 U ml −1 ), interleukin-4 (IL-4, 500 U ml −1 ), transforming growth factor β (TGF- β , 500 U ml −1 ) or insulin growth factor I (IGF-I, 500 U ml −1 ). Enzyme activity is expressed in nanomoles of Pi per mg of protein per min. Columns represent the mean of five experiments per group; vertical lines show s.e.m. Values are percent of control for Na + –K + –ATPase activity (absolute levels in nmol Pi per mg protein per min were 44.7±1.2, n =5). * Significantly different from control values ( P

    Journal: British Journal of Pharmacology

    Article Title: Intestinal Na+-K+-ATPase activity and molecular events downstream of interferon-γ receptor stimulation

    doi: 10.1038/sj.bjp.0705895

    Figure Lengend Snippet: Na + –K + –ATPase activity in Caco-2 cells treated for 24 h with vehicle, interleukin-12 (IL-12, 500 U ml −1 ), interferon- γ (IFN- γ , 500 U ml −1 ), interleukin-10 (IL-10, 500 U ml −1 ), interleukin-4 (IL-4, 500 U ml −1 ), transforming growth factor β (TGF- β , 500 U ml −1 ) or insulin growth factor I (IGF-I, 500 U ml −1 ). Enzyme activity is expressed in nanomoles of Pi per mg of protein per min. Columns represent the mean of five experiments per group; vertical lines show s.e.m. Values are percent of control for Na + –K + –ATPase activity (absolute levels in nmol Pi per mg protein per min were 44.7±1.2, n =5). * Significantly different from control values ( P

    Article Snippet: Anisomycin, EGCG, EGC, human IFN- γ , human IL-4, human IL-10, human IL-12, human TGF- β , and insulin growth factor I (IGF-I) were obtained from Sigma Chemical Company (St Louis, MO, U.S.A.).

    Techniques: Activity Assay

    Serum concentrations of IL-2, IL-4, IL-10 and IL-21 were determined by ELISA. Normalized values were presented as the mean ± standard deviation.*P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Altered circulating T follicular helper cells in patients with chronic immune thrombocytopenia

    doi: 10.3892/etm.2018.6508

    Figure Lengend Snippet: Serum concentrations of IL-2, IL-4, IL-10 and IL-21 were determined by ELISA. Normalized values were presented as the mean ± standard deviation.*P

    Article Snippet: The ELISA kits used in the experiment were Human IL-2 ELISA MAX™ Deluxe (cat. no. 431804), Human IL-4 ELISA MAX™ Deluxe (cat. no. 430304), Human IL-10 ELISA MAX™ Standard (cat. no. 430601) and Human IL-21 ELISA MAX™ Deluxe (cat. no. 433804), all purchased from BioLegend.

    Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation