interleukin 4  (PeproTech)

 
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    Name:
    Recombinant Rat IL 4
    Description:
    IL 4 is a pleiotropic cytokine that regulates diverse T and B cell responses including cell proliferation survival and gene expression Produced by mast cells T cells and bone marrow stromal cells IL 4 regulates the differentiation of naive CD4 T cells into helper Th2 cells characterized by their cytokine secretion profile that includes secretion of IL 4 IL 5 IL 6 IL 10 and IL 13 which favor a humoral immune response Another dominant function of IL 4 is the regulation of immunoglobulin class switching to the IgG1 and IgE isotypes Excessive IL 4 production by Th2 cells has been associated with elevated IgE production and allergy Recombinant rat IL 4 is a 14 0 kDa globular protein containing 126 amino acid residues
    Catalog Number:
    400-04-100UG
    Price:
    650.00
    Category:
    Recombinant Proteins
    Source:
    E.coli
    Reactivity:
    Mouse
    Purity:
    98.0
    Quantity:
    100UG
    Buy from Supplier


    Structured Review

    PeproTech interleukin 4
    Transduction efficay of an Ad vector at various MOIs and surface marker expression of adenovirus-infected DCs. (A) Recombinant AdGFP was used to transduce day 7 immature DCs, which were then cultured for 48 h in the presence of GM-CSF and <t>IL-4.</t> On day 9, flow cytometric analysis of GFP expression by the Ad-GFP-DCs was carried out. Typically, > 74% of cells were GFP + at a MOI of 200. (B) IL-2 and/or AFP expression by gene-modified DCs is shown. PCR products of β-actin, IL-2 and AFP were visualized by electrophoresis in a 2% agarose gel containing 0.5 μg/ml ethidium bromide. Lane 1 Marker, lane 2 IL-2-DC, lane 3 IL-2/AFP-DC, lane 4 AFP-DC, lane 5 GFP-DC, lane 6 DC group. Ad, adenovirus; MOI, multiplicity of infection; DCs, dendritic cells; GFP, green fluorescent protein; GM-CSF, granulocyte macrophage-colony stimulating factor; IL-2, inter-leukin-2; AFP, α-fetoprotein; PCR, polymerase chain reaction.
    IL 4 is a pleiotropic cytokine that regulates diverse T and B cell responses including cell proliferation survival and gene expression Produced by mast cells T cells and bone marrow stromal cells IL 4 regulates the differentiation of naive CD4 T cells into helper Th2 cells characterized by their cytokine secretion profile that includes secretion of IL 4 IL 5 IL 6 IL 10 and IL 13 which favor a humoral immune response Another dominant function of IL 4 is the regulation of immunoglobulin class switching to the IgG1 and IgE isotypes Excessive IL 4 production by Th2 cells has been associated with elevated IgE production and allergy Recombinant rat IL 4 is a 14 0 kDa globular protein containing 126 amino acid residues
    https://www.bioz.com/result/interleukin 4/product/PeproTech
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    interleukin 4 - by Bioz Stars, 2021-05
    99/100 stars

    Images

    1) Product Images from "Co-transfection of dendritic cells with AFP and IL-2 genes enhances the induction of tumor antigen-specific antitumor immunity"

    Article Title: Co-transfection of dendritic cells with AFP and IL-2 genes enhances the induction of tumor antigen-specific antitumor immunity

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2012.635

    Transduction efficay of an Ad vector at various MOIs and surface marker expression of adenovirus-infected DCs. (A) Recombinant AdGFP was used to transduce day 7 immature DCs, which were then cultured for 48 h in the presence of GM-CSF and IL-4. On day 9, flow cytometric analysis of GFP expression by the Ad-GFP-DCs was carried out. Typically, > 74% of cells were GFP + at a MOI of 200. (B) IL-2 and/or AFP expression by gene-modified DCs is shown. PCR products of β-actin, IL-2 and AFP were visualized by electrophoresis in a 2% agarose gel containing 0.5 μg/ml ethidium bromide. Lane 1 Marker, lane 2 IL-2-DC, lane 3 IL-2/AFP-DC, lane 4 AFP-DC, lane 5 GFP-DC, lane 6 DC group. Ad, adenovirus; MOI, multiplicity of infection; DCs, dendritic cells; GFP, green fluorescent protein; GM-CSF, granulocyte macrophage-colony stimulating factor; IL-2, inter-leukin-2; AFP, α-fetoprotein; PCR, polymerase chain reaction.
    Figure Legend Snippet: Transduction efficay of an Ad vector at various MOIs and surface marker expression of adenovirus-infected DCs. (A) Recombinant AdGFP was used to transduce day 7 immature DCs, which were then cultured for 48 h in the presence of GM-CSF and IL-4. On day 9, flow cytometric analysis of GFP expression by the Ad-GFP-DCs was carried out. Typically, > 74% of cells were GFP + at a MOI of 200. (B) IL-2 and/or AFP expression by gene-modified DCs is shown. PCR products of β-actin, IL-2 and AFP were visualized by electrophoresis in a 2% agarose gel containing 0.5 μg/ml ethidium bromide. Lane 1 Marker, lane 2 IL-2-DC, lane 3 IL-2/AFP-DC, lane 4 AFP-DC, lane 5 GFP-DC, lane 6 DC group. Ad, adenovirus; MOI, multiplicity of infection; DCs, dendritic cells; GFP, green fluorescent protein; GM-CSF, granulocyte macrophage-colony stimulating factor; IL-2, inter-leukin-2; AFP, α-fetoprotein; PCR, polymerase chain reaction.

    Techniques Used: Transduction, Plasmid Preparation, Marker, Expressing, Infection, Recombinant, Cell Culture, Flow Cytometry, Modification, Polymerase Chain Reaction, Electrophoresis, Agarose Gel Electrophoresis

    2) Product Images from "Measurement of trihydroxy-linoleic acids in stratum corneum by tape-stripping: Possible biomarker of barrier function in atopic dermatitis"

    Article Title: Measurement of trihydroxy-linoleic acids in stratum corneum by tape-stripping: Possible biomarker of barrier function in atopic dermatitis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0210013

    The mRNA expression of enzymes involved in CLE formation upon IL-4 stimulation. ALOX12B, ALOXE3 and ABHD9 mRNA levels of primary normal human epidermal keratinocytes were measured by real-time RT-PCR. The mean values of threshold cycle were 27.5 ± 0.5 (ALOX12B), 22.4 ± 0.1 (ALOXE3), 23.1 ± 0.4 (ABHD9), 15.4 ± 0.4 (β-actin), respectively. The amount of mRNA was normalized to that of β-actin using the ΔCt method, as described in the manufacturer’s protocol. Data are expressed as mean ± SD (n = 6). One-way ANOVA with repeated measures was used for the comparison. Because the initial P value was less than 0.05, Scheffe’s test was used to determine the significance between groups. * p
    Figure Legend Snippet: The mRNA expression of enzymes involved in CLE formation upon IL-4 stimulation. ALOX12B, ALOXE3 and ABHD9 mRNA levels of primary normal human epidermal keratinocytes were measured by real-time RT-PCR. The mean values of threshold cycle were 27.5 ± 0.5 (ALOX12B), 22.4 ± 0.1 (ALOXE3), 23.1 ± 0.4 (ABHD9), 15.4 ± 0.4 (β-actin), respectively. The amount of mRNA was normalized to that of β-actin using the ΔCt method, as described in the manufacturer’s protocol. Data are expressed as mean ± SD (n = 6). One-way ANOVA with repeated measures was used for the comparison. Because the initial P value was less than 0.05, Scheffe’s test was used to determine the significance between groups. * p

    Techniques Used: Expressing, Quantitative RT-PCR

    3) Product Images from "Bone Marrow-Derived Mesenchymal Stem Cells Exert Diverse Effects on Different Macrophage Subsets"

    Article Title: Bone Marrow-Derived Mesenchymal Stem Cells Exert Diverse Effects on Different Macrophage Subsets

    Journal: Stem Cells International

    doi: 10.1155/2018/8348121

    Identification of M1 and M2 macrophages. (a) (A) Morphology of macrophages as assessed by light microscopy on day 7 of cell culture (×100); (B) flow cytometry showing that the proportion of CD11b-positive cells was greater than 95% on day 7 of cell culture; (C) macrophages after phagocytic neutral red staining (×400). (b) Expression of CD80 and CD163 on macrophages after treatment with LPS + IFN- γ and IL-4 for 24 hours as assessed by flow cytometry. (c) Expression of iNOS and Arg-1 in macrophages after treatment with LPS + IFN- γ and IL-4 for 24 hours as assessed by Western blot.
    Figure Legend Snippet: Identification of M1 and M2 macrophages. (a) (A) Morphology of macrophages as assessed by light microscopy on day 7 of cell culture (×100); (B) flow cytometry showing that the proportion of CD11b-positive cells was greater than 95% on day 7 of cell culture; (C) macrophages after phagocytic neutral red staining (×400). (b) Expression of CD80 and CD163 on macrophages after treatment with LPS + IFN- γ and IL-4 for 24 hours as assessed by flow cytometry. (c) Expression of iNOS and Arg-1 in macrophages after treatment with LPS + IFN- γ and IL-4 for 24 hours as assessed by Western blot.

    Techniques Used: Light Microscopy, Cell Culture, Flow Cytometry, Cytometry, Staining, Expressing, Western Blot

    4) Product Images from "Chlorogenic acid inhibits glioblastoma growth through repolarizating macrophage from M2 to M1 phenotype"

    Article Title: Chlorogenic acid inhibits glioblastoma growth through repolarizating macrophage from M2 to M1 phenotype

    Journal: Scientific Reports

    doi: 10.1038/srep39011

    Effect of CHA on macrophage marker expression induced by interleukin (IL)-4. Ana-1 and RAW264.7 cells were treated with interleukin (IL)-4 (20 ng/ml) in the presence of DMSO or different concentrations of CHA for 24 h. The mRNA levels of M1-marker gene iNOS and M2-marker gene Arg in Ana-1 ( A ) and RAW264.7 cells ( B ) were measured by real-time RT-PCR. The expression of mRNAs was normalized to GAPDH. The expressions of CD206 in Ana-1 ( C ) and RAW264.7 cells ( D ) were evaluated by flow cytometry. The histogram bars represent three independent experiments. The data are presented as the mean ± SD. *p-value
    Figure Legend Snippet: Effect of CHA on macrophage marker expression induced by interleukin (IL)-4. Ana-1 and RAW264.7 cells were treated with interleukin (IL)-4 (20 ng/ml) in the presence of DMSO or different concentrations of CHA for 24 h. The mRNA levels of M1-marker gene iNOS and M2-marker gene Arg in Ana-1 ( A ) and RAW264.7 cells ( B ) were measured by real-time RT-PCR. The expression of mRNAs was normalized to GAPDH. The expressions of CD206 in Ana-1 ( C ) and RAW264.7 cells ( D ) were evaluated by flow cytometry. The histogram bars represent three independent experiments. The data are presented as the mean ± SD. *p-value

    Techniques Used: Marker, Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry

    Effect of CHA on the STAT signaling pathways in macrophages. Ana-1 ( A ) and RAW264.7 ( B ) cells were treated with lipopolysaccharide (LPS) (10 ng/ml) and interferon (IFN)-γ (20 ng/ml) with or without different concentrations of CHA for 24 h. The expressions of Ser727-phosphorylated STAT1 (p-STAT1 ser727 ), Tyr701-phosphorylated STAT1 (p-STAT1 Tyr701 ) and STAT1were evaluated by a Western blot analysis. Ana-1 ( C ) and RAW264.7 ( D ) cells were treated with interleukin (IL)-4 (20 ng/ml) in the presence of DMSO or indicated concentrations of CHA for 24 h. The expressions of Tyr641-phosphorylated STAT6 (p-STAT6 Tyr641 ) and STAT6 were evaluated by a Western blot analysis. The histogram bars represent three independent experiments. The data are presented as the mean ± SD. # p-value
    Figure Legend Snippet: Effect of CHA on the STAT signaling pathways in macrophages. Ana-1 ( A ) and RAW264.7 ( B ) cells were treated with lipopolysaccharide (LPS) (10 ng/ml) and interferon (IFN)-γ (20 ng/ml) with or without different concentrations of CHA for 24 h. The expressions of Ser727-phosphorylated STAT1 (p-STAT1 ser727 ), Tyr701-phosphorylated STAT1 (p-STAT1 Tyr701 ) and STAT1were evaluated by a Western blot analysis. Ana-1 ( C ) and RAW264.7 ( D ) cells were treated with interleukin (IL)-4 (20 ng/ml) in the presence of DMSO or indicated concentrations of CHA for 24 h. The expressions of Tyr641-phosphorylated STAT6 (p-STAT6 Tyr641 ) and STAT6 were evaluated by a Western blot analysis. The histogram bars represent three independent experiments. The data are presented as the mean ± SD. # p-value

    Techniques Used: Western Blot

    Effect of CHA on tumor cells growth in co-culture of tumor cells with macrophages. Tumor cells (1 × 10 3 ) were mono-cultured in lipopolysaccharide (LPS) (10 ng/ml) and interferon (IFN)-γ (20 ng/ml) (M1 stimulator) or interleukin (IL)-4 (20 ng/ml) (M2 stimulator) alone, or in combination with indicated concentration of CHA for 48 h, or were co-cultured with M1 stimulator or M2 stimulator-treated macrophages in the presence of CHA for 48 h. U87-RFP-Luc glioma cells were co-cultured with RAW264.7 ( A ) or Ana-1 ( D ) cells, and then tumor cellular morphology was visualized by fluorescence microscopy. The proliferation of U87-RFP-Luc cell was represented as fluorescent area ( B , E ) or overall photon counts of cells ( C , F ) by using software of ImageProPlus and EnSpire Multimode Plate Reader, respectively. MFC-GFP forestomach cancer cells were co-cultured with RAW264.7 ( G ) or Ana-1 ( I ) cells. The morphology of MFC-GFP cells were visualized by fluorescence microscopy and the proliferation of MFC-GFP cells were assessed by overall fluorescent area of cells using ImageProPlus software ( H , J ). The histogram bars represent three independent experiments. The data are presented as the mean ± SD. *p-value
    Figure Legend Snippet: Effect of CHA on tumor cells growth in co-culture of tumor cells with macrophages. Tumor cells (1 × 10 3 ) were mono-cultured in lipopolysaccharide (LPS) (10 ng/ml) and interferon (IFN)-γ (20 ng/ml) (M1 stimulator) or interleukin (IL)-4 (20 ng/ml) (M2 stimulator) alone, or in combination with indicated concentration of CHA for 48 h, or were co-cultured with M1 stimulator or M2 stimulator-treated macrophages in the presence of CHA for 48 h. U87-RFP-Luc glioma cells were co-cultured with RAW264.7 ( A ) or Ana-1 ( D ) cells, and then tumor cellular morphology was visualized by fluorescence microscopy. The proliferation of U87-RFP-Luc cell was represented as fluorescent area ( B , E ) or overall photon counts of cells ( C , F ) by using software of ImageProPlus and EnSpire Multimode Plate Reader, respectively. MFC-GFP forestomach cancer cells were co-cultured with RAW264.7 ( G ) or Ana-1 ( I ) cells. The morphology of MFC-GFP cells were visualized by fluorescence microscopy and the proliferation of MFC-GFP cells were assessed by overall fluorescent area of cells using ImageProPlus software ( H , J ). The histogram bars represent three independent experiments. The data are presented as the mean ± SD. *p-value

    Techniques Used: Co-Culture Assay, Cell Culture, Concentration Assay, Fluorescence, Microscopy, Software

    5) Product Images from "Down-Regulated FOXO1 in Refractory/Relapse Childhood B-Cell Acute Lymphoblastic Leukemia"

    Article Title: Down-Regulated FOXO1 in Refractory/Relapse Childhood B-Cell Acute Lymphoblastic Leukemia

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2020.579673

    Identification of oncogenic potential of MEIS1–FOXO1 fusion gene. (A) Schematic DNA representation of novel MEIS1–FOXO1 rearrangement (red, MEIS1 ; blue, FOXO1 ). (B) Fusion protein representation of novel MEIS1–FOXO1 rearrangement (red box, MEIS1 PKNOX N domain; pink box, homeobox KN domain; dark blue box, forkhead domain; light blue box, KIX-binding domain; gray blue box, transactivation domain). (C) MEIS1–FOXO1 potentiated leukemia transformation in Ba/F3 cell model. Effects of MEIS1 – FOXO1 fusion genes on Ba/F3 transformation. Following transduction of empty vector, MEIS1 – FOXO1, NRAS G 12 D , or combination, Ba/F3 cells were cultured in IL-3 depleted medium with cytokine-independent cell growth as a measure of oncogenic transformation. Number of viable cells was evaluated daily. (D) MEIS1–FOXO1 fusion genes and proliferation of mouse hematopoietic progenitor cell Ba/F3. Ba/F3 cells were lentivirally transduced with empty vector (black), NRAS G 12 D (blue), MEIS1–FOXO1 (green), or combination of NRAS G 12 D and MEIS1–FOXO1 (red) and then cultured in the presence of IL-3 (10 ng/ml). After 48 h, cell cycle distribution was evaluated using standard PI staining protocol. Statistical significance, determined using the two-sided unpaired t -test, is indicated by ** P
    Figure Legend Snippet: Identification of oncogenic potential of MEIS1–FOXO1 fusion gene. (A) Schematic DNA representation of novel MEIS1–FOXO1 rearrangement (red, MEIS1 ; blue, FOXO1 ). (B) Fusion protein representation of novel MEIS1–FOXO1 rearrangement (red box, MEIS1 PKNOX N domain; pink box, homeobox KN domain; dark blue box, forkhead domain; light blue box, KIX-binding domain; gray blue box, transactivation domain). (C) MEIS1–FOXO1 potentiated leukemia transformation in Ba/F3 cell model. Effects of MEIS1 – FOXO1 fusion genes on Ba/F3 transformation. Following transduction of empty vector, MEIS1 – FOXO1, NRAS G 12 D , or combination, Ba/F3 cells were cultured in IL-3 depleted medium with cytokine-independent cell growth as a measure of oncogenic transformation. Number of viable cells was evaluated daily. (D) MEIS1–FOXO1 fusion genes and proliferation of mouse hematopoietic progenitor cell Ba/F3. Ba/F3 cells were lentivirally transduced with empty vector (black), NRAS G 12 D (blue), MEIS1–FOXO1 (green), or combination of NRAS G 12 D and MEIS1–FOXO1 (red) and then cultured in the presence of IL-3 (10 ng/ml). After 48 h, cell cycle distribution was evaluated using standard PI staining protocol. Statistical significance, determined using the two-sided unpaired t -test, is indicated by ** P

    Techniques Used: Binding Assay, Transformation Assay, Transduction, Plasmid Preparation, Cell Culture, Staining

    6) Product Images from "Triptolide, a constituent of immunosuppressive Chinese herbal medicine, is a potent suppressor of dendritic-cell maturation and trafficking"

    Article Title: Triptolide, a constituent of immunosuppressive Chinese herbal medicine, is a potent suppressor of dendritic-cell maturation and trafficking

    Journal:

    doi: 10.1182/blood-2005-03-0854

    Effects of TPT on human monocyte–derived DCs. (A-B) Human monocytes were cultured with 50 ng/mL of GM-CSF and 50 ng/mL of IL-4 (G4 medium). After 24 hours, the medium was replaced with fresh G4 medium in the presence or absence of desired concentrations
    Figure Legend Snippet: Effects of TPT on human monocyte–derived DCs. (A-B) Human monocytes were cultured with 50 ng/mL of GM-CSF and 50 ng/mL of IL-4 (G4 medium). After 24 hours, the medium was replaced with fresh G4 medium in the presence or absence of desired concentrations

    Techniques Used: Derivative Assay, Cell Culture

    Inhibition of maturation of DCs by TPT. (A) Human monocytes were cultured for 7 days with 50 ng/mL GM-CSF and 50 ng/mL IL-4 in the presence of TPT (D2-7, 2.5 nM; TPT-DC) or medium alone (Control-DC). Cells were stained with the designated mAb and analyzed
    Figure Legend Snippet: Inhibition of maturation of DCs by TPT. (A) Human monocytes were cultured for 7 days with 50 ng/mL GM-CSF and 50 ng/mL IL-4 in the presence of TPT (D2-7, 2.5 nM; TPT-DC) or medium alone (Control-DC). Cells were stained with the designated mAb and analyzed

    Techniques Used: Inhibition, Cell Culture, Staining

    Inhibition of TPT on the allostimulatory activity of DCs and LPS-stimulated DCs. (A) Human monocytes were cultured with 50 ng/mL GM-CSF and 50 ng/mL IL-4 in the presence of TPT (TPT[D2-7], 2.5 nM) or medium alone (control). After 7 days, DCs were extensively
    Figure Legend Snippet: Inhibition of TPT on the allostimulatory activity of DCs and LPS-stimulated DCs. (A) Human monocytes were cultured with 50 ng/mL GM-CSF and 50 ng/mL IL-4 in the presence of TPT (TPT[D2-7], 2.5 nM) or medium alone (control). After 7 days, DCs were extensively

    Techniques Used: Inhibition, Activity Assay, Cell Culture

    7) Product Images from "Discrimination of the heterogeneity of bone marrow-derived dendritic cells"

    Article Title: Discrimination of the heterogeneity of bone marrow-derived dendritic cells

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2017.7448

    Cytokine secretion levels in the non-adherent, adherent and mixed cells throughout the dendritic cell culture process. On days 3, 6 and 8, cell subsets (7×10 6 cells) were harvested, reseeded following an overnight incubation, and the cell suspension was collected to quantify the cytokine levels of IL-2, IL-12p70, IFN-γ, IL-4 and IL-10 using sandwich enzyme-linked immunosorbent assay kits. IFN, interferon; IL, interleukin.
    Figure Legend Snippet: Cytokine secretion levels in the non-adherent, adherent and mixed cells throughout the dendritic cell culture process. On days 3, 6 and 8, cell subsets (7×10 6 cells) were harvested, reseeded following an overnight incubation, and the cell suspension was collected to quantify the cytokine levels of IL-2, IL-12p70, IFN-γ, IL-4 and IL-10 using sandwich enzyme-linked immunosorbent assay kits. IFN, interferon; IL, interleukin.

    Techniques Used: Cell Culture, Incubation, Sandwich ELISA

    8) Product Images from "Nocardia brasiliensis Induces Formation of Foamy Macrophages and Dendritic Cells In Vitro and In Vivo"

    Article Title: Nocardia brasiliensis Induces Formation of Foamy Macrophages and Dendritic Cells In Vitro and In Vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0100064

    Morphology and immunophenotype of bone marrow-derived macrophages (BMDM) and dendritic cells (BMDC). Five-day cultures of BMDM generated with 30% L929-conditioned medium (A) or BMDC generated with 20 ng/mL GM-CSF and IL-4 (B); images were taken at 200x+digital zoom. Over 98% of the BMDM were F4/80+ (C), but less than 4.5% of the macrophage-depleted BMDC were F4/80+ (D).
    Figure Legend Snippet: Morphology and immunophenotype of bone marrow-derived macrophages (BMDM) and dendritic cells (BMDC). Five-day cultures of BMDM generated with 30% L929-conditioned medium (A) or BMDC generated with 20 ng/mL GM-CSF and IL-4 (B); images were taken at 200x+digital zoom. Over 98% of the BMDM were F4/80+ (C), but less than 4.5% of the macrophage-depleted BMDC were F4/80+ (D).

    Techniques Used: Derivative Assay, Generated

    9) Product Images from "TRAIL-functionalized gold nanoparticles selectively trigger apoptosis in polarized macrophages"

    Article Title: TRAIL-functionalized gold nanoparticles selectively trigger apoptosis in polarized macrophages

    Journal: Nanotheranostics

    doi: 10.7150/ntno.20233

    Characterization of polarized macrophages. (A) The morphologies of polarized macrophages after incubation with LPS/IFN-γ or IL-4/IL-13 for 48 h. The scale bar represents 20 μm. (B) The protein expressions of CD markers for M1 macrophages or M2 macrophages after incubation with LPS/IFN-γ or IL-4/IL-13 for 48 h. (C, D) The protein expressions of cytokines for M1 macrophages or M2 macrophages after incubation with LPS/IFN-γ or IL-4/IL-13 for 48 h. * p
    Figure Legend Snippet: Characterization of polarized macrophages. (A) The morphologies of polarized macrophages after incubation with LPS/IFN-γ or IL-4/IL-13 for 48 h. The scale bar represents 20 μm. (B) The protein expressions of CD markers for M1 macrophages or M2 macrophages after incubation with LPS/IFN-γ or IL-4/IL-13 for 48 h. (C, D) The protein expressions of cytokines for M1 macrophages or M2 macrophages after incubation with LPS/IFN-γ or IL-4/IL-13 for 48 h. * p

    Techniques Used: Incubation

    10) Product Images from "Niacin Promotes Cardiac Healing after Myocardial Infarction through Activation of the Myeloid Prostaglandin D2"

    Article Title: Niacin Promotes Cardiac Healing after Myocardial Infarction through Activation of the Myeloid Prostaglandin D2

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    doi: 10.1124/jpet.116.238261

    Niacin stimulates M2 macrophage polarization in vitro. (A) Cultured primary peritoneal mouse macrophages were treated with LPS/IFN γ or IL-4 for 24 hours after washing, then the cells were exposed to niacin (3 mM) for 30 minutes, the supernatants were collected, and PGD 2 was analyzed by mass spectrometry. * P
    Figure Legend Snippet: Niacin stimulates M2 macrophage polarization in vitro. (A) Cultured primary peritoneal mouse macrophages were treated with LPS/IFN γ or IL-4 for 24 hours after washing, then the cells were exposed to niacin (3 mM) for 30 minutes, the supernatants were collected, and PGD 2 was analyzed by mass spectrometry. * P

    Techniques Used: In Vitro, Cell Culture, Mass Spectrometry

    11) Product Images from "Maturation of dendritic cells in vitro and immunological enhancement of mice in vivo by pachyman- and/or OVA-encapsulated poly(d,l-lactic acid) nanospheres"

    Article Title: Maturation of dendritic cells in vitro and immunological enhancement of mice in vivo by pachyman- and/or OVA-encapsulated poly(d,l-lactic acid) nanospheres

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S153567

    Cytokine secretion levels in splenocytes. Notes: Splenocytes were restimulated with OVA (50 µg mL −1 ) and harvested 7 days after the second immunization. IFN-γ ( A ), IL-2 ( B ), IL-4 ( C ), and IL-6 ( D ) levels in the supernatant were measured by ELISA. ## p
    Figure Legend Snippet: Cytokine secretion levels in splenocytes. Notes: Splenocytes were restimulated with OVA (50 µg mL −1 ) and harvested 7 days after the second immunization. IFN-γ ( A ), IL-2 ( B ), IL-4 ( C ), and IL-6 ( D ) levels in the supernatant were measured by ELISA. ## p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    12) Product Images from "Trem2 promotes anti-inflammatory responses in microglia and is suppressed under pro-inflammatory conditions"

    Article Title: Trem2 promotes anti-inflammatory responses in microglia and is suppressed under pro-inflammatory conditions

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddaa209

    LPS suppression of Trem2 in primary microglia. ( A ) Left: primary microglia stained with an antibody against IBA1 and DAPI. Right: a higher-magnification image showing the morphology of microglia in vitro . ( B ) Pro- and anti-inflammatory gene expression changes in primary microglia with either LPS or IL-4 treatment. N = 3–6 independent experiments. Data shown as mean ± SEM. One-way ANOVA showed a significant effect of treatment for Tnf , Il1b and Tgfb1 . Arg1 was not detected under control conditions, and so Student’s t -test was used to test pairwise significance between 24 and 48 h of IL-4 treatment; * P
    Figure Legend Snippet: LPS suppression of Trem2 in primary microglia. ( A ) Left: primary microglia stained with an antibody against IBA1 and DAPI. Right: a higher-magnification image showing the morphology of microglia in vitro . ( B ) Pro- and anti-inflammatory gene expression changes in primary microglia with either LPS or IL-4 treatment. N = 3–6 independent experiments. Data shown as mean ± SEM. One-way ANOVA showed a significant effect of treatment for Tnf , Il1b and Tgfb1 . Arg1 was not detected under control conditions, and so Student’s t -test was used to test pairwise significance between 24 and 48 h of IL-4 treatment; * P

    Techniques Used: Staining, In Vitro, Expressing

    Cluster of genes showing the highest connectivity to Arg1 from the genetic network associated with IL-4. ( A ) Heat map showing the genes with the highest connectivity to Arg1 from the co-expression network associated with IL-4 (TOM > 0.465). Selection of genes presented with expression tested by two-way ANOVA, all genes showing a significant main effect of IL-4-treatment and genotype, and a significant interaction. The expression of these genes is represented in primary microglia from HO- Trem2 R47H KI versus WT mice treated with IL-4 or untreated. Colors represent the z -score of the expression level for each gene (red is high expression and blue is low expression). ( B ) The promoters of genes that show the highest connectivity to Arg1 (TOM > 0.465) contain an enrichment of consensus binding sites for a number of transcription factors including SPI1/PU.1 and STAT6 determined using i- cis Target. Consensus matrices for SPI1/PU.1 and STAT6 are shown. N = 3 mice per genotype per condition.
    Figure Legend Snippet: Cluster of genes showing the highest connectivity to Arg1 from the genetic network associated with IL-4. ( A ) Heat map showing the genes with the highest connectivity to Arg1 from the co-expression network associated with IL-4 (TOM > 0.465). Selection of genes presented with expression tested by two-way ANOVA, all genes showing a significant main effect of IL-4-treatment and genotype, and a significant interaction. The expression of these genes is represented in primary microglia from HO- Trem2 R47H KI versus WT mice treated with IL-4 or untreated. Colors represent the z -score of the expression level for each gene (red is high expression and blue is low expression). ( B ) The promoters of genes that show the highest connectivity to Arg1 (TOM > 0.465) contain an enrichment of consensus binding sites for a number of transcription factors including SPI1/PU.1 and STAT6 determined using i- cis Target. Consensus matrices for SPI1/PU.1 and STAT6 are shown. N = 3 mice per genotype per condition.

    Techniques Used: Expressing, Selection, Mouse Assay, Binding Assay

    Trem2 knockdown resulted in significantly decreased total STAT6 levels and AKT phosphorylation in primary microglia in vitro . ( A and B ) Total and phosphorylated STAT6 protein levels in primary microglia treated with IL-4 and Trem2 siRNA were analyzed with western blot. N = 4 independent experiments. ( C ) Stat6 gene expression levels in primary microglia, assessed by RT-qPCR ( N = 3 independent experiments). ( D and E ) Total and phosphorylated (at Thr308 and Ser473) AKT protein levels analyzed by western blot. N = 4 independent experiments. Protein levels were normalized to beta-actin and gene expression was normalized to Rps28 . Fold change was calculated relative to the negative control without IL-4 treatment in each individual culture preparation. Note that phosphorylated STAT6 was not detectable under control conditions and so under IL-4 conditions the effect of Trem2 knockdown was calculated as fold change relative to control cells. Data were shown as mean ± SEM. With the exception of phosphorylated STAT6, data were analyzed by two-way ANOVA; significant main effects of IL-4 treatment and Trem2 -knockdown indicated by horizontal and vertical lines respectively, no significant interactions were seen between IL-4 treatment and Trem2 knockdown. Phosphorylated STAT6 was analyzed by a one-sample t -test. * P
    Figure Legend Snippet: Trem2 knockdown resulted in significantly decreased total STAT6 levels and AKT phosphorylation in primary microglia in vitro . ( A and B ) Total and phosphorylated STAT6 protein levels in primary microglia treated with IL-4 and Trem2 siRNA were analyzed with western blot. N = 4 independent experiments. ( C ) Stat6 gene expression levels in primary microglia, assessed by RT-qPCR ( N = 3 independent experiments). ( D and E ) Total and phosphorylated (at Thr308 and Ser473) AKT protein levels analyzed by western blot. N = 4 independent experiments. Protein levels were normalized to beta-actin and gene expression was normalized to Rps28 . Fold change was calculated relative to the negative control without IL-4 treatment in each individual culture preparation. Note that phosphorylated STAT6 was not detectable under control conditions and so under IL-4 conditions the effect of Trem2 knockdown was calculated as fold change relative to control cells. Data were shown as mean ± SEM. With the exception of phosphorylated STAT6, data were analyzed by two-way ANOVA; significant main effects of IL-4 treatment and Trem2 -knockdown indicated by horizontal and vertical lines respectively, no significant interactions were seen between IL-4 treatment and Trem2 knockdown. Phosphorylated STAT6 was analyzed by a one-sample t -test. * P

    Techniques Used: In Vitro, Western Blot, Expressing, Quantitative RT-PCR, Negative Control

    The in vitro primary microglial models. Schedule of primary microglia culture from WT or Trem2 R47H KI mice, and treatment with Trem2 siRNA, control non-targeting siRNA, LPS or IL-4.
    Figure Legend Snippet: The in vitro primary microglial models. Schedule of primary microglia culture from WT or Trem2 R47H KI mice, and treatment with Trem2 siRNA, control non-targeting siRNA, LPS or IL-4.

    Techniques Used: In Vitro, Mouse Assay

    Primary microglia from Trem2 R47H KI mice show decreased Trem2 expression, and an impaired IL-4-induced anti-inflammatory response. ( A ) Expression of Trem2 . ( B ) Expression of anti-inflammatory markers, Arg1 and Tgf1b . ( C ) Expression of other microglial genes . Gene expression was normalized to Rps28 and calculated as fold change relative to the WT control without IL-4 treatment. N = 8–15 mice per genotype. Data shown as mean ± SEM. Two-way ANOVA (for all genes except Arg1 ). Significant main effect of IL-4-treatment and genotype indicated as horizontal and vertical lines respectively, no significant interactions were seen between IL-4-treatment and Trem2 knockdown. Sidak’s post hoc tests were performed to test pairwise significance between the three genotypes within a treatment group. Arg1 was analyzed by one-way ANOVA ( P = 0.002) followed by Sidak’s multiple comparisons to test pairwise significance between the three genotypes within a treatment group. * P
    Figure Legend Snippet: Primary microglia from Trem2 R47H KI mice show decreased Trem2 expression, and an impaired IL-4-induced anti-inflammatory response. ( A ) Expression of Trem2 . ( B ) Expression of anti-inflammatory markers, Arg1 and Tgf1b . ( C ) Expression of other microglial genes . Gene expression was normalized to Rps28 and calculated as fold change relative to the WT control without IL-4 treatment. N = 8–15 mice per genotype. Data shown as mean ± SEM. Two-way ANOVA (for all genes except Arg1 ). Significant main effect of IL-4-treatment and genotype indicated as horizontal and vertical lines respectively, no significant interactions were seen between IL-4-treatment and Trem2 knockdown. Sidak’s post hoc tests were performed to test pairwise significance between the three genotypes within a treatment group. Arg1 was analyzed by one-way ANOVA ( P = 0.002) followed by Sidak’s multiple comparisons to test pairwise significance between the three genotypes within a treatment group. * P

    Techniques Used: Mouse Assay, Expressing

    Trem2 knockdown impairs the IL-4-induced anti-inflammatory response of primary microglia. ( A ) Trem2 gene expression was not significantly influenced by IL-4 stimulation. ( B ) Arg1 and Tgfb1 expression, as markers of the anti-inflammatory response, showed significant up-regulation with time after IL-4 application. Particularly, Trem2 knockdown greatly decreased IL-4-induced Arg1 expression compared with negative controls. Gene expression levels were normalized to Rps28 and calculated as fold change relative to the negative control without IL-4 treatment in each individual culture preparation, except for Arg1 . Two-way ANOVA with significant main effect of IL-4 incubation time and Trem2 knockdown indicated as horizontal and vertical lines respectively. A significant interaction between IL-4 treatment length and Trem2 knockdown was seen only in Arg1 expression (B), and so Sidak’s post hoc tests were performed to test pairwise significance between the negative siRNA control and Trem2 knockdown at each time-point. ( C ) Expression of the pro-inflammatory genes ( Tnf and Il1b) and other microglial genes. Gene expression levels were normalized to Rps28 and calculated as fold change relative to the negative control without IL-4 treatment in each individual culture preparation. N = 7–9 independent experiments. Data shown as mean ± SEM. Two-way ANOVA; significant main effects of IL-4 treatment time and Trem2 -knockdown indicated by horizontal and vertical lines respectively, no significant interactions were seen between IL-4 treatment and Trem2 knockdown; * P
    Figure Legend Snippet: Trem2 knockdown impairs the IL-4-induced anti-inflammatory response of primary microglia. ( A ) Trem2 gene expression was not significantly influenced by IL-4 stimulation. ( B ) Arg1 and Tgfb1 expression, as markers of the anti-inflammatory response, showed significant up-regulation with time after IL-4 application. Particularly, Trem2 knockdown greatly decreased IL-4-induced Arg1 expression compared with negative controls. Gene expression levels were normalized to Rps28 and calculated as fold change relative to the negative control without IL-4 treatment in each individual culture preparation, except for Arg1 . Two-way ANOVA with significant main effect of IL-4 incubation time and Trem2 knockdown indicated as horizontal and vertical lines respectively. A significant interaction between IL-4 treatment length and Trem2 knockdown was seen only in Arg1 expression (B), and so Sidak’s post hoc tests were performed to test pairwise significance between the negative siRNA control and Trem2 knockdown at each time-point. ( C ) Expression of the pro-inflammatory genes ( Tnf and Il1b) and other microglial genes. Gene expression levels were normalized to Rps28 and calculated as fold change relative to the negative control without IL-4 treatment in each individual culture preparation. N = 7–9 independent experiments. Data shown as mean ± SEM. Two-way ANOVA; significant main effects of IL-4 treatment time and Trem2 -knockdown indicated by horizontal and vertical lines respectively, no significant interactions were seen between IL-4 treatment and Trem2 knockdown; * P

    Techniques Used: Expressing, Negative Control, Incubation

    Gene expression profile in primary microglia from Trem2 R47H KI and WT mice with IL-4 treatment by RNA-seq. ( A ) Volcano plot showing differentially expressed genes for microglia from HO- Trem2 R47H KI versus WT mice by DESeq2 (FDR
    Figure Legend Snippet: Gene expression profile in primary microglia from Trem2 R47H KI and WT mice with IL-4 treatment by RNA-seq. ( A ) Volcano plot showing differentially expressed genes for microglia from HO- Trem2 R47H KI versus WT mice by DESeq2 (FDR

    Techniques Used: Expressing, Mouse Assay, RNA Sequencing Assay

    Proposed model of the involvement of TREM2 in the IL-4-induced anti-inflammatory response . LPS induces pro-inflammatory activation of microglia, which is accompanied by down-regulated Trem2 expression, and so microglia with reduced TREM2 activity generally show normal activation in response to the pro-inflammatory LPS stimulus. Under non-stimulated conditions, a number of microglial genes are down-regulated when TREM2 activity is decreased, such as Csf1r expression, which may result in the seen reduced survival of microglia. However, IL-4 induces an anti-inflammatory phenotype of microglia. As our data suggest, TREM2 signaling is involved in maintenance of microglial STAT6 levels, which in the IL-4 pathway is phosphorylated and translocates to the nucleus and functions as a key transcription factor for IL-4-induced gene expression changes, such as for Arg1 and Ap1b1 . Thus, Trem2 deficiency results in decreased STAT6 levels in microglia, which leads to an impairment of IL-4-induced signaling. TREM2 also regulates the levels of a number of genes that are up-regulated by IL-4 (suppressed by LPS), and considered a component of the transcriptional response to AD-associated pathology, such as Igf1 , C1qa , Itgax and Clec7a .
    Figure Legend Snippet: Proposed model of the involvement of TREM2 in the IL-4-induced anti-inflammatory response . LPS induces pro-inflammatory activation of microglia, which is accompanied by down-regulated Trem2 expression, and so microglia with reduced TREM2 activity generally show normal activation in response to the pro-inflammatory LPS stimulus. Under non-stimulated conditions, a number of microglial genes are down-regulated when TREM2 activity is decreased, such as Csf1r expression, which may result in the seen reduced survival of microglia. However, IL-4 induces an anti-inflammatory phenotype of microglia. As our data suggest, TREM2 signaling is involved in maintenance of microglial STAT6 levels, which in the IL-4 pathway is phosphorylated and translocates to the nucleus and functions as a key transcription factor for IL-4-induced gene expression changes, such as for Arg1 and Ap1b1 . Thus, Trem2 deficiency results in decreased STAT6 levels in microglia, which leads to an impairment of IL-4-induced signaling. TREM2 also regulates the levels of a number of genes that are up-regulated by IL-4 (suppressed by LPS), and considered a component of the transcriptional response to AD-associated pathology, such as Igf1 , C1qa , Itgax and Clec7a .

    Techniques Used: Activation Assay, Expressing, Activity Assay

    Co-expression genetic networks associated with Trem2 expression and IL-4-treatment. ( A ) Co-expression network associated with Trem2 expression, using RNA-seq derived gene expression from primary microglia of Trem2 R47H KI and WT mice. Network contains key genes mediating microglial functions, and includes Trem2 , Spi1 /PU.1, Tyrobp and Stat6 . Larger green spheres represent ‘hub’ genes, those showing the greatest number of connections to other genes in the network, which are likely to play important roles in driving microglial functions alongside Trem2 . ( B ) Co-expression network associated with IL-4-treatment, using RNA-seq derived gene expression from primary microglia treated with IL-4 and untreated. Network includes key genes involved in anti-inflammatory processes, such Arg1 , Retnla and Chil3 . Larger red spheres represent ‘hub’ genes, which are likely to play important roles in driving the microglial response to IL-4. N = 3 mice per genotype per condition.
    Figure Legend Snippet: Co-expression genetic networks associated with Trem2 expression and IL-4-treatment. ( A ) Co-expression network associated with Trem2 expression, using RNA-seq derived gene expression from primary microglia of Trem2 R47H KI and WT mice. Network contains key genes mediating microglial functions, and includes Trem2 , Spi1 /PU.1, Tyrobp and Stat6 . Larger green spheres represent ‘hub’ genes, those showing the greatest number of connections to other genes in the network, which are likely to play important roles in driving microglial functions alongside Trem2 . ( B ) Co-expression network associated with IL-4-treatment, using RNA-seq derived gene expression from primary microglia treated with IL-4 and untreated. Network includes key genes involved in anti-inflammatory processes, such Arg1 , Retnla and Chil3 . Larger red spheres represent ‘hub’ genes, which are likely to play important roles in driving the microglial response to IL-4. N = 3 mice per genotype per condition.

    Techniques Used: Expressing, RNA Sequencing Assay, Derivative Assay, Mouse Assay

    13) Product Images from "Indolamine 2,3-dioxygenase expression by monocytes and dendritic cell populations in hepatitis C patients"

    Article Title: Indolamine 2,3-dioxygenase expression by monocytes and dendritic cell populations in hepatitis C patients

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/cei.12586

    Monocytes, isolated from healthy individuals, were transduced with JFH-1 T for 48 h, and then treated with granulocyte–macrophage colony-stimulating growth factor (GM-CSF) and interleukin (IL)-4, in the absence or presence of levo-1-methyl tryptophan
    Figure Legend Snippet: Monocytes, isolated from healthy individuals, were transduced with JFH-1 T for 48 h, and then treated with granulocyte–macrophage colony-stimulating growth factor (GM-CSF) and interleukin (IL)-4, in the absence or presence of levo-1-methyl tryptophan

    Techniques Used: Isolation, Transduction

    14) Product Images from "8,9-Epoxyeicosatrienoic Acid Inhibits Antibody Production of B Lymphocytes in Mice"

    Article Title: 8,9-Epoxyeicosatrienoic Acid Inhibits Antibody Production of B Lymphocytes in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0040258

    8,9-EET inhibited plasma-cell differentiation of B cells. A. Splenic B cells were stimulated with 5 µg/ml LPS and 50 ng/ml IL-4 with or without 8,9-EET (1 µM) for 3 days, then CD138 expression was analyzed by flow cytometry. B. Quantification of 3 independent experiments of plasma cell differentiation. Quantification of real time PCR and western blot analysis of mRNA (C) and protein (D) expressions of IRF-4 and XBP-1 in B cells cultured with 8,9-EET, 5 µg/ml LPS and 50 ng/ml IL-4 for 3 days. GAPDH was used as the control. Data are means ± SEM of three independent experiments. *, P
    Figure Legend Snippet: 8,9-EET inhibited plasma-cell differentiation of B cells. A. Splenic B cells were stimulated with 5 µg/ml LPS and 50 ng/ml IL-4 with or without 8,9-EET (1 µM) for 3 days, then CD138 expression was analyzed by flow cytometry. B. Quantification of 3 independent experiments of plasma cell differentiation. Quantification of real time PCR and western blot analysis of mRNA (C) and protein (D) expressions of IRF-4 and XBP-1 in B cells cultured with 8,9-EET, 5 µg/ml LPS and 50 ng/ml IL-4 for 3 days. GAPDH was used as the control. Data are means ± SEM of three independent experiments. *, P

    Techniques Used: Cell Differentiation, Expressing, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture

    8,9-EET inhibited class switch recombination of B cells. A. Splenic B cells were stimulated with 5 µg/ml LPS and 50 ng/ml IL-4 with or without 8,9-EET (1 µM) for 3 days. Flow cytometry of surface IgG 1 (sIgG 1 ) expression in cells. B. Quantification of 3 independent experiments of sIgG 1 expression in B cells. C. Real-time RT-PCR analysis of mRNA levels of AICDA, Iγ 1 -Cγ 1 , Iμ-Cγ 1 , Iγ 2b -Cγ 2b and Iμ-Cγ 2b in 3-day culture. GAPDH was used as the control. Data are means ± SEM of three independent experiments. *, P
    Figure Legend Snippet: 8,9-EET inhibited class switch recombination of B cells. A. Splenic B cells were stimulated with 5 µg/ml LPS and 50 ng/ml IL-4 with or without 8,9-EET (1 µM) for 3 days. Flow cytometry of surface IgG 1 (sIgG 1 ) expression in cells. B. Quantification of 3 independent experiments of sIgG 1 expression in B cells. C. Real-time RT-PCR analysis of mRNA levels of AICDA, Iγ 1 -Cγ 1 , Iμ-Cγ 1 , Iγ 2b -Cγ 2b and Iμ-Cγ 2b in 3-day culture. GAPDH was used as the control. Data are means ± SEM of three independent experiments. *, P

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Quantitative RT-PCR

    8,9-epoxyeicosatrienoic acid (EET) inhibited B-cell antibody production in C57BL/6 and ApoE−/− mice. ELISA of levels of IgM and IgG (A and B) in the supernatant of cultured B cells from C57BL/6 mice after incubation for 3 days with 1 µM EETs plus 5 µg/ml lipopolysaccharide (LPS) with or without 50 ng/ml IL-4. ELISA of levels of IgM (C and E) and IgG (D and F) in the supernatant of cultured B cells from C57BL/6 mice with indicated doses of 8,9-EET (C and D) or with 1 µM 8,9-EET for the indicated times (E and F) with 5 µg/ml LPS with or without 50 ng/ml IL-4. (G and H) ELISA of production of IgM and IgG by B cells from ApoE−/− mice and age- and sex-matched C57BL/6 control mice after incubation for 3 days with the indicated doses of LPS with or without IL-4 (50 ng/ml). *, P
    Figure Legend Snippet: 8,9-epoxyeicosatrienoic acid (EET) inhibited B-cell antibody production in C57BL/6 and ApoE−/− mice. ELISA of levels of IgM and IgG (A and B) in the supernatant of cultured B cells from C57BL/6 mice after incubation for 3 days with 1 µM EETs plus 5 µg/ml lipopolysaccharide (LPS) with or without 50 ng/ml IL-4. ELISA of levels of IgM (C and E) and IgG (D and F) in the supernatant of cultured B cells from C57BL/6 mice with indicated doses of 8,9-EET (C and D) or with 1 µM 8,9-EET for the indicated times (E and F) with 5 µg/ml LPS with or without 50 ng/ml IL-4. (G and H) ELISA of production of IgM and IgG by B cells from ApoE−/− mice and age- and sex-matched C57BL/6 control mice after incubation for 3 days with the indicated doses of LPS with or without IL-4 (50 ng/ml). *, P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Incubation

    15) Product Images from "Tissue-Infiltrating Neutrophils Constitute the Major In Vivo Source of Angiogenesis-Inducing MMP-9 in the Tumor Microenvironment"

    Article Title: Tissue-Infiltrating Neutrophils Constitute the Major In Vivo Source of Angiogenesis-Inducing MMP-9 in the Tumor Microenvironment

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2014.08.013

    Re-enforced polarization toward M2 phenotype increases proMMP-9 production and angiogenesis-inducing capacity of LLC TAMs. (A) Forty-eight-hour SF CM from WT LLC TAMs, incubated with IL-4 to “re-enforce” the M2 phenotype or left not treated
    Figure Legend Snippet: Re-enforced polarization toward M2 phenotype increases proMMP-9 production and angiogenesis-inducing capacity of LLC TAMs. (A) Forty-eight-hour SF CM from WT LLC TAMs, incubated with IL-4 to “re-enforce” the M2 phenotype or left not treated

    Techniques Used: Incubation

    16) Product Images from "Intrahepatic cholangiocarcinoma induced M2-polarized tumor-associated macrophages facilitate tumor growth and invasiveness"

    Article Title: Intrahepatic cholangiocarcinoma induced M2-polarized tumor-associated macrophages facilitate tumor growth and invasiveness

    Journal: Cancer Cell International

    doi: 10.1186/s12935-020-01687-w

    ICC cell-induced M2 macrophages polarized from monocytic THP-1 cells. a Schematic of published methods to induce M2-polarized macrophages from THP-1 cells. The left panel shows that THP-1 cells treated with PMA, IL-4 and IL-13 polarized into M2 macrophages. The relative expression of cytokines (CD206, CD163, Arg1, iNOS and IL-6) in M2 macrophages between THP-1 cells and M2 macrophages was analyzed by qRT-PCR. b Schematic of M2-like polarized macrophages induced by ICC cells. After treatment with 200 nM PMA for 24 h, THP-1 cells differentiated into unpolarized (M0) macrophages. M0 macrophages cocultured with ICC cells for 48 h polarized into M2-phenotype macrophages. c Representative images of M0 macrophages and M2 macrophages. d The relative expression of CD206, CD163, Arg1, iNOS and IL-6 in M0 macrophages after coculture with ICC cell lines was analyzed by qRT-PCR. The data are presented as the mean ± SD; * p
    Figure Legend Snippet: ICC cell-induced M2 macrophages polarized from monocytic THP-1 cells. a Schematic of published methods to induce M2-polarized macrophages from THP-1 cells. The left panel shows that THP-1 cells treated with PMA, IL-4 and IL-13 polarized into M2 macrophages. The relative expression of cytokines (CD206, CD163, Arg1, iNOS and IL-6) in M2 macrophages between THP-1 cells and M2 macrophages was analyzed by qRT-PCR. b Schematic of M2-like polarized macrophages induced by ICC cells. After treatment with 200 nM PMA for 24 h, THP-1 cells differentiated into unpolarized (M0) macrophages. M0 macrophages cocultured with ICC cells for 48 h polarized into M2-phenotype macrophages. c Representative images of M0 macrophages and M2 macrophages. d The relative expression of CD206, CD163, Arg1, iNOS and IL-6 in M0 macrophages after coculture with ICC cell lines was analyzed by qRT-PCR. The data are presented as the mean ± SD; * p

    Techniques Used: Immunocytochemistry, Expressing, Quantitative RT-PCR

    17) Product Images from "Targeted delivery of chlorogenic acid by mannosylated liposomes to effectively promote the polarization of TAMs for the treatment of glioblastoma"

    Article Title: Targeted delivery of chlorogenic acid by mannosylated liposomes to effectively promote the polarization of TAMs for the treatment of glioblastoma

    Journal: Bioactive Materials

    doi: 10.1016/j.bioactmat.2020.05.001

    In vitro cellular uptake profile of liposomes in BMDMs. (A) Scheme for the isolation, formation, and stimulation of mouse BMDMs. (B and C) Flow cytometric analysis of purity of cultured BMDMs. (D) Flow cytometric analysis of CD206 expression level of IL-4-treated BMDMs. (E and G) The cellular uptake profile of coumarin-6 labeled liposomes in M2-type BMDMs. (F and H) The cellular uptake profile of coumarin-6 labeled Man-PEG-Lipo in naïve BMDMs (Control) or M2-type BMDMs alone (Man-PEG-Lipo) or co-incubation with excess mannose (Mannose). Each value represents the mean ± SEM (n = 3). *p
    Figure Legend Snippet: In vitro cellular uptake profile of liposomes in BMDMs. (A) Scheme for the isolation, formation, and stimulation of mouse BMDMs. (B and C) Flow cytometric analysis of purity of cultured BMDMs. (D) Flow cytometric analysis of CD206 expression level of IL-4-treated BMDMs. (E and G) The cellular uptake profile of coumarin-6 labeled liposomes in M2-type BMDMs. (F and H) The cellular uptake profile of coumarin-6 labeled Man-PEG-Lipo in naïve BMDMs (Control) or M2-type BMDMs alone (Man-PEG-Lipo) or co-incubation with excess mannose (Mannose). Each value represents the mean ± SEM (n = 3). *p

    Techniques Used: In Vitro, Isolation, Cell Culture, Expressing, Labeling, Incubation

    Polarization of M2-type BMDMs in vitro. (A and B) In vitro cytotoxicity of liposomes in RAW264.7 and BMDMs. Each value represents the mean ± SEM (n = 3). (C and D) CD206 expression level of M2-type BMDMs after incubation with culture medium (Untreated), culture medium containing IL-4 (Control), free CHA containing IL-4 (CHA), and CHA-encapsulated Man-PEG-Lipo containing IL-4 (Man-PEG-Lipo).
    Figure Legend Snippet: Polarization of M2-type BMDMs in vitro. (A and B) In vitro cytotoxicity of liposomes in RAW264.7 and BMDMs. Each value represents the mean ± SEM (n = 3). (C and D) CD206 expression level of M2-type BMDMs after incubation with culture medium (Untreated), culture medium containing IL-4 (Control), free CHA containing IL-4 (CHA), and CHA-encapsulated Man-PEG-Lipo containing IL-4 (Man-PEG-Lipo).

    Techniques Used: In Vitro, Expressing, Incubation

    18) Product Images from "Zoledronic acid impairs stromal reactivity by inhibiting M2-macrophages polarization and prostate cancer-associated fibroblasts"

    Article Title: Zoledronic acid impairs stromal reactivity by inhibiting M2-macrophages polarization and prostate cancer-associated fibroblasts

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9497

    M2 and M2-like macrophages-dependent increase of PC3 cells invasiveness is impaired by ZA treatment A. Monocytes were differentiated for 7 days with M-CSF and then polarized into M2 macrophages by stimulating with IL-4 for 24h. Alternatively, monocytes were stimulated with CM from PC3 for 7 days to obtain M2-like macrophages. ZA was administrated during differentiation at different concentrations and then macrophages were serum-starved for 48h to obtain the corresponding CM. PC3 cells were incubated for 24h with CM from the above differentiated macrophages (treated or not with ZA), or serum starved as a control, and then allowed to invade toward medium containing 10% serum as chemoattractant for additional 24 h. (St. Med. Starvation Medium) B. Invading cells were counted and the mean of six randomly chosen fields was plotted in the bar graph. 1-way ANOVA, Dunnett's corrected, ***p
    Figure Legend Snippet: M2 and M2-like macrophages-dependent increase of PC3 cells invasiveness is impaired by ZA treatment A. Monocytes were differentiated for 7 days with M-CSF and then polarized into M2 macrophages by stimulating with IL-4 for 24h. Alternatively, monocytes were stimulated with CM from PC3 for 7 days to obtain M2-like macrophages. ZA was administrated during differentiation at different concentrations and then macrophages were serum-starved for 48h to obtain the corresponding CM. PC3 cells were incubated for 24h with CM from the above differentiated macrophages (treated or not with ZA), or serum starved as a control, and then allowed to invade toward medium containing 10% serum as chemoattractant for additional 24 h. (St. Med. Starvation Medium) B. Invading cells were counted and the mean of six randomly chosen fields was plotted in the bar graph. 1-way ANOVA, Dunnett's corrected, ***p

    Techniques Used: Incubation

    Treatment with ZA inhibits the M2/M2-like macrophages-induced pro-angiogenic effect M2 and M2-like macrophages were obtained by treating monocytes for 7 days with M-CSF (then treated with IL-4 for additional 24h) or with CM from PC3, respectively. M2 and M2-like macrophages were treated with different concentrations of ZA during differentiation and then were serum-starved for 48h to obtain the corresponding CM. HUVEC A. and EPC B. cells were treated with CM from the above differentiated macrophages (treated or not with ZA) and in vitro angiogenesis was evaluated by capillary morphogenesis assay. The number of joints was quantified and plotted in the bar graph. 1-way ANOVA, Dunnett's corrected, ***p
    Figure Legend Snippet: Treatment with ZA inhibits the M2/M2-like macrophages-induced pro-angiogenic effect M2 and M2-like macrophages were obtained by treating monocytes for 7 days with M-CSF (then treated with IL-4 for additional 24h) or with CM from PC3, respectively. M2 and M2-like macrophages were treated with different concentrations of ZA during differentiation and then were serum-starved for 48h to obtain the corresponding CM. HUVEC A. and EPC B. cells were treated with CM from the above differentiated macrophages (treated or not with ZA) and in vitro angiogenesis was evaluated by capillary morphogenesis assay. The number of joints was quantified and plotted in the bar graph. 1-way ANOVA, Dunnett's corrected, ***p

    Techniques Used: In Vitro

    ZA counteracts macrophage-dependent fibroblasts activation A. M2 and M2-like macrophages were obtained by treating monocytes for 7 days with M-CSF (then treated with IL-4 for additional 24h) or with CM from PC3, respectively. M2 and M2-like macrophages were treated with different concentrations of ZA during differentiation and then were serum-starved for 48h to obtain the corresponding CM. Subconfluent HPFs were treated for 24 h with CM from the above differentiated macrophages (treated or not with ZA) or with 10 ng/ml TGF-β1 as a positive control, and serum starved for additional 24 h. Fibroblasts were lysed and immunoblots for α-SMA and actin were performed. B . M2 and M2-like macrophages were obtained as in A and were treated with ZA 200nM during differentiation and then were serum-starved for 48 h to obtain the corresponding CM. Subconfluent HPFs were treated for 24 h with CM from the above differentiated macrophages (treated or not with ZA) or with 10 ng/ml TGF-β1 as a positive control, α-SMA was quantified by confocal microscopy. Bar, 50 μm. C. PC3 cells were incubated for 24 h with CM from HPFs treated as in A (CM M2 AFs or CM M2-like AFs; AFs: Activated fibroblasts) and then allowed to invade for additional 24 h toward medium containing 10% serum as chemoattractant. Invading cells were counted and the mean of six randomly chosen fields was plotted in the bar graph. Representative photographs for each sample are shown under the corresponding bar. 1-way ANOVA, Dunnett's corrected, ***p
    Figure Legend Snippet: ZA counteracts macrophage-dependent fibroblasts activation A. M2 and M2-like macrophages were obtained by treating monocytes for 7 days with M-CSF (then treated with IL-4 for additional 24h) or with CM from PC3, respectively. M2 and M2-like macrophages were treated with different concentrations of ZA during differentiation and then were serum-starved for 48h to obtain the corresponding CM. Subconfluent HPFs were treated for 24 h with CM from the above differentiated macrophages (treated or not with ZA) or with 10 ng/ml TGF-β1 as a positive control, and serum starved for additional 24 h. Fibroblasts were lysed and immunoblots for α-SMA and actin were performed. B . M2 and M2-like macrophages were obtained as in A and were treated with ZA 200nM during differentiation and then were serum-starved for 48 h to obtain the corresponding CM. Subconfluent HPFs were treated for 24 h with CM from the above differentiated macrophages (treated or not with ZA) or with 10 ng/ml TGF-β1 as a positive control, α-SMA was quantified by confocal microscopy. Bar, 50 μm. C. PC3 cells were incubated for 24 h with CM from HPFs treated as in A (CM M2 AFs or CM M2-like AFs; AFs: Activated fibroblasts) and then allowed to invade for additional 24 h toward medium containing 10% serum as chemoattractant. Invading cells were counted and the mean of six randomly chosen fields was plotted in the bar graph. Representative photographs for each sample are shown under the corresponding bar. 1-way ANOVA, Dunnett's corrected, ***p

    Techniques Used: Activation Assay, Positive Control, Western Blot, Confocal Microscopy, Incubation

    ZA suppresses monocyte differentiation toward M2 macrophages A, B. Human monocytes isolated from normal donor buffy coat were cultured for 7 days with M-CSF (50ng/ml). Differentiation toward the M1 phenotype was induced by treatment with LPS (100 ng/ml) and IFNγ (100 ng/ml) for 24h. M2 macrophages were polarized by stimulating with IL-4 (20 ng/ml) for 24h. Alternatively, M2-like macrophages were obtained by treating isolated monocytes with CM from PC3 for 7 days. During differentiation macrophages were treated with different concentrations of ZA and the levels of IL-12 or IL-10 were measured by ELISA test. 1-way ANOVA, Dunnett's corrected, ***p
    Figure Legend Snippet: ZA suppresses monocyte differentiation toward M2 macrophages A, B. Human monocytes isolated from normal donor buffy coat were cultured for 7 days with M-CSF (50ng/ml). Differentiation toward the M1 phenotype was induced by treatment with LPS (100 ng/ml) and IFNγ (100 ng/ml) for 24h. M2 macrophages were polarized by stimulating with IL-4 (20 ng/ml) for 24h. Alternatively, M2-like macrophages were obtained by treating isolated monocytes with CM from PC3 for 7 days. During differentiation macrophages were treated with different concentrations of ZA and the levels of IL-12 or IL-10 were measured by ELISA test. 1-way ANOVA, Dunnett's corrected, ***p

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

    19) Product Images from "Measurement of trihydroxy-linoleic acids in stratum corneum by tape-stripping: Possible biomarker of barrier function in atopic dermatitis"

    Article Title: Measurement of trihydroxy-linoleic acids in stratum corneum by tape-stripping: Possible biomarker of barrier function in atopic dermatitis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0210013

    The mRNA expression of enzymes involved in CLE formation upon IL-4 stimulation. ALOX12B, ALOXE3 and ABHD9 mRNA levels of primary normal human epidermal keratinocytes were measured by real-time RT-PCR. The mean values of threshold cycle were 27.5 ± 0.5 (ALOX12B), 22.4 ± 0.1 (ALOXE3), 23.1 ± 0.4 (ABHD9), 15.4 ± 0.4 (β-actin), respectively. The amount of mRNA was normalized to that of β-actin using the ΔCt method, as described in the manufacturer’s protocol. Data are expressed as mean ± SD (n = 6). One-way ANOVA with repeated measures was used for the comparison. Because the initial P value was less than 0.05, Scheffe’s test was used to determine the significance between groups. * p
    Figure Legend Snippet: The mRNA expression of enzymes involved in CLE formation upon IL-4 stimulation. ALOX12B, ALOXE3 and ABHD9 mRNA levels of primary normal human epidermal keratinocytes were measured by real-time RT-PCR. The mean values of threshold cycle were 27.5 ± 0.5 (ALOX12B), 22.4 ± 0.1 (ALOXE3), 23.1 ± 0.4 (ABHD9), 15.4 ± 0.4 (β-actin), respectively. The amount of mRNA was normalized to that of β-actin using the ΔCt method, as described in the manufacturer’s protocol. Data are expressed as mean ± SD (n = 6). One-way ANOVA with repeated measures was used for the comparison. Because the initial P value was less than 0.05, Scheffe’s test was used to determine the significance between groups. * p

    Techniques Used: Expressing, Quantitative RT-PCR

    20) Product Images from "Characteristics of immunogenic and tolerogenic dendritic cells within the arterial wall in atherosclerosis and in vitro"

    Article Title: Characteristics of immunogenic and tolerogenic dendritic cells within the arterial wall in atherosclerosis and in vitro

    Journal: International Journal of Clinical and Experimental Medicine

    doi:

    CD11c + CD11b + TDC and CD11c + CD83 + mDC were induced successfully and displayed tolerogenic phenotype. A. Left dots plot showed that the ratio of rmGM-CSF and IL-4 induced CD11c + CD11b + TDC was approximately 41.5%, and right dots plot showed that the ratio of
    Figure Legend Snippet: CD11c + CD11b + TDC and CD11c + CD83 + mDC were induced successfully and displayed tolerogenic phenotype. A. Left dots plot showed that the ratio of rmGM-CSF and IL-4 induced CD11c + CD11b + TDC was approximately 41.5%, and right dots plot showed that the ratio of

    Techniques Used:

    21) Product Images from "Morphine Suppresses MHC-II Expression on Circulating B Lymphocytes via Activation of the HPA"

    Article Title: Morphine Suppresses MHC-II Expression on Circulating B Lymphocytes via Activation of the HPA

    Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

    doi: 10.1007/s11481-010-9218-7

    Effects of chronic morphine exposure on IL-4-induced MHC-II expression in B lymphocytes. Sprague-Dawley rats ( n =8) were administered either saline or morphine twice daily using the dosing schedule as described in “Materials and methods”. Acutely treated animals received morphine (10 mg/kg, s.c.) on the day of the experiment. Two hours after the final injection, peripheral blood mononuclear cells were isolated and cultured with rrIL-4 (0.1– 1,000 ng/ml). Stained cells were fixed using 1% paraformaldehyde prior to analysis using a FACSort flow cytometer. Data are expressed as MFI ± SEM. Data repeated in a subsequent experiment. * p
    Figure Legend Snippet: Effects of chronic morphine exposure on IL-4-induced MHC-II expression in B lymphocytes. Sprague-Dawley rats ( n =8) were administered either saline or morphine twice daily using the dosing schedule as described in “Materials and methods”. Acutely treated animals received morphine (10 mg/kg, s.c.) on the day of the experiment. Two hours after the final injection, peripheral blood mononuclear cells were isolated and cultured with rrIL-4 (0.1– 1,000 ng/ml). Stained cells were fixed using 1% paraformaldehyde prior to analysis using a FACSort flow cytometer. Data are expressed as MFI ± SEM. Data repeated in a subsequent experiment. * p

    Techniques Used: Expressing, Injection, Isolation, Cell Culture, Staining, Flow Cytometry, Cytometry

    22) Product Images from "Effect of Mannose Targeting of Hydroxyl PAMAM Dendrimers on Cellular and Organ Biodistribution in a Neonatal Brain Injury Model"

    Article Title: Effect of Mannose Targeting of Hydroxyl PAMAM Dendrimers on Cellular and Organ Biodistribution in a Neonatal Brain Injury Model

    Journal: Journal of controlled release : official journal of the Controlled Release Society

    doi: 10.1016/j.jconrel.2018.06.003

    Cells were treated with LPS to activate, mannan to block mannose receptor, both, or neither. A. RT-qPCR for CD206 expression of LPS and mannan treated cells. (n=4) B. Dendrimer internalization of cells given the previously stated treatments. (n=6). The same treatment scheme was then replicated with IL-4 replacing LPS. C. RT-qPCR for CD206 expression of IL-4 and mannan treated cells. (n=3) D. Internalized dendrimer content of cells undergoing IL-4 and mannan treatments. (n=3) E. Representative confocal microscopy images of cells undergoing the same treatments as D , showing expression of CD206 (green) and localization of dendrimer (red) within the cells. Nuclei are stained with DAPI and shown in blue.
    Figure Legend Snippet: Cells were treated with LPS to activate, mannan to block mannose receptor, both, or neither. A. RT-qPCR for CD206 expression of LPS and mannan treated cells. (n=4) B. Dendrimer internalization of cells given the previously stated treatments. (n=6). The same treatment scheme was then replicated with IL-4 replacing LPS. C. RT-qPCR for CD206 expression of IL-4 and mannan treated cells. (n=3) D. Internalized dendrimer content of cells undergoing IL-4 and mannan treatments. (n=3) E. Representative confocal microscopy images of cells undergoing the same treatments as D , showing expression of CD206 (green) and localization of dendrimer (red) within the cells. Nuclei are stained with DAPI and shown in blue.

    Techniques Used: Blocking Assay, Quantitative RT-PCR, Expressing, Confocal Microscopy, Staining

    Related Articles

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    Article Title: RBP-J is required for M2 macrophage polarization in response to chitin and mediates expression of a subset of M2 genes
    Article Snippet: .. Recombinant mouse IL-4 was from Peprotech and used at 10 ng/mL unless otherwise noted. .. mRNA isolation and real time PCR RNA was extracted from whole cell lysates with an RNeasy Mini kit (Qiagen) and 0.5 μg of total RNA was reverse transcribed with a First Strand cDNA synthesis kit (Fermentas).

    Article Title: Stat6 and IRS-2 Cooperate in Interleukin 4 (IL-4)-Induced Proliferation and Differentiation but Are Dispensable for IL-4-Dependent Rescue from Apoptosis
    Article Snippet: Antibodies specific for phosphorylated Stat6 and phosphorylated akt were obtained from New England Biolabs. .. Recombinant IL-4 was obtained from Peprotech. .. Recombinant IL-2 was provided by Chiron.

    Article Title: IL-4 Receptor-Alpha-Dependent Control of Cryptococcus neoformans in the Early Phase of Pulmonary Infection
    Article Snippet: Conventional dendritic cells were generated by cultivation of bone marrow cells (2×105 /ml) for 8 days in RPMI 1640 supplemented with 10% (v/v) FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1% (v/v) essential and non-essential amino acids (GE Healthcare Europe) 1 mM sodium pyruvate, 50 µM β-mercaptoethanol (Sigma-Aldrich) and 10% (v/v) GM-CSF containing supernatant at 37°C in a humidified atmosphere containing 5% (v/v) CO2 . .. After harvesting, the cells were adjusted to 5×105 /ml and stimulated with C. neoformans , strain 1841 in the presence or absence of 25 U/ml recombinant IL-4 (Peprotech, Hamburg, Germany). .. Detection of cytokines and nitric oxide in cell culture supernatants Cytokines in cell culture supernatants were measured by sandwich ELISA.

    Article Title: Interleukin 4 promotes the development of ex-Foxp3 Th2 cells during immunity to intestinal helminths
    Article Snippet: Adherent cells were harvested at day 7 and resuspended in 1% DMEM with GlutaMAX (Thermo Fisher Scientific), 1% FBS, 10 mM Hepes, 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco), 2.7 mM l -glutamine (Gibco), 0.05 mM 2-mercaptoethanol (Gibco), and 1 mM sodium pyruvate (Lonza). .. 106 BMDMs were plated in 24-well flat-bottom tissue culture–treated plates and left to rest for 24 h. 105 sort-purified T cells were resuspended in 1% DMEM containing 1 µg/ml soluble CD3 and 10 µg/ml soluble CD28 antibody (Bio X Cell) and cultured with the BMDMs for 24 h. As a control, BMDMs were co-cultured in the presence of 20 ng/ml recombinant IL-4 (PeproTech) and 20 ng/ml IL-13 (PeproTech) or media alone. ..

    other:

    Article Title: A Novel Osteoclast Precursor Cell Line, 4B12, Recapitulates the Features of Primary Osteoclast Differentiation and Function: Enhanced Transfection Efficiency Before and After Differentiation
    Article Snippet: Mouse SCF (mSCF), mouse GM-CSF (mGM-CSF), and mouse IL-4 (mIL-4) were purchased from Peprotech (Rocky Hill, NJ).

    Incubation:

    Article Title: Co-transfection of dendritic cells with AFP and IL-2 genes enhances the induction of tumor antigen-specific antitumor immunity
    Article Snippet: .. Adherent cells were replenished with 30 ml X-VIVO 15 medium containing 100 ng/ml granulocyte macrophage-colony stimulating factor (GM-CSF; Peprotech, Inc., Rocky Hill, NJ, USA) and 10 ng/ml interleukin-4 (IL-4; Peprotech) and incubated for 7 days at 37°C with 5% CO2 . .. Generation and genetic modification of DCs Ad vectors were purified by two rounds of CsCl density centrifugation, dialysed and stored at −70°C in 3% sucrose.

    Cell Culture:

    Article Title: Interleukin 4 promotes the development of ex-Foxp3 Th2 cells during immunity to intestinal helminths
    Article Snippet: Adherent cells were harvested at day 7 and resuspended in 1% DMEM with GlutaMAX (Thermo Fisher Scientific), 1% FBS, 10 mM Hepes, 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco), 2.7 mM l -glutamine (Gibco), 0.05 mM 2-mercaptoethanol (Gibco), and 1 mM sodium pyruvate (Lonza). .. 106 BMDMs were plated in 24-well flat-bottom tissue culture–treated plates and left to rest for 24 h. 105 sort-purified T cells were resuspended in 1% DMEM containing 1 µg/ml soluble CD3 and 10 µg/ml soluble CD28 antibody (Bio X Cell) and cultured with the BMDMs for 24 h. As a control, BMDMs were co-cultured in the presence of 20 ng/ml recombinant IL-4 (PeproTech) and 20 ng/ml IL-13 (PeproTech) or media alone. ..

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    PeproTech th1 polarizing reagents
    CSE acts directly on CD4 T cells to reduce expansion and increase <t>Th1-polarization</t> Naïve CD4 T cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies in the presence or absence of CSE. The recovery of effectors at day 4 ( A ), CFSE dilution ( B ), FSC and CD69 MFI values (C) , analysis of apoptosis ( D and E ), Th1 cytokine production ( F and G ), and T-bet expression ( H and I ) is shown from one of 3 experiments.
    Th1 Polarizing Reagents, supplied by PeproTech, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PeproTech bm il 4 mϕ
    <t>IL-4-induced</t> pulmonary inflammation requires gV-sPLA 2
    Bm Il 4 Mϕ, supplied by PeproTech, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CSE acts directly on CD4 T cells to reduce expansion and increase Th1-polarization Naïve CD4 T cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies in the presence or absence of CSE. The recovery of effectors at day 4 ( A ), CFSE dilution ( B ), FSC and CD69 MFI values (C) , analysis of apoptosis ( D and E ), Th1 cytokine production ( F and G ), and T-bet expression ( H and I ) is shown from one of 3 experiments.

    Journal: Cellular immunology

    Article Title: Cigarette smoke extract acts directly on CD4 T cells to enhance Th1 polarization and reduce memory potential

    doi: 10.1016/j.cellimm.2018.06.005

    Figure Lengend Snippet: CSE acts directly on CD4 T cells to reduce expansion and increase Th1-polarization Naïve CD4 T cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies in the presence or absence of CSE. The recovery of effectors at day 4 ( A ), CFSE dilution ( B ), FSC and CD69 MFI values (C) , analysis of apoptosis ( D and E ), Th1 cytokine production ( F and G ), and T-bet expression ( H and I ) is shown from one of 3 experiments.

    Article Snippet: Exogenous recombinant murine IL-2 (11 ng/mL, Peprotech), and Th1-polarizing reagents (2 ng/ml recombinant IL-12, Peprotech, 15 μg/mL anti-IL-4 clone 11B11, BioXcell) were added at the initiation of cultures.

    Techniques: Expressing

    Exposure to CSE during priming enhances Th1 polarization Naïve CD4 T cells were stimulated with APC and peptide under Th1 conditions in the presence or absence of CSE. Effector cells were harvested on day 4 of culture and analyzed for forward scatter (FSC), CD25, and CD69 with ( A ) representative staining and ( B ) mean fluorescence intensity (MFI) analysis from triplicate cultures shown. Values from naïve CD4 T cells are shown as grey histograms in ( A ) and as dotted line in ( B ). Effector cells were assessed for their capacity to produce of IFNγ and TNF by intracellular cytokine staining. Shown is ( C ) representative staining in the absence or presence of stimulation by PMA and Ionomycin by effectors generated in the presence or absence of CSE, as well as ( D ) the frequency of cells from each condition capable of co-producing TNF and IFNγ and ( E ) the MFI of IFNγ + cells from triplicate wells (one of 4 experiments). Expression of T-bet in effectors was determined by intracellular staining with MFI analysis and representative staining shown (F and G) . Representative staining of re-stimulated cells for production of IL-4 ( H ) and IL-17 ( I ) (one of 2 experiments).

    Journal: Cellular immunology

    Article Title: Cigarette smoke extract acts directly on CD4 T cells to enhance Th1 polarization and reduce memory potential

    doi: 10.1016/j.cellimm.2018.06.005

    Figure Lengend Snippet: Exposure to CSE during priming enhances Th1 polarization Naïve CD4 T cells were stimulated with APC and peptide under Th1 conditions in the presence or absence of CSE. Effector cells were harvested on day 4 of culture and analyzed for forward scatter (FSC), CD25, and CD69 with ( A ) representative staining and ( B ) mean fluorescence intensity (MFI) analysis from triplicate cultures shown. Values from naïve CD4 T cells are shown as grey histograms in ( A ) and as dotted line in ( B ). Effector cells were assessed for their capacity to produce of IFNγ and TNF by intracellular cytokine staining. Shown is ( C ) representative staining in the absence or presence of stimulation by PMA and Ionomycin by effectors generated in the presence or absence of CSE, as well as ( D ) the frequency of cells from each condition capable of co-producing TNF and IFNγ and ( E ) the MFI of IFNγ + cells from triplicate wells (one of 4 experiments). Expression of T-bet in effectors was determined by intracellular staining with MFI analysis and representative staining shown (F and G) . Representative staining of re-stimulated cells for production of IL-4 ( H ) and IL-17 ( I ) (one of 2 experiments).

    Article Snippet: Exogenous recombinant murine IL-2 (11 ng/mL, Peprotech), and Th1-polarizing reagents (2 ng/ml recombinant IL-12, Peprotech, 15 μg/mL anti-IL-4 clone 11B11, BioXcell) were added at the initiation of cultures.

    Techniques: Staining, Fluorescence, Generated, Expressing

    Delayed exposure to CSE reduces effector expansion CSE was introduced to Th1 cultures at either day 0 or day 2 and ( A ) the total number of effector cells recovered at day 4 is shown from quadruplicate wells (one of 3 experiments). ( B ) Representative analysis of CFSE dilution from effectors either not exposed to CSE (black) or exposed to CSE for only from days 2–4 of culture. Effector cells were analyzed for apoptotic cells with ( C ) representative staining and ( D ) analysis from triplicate wells shown (one of 2 experiments).

    Journal: Cellular immunology

    Article Title: Cigarette smoke extract acts directly on CD4 T cells to enhance Th1 polarization and reduce memory potential

    doi: 10.1016/j.cellimm.2018.06.005

    Figure Lengend Snippet: Delayed exposure to CSE reduces effector expansion CSE was introduced to Th1 cultures at either day 0 or day 2 and ( A ) the total number of effector cells recovered at day 4 is shown from quadruplicate wells (one of 3 experiments). ( B ) Representative analysis of CFSE dilution from effectors either not exposed to CSE (black) or exposed to CSE for only from days 2–4 of culture. Effector cells were analyzed for apoptotic cells with ( C ) representative staining and ( D ) analysis from triplicate wells shown (one of 2 experiments).

    Article Snippet: Exogenous recombinant murine IL-2 (11 ng/mL, Peprotech), and Th1-polarizing reagents (2 ng/ml recombinant IL-12, Peprotech, 15 μg/mL anti-IL-4 clone 11B11, BioXcell) were added at the initiation of cultures.

    Techniques: Staining

    Delayed exposure to CSE enhances Th1-polarization Effectors generated without exposure of CSE or CSE added only from days 2–4 of culture only were analyzed for Th1 cytokine production by intracellular staining with ( A ) representative staining and ( B ) analysis of dual cytokine producers from triplicate conditions shown. T-bet levels in effectors was also determined with ( C ) representative staining and ( D ) MFI analysis from triplicate wells shown (one of 2 experiments).

    Journal: Cellular immunology

    Article Title: Cigarette smoke extract acts directly on CD4 T cells to enhance Th1 polarization and reduce memory potential

    doi: 10.1016/j.cellimm.2018.06.005

    Figure Lengend Snippet: Delayed exposure to CSE enhances Th1-polarization Effectors generated without exposure of CSE or CSE added only from days 2–4 of culture only were analyzed for Th1 cytokine production by intracellular staining with ( A ) representative staining and ( B ) analysis of dual cytokine producers from triplicate conditions shown. T-bet levels in effectors was also determined with ( C ) representative staining and ( D ) MFI analysis from triplicate wells shown (one of 2 experiments).

    Article Snippet: Exogenous recombinant murine IL-2 (11 ng/mL, Peprotech), and Th1-polarizing reagents (2 ng/ml recombinant IL-12, Peprotech, 15 μg/mL anti-IL-4 clone 11B11, BioXcell) were added at the initiation of cultures.

    Techniques: Generated, Staining

    Exposure to CSE during priming reduces CD4 T cell division and increases apoptosis Triplicate wells of equal numbers of naïve HNT cells and irradiated APC were cultured with cognate peptide under Th1-polarizing conditions for 4 days in the presence of stated concentrations of CSE. The number of resulting effector cells was determined in replicate experiments with a broad ( A ) and narrower ( B ) titration of CSE (2 of 5 similar experiments). ( C ) In some experiments naïve CD4 T cells were labeled with CFSE prior to culture. Shown is representative CFSE staining from effectors at 4 days of culture in the presence of 0% (black) or 7.5% (red) CSE versus CFSE staining of undivided naïve cells not exposed to antigen. Day 4 effector cells were analyzed for apoptotic phenotype by staining for Annexin V and 7-AAD. Shown are ( D ) representative staining of effectors cultured with or without CSE and ( E ) summary of 4 wells per condition (one of 2 experiments).

    Journal: Cellular immunology

    Article Title: Cigarette smoke extract acts directly on CD4 T cells to enhance Th1 polarization and reduce memory potential

    doi: 10.1016/j.cellimm.2018.06.005

    Figure Lengend Snippet: Exposure to CSE during priming reduces CD4 T cell division and increases apoptosis Triplicate wells of equal numbers of naïve HNT cells and irradiated APC were cultured with cognate peptide under Th1-polarizing conditions for 4 days in the presence of stated concentrations of CSE. The number of resulting effector cells was determined in replicate experiments with a broad ( A ) and narrower ( B ) titration of CSE (2 of 5 similar experiments). ( C ) In some experiments naïve CD4 T cells were labeled with CFSE prior to culture. Shown is representative CFSE staining from effectors at 4 days of culture in the presence of 0% (black) or 7.5% (red) CSE versus CFSE staining of undivided naïve cells not exposed to antigen. Day 4 effector cells were analyzed for apoptotic phenotype by staining for Annexin V and 7-AAD. Shown are ( D ) representative staining of effectors cultured with or without CSE and ( E ) summary of 4 wells per condition (one of 2 experiments).

    Article Snippet: Exogenous recombinant murine IL-2 (11 ng/mL, Peprotech), and Th1-polarizing reagents (2 ng/ml recombinant IL-12, Peprotech, 15 μg/mL anti-IL-4 clone 11B11, BioXcell) were added at the initiation of cultures.

    Techniques: Irradiation, Cell Culture, Titration, Labeling, Staining

    IL-4-induced pulmonary inflammation requires gV-sPLA 2

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

    Article Title: Group V secretory phospholipase A2 is involved in macrophage activation and is sufficient for macrophage effector functions in allergic pulmonary inflammation

    doi: 10.4049/jimmunol.1203202

    Figure Lengend Snippet: IL-4-induced pulmonary inflammation requires gV-sPLA 2

    Article Snippet: On day 7, the cells were pulsed with 20 ng/ml of IL-4 (PeproTech) for BM-IL-4-Mϕ (a surrogate of AAMϕ), LPS (100 ng/ml) (Sigma-Aldrich) and IFN-γ (200 U/ml) (PeproTech) for BM-LPS/IFNγ-Mϕ (a surrogate of CaMϕ), cultured in medium (BM-Immature-Mϕ, BM-IM-Mϕ), or IL-4 and/or IL-13, IL-33 and GM-CSF (PeproTech).

    Techniques:

    Transfer of WT but not Pla2g5 -null BM-IM-Mϕ into Pla2g5 -null recipient mice amplifies IL-4-induced pulmonary inflammation

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

    Article Title: Group V secretory phospholipase A2 is involved in macrophage activation and is sufficient for macrophage effector functions in allergic pulmonary inflammation

    doi: 10.4049/jimmunol.1203202

    Figure Lengend Snippet: Transfer of WT but not Pla2g5 -null BM-IM-Mϕ into Pla2g5 -null recipient mice amplifies IL-4-induced pulmonary inflammation

    Article Snippet: On day 7, the cells were pulsed with 20 ng/ml of IL-4 (PeproTech) for BM-IL-4-Mϕ (a surrogate of AAMϕ), LPS (100 ng/ml) (Sigma-Aldrich) and IFN-γ (200 U/ml) (PeproTech) for BM-LPS/IFNγ-Mϕ (a surrogate of CaMϕ), cultured in medium (BM-Immature-Mϕ, BM-IM-Mϕ), or IL-4 and/or IL-13, IL-33 and GM-CSF (PeproTech).

    Techniques: Mouse Assay

    Differentiation stages of 4B12 cells along the osteoclast lineage and inhibition by GM-CSF, IL-4 and LPS

    Journal: Journal of cellular physiology

    Article Title: A Novel Osteoclast Precursor Cell Line, 4B12, Recapitulates the Features of Primary Osteoclast Differentiation and Function: Enhanced Transfection Efficiency Before and After Differentiation

    doi: 10.1002/jcp.21827

    Figure Lengend Snippet: Differentiation stages of 4B12 cells along the osteoclast lineage and inhibition by GM-CSF, IL-4 and LPS

    Article Snippet: Mouse SCF (mSCF), mouse GM-CSF (mGM-CSF), and mouse IL-4 (mIL-4) were purchased from Peprotech (Rocky Hill, NJ).

    Techniques: Inhibition

    Adoptively transferred T reg cells lose Foxp3 expression and convert to Il4 -expressing cells after secondary infection with H. polygyrus . (A) Experimental model. T reg cells (HpTR; CD4 + TCRβ + Il4 GFP– Foxp3 RFP+ CD25 high ) were sort purified from male Il4 GFP Foxp3 RFP mice infected with H. polygyrus for 14 d. nT cells (CD4 + TCRβ + CD25 – CD44 low Il4 GFP– Foxp3 RFP– ) were sort purified from the spleen and MLN of naive male Il4 GFP Foxp3 RFP mice. HpTR or nT cells were transferred to male Tcra −/− mice further subjected to a secondary infection with H. polygyrus . (B) Representative FACS plots of Foxp3 RFP , Il4 GFP , and CD25 expression in HpTR cells before and after sort and day 42 after transfer. (C and D) Proportion of Foxp3 RFP+ CD25 high cells in HpTR recipients day 42 after transfer and relative to Foxp3 RFP expression before transfer (C) and proportion of CD4 + TCRβ + Il4 GFP+ Foxp3 RFP– cells in the spleen, MLN, and PPs of nT or HpTR cell recipients day 42 after transfer (D). Data are representative of at least five independent experiments with three to five mice per group. (E) qRT-PCR validation of Foxp3 and Il4 expression relative (rel.) to Hprt in sort-purified nT→ Il4 GFP+ , HpTR→ Il4 GFP+ , or HpTR cells. Data are representative of three experiments with cells pooled from three to five recipients. (F) Frequency of CD4 + CD44 high IL-4 + cells in the MLN of nT and HpTR cell recipients as measured by intracellular cytokine staining. Data are representative of three independent experiments with four mice per group. CD4 + TCRβ + Foxp3 RFP+ CD25 high or CD25 low cells were sort purified from Il4 GFP Foxp3 RFP mice infected with H. polygyrus for 14 d and transferred to Tcra −/− mice subjected to a secondary infection with H. polygyrus . (G) Representative FACS plots of Foxp3 RFP and CD25 expression in donor HpTR cells presort. (H) Proportion of CD4 + TCRβ + Il4 GFP+ cells in the PPs of Hp 2° recipients day 42 after transfer. Data are representative of two independent experiments with four to five mice per group. Adopt., adoptively. Error bars represent SEM.

    Journal: The Journal of Experimental Medicine

    Article Title: Interleukin 4 promotes the development of ex-Foxp3 Th2 cells during immunity to intestinal helminths

    doi: 10.1084/jem.20161104

    Figure Lengend Snippet: Adoptively transferred T reg cells lose Foxp3 expression and convert to Il4 -expressing cells after secondary infection with H. polygyrus . (A) Experimental model. T reg cells (HpTR; CD4 + TCRβ + Il4 GFP– Foxp3 RFP+ CD25 high ) were sort purified from male Il4 GFP Foxp3 RFP mice infected with H. polygyrus for 14 d. nT cells (CD4 + TCRβ + CD25 – CD44 low Il4 GFP– Foxp3 RFP– ) were sort purified from the spleen and MLN of naive male Il4 GFP Foxp3 RFP mice. HpTR or nT cells were transferred to male Tcra −/− mice further subjected to a secondary infection with H. polygyrus . (B) Representative FACS plots of Foxp3 RFP , Il4 GFP , and CD25 expression in HpTR cells before and after sort and day 42 after transfer. (C and D) Proportion of Foxp3 RFP+ CD25 high cells in HpTR recipients day 42 after transfer and relative to Foxp3 RFP expression before transfer (C) and proportion of CD4 + TCRβ + Il4 GFP+ Foxp3 RFP– cells in the spleen, MLN, and PPs of nT or HpTR cell recipients day 42 after transfer (D). Data are representative of at least five independent experiments with three to five mice per group. (E) qRT-PCR validation of Foxp3 and Il4 expression relative (rel.) to Hprt in sort-purified nT→ Il4 GFP+ , HpTR→ Il4 GFP+ , or HpTR cells. Data are representative of three experiments with cells pooled from three to five recipients. (F) Frequency of CD4 + CD44 high IL-4 + cells in the MLN of nT and HpTR cell recipients as measured by intracellular cytokine staining. Data are representative of three independent experiments with four mice per group. CD4 + TCRβ + Foxp3 RFP+ CD25 high or CD25 low cells were sort purified from Il4 GFP Foxp3 RFP mice infected with H. polygyrus for 14 d and transferred to Tcra −/− mice subjected to a secondary infection with H. polygyrus . (G) Representative FACS plots of Foxp3 RFP and CD25 expression in donor HpTR cells presort. (H) Proportion of CD4 + TCRβ + Il4 GFP+ cells in the PPs of Hp 2° recipients day 42 after transfer. Data are representative of two independent experiments with four to five mice per group. Adopt., adoptively. Error bars represent SEM.

    Article Snippet: 106 BMDMs were plated in 24-well flat-bottom tissue culture–treated plates and left to rest for 24 h. 105 sort-purified T cells were resuspended in 1% DMEM containing 1 µg/ml soluble CD3 and 10 µg/ml soluble CD28 antibody (Bio X Cell) and cultured with the BMDMs for 24 h. As a control, BMDMs were co-cultured in the presence of 20 ng/ml recombinant IL-4 (PeproTech) and 20 ng/ml IL-13 (PeproTech) or media alone.

    Techniques: Expressing, Infection, Purification, Mouse Assay, FACS, Quantitative RT-PCR, Staining

    Ex-Foxp3 Th2 cells secrete type-2 cytokines, promote AAMφ in vitro, and are sufficient to drive the expulsion of H. polygyrus . (A–J) T reg, Th2, and ex-Foxp3 Th2 cells were sort purified from Hp 1° and Hp 2° Il4 GFP Foxp3 YFP/Cre R26R FP635 mice and stimulated with PMA/ionomycin for 24 h. (A) Concentration of IL-4, IL-13, IL-5, and IL-2 in the supernatant of restimulated cells. Three technical replicates were used. (B) BMDMs were cultured with FACS-purified T cells for 24 h or with media and recombinant IL-4 + IL-13. (C) Expression of Arg1 and Retnla in stimulated BMDMs. Data are representative of two to three independent experiments with three technical replicates. Sort-purified T cells were pooled from three to four donor mice. rel., relative. (D) Th2 and ex-Foxp3 Th2 cells were sort purified from Hp 2° Il4 GFP Foxp3 YFP/Cre R26R FP635 mice at day 14 after infection and transferred to Hp 1°C57BL/6 recipients 2 d after infection. (E) Intestinal worm burden at day 21 after infection. Data represent two pooled experiments with 6–10 mice per group. (F) Experimental model (see model in Fig. 6 D ). Absolute number of CD4 + TCRβ + Il4 GFP+ Foxp3 FATE– and CD4 + TCRβ + Il4 GFP+ Foxp3 FATE+ cells in the spleens is shown. Four mice per group were used. (G) Small intestine expression of key type-2 response genes, expressed relative (rel.) to Hprt . Four mice per group were used. (H and I) IgE (H)- and H. polygyrus (I)–specific IgG1 levels in the serum. Data represent two independent experiments with three to four mice per group. a.u., arbitrary units. (J) Intestinal worm count day 14 after infection. Data represent two independent experiments with five to seven mice per group. *, P ≤ 0.05; Mann-Whitney test. Error bars represent SEM.

    Journal: The Journal of Experimental Medicine

    Article Title: Interleukin 4 promotes the development of ex-Foxp3 Th2 cells during immunity to intestinal helminths

    doi: 10.1084/jem.20161104

    Figure Lengend Snippet: Ex-Foxp3 Th2 cells secrete type-2 cytokines, promote AAMφ in vitro, and are sufficient to drive the expulsion of H. polygyrus . (A–J) T reg, Th2, and ex-Foxp3 Th2 cells were sort purified from Hp 1° and Hp 2° Il4 GFP Foxp3 YFP/Cre R26R FP635 mice and stimulated with PMA/ionomycin for 24 h. (A) Concentration of IL-4, IL-13, IL-5, and IL-2 in the supernatant of restimulated cells. Three technical replicates were used. (B) BMDMs were cultured with FACS-purified T cells for 24 h or with media and recombinant IL-4 + IL-13. (C) Expression of Arg1 and Retnla in stimulated BMDMs. Data are representative of two to three independent experiments with three technical replicates. Sort-purified T cells were pooled from three to four donor mice. rel., relative. (D) Th2 and ex-Foxp3 Th2 cells were sort purified from Hp 2° Il4 GFP Foxp3 YFP/Cre R26R FP635 mice at day 14 after infection and transferred to Hp 1°C57BL/6 recipients 2 d after infection. (E) Intestinal worm burden at day 21 after infection. Data represent two pooled experiments with 6–10 mice per group. (F) Experimental model (see model in Fig. 6 D ). Absolute number of CD4 + TCRβ + Il4 GFP+ Foxp3 FATE– and CD4 + TCRβ + Il4 GFP+ Foxp3 FATE+ cells in the spleens is shown. Four mice per group were used. (G) Small intestine expression of key type-2 response genes, expressed relative (rel.) to Hprt . Four mice per group were used. (H and I) IgE (H)- and H. polygyrus (I)–specific IgG1 levels in the serum. Data represent two independent experiments with three to four mice per group. a.u., arbitrary units. (J) Intestinal worm count day 14 after infection. Data represent two independent experiments with five to seven mice per group. *, P ≤ 0.05; Mann-Whitney test. Error bars represent SEM.

    Article Snippet: 106 BMDMs were plated in 24-well flat-bottom tissue culture–treated plates and left to rest for 24 h. 105 sort-purified T cells were resuspended in 1% DMEM containing 1 µg/ml soluble CD3 and 10 µg/ml soluble CD28 antibody (Bio X Cell) and cultured with the BMDMs for 24 h. As a control, BMDMs were co-cultured in the presence of 20 ng/ml recombinant IL-4 (PeproTech) and 20 ng/ml IL-13 (PeproTech) or media alone.

    Techniques: In Vitro, Purification, Mouse Assay, Concentration Assay, Cell Culture, FACS, Recombinant, Expressing, Infection, MANN-WHITNEY

    IL-4 is sufficient to promote T reg to Th2 cell conversion in vitro. (A–G) HpTR cells and nT cells were sort purified from Il4 GFP Foxp3 RFP reporter mice as described in Fig. 2 . Sorted cells were stimulated with recombinant IL-4 at 37°C for 15 min or with media as a control. (A) Levels of pSTAT6, total STAT6, and α-tubulin protein in restimulated cells. Data are representative of three independent experiments. Sorted T cells were pooled from three to four mice. mW, molecular weight. (B and C) nT or HpTR cells were cultured with anti-CD3/CD28 and IL-2, with and without the addition of IL-4. Representative FACS plots (B) and graph (C) showing the frequency of CD4 + TCRβ + Foxp3 RFP+ and CD4 + TCRβ + Il4 GFP+ cells in day 7 cultures are shown. HpTR cells were cultured with anti-CD3/CD28 and IL-2, with increasing concentrations of IL-4, and cells were harvested for FACS or qRT-PCR at day 7. (D and E) Frequency of CD4 + TCRβ + Il4 GFP+ (D) and CD4 + TCRβ + Foxp3 RFP+ (E) cells in day 7 cultures. (F and G) Gene expression of Gata3 (relative [rel] to Hprt ; F) and mir182 (relative to rnu6b ; G) in cultured cells at day 7. Data are representative of two independent experiments. Cells were sort purified from four to six mice. *, P ≤ 0.05; Mann-Whitney test. Error bars represent SEM.

    Journal: The Journal of Experimental Medicine

    Article Title: Interleukin 4 promotes the development of ex-Foxp3 Th2 cells during immunity to intestinal helminths

    doi: 10.1084/jem.20161104

    Figure Lengend Snippet: IL-4 is sufficient to promote T reg to Th2 cell conversion in vitro. (A–G) HpTR cells and nT cells were sort purified from Il4 GFP Foxp3 RFP reporter mice as described in Fig. 2 . Sorted cells were stimulated with recombinant IL-4 at 37°C for 15 min or with media as a control. (A) Levels of pSTAT6, total STAT6, and α-tubulin protein in restimulated cells. Data are representative of three independent experiments. Sorted T cells were pooled from three to four mice. mW, molecular weight. (B and C) nT or HpTR cells were cultured with anti-CD3/CD28 and IL-2, with and without the addition of IL-4. Representative FACS plots (B) and graph (C) showing the frequency of CD4 + TCRβ + Foxp3 RFP+ and CD4 + TCRβ + Il4 GFP+ cells in day 7 cultures are shown. HpTR cells were cultured with anti-CD3/CD28 and IL-2, with increasing concentrations of IL-4, and cells were harvested for FACS or qRT-PCR at day 7. (D and E) Frequency of CD4 + TCRβ + Il4 GFP+ (D) and CD4 + TCRβ + Foxp3 RFP+ (E) cells in day 7 cultures. (F and G) Gene expression of Gata3 (relative [rel] to Hprt ; F) and mir182 (relative to rnu6b ; G) in cultured cells at day 7. Data are representative of two independent experiments. Cells were sort purified from four to six mice. *, P ≤ 0.05; Mann-Whitney test. Error bars represent SEM.

    Article Snippet: 106 BMDMs were plated in 24-well flat-bottom tissue culture–treated plates and left to rest for 24 h. 105 sort-purified T cells were resuspended in 1% DMEM containing 1 µg/ml soluble CD3 and 10 µg/ml soluble CD28 antibody (Bio X Cell) and cultured with the BMDMs for 24 h. As a control, BMDMs were co-cultured in the presence of 20 ng/ml recombinant IL-4 (PeproTech) and 20 ng/ml IL-13 (PeproTech) or media alone.

    Techniques: In Vitro, Purification, Mouse Assay, Recombinant, Molecular Weight, Cell Culture, FACS, Quantitative RT-PCR, Expressing, MANN-WHITNEY

    IL-4 signaling in T reg cells is required for the development of ex-Foxp3 Th2 cells in vivo. (A and B) nT, Il4ra fl/fl , Il4ra fl/wt , or Il4ra wt/wt HpTR cells were sort purified from naive (A) or Hp 1° (B) Il4ra wt/wt or Il4ra fl/fl fate-reporter mice day 14 after infection and stimulated with recombinant IL-4 at 37°C for 15 min or media as a control. Levels of pSTAT6, total STAT6, and α-tubulin protein in restimulated cells are shown. Data are representative of two independent experiments. Sorted T cells were pooled from two to three mice. mW, molecular weight. (C) Proportion and absolute number of Foxp3 YFP+ cells in the spleen of naive Il4ra fl/fl , Il4ra fl/wt , or Il4ra wt/wt fate-reporter mice. Data are representative of two independent experiments. (D) Experimental model. Il4 GFP Foxp3 YFP/Cre R26R FP635 Il4ra fl/fl mice were infected with 200 H. polygyrus larvae. Hp 2° mice were treated with pyrantel embonate on day 14–15 and reinfected on day 28. Mice were harvested at day 7 after infection. (E) Representative FACS plots of Foxp3 FATE expression within CD4 + TCRβ + Il4 GFP+ cells in Il4ra fl/fl or Il4ra wt/wt Hp 2° mice day 7 after infection. (F and G) Proportion (F) and absolute number (G) of CD4 + TCRβ + Il4 GFP+ Foxp3 FATE+ cells in the spleen. Data represent two independent experiments with three to four mice per group. (H) Experimental model. T reg cells were sort purified from Il4ra wt/wt or Il4ra fl/fl Hp 1° mice day 14 after infection. Il4ra wt/wt or Il4ra fl/fl T reg cells were transferred to Tcra −/− mice. Recipient mice were infected with H. polygyrus , treated with pyrantel embonate at days 14–15, and then infected with H. polygyrus at day 35 and harvested at day 42 after transfer (see experimental model in Fig. 2 A ). (I and J) Representative FACS plots of CD4 + TCRβ + Il4 GFP+ cells (I) and frequency of Il4 GFP+ cells (J) in the spleen and MLN of Il4ra fl/fl or Il4ra wt/wt HpTR-recipient mice day 14 after infection. Data are representative of two experiments with three to five mice per group. Adopt., adoptively. (K) Experimental model. In brief, Il4ra fl/fl or Il4ra wt/wt mice were subjected to a model of HDM/alum sensitization. i.t., intratracheally. (L) Frequency of CD4 + TCRβ + Il4 GFP+ Foxp3 FATE+ cells in mediastinal LNs (medLN) and lungs 24 h after challenge. (M) Proportion of eosinophils in the BAL fluid. Data represent two independent experiments with four to nine mice per group. *, P ≤ 0.05; Mann-Whitney test. Error bars represent SEM.

    Journal: The Journal of Experimental Medicine

    Article Title: Interleukin 4 promotes the development of ex-Foxp3 Th2 cells during immunity to intestinal helminths

    doi: 10.1084/jem.20161104

    Figure Lengend Snippet: IL-4 signaling in T reg cells is required for the development of ex-Foxp3 Th2 cells in vivo. (A and B) nT, Il4ra fl/fl , Il4ra fl/wt , or Il4ra wt/wt HpTR cells were sort purified from naive (A) or Hp 1° (B) Il4ra wt/wt or Il4ra fl/fl fate-reporter mice day 14 after infection and stimulated with recombinant IL-4 at 37°C for 15 min or media as a control. Levels of pSTAT6, total STAT6, and α-tubulin protein in restimulated cells are shown. Data are representative of two independent experiments. Sorted T cells were pooled from two to three mice. mW, molecular weight. (C) Proportion and absolute number of Foxp3 YFP+ cells in the spleen of naive Il4ra fl/fl , Il4ra fl/wt , or Il4ra wt/wt fate-reporter mice. Data are representative of two independent experiments. (D) Experimental model. Il4 GFP Foxp3 YFP/Cre R26R FP635 Il4ra fl/fl mice were infected with 200 H. polygyrus larvae. Hp 2° mice were treated with pyrantel embonate on day 14–15 and reinfected on day 28. Mice were harvested at day 7 after infection. (E) Representative FACS plots of Foxp3 FATE expression within CD4 + TCRβ + Il4 GFP+ cells in Il4ra fl/fl or Il4ra wt/wt Hp 2° mice day 7 after infection. (F and G) Proportion (F) and absolute number (G) of CD4 + TCRβ + Il4 GFP+ Foxp3 FATE+ cells in the spleen. Data represent two independent experiments with three to four mice per group. (H) Experimental model. T reg cells were sort purified from Il4ra wt/wt or Il4ra fl/fl Hp 1° mice day 14 after infection. Il4ra wt/wt or Il4ra fl/fl T reg cells were transferred to Tcra −/− mice. Recipient mice were infected with H. polygyrus , treated with pyrantel embonate at days 14–15, and then infected with H. polygyrus at day 35 and harvested at day 42 after transfer (see experimental model in Fig. 2 A ). (I and J) Representative FACS plots of CD4 + TCRβ + Il4 GFP+ cells (I) and frequency of Il4 GFP+ cells (J) in the spleen and MLN of Il4ra fl/fl or Il4ra wt/wt HpTR-recipient mice day 14 after infection. Data are representative of two experiments with three to five mice per group. Adopt., adoptively. (K) Experimental model. In brief, Il4ra fl/fl or Il4ra wt/wt mice were subjected to a model of HDM/alum sensitization. i.t., intratracheally. (L) Frequency of CD4 + TCRβ + Il4 GFP+ Foxp3 FATE+ cells in mediastinal LNs (medLN) and lungs 24 h after challenge. (M) Proportion of eosinophils in the BAL fluid. Data represent two independent experiments with four to nine mice per group. *, P ≤ 0.05; Mann-Whitney test. Error bars represent SEM.

    Article Snippet: 106 BMDMs were plated in 24-well flat-bottom tissue culture–treated plates and left to rest for 24 h. 105 sort-purified T cells were resuspended in 1% DMEM containing 1 µg/ml soluble CD3 and 10 µg/ml soluble CD28 antibody (Bio X Cell) and cultured with the BMDMs for 24 h. As a control, BMDMs were co-cultured in the presence of 20 ng/ml recombinant IL-4 (PeproTech) and 20 ng/ml IL-13 (PeproTech) or media alone.

    Techniques: In Vivo, Purification, Mouse Assay, Infection, Recombinant, Molecular Weight, FACS, Expressing, MANN-WHITNEY