interleukin 6  (R&D Systems)

 
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    Name:
    Recombinant Mouse IL 6 Protein CF
    Description:
    The Recombinant Mouse IL 6 Protein from R D Systems is derived from E coli The Recombinant Mouse IL 6 Protein has been validated for the following applications Bioactivity
    Catalog Number:
    406-ML-005/CF
    Price:
    239
    Category:
    Proteins and Enzymes
    Source:
    E. coli-derived Recombinant Mouse IL-6 Protein
    Applications:
    Bioactivity
    Purity:
    >97%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie« Blue Staining.
    Conjugate:
    Unconjugated
    Size:
    5 ug
    Buy from Supplier


    Structured Review

    R&D Systems interleukin 6
    Recombinant Mouse IL 6 Protein CF
    The Recombinant Mouse IL 6 Protein from R D Systems is derived from E coli The Recombinant Mouse IL 6 Protein has been validated for the following applications Bioactivity
    https://www.bioz.com/result/interleukin 6/product/R&D Systems
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    interleukin 6 - by Bioz Stars, 2021-05
    99/100 stars

    Images

    1) Product Images from "Activated brain mast cells contribute to postoperative cognitive dysfunction by evoking microglia activation and neuronal apoptosis"

    Article Title: Activated brain mast cells contribute to postoperative cognitive dysfunction by evoking microglia activation and neuronal apoptosis

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-016-0592-9

    MAPK signaling pathways were involved in microglia activation and the following cytokine production. a The activated levels of ERK and JNK, which was assessed by increased phosphorylation of tyrosine residues of these kinases, was detected by Western blotting using specific antibodies. Each blot is representative of three experiments. b Phosphorylated levels of ERK and JNK were quantified and normalized to total levels. Each value was then expressed relative to the control, which was set to 1. c – d The levels of proinflammatory factors TNF-α and IL-6 were detected by ELISA. * P
    Figure Legend Snippet: MAPK signaling pathways were involved in microglia activation and the following cytokine production. a The activated levels of ERK and JNK, which was assessed by increased phosphorylation of tyrosine residues of these kinases, was detected by Western blotting using specific antibodies. Each blot is representative of three experiments. b Phosphorylated levels of ERK and JNK were quantified and normalized to total levels. Each value was then expressed relative to the control, which was set to 1. c – d The levels of proinflammatory factors TNF-α and IL-6 were detected by ELISA. * P

    Techniques Used: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay

    Cromolyn inhibited surgery-induced MAPK activation and cytokine secretion in the hippocampus. a The activated levels of ERK and JNK in the area CA1 of the hippocampus, which was assessed by increased phosphorylation of tyrosine residues of these kinases, were detected by Western blotting using specific antibodies. Each blot is representative of three experiments. b Phosphorylated levels of ERK and JNK were quantified and normalized to total levels. Each value was then expressed relative to the control, which was set to 1. c – d The levels of proinflammatory factors TNF-α and IL-6 were detected by ELISA. * P
    Figure Legend Snippet: Cromolyn inhibited surgery-induced MAPK activation and cytokine secretion in the hippocampus. a The activated levels of ERK and JNK in the area CA1 of the hippocampus, which was assessed by increased phosphorylation of tyrosine residues of these kinases, were detected by Western blotting using specific antibodies. Each blot is representative of three experiments. b Phosphorylated levels of ERK and JNK were quantified and normalized to total levels. Each value was then expressed relative to the control, which was set to 1. c – d The levels of proinflammatory factors TNF-α and IL-6 were detected by ELISA. * P

    Techniques Used: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay

    2) Product Images from "Opposite Effect Of Inflammation on SVZ vs. Hippocampal Precursors In Brain Injury"

    Article Title: Opposite Effect Of Inflammation on SVZ vs. Hippocampal Precursors In Brain Injury

    Journal: Annals of neurology

    doi: 10.1002/ana.22473

    IL-6 enhances neural stem cell expansion/maintenance, which can be inhibited by COX inhibitors. Primary neurospheres were dissociated and replated into either control media or media supplemented with 5ng/mL of rmIL-6. The number (A) and size (B) of spheres was quantified after 6 days. IL-6 increased the number of secondary neurospheres (*, p
    Figure Legend Snippet: IL-6 enhances neural stem cell expansion/maintenance, which can be inhibited by COX inhibitors. Primary neurospheres were dissociated and replated into either control media or media supplemented with 5ng/mL of rmIL-6. The number (A) and size (B) of spheres was quantified after 6 days. IL-6 increased the number of secondary neurospheres (*, p

    Techniques Used:

    3) Product Images from "Catalog of Differentially Expressed Long Non-Coding RNA following Activation of Human and Mouse Innate Immune Response"

    Article Title: Catalog of Differentially Expressed Long Non-Coding RNA following Activation of Human and Mouse Innate Immune Response

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01038

    IL7-AS regulates the expression and release of IL6 from interleukin-1β (IL1β)- and lipopolysaccharides (LPS)-stimulated human and mouse cells. (A) Comparison of fold change in expression of differentially expressed lncRNAs using RNA sequencing and qRT-PCR. Structure and profile of IL7-AS expression in (B) human and (C) mouse cells visualized using the Integrated Genomics Viewer (IGV). (D) Time course of IL7-AS and IL7 mRNA production in IL1β-stimulated human alveolar A549 epithelium and LPS-stimulated human THP1 monocytes and mouse RAW 264.7 macrophages ( n = 3 independent experiments). (E) Subcellular distribution of IL7-AS in IL1β-stimulated A549 epithelium and LPS-stimulated THP1 monocytes in which NEAT-1 and mitochondrial-cytochrome b ( MT-CYB ) are employed as markers of nuclear and cytoplasmic fractions, respectively ( n = 4 independent experiments). (F) Effect of transfection with a negative control LNA (scramble) or two antisense LNA (LNA 1 or 2) targeting, respectively, exons 1 and 4 (human cells) or exons 1 and 3 (mouse cells) of IL7-AS at a final concentration of 30 nM. Cells were then treated with either IL1β (30 ng/ml) or LPS (1 µg/ml), or left untreated for 24 h, prior to measurement of levels of the stated gene (by q-PCR) or proteins (by ELISA) ( n = 7–8 independent experiments). Statistical significance was performed using either two-way analysis of variance (ANOVA) for time courses or repeated measure one-way ANOVA with both a Dunnett’s post-test correction, where * p
    Figure Legend Snippet: IL7-AS regulates the expression and release of IL6 from interleukin-1β (IL1β)- and lipopolysaccharides (LPS)-stimulated human and mouse cells. (A) Comparison of fold change in expression of differentially expressed lncRNAs using RNA sequencing and qRT-PCR. Structure and profile of IL7-AS expression in (B) human and (C) mouse cells visualized using the Integrated Genomics Viewer (IGV). (D) Time course of IL7-AS and IL7 mRNA production in IL1β-stimulated human alveolar A549 epithelium and LPS-stimulated human THP1 monocytes and mouse RAW 264.7 macrophages ( n = 3 independent experiments). (E) Subcellular distribution of IL7-AS in IL1β-stimulated A549 epithelium and LPS-stimulated THP1 monocytes in which NEAT-1 and mitochondrial-cytochrome b ( MT-CYB ) are employed as markers of nuclear and cytoplasmic fractions, respectively ( n = 4 independent experiments). (F) Effect of transfection with a negative control LNA (scramble) or two antisense LNA (LNA 1 or 2) targeting, respectively, exons 1 and 4 (human cells) or exons 1 and 3 (mouse cells) of IL7-AS at a final concentration of 30 nM. Cells were then treated with either IL1β (30 ng/ml) or LPS (1 µg/ml), or left untreated for 24 h, prior to measurement of levels of the stated gene (by q-PCR) or proteins (by ELISA) ( n = 7–8 independent experiments). Statistical significance was performed using either two-way analysis of variance (ANOVA) for time courses or repeated measure one-way ANOVA with both a Dunnett’s post-test correction, where * p

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Transfection, Negative Control, Concentration Assay, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Related Articles

    Enzyme-linked Immunosorbent Assay:

    Article Title: Regulation of inflammatory responses by dynamic subcellular localization of RNA-binding protein Arid5a
    Article Snippet: Anti-Flag (F1804; Sigma-Aldrich), anti-Arid5a (customized antibody; Sigma-Aldrich), anti–Regnase-1 (sc-515275; Santa Cruz Biotechnology), anti-KPNA2 (ab84440; Abcam), anti–Lamin-B (PM064; MBL), anti–α-tubulin (M175; MBL), anti-UPF1 (sc-166092; Santa Cruz Biotechnology), anti-UPF2 (D3B10; Cell Signaling Technology), anti-UPF3A (MBS712426; My Bio Source), anti-UPF3B (HPA001882; Atlas), and anti–c-Myc (Nacalai Tesque) antibodies were used in this study. .. The levels of mouse IL-6 and TNF in serum were measured by ELISA using commercial kits according to the manufacturer’s protocol (R & D Systems). ..

    Article Title: Killing of Pseudomonas aeruginosa by Chicken Cathelicidin-2 Is Immunogenically Silent, Preventing Lung Inflammation In Vivo
    Article Snippet: .. The levels of mouse IL-6, KC, and TNF-α were measured using ELISA kits per the manufacturer's instructions (R & D Systems, Minneapolis, MN). .. A Bio-Plex 200 readout system was used (Bio-Rad), and cytokine levels were automatically calculated from standard curves using Bio-Plex Manager software (v.4.1.1, Bio-Rad).

    Article Title: Niclosamide suppresses the expansion of follicular helper T cells and alleviates disease severity in two murine models of lupus via STAT3
    Article Snippet: .. Enzyme-linked immunosorbent assay (ELISA)Cytokines in sera or spleen lysates were assayed using mouse IL-6 and IL-21 Duoset ELISA kits (R & D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. .. The serum levels of anti-double-stranded DNA (dsDNA) IgG antibodies were measured by ELISA following the manufacturer’s instructions (Alpha Diagnostics, San Antonio, TX, USA).

    Article Title: Interleukin-18 mediates interleukin-1-induced cardiac dysfunction
    Article Snippet: Alternatively, blood was obtained in heparin tubes (BD) and then centrifuged at 2,000 rpm at 4°C for 10 min for plasma. .. Samples were stored at −80°C and subsequently analyzed with a specific ELISA for murine IL-18 (serum, MBL) and for mouse IL-6 (plasma, R & D Systems) accordingly to the supplier's instructions to assess the induction of IL-18 and IL-6 in plasma after IL-1β challenge. ..

    Article Title: Mast Cells Are Activated by Streptococcus pneumoniae In Vitro but Dispensable for the Host Defense Against Pneumococcal Central Nervous System Infection In Vivo
    Article Snippet: For screening purposes, supernatants from control and S. pneumoniae -challenged cells were subjected to a mouse Multi-Analyte ELISA Array Kit (M-005a; Qiagen, Hilden, Germany). .. In all other experiments, supernatants were assessed for the presence of mouse IL-6 and CCL2 using mouse DuoSet ELISA Kits (R & D Systems, Bio-Techne, Wiesbaden, Germany). .. Moreover, the LDH activity, a marker of cell damage, was determined in supernatants (S), centrifuged supernatants of control cells after lysis with Triton X-100 (PC), and in control medium [negative control (NC)] using a colorimetric assay kit (BioVision, Biocat GmbH, Heidelberg, Germany).

    Sandwich ELISA:

    Article Title: Neutrophil extracellular traps exacerbate Th1-mediated autoimmune responses in rheumatoid arthritis by promoting DC maturation
    Article Snippet: Detection was performed using HRP-linked Abs (Cell Signaling Technology) and enhanced chemiluminescent (ECL) detection reagents (Amersham Biosciences). .. Detection of mouse IL-6 and TNF-α Duo set (R & D systems) and human IL-6 and TNF-α (eBioscience) in culture supernatants harvested at the indicated time, were performed by sandwich ELISA following the manufacturer’s recommendations. .. Light absorbance at 450 nm was measured using the ELx800 Biotek.

    Cell Culture:

    Article Title: Unique Macrophages Different from M1/M2 Macrophages Inhibit T Cell Mitogenesis while Upregulating Th17 Polarization
    Article Snippet: PI-stained cells were subjected to sub-G1 analysis by flow cytometry using a FACSCalibur cytometer (BD Biosciences). .. Cytokine production by test T cells and MΦs TCR-stimulated splenic T cells were cultured in the presence or absence of test MΦs (MΦ:T cell ratio = 1:14) for up to 7 days under the Th17 polarizing condition using RPMI medium containing mouse IL-6 (20 ng/ml; R & D Systems), human TGF-β (1 ng/ml; PeproTech), anti-mouse IFN-γ Ab (1 μg/ml; eBioscience), and anti-mouse IL-4 Ab (1 μg/ml; eBioscience) or the non-Th17 skewing condition using RPMI medium free from the above supplements. .. At intervals, culture fluids were harvested and measured for cytokine concentration by ELISA using the DuoSet ELISA Development System kit (R & D Systems) according to the manufacturer's instructions.

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    R&D Systems il6 protein
    The oxysterol 25HC affects inflammatory signaling mediated by several PRRs via a non–LXR-dependent pathway. ( A ) Macrophages derived from three WT and three Ch25h −/− mice were stimulated with the indicated agonists at the indicated time points and RNA was analyzed by real-time PCR. Data shown are the average from the three biological replicates. ( B ) Expression of <t>Il6</t> in Lxrαβ −/− BMDMs treated with 5 μM 25HC for 1 h and stimulated with 6 μg/mL PIC for 6 or 18 h were determined by real-time PCR. Data shown are the average of three replicates.
    Il6 Protein, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/il6 protein/product/R&D Systems
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    93
    R&D Systems recombinant murine il 6
    Signaling in cultured fps K588R homozygous mutant BMM. BMM from wild-type (+/+) or homozygous mutant (−/−) mice were starved for 48 h in 0.5% FBS prior to stimulation with either GM-CSF (30 ng/ml) or <t>IL-6</t> (30 ng/ml). Cells were exposed to each cytokine for 15 min at 37°C, scraped in 2× SDS sample buffer, run out on an SDS–7.5% polyacrylamide gel, transferred to Immobilon-P membrane, and then probed successively with the indicated antibodies: (A) anti-pStat3 (top) followed by anti-Stat3 (bottom); or (B) anti-pStat5A/B (top) followed by anti-Stat5A (bottom). Stat5B (90 to 92 kDa) is constitutively phosphorylated in these BMM cultures, even under starvation conditions, whereas Stat5A (95 kDa) is phosphorylated only following GM-CSF stimulation. (C) BMM were starved for 48 h in 0.5% FBS prior to exposure to various doses of LPS for 30 min at 37°C. Whole-cell lysates were run out on SDS–11% polyacrylamide gels, transferred to Immobilon-P membrane, and then probed with anti-Erk antibody (bottom) followed by anti-pErk antibody (top).
    Recombinant Murine Il 6, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant murine il 6/product/R&D Systems
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant murine il 6 - by Bioz Stars, 2021-05
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    95
    R&D Systems mouse il 6
    Blockade of IL-18 signaling does not prevent the <t>IL-6</t> increase after IL-1β administration. A : plasma levels of IL-6 in C57BL6 WT, IL-18 KO, and IL-18R KO mice 4 h after IL-1β (3 μg/kg) injection. n = 6 mice/group. * P
    Mouse Il 6, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse il 6/product/R&D Systems
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse il 6 - by Bioz Stars, 2021-05
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    99
    R&D Systems il 17a
    T cell–derived IL-3 is essential to cardiac inflammation in myocarditis. (A) Il3 mRNA levels in the heart (HT), BM, spleen (Sp), draining LN, thymus (TH), and lung (LG) before and 8, 14, and 21 d after the first immunization ( n = 6–9 per group representing two independent experiments). nd, not detected. (B) Representative flow dot plots of heart tissue cell suspensions to identify IL-3 + cells on day 21. (C) Further flow cytometric characterization of IL-3–producing CD4 + T cells by costaining for IFN-γ, <t>IL-17A,</t> and IL-4 in the inflamed heart. (D) T cells were isolated by draining LNs of either WT or Il3 −/− immunized mice on day 14 and culturing with WT BMDCs in the presence or absence of the indicated peptide (10 µg/ml) for 72 h. Culture supernatants were collected, and IL-3 levels were measured by ELISA. MOG, myelin oligodendrocyte glycoprotein. (E) Schematic diagram of T cell adoptive transfer–induced EAM. (F) Quantification of total leukocyte numbers in the hearts of recipient Scid mice ( n = 6–7 per group of two independent experiments). (G and H) WT mice were lethally irradiated and reconstituted with a mixture of BM cells obtained from Rag1 −/− and either WT or Il3 −/− mice at 1:1 ratio to generate Rag1 −/− /WT and Rag1 −/− / Il3 −/− BM mixed chimeras (G). After 6–7 wk to allow for reconstitution, the mice were subjected to EAM induction, and leukocyte subsets in the heart were evaluated by flow cytometry on day 21 (H; n = 7–8 per group of two independent experiments). *, P
    Il 17a, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/il 17a/product/R&D Systems
    Average 99 stars, based on 1 article reviews
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    il 17a - by Bioz Stars, 2021-05
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    Image Search Results


    The oxysterol 25HC affects inflammatory signaling mediated by several PRRs via a non–LXR-dependent pathway. ( A ) Macrophages derived from three WT and three Ch25h −/− mice were stimulated with the indicated agonists at the indicated time points and RNA was analyzed by real-time PCR. Data shown are the average from the three biological replicates. ( B ) Expression of Il6 in Lxrαβ −/− BMDMs treated with 5 μM 25HC for 1 h and stimulated with 6 μg/mL PIC for 6 or 18 h were determined by real-time PCR. Data shown are the average of three replicates.

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

    Article Title: 25-Hydroxycholesterol acts as an amplifier of inflammatory signaling

    doi: 10.1073/pnas.1404271111

    Figure Lengend Snippet: The oxysterol 25HC affects inflammatory signaling mediated by several PRRs via a non–LXR-dependent pathway. ( A ) Macrophages derived from three WT and three Ch25h −/− mice were stimulated with the indicated agonists at the indicated time points and RNA was analyzed by real-time PCR. Data shown are the average from the three biological replicates. ( B ) Expression of Il6 in Lxrαβ −/− BMDMs treated with 5 μM 25HC for 1 h and stimulated with 6 μg/mL PIC for 6 or 18 h were determined by real-time PCR. Data shown are the average of three replicates.

    Article Snippet: Levels of IL6 protein were measured using a commercially available ELISA (R & D Systems; DY406) according to the manufacturer’s instructions.

    Techniques: Derivative Assay, Mouse Assay, Real-time Polymerase Chain Reaction, Expressing

    Signaling in cultured fps K588R homozygous mutant BMM. BMM from wild-type (+/+) or homozygous mutant (−/−) mice were starved for 48 h in 0.5% FBS prior to stimulation with either GM-CSF (30 ng/ml) or IL-6 (30 ng/ml). Cells were exposed to each cytokine for 15 min at 37°C, scraped in 2× SDS sample buffer, run out on an SDS–7.5% polyacrylamide gel, transferred to Immobilon-P membrane, and then probed successively with the indicated antibodies: (A) anti-pStat3 (top) followed by anti-Stat3 (bottom); or (B) anti-pStat5A/B (top) followed by anti-Stat5A (bottom). Stat5B (90 to 92 kDa) is constitutively phosphorylated in these BMM cultures, even under starvation conditions, whereas Stat5A (95 kDa) is phosphorylated only following GM-CSF stimulation. (C) BMM were starved for 48 h in 0.5% FBS prior to exposure to various doses of LPS for 30 min at 37°C. Whole-cell lysates were run out on SDS–11% polyacrylamide gels, transferred to Immobilon-P membrane, and then probed with anti-Erk antibody (bottom) followed by anti-pErk antibody (top).

    Journal: Molecular and Cellular Biology

    Article Title: Targeted Disruption of the Murine fps/fes Proto-Oncogene Reveals that Fps/Fes Kinase Activity Is Dispensable for Hematopoiesis

    doi:

    Figure Lengend Snippet: Signaling in cultured fps K588R homozygous mutant BMM. BMM from wild-type (+/+) or homozygous mutant (−/−) mice were starved for 48 h in 0.5% FBS prior to stimulation with either GM-CSF (30 ng/ml) or IL-6 (30 ng/ml). Cells were exposed to each cytokine for 15 min at 37°C, scraped in 2× SDS sample buffer, run out on an SDS–7.5% polyacrylamide gel, transferred to Immobilon-P membrane, and then probed successively with the indicated antibodies: (A) anti-pStat3 (top) followed by anti-Stat3 (bottom); or (B) anti-pStat5A/B (top) followed by anti-Stat5A (bottom). Stat5B (90 to 92 kDa) is constitutively phosphorylated in these BMM cultures, even under starvation conditions, whereas Stat5A (95 kDa) is phosphorylated only following GM-CSF stimulation. (C) BMM were starved for 48 h in 0.5% FBS prior to exposure to various doses of LPS for 30 min at 37°C. Whole-cell lysates were run out on SDS–11% polyacrylamide gels, transferred to Immobilon-P membrane, and then probed with anti-Erk antibody (bottom) followed by anti-pErk antibody (top).

    Article Snippet: Briefly, 50,000 unfractionated BM cells were cultured in 1.2 ml of semisolid medium consisting of 1% methylcellulose (Fluka), 10% fetal bovine serum (FBS), 2% bovine serum albumin (BSA), 200 μg of human plasma transferrin (iron saturated; (Calbiochem) per ml, 50 μM α-monothioglycerol (Sigma), and either (i) a cytokine cocktail consisting of recombinant human Epo (1 U/ml; R & D Systems), recombinant murine IL-3 (5 ng/ml; (R & D Systems), recombinant murine Steel factor (SF) (50 ng/ml; R & D Systems), and recombinant murine IL-6 (10 ng/ml; R & D Systems); (ii) recombinant murine GM-CSF (5 ng/ml; R & D Systems) and recombinant human Epo (1 U/ml); or (iii) recombinant human Epo (1 U/ml) alone.

    Techniques: Cell Culture, Mutagenesis, Mouse Assay

    BM hematopoietic progenitor cell colony assays. (A) BM cells were grown in the presence of a cocktail of cytokines, consisting of IL-3 (5 ng/ml), IL-6 (10 ng/ml), SF (50 ng/ml), and Epo (1 U/ml). (B) BM cells were grown in the presence of GM-CSF (5 ng/ml) and Epo (1 U/ml). Bars in panels A and B: white, total number of colonies; hatched, CFU-GM and CFU-M colonies combined; stippled, BFU-E colonies; black, CFU-GEMM colonies. Colony counts were made on day 8 postplating. In panel C, CFU-E colonies were grown from BM cells under the following conditions: the cytokine cocktail (white bars), GM-CSF and Epo (hatched bars), and Epo alone (black bars). CFU-E colony counts were made 2 days postplating. The sample sizes were six mice per genotype for each panel. The height of each bar represents the average number of colonies for the six mice analyzed, and the error bars represent SD. In panel D, 20 CFU-GM colonies were randomly picked from 1.2-ml methylcellulose cultures which had been seeded with 50,000 nucleated BM cells. Colonies were picked 9 days postplating, and cells were pooled and counted. The height of each bar represents the average number of cells per CFU-GM colony, and error bars indicate SD. The sample sizes were four mice of each genotype.

    Journal: Molecular and Cellular Biology

    Article Title: Targeted Disruption of the Murine fps/fes Proto-Oncogene Reveals that Fps/Fes Kinase Activity Is Dispensable for Hematopoiesis

    doi:

    Figure Lengend Snippet: BM hematopoietic progenitor cell colony assays. (A) BM cells were grown in the presence of a cocktail of cytokines, consisting of IL-3 (5 ng/ml), IL-6 (10 ng/ml), SF (50 ng/ml), and Epo (1 U/ml). (B) BM cells were grown in the presence of GM-CSF (5 ng/ml) and Epo (1 U/ml). Bars in panels A and B: white, total number of colonies; hatched, CFU-GM and CFU-M colonies combined; stippled, BFU-E colonies; black, CFU-GEMM colonies. Colony counts were made on day 8 postplating. In panel C, CFU-E colonies were grown from BM cells under the following conditions: the cytokine cocktail (white bars), GM-CSF and Epo (hatched bars), and Epo alone (black bars). CFU-E colony counts were made 2 days postplating. The sample sizes were six mice per genotype for each panel. The height of each bar represents the average number of colonies for the six mice analyzed, and the error bars represent SD. In panel D, 20 CFU-GM colonies were randomly picked from 1.2-ml methylcellulose cultures which had been seeded with 50,000 nucleated BM cells. Colonies were picked 9 days postplating, and cells were pooled and counted. The height of each bar represents the average number of cells per CFU-GM colony, and error bars indicate SD. The sample sizes were four mice of each genotype.

    Article Snippet: Briefly, 50,000 unfractionated BM cells were cultured in 1.2 ml of semisolid medium consisting of 1% methylcellulose (Fluka), 10% fetal bovine serum (FBS), 2% bovine serum albumin (BSA), 200 μg of human plasma transferrin (iron saturated; (Calbiochem) per ml, 50 μM α-monothioglycerol (Sigma), and either (i) a cytokine cocktail consisting of recombinant human Epo (1 U/ml; R & D Systems), recombinant murine IL-3 (5 ng/ml; (R & D Systems), recombinant murine Steel factor (SF) (50 ng/ml; R & D Systems), and recombinant murine IL-6 (10 ng/ml; R & D Systems); (ii) recombinant murine GM-CSF (5 ng/ml; R & D Systems) and recombinant human Epo (1 U/ml); or (iii) recombinant human Epo (1 U/ml) alone.

    Techniques: Mouse Assay

    Blockade of IL-18 signaling does not prevent the IL-6 increase after IL-1β administration. A : plasma levels of IL-6 in C57BL6 WT, IL-18 KO, and IL-18R KO mice 4 h after IL-1β (3 μg/kg) injection. n = 6 mice/group. * P

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Interleukin-18 mediates interleukin-1-induced cardiac dysfunction

    doi: 10.1152/ajpheart.00795.2013

    Figure Lengend Snippet: Blockade of IL-18 signaling does not prevent the IL-6 increase after IL-1β administration. A : plasma levels of IL-6 in C57BL6 WT, IL-18 KO, and IL-18R KO mice 4 h after IL-1β (3 μg/kg) injection. n = 6 mice/group. * P

    Article Snippet: Samples were stored at −80°C and subsequently analyzed with a specific ELISA for murine IL-18 (serum, MBL) and for mouse IL-6 (plasma, R & D Systems) accordingly to the supplier's instructions to assess the induction of IL-18 and IL-6 in plasma after IL-1β challenge.

    Techniques: Mouse Assay, Injection

    T cell–derived IL-3 is essential to cardiac inflammation in myocarditis. (A) Il3 mRNA levels in the heart (HT), BM, spleen (Sp), draining LN, thymus (TH), and lung (LG) before and 8, 14, and 21 d after the first immunization ( n = 6–9 per group representing two independent experiments). nd, not detected. (B) Representative flow dot plots of heart tissue cell suspensions to identify IL-3 + cells on day 21. (C) Further flow cytometric characterization of IL-3–producing CD4 + T cells by costaining for IFN-γ, IL-17A, and IL-4 in the inflamed heart. (D) T cells were isolated by draining LNs of either WT or Il3 −/− immunized mice on day 14 and culturing with WT BMDCs in the presence or absence of the indicated peptide (10 µg/ml) for 72 h. Culture supernatants were collected, and IL-3 levels were measured by ELISA. MOG, myelin oligodendrocyte glycoprotein. (E) Schematic diagram of T cell adoptive transfer–induced EAM. (F) Quantification of total leukocyte numbers in the hearts of recipient Scid mice ( n = 6–7 per group of two independent experiments). (G and H) WT mice were lethally irradiated and reconstituted with a mixture of BM cells obtained from Rag1 −/− and either WT or Il3 −/− mice at 1:1 ratio to generate Rag1 −/− /WT and Rag1 −/− / Il3 −/− BM mixed chimeras (G). After 6–7 wk to allow for reconstitution, the mice were subjected to EAM induction, and leukocyte subsets in the heart were evaluated by flow cytometry on day 21 (H; n = 7–8 per group of two independent experiments). *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Self-reactive CD4+ IL-3+ T cells amplify autoimmune inflammation in myocarditis by inciting monocyte chemotaxis

    doi: 10.1084/jem.20180722

    Figure Lengend Snippet: T cell–derived IL-3 is essential to cardiac inflammation in myocarditis. (A) Il3 mRNA levels in the heart (HT), BM, spleen (Sp), draining LN, thymus (TH), and lung (LG) before and 8, 14, and 21 d after the first immunization ( n = 6–9 per group representing two independent experiments). nd, not detected. (B) Representative flow dot plots of heart tissue cell suspensions to identify IL-3 + cells on day 21. (C) Further flow cytometric characterization of IL-3–producing CD4 + T cells by costaining for IFN-γ, IL-17A, and IL-4 in the inflamed heart. (D) T cells were isolated by draining LNs of either WT or Il3 −/− immunized mice on day 14 and culturing with WT BMDCs in the presence or absence of the indicated peptide (10 µg/ml) for 72 h. Culture supernatants were collected, and IL-3 levels were measured by ELISA. MOG, myelin oligodendrocyte glycoprotein. (E) Schematic diagram of T cell adoptive transfer–induced EAM. (F) Quantification of total leukocyte numbers in the hearts of recipient Scid mice ( n = 6–7 per group of two independent experiments). (G and H) WT mice were lethally irradiated and reconstituted with a mixture of BM cells obtained from Rag1 −/− and either WT or Il3 −/− mice at 1:1 ratio to generate Rag1 −/− /WT and Rag1 −/− / Il3 −/− BM mixed chimeras (G). After 6–7 wk to allow for reconstitution, the mice were subjected to EAM induction, and leukocyte subsets in the heart were evaluated by flow cytometry on day 21 (H; n = 7–8 per group of two independent experiments). *, P

    Article Snippet: IL-2, IL-4, IL-6, IL-17A, IFN-γ, TNF-α, and GM-CSF levels were also measured in cell culture supernatants with ELISA Kits from R & D Systems according to the manufacturer’s instructions.

    Techniques: Derivative Assay, Flow Cytometry, Isolation, Mouse Assay, Enzyme-linked Immunosorbent Assay, Adoptive Transfer Assay, Irradiation, Cytometry

    IL-3 is dispensable for T cell sensitization. (A) In vivo T cell proliferation in the draining LNs was measured by BrdU incorporation before and 10 and 21 d after the first immunization ( n = 4–8 per group of two independent experiments). BrdU was injected intraperitoneally 2 h before the sacrifice. (B) In vitro T cell proliferation was assessed by a cell tracer dye, Cell Trace Violet. CD4 + T cells obtained from LNs of immunized WT or Il3 −/− mice were stained with Cell Trace Violet and cultured at indicated conditions for 72 h ( n = 4–8 per group of three independent experiments). (C) Enumeration of CD4 + T cell numbers in the draining LNs before and 10 d after the first immunization ( n = 4–8 per group of two independent experiments). (D) Representative flow dot plots to identify DC subsets in the draining LNs. (E) Quantification of migratory cDCs, resident cDCs, and moDCs in WT and Il3 −/− draining LNs on days 0 and 10 ( n = 4 per group of two independent experiments). (F) Production of IFN-γ and IL-17A by WT and Il3 −/− CD4 + T cells in the draining LNs on day 21. (G) Percentage of IFN-γ + or IL-17A + CD4 + T cells in the draining LNs on day 21 ( n = 4–8 per group of two independent experiments). (H) CD4 + T cells collected from draining LNs of immunized WT or Il3 −/− mice were cultured with BMDC in the presence of 10 µg/ml αMHC for 3 d, and indicated cytokines were measured in the supernatants ( n = 4–8 per group of two independent experiments). (I) Schematic diagram of the experimental design for BMDC-induced EAM. (J) Flow cytometry–based quantification of indicated cells in the hearts of WT and Il3 −/− mice 10 d after the first BMDC injection ( n = 4–14 per group grouped from at least two independent experiments). *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Self-reactive CD4+ IL-3+ T cells amplify autoimmune inflammation in myocarditis by inciting monocyte chemotaxis

    doi: 10.1084/jem.20180722

    Figure Lengend Snippet: IL-3 is dispensable for T cell sensitization. (A) In vivo T cell proliferation in the draining LNs was measured by BrdU incorporation before and 10 and 21 d after the first immunization ( n = 4–8 per group of two independent experiments). BrdU was injected intraperitoneally 2 h before the sacrifice. (B) In vitro T cell proliferation was assessed by a cell tracer dye, Cell Trace Violet. CD4 + T cells obtained from LNs of immunized WT or Il3 −/− mice were stained with Cell Trace Violet and cultured at indicated conditions for 72 h ( n = 4–8 per group of three independent experiments). (C) Enumeration of CD4 + T cell numbers in the draining LNs before and 10 d after the first immunization ( n = 4–8 per group of two independent experiments). (D) Representative flow dot plots to identify DC subsets in the draining LNs. (E) Quantification of migratory cDCs, resident cDCs, and moDCs in WT and Il3 −/− draining LNs on days 0 and 10 ( n = 4 per group of two independent experiments). (F) Production of IFN-γ and IL-17A by WT and Il3 −/− CD4 + T cells in the draining LNs on day 21. (G) Percentage of IFN-γ + or IL-17A + CD4 + T cells in the draining LNs on day 21 ( n = 4–8 per group of two independent experiments). (H) CD4 + T cells collected from draining LNs of immunized WT or Il3 −/− mice were cultured with BMDC in the presence of 10 µg/ml αMHC for 3 d, and indicated cytokines were measured in the supernatants ( n = 4–8 per group of two independent experiments). (I) Schematic diagram of the experimental design for BMDC-induced EAM. (J) Flow cytometry–based quantification of indicated cells in the hearts of WT and Il3 −/− mice 10 d after the first BMDC injection ( n = 4–14 per group grouped from at least two independent experiments). *, P

    Article Snippet: IL-2, IL-4, IL-6, IL-17A, IFN-γ, TNF-α, and GM-CSF levels were also measured in cell culture supernatants with ELISA Kits from R & D Systems according to the manufacturer’s instructions.

    Techniques: In Vivo, BrdU Incorporation Assay, Injection, In Vitro, Mouse Assay, Staining, Cell Culture, Flow Cytometry, Cytometry

    Monocyte-derived APCs promote local T cell proliferation in the inflamed heart. (A) CD4 + T cell proliferation was measured by BrdU incorporation in the inflamed hearts (HT) and blood of WT and Il3 −/− mice 21 d after the first immunization. BrdU was injected 2 h before the sacrifice. (B) Percentage of BrdU + cells in cardiac and blood (BL) CD4 + T cells of WT and Il3 −/− mice at the indicated time points ( n = 4–8 per group of two independent experiments). (C) Representative flow cytometric dot plots of activated caspase-3 expression in WT and Il3 −/− CD4 + T cells in the heart 21 d after the first immunization. (D) Percentage of activated caspase-3 + cells shown in C ( n = 4 per group of two independent experiments). (E) Quantification of IFN-γ + , IL-17A + , and GM-CSF + CD4 + T cells in the hearts on days 0 and 21 ( n = 4–8 per group of two independent experiments). (F) 5 × 10 4 of autoreactive T cells and 10 4 of each of the sorted cardiac populations were cultured at indicated conditions for 3 d. The percentage of proliferating T cells was evaluated by Cell Trace Violet dye and normalized to T cells cultured with moDCs in the presence of αMHC peptide ( n = 4–7 per group of three independent experiments). (G) T cells sorted from day 14 draining LNs were stained with Cell Trace Violet and cultured with sorted cardiac moDCs at the indicated ratio. T cell proliferation was assessed 3 d later ( n = 4 per group of two independent experiments). (H) IL-3 protein levels were measured by ELISA in the supernatant of the culture as in G ( n = 4 per group of two independent experiments). (I) Correlation between T cell proliferation and the number of moDCs or MHCII + macrophages in WT hearts at peak of inflammation. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Self-reactive CD4+ IL-3+ T cells amplify autoimmune inflammation in myocarditis by inciting monocyte chemotaxis

    doi: 10.1084/jem.20180722

    Figure Lengend Snippet: Monocyte-derived APCs promote local T cell proliferation in the inflamed heart. (A) CD4 + T cell proliferation was measured by BrdU incorporation in the inflamed hearts (HT) and blood of WT and Il3 −/− mice 21 d after the first immunization. BrdU was injected 2 h before the sacrifice. (B) Percentage of BrdU + cells in cardiac and blood (BL) CD4 + T cells of WT and Il3 −/− mice at the indicated time points ( n = 4–8 per group of two independent experiments). (C) Representative flow cytometric dot plots of activated caspase-3 expression in WT and Il3 −/− CD4 + T cells in the heart 21 d after the first immunization. (D) Percentage of activated caspase-3 + cells shown in C ( n = 4 per group of two independent experiments). (E) Quantification of IFN-γ + , IL-17A + , and GM-CSF + CD4 + T cells in the hearts on days 0 and 21 ( n = 4–8 per group of two independent experiments). (F) 5 × 10 4 of autoreactive T cells and 10 4 of each of the sorted cardiac populations were cultured at indicated conditions for 3 d. The percentage of proliferating T cells was evaluated by Cell Trace Violet dye and normalized to T cells cultured with moDCs in the presence of αMHC peptide ( n = 4–7 per group of three independent experiments). (G) T cells sorted from day 14 draining LNs were stained with Cell Trace Violet and cultured with sorted cardiac moDCs at the indicated ratio. T cell proliferation was assessed 3 d later ( n = 4 per group of two independent experiments). (H) IL-3 protein levels were measured by ELISA in the supernatant of the culture as in G ( n = 4 per group of two independent experiments). (I) Correlation between T cell proliferation and the number of moDCs or MHCII + macrophages in WT hearts at peak of inflammation. *, P

    Article Snippet: IL-2, IL-4, IL-6, IL-17A, IFN-γ, TNF-α, and GM-CSF levels were also measured in cell culture supernatants with ELISA Kits from R & D Systems according to the manufacturer’s instructions.

    Techniques: Derivative Assay, BrdU Incorporation Assay, Mouse Assay, Injection, Flow Cytometry, Expressing, Cell Culture, Staining, Enzyme-linked Immunosorbent Assay