tnfα  (BioLegend)

 
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    Name:
    Recombinant Human TNF α Animal Free
    Description:
    Recombinant Human TNF α Animal Free Apps BA Size 50 μg
    Catalog Number:
    717904
    Price:
    215
    Applications:
    BA
    Conjugate:
    RECOM
    Size:
    50 μg
    Category:
    Recombinant Protein
    Score:
    85
    Quantity:
    1
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    Structured Review

    BioLegend tnfα
    HMGB1 does not induce NF-κB translocation in HCEC. hTCEpi were cultured in the presence of HMGB1 (50 ng/mL) for 2 hours and immunostained for NF-κB p65 expression. <t>TNFα</t> (10 ng/mL) was used as a positive control for NF-κB p65 cytoplasm-nucleus translocation. No translocation of NF-κB p65 was observed with HMGB1 treatment. Images are representative of n = 2 independent experiments; scale bar: 50 μm.
    Recombinant Human TNF α Animal Free Apps BA Size 50 μg
    https://www.bioz.com/result/tnfα/product/BioLegend
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tnfα - by Bioz Stars, 2020-01
    99/100 stars

    Related Products / Commonly Used Together

    il-1β

    Images

    1) Product Images from "High-Mobility Group Box 1 in Dry Eye Inflammation"

    Article Title: High-Mobility Group Box 1 in Dry Eye Inflammation

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.17-23363

    HMGB1 does not induce NF-κB translocation in HCEC. hTCEpi were cultured in the presence of HMGB1 (50 ng/mL) for 2 hours and immunostained for NF-κB p65 expression. TNFα (10 ng/mL) was used as a positive control for NF-κB p65 cytoplasm-nucleus translocation. No translocation of NF-κB p65 was observed with HMGB1 treatment. Images are representative of n = 2 independent experiments; scale bar: 50 μm.
    Figure Legend Snippet: HMGB1 does not induce NF-κB translocation in HCEC. hTCEpi were cultured in the presence of HMGB1 (50 ng/mL) for 2 hours and immunostained for NF-κB p65 expression. TNFα (10 ng/mL) was used as a positive control for NF-κB p65 cytoplasm-nucleus translocation. No translocation of NF-κB p65 was observed with HMGB1 treatment. Images are representative of n = 2 independent experiments; scale bar: 50 μm.

    Techniques Used: Translocation Assay, Cell Culture, Expressing, Positive Control

    IFNγ does not improve HCEC responsiveness to LPS or HMGB1. Primary HCEC were stimulated with IFNγ (200 U/mL) prior to treatment with HMGB1, LPS, or both for 8 hours. Cell lysates were collected for RNA extraction and qPCR determination of relative IL-6, IL-8, and TNFα mRNA expression. Graphs represent mean ± SEM of n = 2 independent experiments.
    Figure Legend Snippet: IFNγ does not improve HCEC responsiveness to LPS or HMGB1. Primary HCEC were stimulated with IFNγ (200 U/mL) prior to treatment with HMGB1, LPS, or both for 8 hours. Cell lysates were collected for RNA extraction and qPCR determination of relative IL-6, IL-8, and TNFα mRNA expression. Graphs represent mean ± SEM of n = 2 independent experiments.

    Techniques Used: RNA Extraction, Real-time Polymerase Chain Reaction, Expressing

    HMGB1 does not induce secretion of inflammatory cytokines in HCEC. hTCEpi (A, B) and macrophage-differentiated U937 (Mϕ-U937) cells (C, D) were stimulated with HMGB1 (10 μg/mL) for 4 or 8 hours. mRNA expression (left graphs) and secreted IL-6, IL-8, and TNFα (right graphs) were measured by qPCR and ELISA, respectively. Graphs represent mean ± SEM of n = 2 (Mϕ-U937) and n = 4 (HTCEpi) independent experiments. ANOVA was used to test for statistical significance with Bonferroni's test for multiple comparisons. ***P ≤ 0.0001; **P ≤ 0.001; *P ≤ 0.01.
    Figure Legend Snippet: HMGB1 does not induce secretion of inflammatory cytokines in HCEC. hTCEpi (A, B) and macrophage-differentiated U937 (Mϕ-U937) cells (C, D) were stimulated with HMGB1 (10 μg/mL) for 4 or 8 hours. mRNA expression (left graphs) and secreted IL-6, IL-8, and TNFα (right graphs) were measured by qPCR and ELISA, respectively. Graphs represent mean ± SEM of n = 2 (Mϕ-U937) and n = 4 (HTCEpi) independent experiments. ANOVA was used to test for statistical significance with Bonferroni's test for multiple comparisons. ***P ≤ 0.0001; **P ≤ 0.001; *P ≤ 0.01.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Hyperosmolar stress and TNFα increase HMGB1 cellular expression and secretion in HCEC. (A) hTCEpi were cultured with 450 mOsM media or in the presence of TNFα (10 ng/mL). After 6 hours, both hyperosmolar stress and TNFα induced increase of nuclear and cytoplasmic HMGB1 expression when compared to the UT control. Images are representative of n = 2 independent experiments; scale bar: 50 μm. (B) SV40 HCEC were cultured for 6, 12, or 24 hours in hyperosmolar media (400, 450, or 500 mOsM) (left graph). hTCEpi were stimulated with TNFα (10 ng/mL) for 1 or 6 hours (right graph). HMGB1 was measured in cell culture supernatants by ELISA. Graphs represent mean ± SEM of n = 2 independent experiments. ANOVA was used to test for statistical significance with Bonferroni's test for multiple comparisons. ***P ≤ 0.0001; **P ≤ 0.001; *P ≤ 0.05.
    Figure Legend Snippet: Hyperosmolar stress and TNFα increase HMGB1 cellular expression and secretion in HCEC. (A) hTCEpi were cultured with 450 mOsM media or in the presence of TNFα (10 ng/mL). After 6 hours, both hyperosmolar stress and TNFα induced increase of nuclear and cytoplasmic HMGB1 expression when compared to the UT control. Images are representative of n = 2 independent experiments; scale bar: 50 μm. (B) SV40 HCEC were cultured for 6, 12, or 24 hours in hyperosmolar media (400, 450, or 500 mOsM) (left graph). hTCEpi were stimulated with TNFα (10 ng/mL) for 1 or 6 hours (right graph). HMGB1 was measured in cell culture supernatants by ELISA. Graphs represent mean ± SEM of n = 2 independent experiments. ANOVA was used to test for statistical significance with Bonferroni's test for multiple comparisons. ***P ≤ 0.0001; **P ≤ 0.001; *P ≤ 0.05.

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

    2) Product Images from "Methylthioadenosine Reprograms Macrophage Activation through Adenosine Receptor Stimulation"

    Article Title: Methylthioadenosine Reprograms Macrophage Activation through Adenosine Receptor Stimulation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0104210

    MTA inhibits TLR-induced TNFα mRNA production and NF-KB induction. (A) BMDM were incubated for 4 h with either DMSO or 200 µM MTA in the absence or presence of either 100 EU/mL LPS or 1 µg/mL Pam3CSK4. Total RNA was extracted from cells. TNFα expression relative to β-actin was determined by Δ(ΔCT) method using real-time PCR. (B) RAW NF-KB reporter cells were treated with DMSO, 200 µM MTA or 200 µM adenosine (Ado) in the absence or presence of the indicated TLR ligands for 4 h at 37°C. Supernatants were assayed for luciferase, which was normalized to DMSO-treated cells that received no TLR stimulation. The graphs represent mean ±sem of 4 (A) or 5 (B) experiments. ** p
    Figure Legend Snippet: MTA inhibits TLR-induced TNFα mRNA production and NF-KB induction. (A) BMDM were incubated for 4 h with either DMSO or 200 µM MTA in the absence or presence of either 100 EU/mL LPS or 1 µg/mL Pam3CSK4. Total RNA was extracted from cells. TNFα expression relative to β-actin was determined by Δ(ΔCT) method using real-time PCR. (B) RAW NF-KB reporter cells were treated with DMSO, 200 µM MTA or 200 µM adenosine (Ado) in the absence or presence of the indicated TLR ligands for 4 h at 37°C. Supernatants were assayed for luciferase, which was normalized to DMSO-treated cells that received no TLR stimulation. The graphs represent mean ±sem of 4 (A) or 5 (B) experiments. ** p

    Techniques Used: Incubation, Expressing, Real-time Polymerase Chain Reaction, Luciferase

    MTA inhibition of TLR ligands acts via Adenosine Receptors. BMDM were incubated overnight with DMSO, the indicated concentrations of MTA or adenosine (Ado), presence or absence of 10 µM SCH442416 and 10 µM PSB1115 and the presence or absence of 10 ng/mL LPS. Supernatants were analyzed for TNFα production by ELISA (A) while cells were harvested, stained and analyzed for surface CD86 (B) expression by FACS. Data represent mean ±sem of at least 3 experiments. * p
    Figure Legend Snippet: MTA inhibition of TLR ligands acts via Adenosine Receptors. BMDM were incubated overnight with DMSO, the indicated concentrations of MTA or adenosine (Ado), presence or absence of 10 µM SCH442416 and 10 µM PSB1115 and the presence or absence of 10 ng/mL LPS. Supernatants were analyzed for TNFα production by ELISA (A) while cells were harvested, stained and analyzed for surface CD86 (B) expression by FACS. Data represent mean ±sem of at least 3 experiments. * p

    Techniques Used: Inhibition, Incubation, Enzyme-linked Immunosorbent Assay, Staining, Expressing, FACS

    MTA alters LPS tolerance. BMDM were treated overnight with either 0 ng/mL, 10 ng/mL, or 100 ng/mL (A–C) or 100 EU/mL (D) LPS in the presence or absence of 200 µM MTA, and 10 µM SCH442416 with 10 µM PSB1115, washed and rested for 6 h. BMDM were then restimulated with either 0, 10 ng/mL, or 100 EU/mL LPS overnight. Supernatants were collected and analyzed for TNFα (A) by ELISA while cells were harvested, stained and analyzed for surface CD69 (B) or CD86 (C, D) expression by FACS. Data represent mean ±sem of 3 (A) or 4 (B–D) experiments. * p
    Figure Legend Snippet: MTA alters LPS tolerance. BMDM were treated overnight with either 0 ng/mL, 10 ng/mL, or 100 ng/mL (A–C) or 100 EU/mL (D) LPS in the presence or absence of 200 µM MTA, and 10 µM SCH442416 with 10 µM PSB1115, washed and rested for 6 h. BMDM were then restimulated with either 0, 10 ng/mL, or 100 EU/mL LPS overnight. Supernatants were collected and analyzed for TNFα (A) by ELISA while cells were harvested, stained and analyzed for surface CD69 (B) or CD86 (C, D) expression by FACS. Data represent mean ±sem of 3 (A) or 4 (B–D) experiments. * p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Staining, Expressing, FACS

    MTA inhibits TLR responses. BMDM were incubated overnight with DMSO or 200 µM MTA in the absence or presence of the following TLR ligands: 10 ng/mL LPS, 10 µg/mL polyI:C, 1 µg/mL Pam3CSK4, 6.67 µM CpG or 2 µg/mL Imiquimod. Supernatants were assayed for TNFα (A), IL-6 (B) or IL-10 (C) production by ELISA while cells were harvested, stained and analyzed for surface CD69 (D,E) or CD86 (D, F) expression by FACS. Graphs represent mean ±sem of 3 (A, E, F), 4 (C) or 5 (B) experiments. * p
    Figure Legend Snippet: MTA inhibits TLR responses. BMDM were incubated overnight with DMSO or 200 µM MTA in the absence or presence of the following TLR ligands: 10 ng/mL LPS, 10 µg/mL polyI:C, 1 µg/mL Pam3CSK4, 6.67 µM CpG or 2 µg/mL Imiquimod. Supernatants were assayed for TNFα (A), IL-6 (B) or IL-10 (C) production by ELISA while cells were harvested, stained and analyzed for surface CD69 (D,E) or CD86 (D, F) expression by FACS. Graphs represent mean ±sem of 3 (A, E, F), 4 (C) or 5 (B) experiments. * p

    Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay, Staining, Expressing, FACS

    3) Product Images from "Specialized proresolving mediators enhance human B cell differentiation to antibody secreting cells"

    Article Title: Specialized proresolving mediators enhance human B cell differentiation to antibody secreting cells

    Journal:

    doi: 10.4049/jimmunol.1103483

    17-HDHA decreases IL-6 and IL-10 production, but not TNFα. Positively selected CD19 + human B cells (3 × 10 6 cells/ml) were treated with 17-HDHA or vehicle 30 minutes prior to B cell stimulation with CpG ODN 2395 (1 μg/ml) plus
    Figure Legend Snippet: 17-HDHA decreases IL-6 and IL-10 production, but not TNFα. Positively selected CD19 + human B cells (3 × 10 6 cells/ml) were treated with 17-HDHA or vehicle 30 minutes prior to B cell stimulation with CpG ODN 2395 (1 μg/ml) plus

    Techniques Used: Cell Stimulation

    4) Product Images from "Regulation of Asymmetric Division by Atypical Protein Kinase C Influences Early Specification of CD8+ T Lymphocyte Fates"

    Article Title: Regulation of Asymmetric Division by Atypical Protein Kinase C Influences Early Specification of CD8+ T Lymphocyte Fates

    Journal: Scientific Reports

    doi: 10.1038/srep19182

    Deficiency of aPKC increases differentiation into the terminal effector fate early during the immune response. ( a ) BrdU incorporation after a 1 hour pulse-chase by wild-type or aPKC-deficient OT-I CD8 + T cells on day 5 post-infection in the spleens of mice that received 5 × 10 3 wild-type or aPKC-deficient OT-I CD45.1 + CD8 + T cells and were infected with Lm-OVA. ( b ) 7-AAD uptake (top), active caspase-3/-7 (middle), and mitochondrial membrane potential (Δψm) (bottom) of wild-type or aPKC-deficient OT-I CD8 + T cells on day 5 post-infection in the spleens of mice that received cells as in ( a ). ( c ) Frequencies of apoptotic (7-AAD + , Caspase 3/7 + , or Δψm lo ) wild-type or aPKC-deficient OT-I CD8 + T cells from mice shown in (b). Bars represent mean ± SEM, n = 4/group. ( d,e ) Left, expression of T-bet, Granzyme B, Eomes, and Bcl2 by wild-type (solid black) or aPKC-deficient (dashed black) CD45.1 + CD8 + T cells on days ( d ) 5 or ( e ) 7 post-infection in the spleens of mice as in ( a ). Isotype controls (gray filled) are shown with the associated gates used to measure the percentage of cells expressing the indicated protein. Right, frequencies of expression of T-bet, Granzyme B, Eomes, and Bcl2 from mice shown on the left. Bars represent mean ± SEM, n = 3–4/group. ( f ) Expression of IFNγ, TNFα, and IL-2 by wild-type or aPKC-deficient OT-I CD45.1 + CD8 + T cells following restimulation ex vivo for 6 hours with ovalbumin peptide on day 7 post-infection. ( g ) Frequencies of expression of IFNγ, TNFα, or IL-2 from mice shown in ( f ). Bars represent mean ± SEM, n = 3–4/group. *P
    Figure Legend Snippet: Deficiency of aPKC increases differentiation into the terminal effector fate early during the immune response. ( a ) BrdU incorporation after a 1 hour pulse-chase by wild-type or aPKC-deficient OT-I CD8 + T cells on day 5 post-infection in the spleens of mice that received 5 × 10 3 wild-type or aPKC-deficient OT-I CD45.1 + CD8 + T cells and were infected with Lm-OVA. ( b ) 7-AAD uptake (top), active caspase-3/-7 (middle), and mitochondrial membrane potential (Δψm) (bottom) of wild-type or aPKC-deficient OT-I CD8 + T cells on day 5 post-infection in the spleens of mice that received cells as in ( a ). ( c ) Frequencies of apoptotic (7-AAD + , Caspase 3/7 + , or Δψm lo ) wild-type or aPKC-deficient OT-I CD8 + T cells from mice shown in (b). Bars represent mean ± SEM, n = 4/group. ( d,e ) Left, expression of T-bet, Granzyme B, Eomes, and Bcl2 by wild-type (solid black) or aPKC-deficient (dashed black) CD45.1 + CD8 + T cells on days ( d ) 5 or ( e ) 7 post-infection in the spleens of mice as in ( a ). Isotype controls (gray filled) are shown with the associated gates used to measure the percentage of cells expressing the indicated protein. Right, frequencies of expression of T-bet, Granzyme B, Eomes, and Bcl2 from mice shown on the left. Bars represent mean ± SEM, n = 3–4/group. ( f ) Expression of IFNγ, TNFα, and IL-2 by wild-type or aPKC-deficient OT-I CD45.1 + CD8 + T cells following restimulation ex vivo for 6 hours with ovalbumin peptide on day 7 post-infection. ( g ) Frequencies of expression of IFNγ, TNFα, or IL-2 from mice shown in ( f ). Bars represent mean ± SEM, n = 3–4/group. *P

    Techniques Used: BrdU Incorporation Assay, Pulse Chase, Infection, Mouse Assay, Expressing, Ex Vivo

    aPKC regulates long-lived CD8 + T cell differentiation. ( a ) Frequencies of CD45.1 + CD8 + T cells in the blood (left), spleen (middle), and lymph nodes (right) on day 50 post-infection; each point represents an individual mouse and lines indicate the mean ± SEM; n = 7–8/group. ( b ) Total number of OT-I (left), CD62L hi T CM (middle), and CD62L lo T EM (right) CD45.1 + CD8 + T cells in the spleen on day 50 post-infection; bars represent mean ± SEM; n = 7–8/group. ( c ) Expression of T-bet, Eomes, Tcf-1, and Bcl2 in wild-type (gray filled) and aPKC-deficient (dashed black) CD45.1 + CD8 + cells in the spleen on day 50 post-infection. ( d ) Expression of IFNγ, TNFα, and IL-2 by wild-type or aPKC-deficient OT-I CD45.1 + CD8 + T cells following restimulation ex vivo for 6 hours with ovalbumin peptide on day 50 post-infection. ( e ) Frequencies of CD45.1 + CD8 + T cells on days 5, 6, 7, and 8 in the blood of mice that received 10 4 wild-type or aPKC-deficient CD45.1 + CD8 + T cells on day 50 post-infection and were subsequently challenged with 10 5 CFU Lm-OVA. Points represent mean ± SEM; n = 4/group. For ( a,b ) *P
    Figure Legend Snippet: aPKC regulates long-lived CD8 + T cell differentiation. ( a ) Frequencies of CD45.1 + CD8 + T cells in the blood (left), spleen (middle), and lymph nodes (right) on day 50 post-infection; each point represents an individual mouse and lines indicate the mean ± SEM; n = 7–8/group. ( b ) Total number of OT-I (left), CD62L hi T CM (middle), and CD62L lo T EM (right) CD45.1 + CD8 + T cells in the spleen on day 50 post-infection; bars represent mean ± SEM; n = 7–8/group. ( c ) Expression of T-bet, Eomes, Tcf-1, and Bcl2 in wild-type (gray filled) and aPKC-deficient (dashed black) CD45.1 + CD8 + cells in the spleen on day 50 post-infection. ( d ) Expression of IFNγ, TNFα, and IL-2 by wild-type or aPKC-deficient OT-I CD45.1 + CD8 + T cells following restimulation ex vivo for 6 hours with ovalbumin peptide on day 50 post-infection. ( e ) Frequencies of CD45.1 + CD8 + T cells on days 5, 6, 7, and 8 in the blood of mice that received 10 4 wild-type or aPKC-deficient CD45.1 + CD8 + T cells on day 50 post-infection and were subsequently challenged with 10 5 CFU Lm-OVA. Points represent mean ± SEM; n = 4/group. For ( a,b ) *P

    Techniques Used: Cell Differentiation, Infection, Electron Microscopy, Expressing, Ex Vivo, Mouse Assay

    5) Product Images from "Inhibition of a novel fibrogenic factor Tl1a reverses established colonic fibrosis"

    Article Title: Inhibition of a novel fibrogenic factor Tl1a reverses established colonic fibrosis

    Journal: Mucosal immunology

    doi: 10.1038/mi.2014.37

    Intestinal fibroblasts express Dr3 and respond to Tl1a stimulation. ( a ) Primary intestinal fibroblasts were stained with Dr3, αSMA and vimentin and analyzed by flow cytometry. Fibroblasts expressing high, intermediate, and low αSMA were gated as shown and Dr3 staining is preferentially found in αSMA high > intermediate > low. Three independent experiments were performed. ( b ) Data are representative of 3 independent sorted αSMA positive myofibroblasts at 200× magnification. There was co-staining of Dr3 in WT, but not in Dr3 deficient αSMA positive myofibroblasts. ( c ) Expression of Col1a2 and Il31Ra mRNA in WT primary intestinal fibroblasts with increasing Tl1a stimulation (0–200 ng/mL) and represented as mean ± SD are shown (n=3). ( d ) Induction of Col1a2 and Il31Ra mRNA by Tl1a, Tgfβ/Igf1, and Tnfα in WT and Dr3 −/− intestinal are shown and represented as mean ± SD (n=3). * P
    Figure Legend Snippet: Intestinal fibroblasts express Dr3 and respond to Tl1a stimulation. ( a ) Primary intestinal fibroblasts were stained with Dr3, αSMA and vimentin and analyzed by flow cytometry. Fibroblasts expressing high, intermediate, and low αSMA were gated as shown and Dr3 staining is preferentially found in αSMA high > intermediate > low. Three independent experiments were performed. ( b ) Data are representative of 3 independent sorted αSMA positive myofibroblasts at 200× magnification. There was co-staining of Dr3 in WT, but not in Dr3 deficient αSMA positive myofibroblasts. ( c ) Expression of Col1a2 and Il31Ra mRNA in WT primary intestinal fibroblasts with increasing Tl1a stimulation (0–200 ng/mL) and represented as mean ± SD are shown (n=3). ( d ) Induction of Col1a2 and Il31Ra mRNA by Tl1a, Tgfβ/Igf1, and Tnfα in WT and Dr3 −/− intestinal are shown and represented as mean ± SD (n=3). * P

    Techniques Used: Staining, Flow Cytometry, Cytometry, Expressing

    6) Product Images from "TRIM32-TAX1BP1-dependent selective autophagic degradation of TRIF negatively regulates TLR3/4-mediated innate immune responses"

    Article Title: TRIM32-TAX1BP1-dependent selective autophagic degradation of TRIF negatively regulates TLR3/4-mediated innate immune responses

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006600

    TRIM32-deficiency potentiates TLR3/4-mediated immune responses in vivo . (A) Serum cytokine concentrations in Trim32 +/+ and Trim32 -/- mice. Sex- and age-matched Trim32 +/+ and Trim32 -/- mice (n = 6) were injected intraperitoneally with poly(I:C) plus D-galactosamine or LPS for the indicated times and the concentrations of IFN-β, TNFα and IL-6 in the serum were determined by ELISA. (B) Effects of TRIM32-deficiency on poly(I:C) and LPS-induced inflammation in the lungs of mice. Sex- and age-matched Trim32 +/+ and Trim32 -/- mice were injected intraperitoneally with poly(I:C) (2 μg/g) plus D-galactosamine (1 mg/g) or LPS (10 μg /g) for 6 hours and lung sections were used for histological analysis (H E staining). (C-D) Effects of TRIM32-deficiency on poly(I:C)- and LPS-induced inflammatory death of mice. Wild-type and Trim32 −/− littermates (n = 6) were treated with poly(I:C) plus D-galactosamine (C) or LPS (D) as in (A). The survival rates of the mice were recorded every 4 hours in the following 44 hours. (E) Serum cytokine concentrations in Trim32 +/+ and Trim32 -/- mice. Sex- and age-matched Trim32 +/+ and Trim32 -/- mice (n = 5) were injected intraperitoneally with MPLA (3 mg/g) plus D-galactosamine (1 mg/g) for 2 hours and the concentrations of TNFα and IL-6 in the sera were measured by ELISA. (F) Effects of TRIM32-deficiency on MPLA plus D-galactosamine-induced inflammatory death of mice. Sex- and age-matched Trim32 +/+ (n = 8) and Trim32 -/- mice (n = 6) were injected intraperitoneally with MPLA (3 mg/g) plus D-galactosamine (1 mg/g). The survival rates of the mice were recorded every 4 hours in the following 44 hours. *, p
    Figure Legend Snippet: TRIM32-deficiency potentiates TLR3/4-mediated immune responses in vivo . (A) Serum cytokine concentrations in Trim32 +/+ and Trim32 -/- mice. Sex- and age-matched Trim32 +/+ and Trim32 -/- mice (n = 6) were injected intraperitoneally with poly(I:C) plus D-galactosamine or LPS for the indicated times and the concentrations of IFN-β, TNFα and IL-6 in the serum were determined by ELISA. (B) Effects of TRIM32-deficiency on poly(I:C) and LPS-induced inflammation in the lungs of mice. Sex- and age-matched Trim32 +/+ and Trim32 -/- mice were injected intraperitoneally with poly(I:C) (2 μg/g) plus D-galactosamine (1 mg/g) or LPS (10 μg /g) for 6 hours and lung sections were used for histological analysis (H E staining). (C-D) Effects of TRIM32-deficiency on poly(I:C)- and LPS-induced inflammatory death of mice. Wild-type and Trim32 −/− littermates (n = 6) were treated with poly(I:C) plus D-galactosamine (C) or LPS (D) as in (A). The survival rates of the mice were recorded every 4 hours in the following 44 hours. (E) Serum cytokine concentrations in Trim32 +/+ and Trim32 -/- mice. Sex- and age-matched Trim32 +/+ and Trim32 -/- mice (n = 5) were injected intraperitoneally with MPLA (3 mg/g) plus D-galactosamine (1 mg/g) for 2 hours and the concentrations of TNFα and IL-6 in the sera were measured by ELISA. (F) Effects of TRIM32-deficiency on MPLA plus D-galactosamine-induced inflammatory death of mice. Sex- and age-matched Trim32 +/+ (n = 8) and Trim32 -/- mice (n = 6) were injected intraperitoneally with MPLA (3 mg/g) plus D-galactosamine (1 mg/g). The survival rates of the mice were recorded every 4 hours in the following 44 hours. *, p

    Techniques Used: In Vivo, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Staining

    7) Product Images from "Recombinant IL-7/HGF? Hybrid Cytokine Enhances T Cell Recovery in Mice Following Allogeneic Bone Marrow Transplantation"

    Article Title: Recombinant IL-7/HGF? Hybrid Cytokine Enhances T Cell Recovery in Mice Following Allogeneic Bone Marrow Transplantation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082998

    Peripheral T cells in the rIL-7/HGFβ-treated allo-BMT recipients are functional. Lethally irradiated BALB/c mice were injected with TCD-BM from B6 mice and treated with cytokines as in Figure 1 . On day 30 after BMT, (A) splenic CD3 + T cells were stimulated with anti-CD3 and anti-CD28 antibodies (5 μg/ml), and cell proliferation was determined by BrdU incorporation 4 days later. (B) splenic CD3 + T cells were stimulated with Con A (4 μg/ml) and cell proliferation was determined by BrdU incorporation 4 days later. (A and B) Data are shown as stimulation index. (C and D) Splenocytes were stimulated with phorbol myristate acetate and ionomycin, and stained with antibodies for cell surface markers and intercellular cytokines (H-2 b , CD4, CD8, IL-2, IFN-γ, and TNFα). The percentage of IL-2, IFN-γ and TNFα positive cells in donor-origin CD4 + and CD8 + T cells was determined by flow cytometry. The data are representative of 2 independent experiments with 4-6 mice per group. * P
    Figure Legend Snippet: Peripheral T cells in the rIL-7/HGFβ-treated allo-BMT recipients are functional. Lethally irradiated BALB/c mice were injected with TCD-BM from B6 mice and treated with cytokines as in Figure 1 . On day 30 after BMT, (A) splenic CD3 + T cells were stimulated with anti-CD3 and anti-CD28 antibodies (5 μg/ml), and cell proliferation was determined by BrdU incorporation 4 days later. (B) splenic CD3 + T cells were stimulated with Con A (4 μg/ml) and cell proliferation was determined by BrdU incorporation 4 days later. (A and B) Data are shown as stimulation index. (C and D) Splenocytes were stimulated with phorbol myristate acetate and ionomycin, and stained with antibodies for cell surface markers and intercellular cytokines (H-2 b , CD4, CD8, IL-2, IFN-γ, and TNFα). The percentage of IL-2, IFN-γ and TNFα positive cells in donor-origin CD4 + and CD8 + T cells was determined by flow cytometry. The data are representative of 2 independent experiments with 4-6 mice per group. * P

    Techniques Used: Functional Assay, Irradiation, Mouse Assay, Injection, BrdU Incorporation Assay, Staining, Flow Cytometry, Cytometry

    8) Product Images from "CCL5 promotes breast cancer recurrence through macrophage recruitment in residual tumors"

    Article Title: CCL5 promotes breast cancer recurrence through macrophage recruitment in residual tumors

    Journal: eLife

    doi: 10.7554/eLife.43653

    Gene expression changes following Her2 inhibition. ( a ) qRT-PCR analysis of Erbb2 expression in primary cells with Her2 on (+dox) or Her2 off (-dox). ( b ) Gene set enrichment analysis (GSEA) of RNA-seq data showing an E2F gene signature is enriched in cells with Her2 signaling on. p-Values and normalized enrichment scores (NES) are shown. ( c ) Western blot showing p65 phosphorylation in primary tumor cells treated with the indicated concentration of Neratinib for 24 hr, or 24 hr following dox withdrawal. ( d–f ) qRT-PCR analysis of TNFα, CCL5, and CXCL5 expression 24 hr after treatment with 0.1 μM Neratinib. ( g ) qRT-PCR analysis of CCL2, CCL5, and CXCL5 expression in NIH-3T3 treated with 2 μg/mL dox, 10 ng/mL TNFα, or both for 24 hr. ( h ) qRT-PCR analysis of Erbb2 expression of cells treated with -dox conditioned media with dox supplementation. ( i ) Primary tumor cells were treated with +dox conditioned media and activation of the NF-κB pathway was assessed by Western blot analysis of total and phospho-p65. Results show two biological replicates per time point.
    Figure Legend Snippet: Gene expression changes following Her2 inhibition. ( a ) qRT-PCR analysis of Erbb2 expression in primary cells with Her2 on (+dox) or Her2 off (-dox). ( b ) Gene set enrichment analysis (GSEA) of RNA-seq data showing an E2F gene signature is enriched in cells with Her2 signaling on. p-Values and normalized enrichment scores (NES) are shown. ( c ) Western blot showing p65 phosphorylation in primary tumor cells treated with the indicated concentration of Neratinib for 24 hr, or 24 hr following dox withdrawal. ( d–f ) qRT-PCR analysis of TNFα, CCL5, and CXCL5 expression 24 hr after treatment with 0.1 μM Neratinib. ( g ) qRT-PCR analysis of CCL2, CCL5, and CXCL5 expression in NIH-3T3 treated with 2 μg/mL dox, 10 ng/mL TNFα, or both for 24 hr. ( h ) qRT-PCR analysis of Erbb2 expression of cells treated with -dox conditioned media with dox supplementation. ( i ) Primary tumor cells were treated with +dox conditioned media and activation of the NF-κB pathway was assessed by Western blot analysis of total and phospho-p65. Results show two biological replicates per time point.

    Techniques Used: Expressing, Inhibition, Quantitative RT-PCR, RNA Sequencing Assay, Western Blot, Concentration Assay, Activation Assay

    9) Product Images from "Cordycepin and a preparation from Cordyceps militaris inhibit malignant transformation and proliferation by decreasing EGFR and IL-17RA signaling in a murine oral cancer model"

    Article Title: Cordycepin and a preparation from Cordyceps militaris inhibit malignant transformation and proliferation by decreasing EGFR and IL-17RA signaling in a murine oral cancer model

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21477

    CMP and its ingredient enhanced anti-tumor immunity ( A, B ) Tumoricidal activities of splenocytes and peritoneal macrophages to a mouse cancer cell line at different ratios of effector cells to target cells. Data are presented as mean ± SEM and statistical analysis is performed with one-way ANOVA following Duncan’s multiple range test. ( C, D ) Levels of IFN-γ and TNFα in the culture supernatant of splenocytes and in the serum. Data are presented as mean ± SEM and statistical analysis is effectuated with one-way ANOVA following Tukey’s test. ( E ) Messenger RNA levels of IFN-γ ( Ifng ) and transcription factor t-bet ( Tbx21 ) in the lymph nodes and spleen. Data were normalized to β-actin ( Actb ) expression levels and presented as n-fold changes to control group by 2 -∆∆CT method. Data are depicted as mean ± SEM and statistical analysis is executed with one-way ANOVA following Tukey’s test. ( F ) Extracellular polysaccharides (EPS) induced TNFα production in the peritoneal macrophages, but cordycepin could not. Lipopolysaccharides (LPS) at 1 μg/ ml was used as a positive control. Total protein levels were used for correction the total cell numbers. Data are presented as mean ± SD and N.D. means not detection. ( G ) IFN-γ and TNFα inhibited proliferation of 4NAOC-1 cells after 96 hours treatment. Data are presented as percentage relative to vehicle control and statistical analysis is conducted with one-way ANOVA following Tukey’s test. ( H ) Cordycepin decreased PD-L1 and PD-L2 expression in 4NAOC-1 cells. Representative results from three independent tests are shown.
    Figure Legend Snippet: CMP and its ingredient enhanced anti-tumor immunity ( A, B ) Tumoricidal activities of splenocytes and peritoneal macrophages to a mouse cancer cell line at different ratios of effector cells to target cells. Data are presented as mean ± SEM and statistical analysis is performed with one-way ANOVA following Duncan’s multiple range test. ( C, D ) Levels of IFN-γ and TNFα in the culture supernatant of splenocytes and in the serum. Data are presented as mean ± SEM and statistical analysis is effectuated with one-way ANOVA following Tukey’s test. ( E ) Messenger RNA levels of IFN-γ ( Ifng ) and transcription factor t-bet ( Tbx21 ) in the lymph nodes and spleen. Data were normalized to β-actin ( Actb ) expression levels and presented as n-fold changes to control group by 2 -∆∆CT method. Data are depicted as mean ± SEM and statistical analysis is executed with one-way ANOVA following Tukey’s test. ( F ) Extracellular polysaccharides (EPS) induced TNFα production in the peritoneal macrophages, but cordycepin could not. Lipopolysaccharides (LPS) at 1 μg/ ml was used as a positive control. Total protein levels were used for correction the total cell numbers. Data are presented as mean ± SD and N.D. means not detection. ( G ) IFN-γ and TNFα inhibited proliferation of 4NAOC-1 cells after 96 hours treatment. Data are presented as percentage relative to vehicle control and statistical analysis is conducted with one-way ANOVA following Tukey’s test. ( H ) Cordycepin decreased PD-L1 and PD-L2 expression in 4NAOC-1 cells. Representative results from three independent tests are shown.

    Techniques Used: Expressing, Positive Control

    10) Product Images from "Mammalian Target of Rapamycin Inhibition in Trypanosoma cruzi-Infected Macrophages Leads to an Intracellular Profile That Is Detrimental for Infection"

    Article Title: Mammalian Target of Rapamycin Inhibition in Trypanosoma cruzi-Infected Macrophages Leads to an Intracellular Profile That Is Detrimental for Infection

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00313

    Mammalian target of rapamycin inhibition alters cytokine balance in macrophages toward a pro-inflammatory phenotype upon Trypanosoma cruzi infection. Bone marrow-derived macrophages (BMDMs) from C57BL/6 mice were pretreated with DMSO as control or with rapamycin (100 nM) during 90 min. After pretreatment, BMDM uninfected or infected with T. cruzi trypomastigotes (1:5, cell:parasite ratio) were cultured at different times. At 12, 18, and 24 h postinfection, supernatants were collected and processed to determine the IL-10 (A) , IL-12p70 (B) , IL-6 (C) , TNFα (D) , and IL-1β (E) production by ELISA Sandwich. Besides, supernatants from BMDM stimulated with IL-4 (80 ng/mL), or with LPS (1 µg/mL) + IFNγ (100 ng/mL), or with LPS (1 µg/mL) + ATP (5 mM) during 24 h were used as positive controls. Bars panels represent mean ± SD from three independent assays (* p
    Figure Legend Snippet: Mammalian target of rapamycin inhibition alters cytokine balance in macrophages toward a pro-inflammatory phenotype upon Trypanosoma cruzi infection. Bone marrow-derived macrophages (BMDMs) from C57BL/6 mice were pretreated with DMSO as control or with rapamycin (100 nM) during 90 min. After pretreatment, BMDM uninfected or infected with T. cruzi trypomastigotes (1:5, cell:parasite ratio) were cultured at different times. At 12, 18, and 24 h postinfection, supernatants were collected and processed to determine the IL-10 (A) , IL-12p70 (B) , IL-6 (C) , TNFα (D) , and IL-1β (E) production by ELISA Sandwich. Besides, supernatants from BMDM stimulated with IL-4 (80 ng/mL), or with LPS (1 µg/mL) + IFNγ (100 ng/mL), or with LPS (1 µg/mL) + ATP (5 mM) during 24 h were used as positive controls. Bars panels represent mean ± SD from three independent assays (* p

    Techniques Used: Inhibition, Infection, Derivative Assay, Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    11) Product Images from "Anti-TNFR1 targeting in humanized mice ameliorates disease in a model of multiple sclerosis"

    Article Title: Anti-TNFR1 targeting in humanized mice ameliorates disease in a model of multiple sclerosis

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31957-7

    Anti-TNFR1 treatment reduces TNFα-induced T cell adhesion and endothelial cell adhesion molecule expression. ( A ) An adhesion assay of DiI-labelled T cells (red) was performed using a human brain endothelial cell line (hCMEC/D3) which was grown to confluency and pre-activated as indicated prior to the assay. ( C ) Quantification revealed that 24 hour pre-treatment with huTNFα significantly increased the number of adherent T cells, but not when co-treated with ATROSAB. ( B ) Similarly, 24 hour treatment of hCMEC/D3 cells with huTNFα resulted in a robust production of VCAM-1 (red), which was essentially blocked by ATROSAB co-treatment, as quantified in ( D ). Dapi counter-staining (blue) indicates the endothelial cell nuclei. Measurement of surface ( E ) VCAM-1 and ( G ) ICAM-1 expression on hCMEC/D3 cells was assessed by flow cytometry, with quantification given in ( F , H ), respectively, showing a significant reduction in TNFα-induced upregulation by co-incubation with ATROSAB. Scale bars = 100 µm. * p
    Figure Legend Snippet: Anti-TNFR1 treatment reduces TNFα-induced T cell adhesion and endothelial cell adhesion molecule expression. ( A ) An adhesion assay of DiI-labelled T cells (red) was performed using a human brain endothelial cell line (hCMEC/D3) which was grown to confluency and pre-activated as indicated prior to the assay. ( C ) Quantification revealed that 24 hour pre-treatment with huTNFα significantly increased the number of adherent T cells, but not when co-treated with ATROSAB. ( B ) Similarly, 24 hour treatment of hCMEC/D3 cells with huTNFα resulted in a robust production of VCAM-1 (red), which was essentially blocked by ATROSAB co-treatment, as quantified in ( D ). Dapi counter-staining (blue) indicates the endothelial cell nuclei. Measurement of surface ( E ) VCAM-1 and ( G ) ICAM-1 expression on hCMEC/D3 cells was assessed by flow cytometry, with quantification given in ( F , H ), respectively, showing a significant reduction in TNFα-induced upregulation by co-incubation with ATROSAB. Scale bars = 100 µm. * p

    Techniques Used: Expressing, Cell Adhesion Assay, Staining, Flow Cytometry, Cytometry, Incubation

    T cell cytokine secretion is unchanged by anti-TNFR1 treatment. T cells were isolated from lymph nodes following the second injection of ATROSAB/control IgG on days 5 or 6 of acute EAE. Following isolation and purification, T cells were re-stimulated with either anti-CD3 or MOG 35−55 and ELISAs performed for IFNγ ( A ), IL17A ( B ) and TNFα ( C ). However, no differences were seen between T cells from both treatment groups in the secretion of either cytokine. n = 6 per treatment. ATR, ATROSAB.
    Figure Legend Snippet: T cell cytokine secretion is unchanged by anti-TNFR1 treatment. T cells were isolated from lymph nodes following the second injection of ATROSAB/control IgG on days 5 or 6 of acute EAE. Following isolation and purification, T cells were re-stimulated with either anti-CD3 or MOG 35−55 and ELISAs performed for IFNγ ( A ), IL17A ( B ) and TNFα ( C ). However, no differences were seen between T cells from both treatment groups in the secretion of either cytokine. n = 6 per treatment. ATR, ATROSAB.

    Techniques Used: Isolation, Injection, Purification

    12) Product Images from "Elucidating immunologic mechanisms of PROSTVAC cancer immunotherapy"

    Article Title: Elucidating immunologic mechanisms of PROSTVAC cancer immunotherapy

    Journal: Journal for Immunotherapy of Cancer

    doi: 10.1186/s40425-014-0034-0

    Heterologous prime-boost improves the quality of PSA-specific T cell responses. BALB/c mice (6/group) were treated as described for Figure 1 . Spleens were harvested 14 days after the last treatment, and pooled splenocytes were restimulated overnight with PSA OPL or controls (controls not shown). The cells were stained for intracellular IFNγ, TNFα, and IL-2 prior to flow cytometric analysis. (A) The pie charts are weighted in size to reflect the numbers of detected cells (total numbers of PSA-specific CD8 per million T cells are indicated below each chart, % of respective T cell populations can be found in Additional file 1 : Table SD1). (B) Amount of IFNγ production on a per cell basis as measured by mean fluorescence intensity (MFI). Graphs show representative data of two independently performed experiments.
    Figure Legend Snippet: Heterologous prime-boost improves the quality of PSA-specific T cell responses. BALB/c mice (6/group) were treated as described for Figure 1 . Spleens were harvested 14 days after the last treatment, and pooled splenocytes were restimulated overnight with PSA OPL or controls (controls not shown). The cells were stained for intracellular IFNγ, TNFα, and IL-2 prior to flow cytometric analysis. (A) The pie charts are weighted in size to reflect the numbers of detected cells (total numbers of PSA-specific CD8 per million T cells are indicated below each chart, % of respective T cell populations can be found in Additional file 1 : Table SD1). (B) Amount of IFNγ production on a per cell basis as measured by mean fluorescence intensity (MFI). Graphs show representative data of two independently performed experiments.

    Techniques Used: Mouse Assay, Staining, Flow Cytometry, Fluorescence

    13) Product Images from "Mucosa-associated lymphoid tissue lymphoma translocation 1 as a novel therapeutic target for rheumatoid arthritis"

    Article Title: Mucosa-associated lymphoid tissue lymphoma translocation 1 as a novel therapeutic target for rheumatoid arthritis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-12349-9

    Effects of MI-2 on inflammatory TNFα production in macrophages. ( a ) TNFα concentrations in the culture supernatants from untreated or LPS-treated macrophages, with or without MI-2 treatment, as measured by ELISA. Pooled results are shown from 3 independent experiments. ** and *** indicate significant differences based on 2-tailed unpaired Student’s t -tests at P
    Figure Legend Snippet: Effects of MI-2 on inflammatory TNFα production in macrophages. ( a ) TNFα concentrations in the culture supernatants from untreated or LPS-treated macrophages, with or without MI-2 treatment, as measured by ELISA. Pooled results are shown from 3 independent experiments. ** and *** indicate significant differences based on 2-tailed unpaired Student’s t -tests at P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    The MALT1 inhibitor MI-2 reduced osteoclast formation in the presence of TNFα. Inhibitory effects of MI-2 on osteoclast formation from monocytes in the presence of 20 ng/mL TNFα, as determined by performing in vitro osteoclast-differentiation assays. ( a ) Representative microscopic images from 3 independent experiments are shown. ( b ) Relative abundances (%) of TRAP-positive osteoclasts per well. Pooled results are shown from 3 independent experiments. ( c ) TRAP enzyme activities (%) in untreated cells and cells treated with the indicated concentrations of MI-2. *, **, and *** indicate significant differences from control (untreated) cells based on 2-tailed unpaired Student’s t -tests at P
    Figure Legend Snippet: The MALT1 inhibitor MI-2 reduced osteoclast formation in the presence of TNFα. Inhibitory effects of MI-2 on osteoclast formation from monocytes in the presence of 20 ng/mL TNFα, as determined by performing in vitro osteoclast-differentiation assays. ( a ) Representative microscopic images from 3 independent experiments are shown. ( b ) Relative abundances (%) of TRAP-positive osteoclasts per well. Pooled results are shown from 3 independent experiments. ( c ) TRAP enzyme activities (%) in untreated cells and cells treated with the indicated concentrations of MI-2. *, **, and *** indicate significant differences from control (untreated) cells based on 2-tailed unpaired Student’s t -tests at P

    Techniques Used: In Vitro

    14) Product Images from "Unexpected positive control of NFκB and miR-155 by DGKα and ζ ensures effector and memory CD8+ T cell differentiation"

    Article Title: Unexpected positive control of NFκB and miR-155 by DGKα and ζ ensures effector and memory CD8+ T cell differentiation

    Journal: Oncotarget

    doi: 10.18632/oncotarget.8164

    Effects of DGKαζ deficiency on CD8 effector/memory lineage differentiation and function Recipient mice injected with WT and DKO OT 1 T cells were similarly infected with LM-OVA and analyzed at the indicated times, as in Figure 1 . a. Representative dot plots showing CD127 and KLRG1 expression in gated donor-derived OT1 T cells from PBLs. b. - c. Percentages of MPECs b. and SLECs c. of donor OT1 T cells at different times. d. Expression of cell surface markers in gated donor-derived OT1 T cells. e. - f. Splenocytes from recipient mice 7 days after LM-OVA infection were left unstimulated or stimulated with SIINFEKL peptide (1μg/ml) in the presence of GolgiPlug for 5 hours. IFNγ and TNFα were detected by intracellular staining. e. Repesentative dot plot of IFNγ and TNFα staining in gated OT1 T cells. f. Percentages of IFNγ- or TNFα-expressing OT1 cells. Data shown in a. - d. and e. - f. are representative of three and two independent experiments, respectively. **, P
    Figure Legend Snippet: Effects of DGKαζ deficiency on CD8 effector/memory lineage differentiation and function Recipient mice injected with WT and DKO OT 1 T cells were similarly infected with LM-OVA and analyzed at the indicated times, as in Figure 1 . a. Representative dot plots showing CD127 and KLRG1 expression in gated donor-derived OT1 T cells from PBLs. b. - c. Percentages of MPECs b. and SLECs c. of donor OT1 T cells at different times. d. Expression of cell surface markers in gated donor-derived OT1 T cells. e. - f. Splenocytes from recipient mice 7 days after LM-OVA infection were left unstimulated or stimulated with SIINFEKL peptide (1μg/ml) in the presence of GolgiPlug for 5 hours. IFNγ and TNFα were detected by intracellular staining. e. Repesentative dot plot of IFNγ and TNFα staining in gated OT1 T cells. f. Percentages of IFNγ- or TNFα-expressing OT1 cells. Data shown in a. - d. and e. - f. are representative of three and two independent experiments, respectively. **, P

    Techniques Used: Mouse Assay, Injection, Infection, Expressing, Derivative Assay, Staining

    Effects of DGKα and ζ double deficiency on memory CD8 T cell responses WT and DKO memory OT1 T cells sorted from splenocytes of recipients on day 35 after LM-OVA infection were transferred into secondary recipients (1 × 10 4 /mouse), which were subsequently infected by LM-OVA 18 hours after transfer. a. Representative dot plots and percentages b. of WT and DKO donor-derived CD45.1 − CD45.2 + memory OT1 cells in PBLs and splenocytes in secondary recipients 7 days postinfection. c. - d. Splenocytes from secondary recipients were stimulated with SIINFEKL peptide in the presence of GolgiPlug for 5 hours, followed by cell surface and intracellular staining and FACS analysis. Representative contour plots c. and percentages of IFNγ + or TNFα + cells d. of donor-derived WT and DKO memory OT1 T cells are shown. Data shown represent two independent experiments. *, P
    Figure Legend Snippet: Effects of DGKα and ζ double deficiency on memory CD8 T cell responses WT and DKO memory OT1 T cells sorted from splenocytes of recipients on day 35 after LM-OVA infection were transferred into secondary recipients (1 × 10 4 /mouse), which were subsequently infected by LM-OVA 18 hours after transfer. a. Representative dot plots and percentages b. of WT and DKO donor-derived CD45.1 − CD45.2 + memory OT1 cells in PBLs and splenocytes in secondary recipients 7 days postinfection. c. - d. Splenocytes from secondary recipients were stimulated with SIINFEKL peptide in the presence of GolgiPlug for 5 hours, followed by cell surface and intracellular staining and FACS analysis. Representative contour plots c. and percentages of IFNγ + or TNFα + cells d. of donor-derived WT and DKO memory OT1 T cells are shown. Data shown represent two independent experiments. *, P

    Techniques Used: Infection, Derivative Assay, Staining, FACS

    15) Product Images from "Differences in the influenza-specific CD4 T cell immunodominance hierarchy and functional potential between children and young adults"

    Article Title: Differences in the influenza-specific CD4 T cell immunodominance hierarchy and functional potential between children and young adults

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37167-5

    Pediatric subjects have a higher frequency of influenza-specific cells producing TNFα compared to IFNγ for all influenza proteins examined, with a global lack of NP-specific cells. Cells were stimulated and stained as described previously and then gated as in Fig. 1 to determine the frequency of activated, cytokine producing CD4 T cells for each stimulation condition. The frequency of influenza specific, cytokine producing CD4 T-cells was then graphed by individual stimulation condition. Statistical testing was completed using a 2-way ANOVA followed by post hoc comparisons with Sidak multiple comparison adjustment to assess for statistical significance between data obtained from children (solid circle) and young adults (open square).
    Figure Legend Snippet: Pediatric subjects have a higher frequency of influenza-specific cells producing TNFα compared to IFNγ for all influenza proteins examined, with a global lack of NP-specific cells. Cells were stimulated and stained as described previously and then gated as in Fig. 1 to determine the frequency of activated, cytokine producing CD4 T cells for each stimulation condition. The frequency of influenza specific, cytokine producing CD4 T-cells was then graphed by individual stimulation condition. Statistical testing was completed using a 2-way ANOVA followed by post hoc comparisons with Sidak multiple comparison adjustment to assess for statistical significance between data obtained from children (solid circle) and young adults (open square).

    Techniques Used: Staining

    Overall cytokine production by influenza-specific CD4 T-cells is biased away from IFNγ in pediatric subjects. Following PBMC stimulation and staining, the frequency of activated IFNγ, IL-2 and TNFα producing cells per million CD4 T-cells was determined for each subject and stimulation condition. Cells producing each cytokine were then summed across stimulation conditions to quantify the total frequency of influenza-specific CD4 T cells for each cytokine examined. Data are presented as boxplots, with each point representing a given subject. In ( B ) these data were normalized by dividing by the frequency of IFNγ+ producing cells to better illustrate the differences in CD4 T cell cytokine production between young adults and children. Statistical analysis was performed by two-group comparison testing with the Wilcoxon rank-sum test after data preprocessing. P-values were adjusted for multiple comparisons using the Benjamini-Hochberg procedure.
    Figure Legend Snippet: Overall cytokine production by influenza-specific CD4 T-cells is biased away from IFNγ in pediatric subjects. Following PBMC stimulation and staining, the frequency of activated IFNγ, IL-2 and TNFα producing cells per million CD4 T-cells was determined for each subject and stimulation condition. Cells producing each cytokine were then summed across stimulation conditions to quantify the total frequency of influenza-specific CD4 T cells for each cytokine examined. Data are presented as boxplots, with each point representing a given subject. In ( B ) these data were normalized by dividing by the frequency of IFNγ+ producing cells to better illustrate the differences in CD4 T cell cytokine production between young adults and children. Statistical analysis was performed by two-group comparison testing with the Wilcoxon rank-sum test after data preprocessing. P-values were adjusted for multiple comparisons using the Benjamini-Hochberg procedure.

    Techniques Used: Staining

    Cytokine production by CD4 T cells isolated from pediatric subjects is characterized by overall less IFNγ production, with the differences in cytokine elaboration between children and adults largely consistent across protein specificities. Cell were stimulated and stained as previously described, with gating as shown in Fig. 1 . Combination gates were then created using FlowJo v10 software (TreeStar) to highlight single, double, and triple cytokine producing cells. Following data preprocessing, arced pie graphs were constructed using SPICE software (NIH), with the arcs demonstrating all cells producing a given cytokine (IFNγ: green, IL-2: blue, TNFα: purple) and pie slices depicting the percentage of cells with a given cytokine expression pattern as indicated in the Figure. Data from pediatric subjects are shown in Panel A, while young adult data are shown in Panel B.
    Figure Legend Snippet: Cytokine production by CD4 T cells isolated from pediatric subjects is characterized by overall less IFNγ production, with the differences in cytokine elaboration between children and adults largely consistent across protein specificities. Cell were stimulated and stained as previously described, with gating as shown in Fig. 1 . Combination gates were then created using FlowJo v10 software (TreeStar) to highlight single, double, and triple cytokine producing cells. Following data preprocessing, arced pie graphs were constructed using SPICE software (NIH), with the arcs demonstrating all cells producing a given cytokine (IFNγ: green, IL-2: blue, TNFα: purple) and pie slices depicting the percentage of cells with a given cytokine expression pattern as indicated in the Figure. Data from pediatric subjects are shown in Panel A, while young adult data are shown in Panel B.

    Techniques Used: Isolation, Staining, Software, Construct, Expressing

    Gating schematic utilized for analysis of intracellular cytokine staining data. Using FlowJo version 10, sequential gating was applied to multiparameter flow cytometric data. Debris was first excluded, followed by gating to exclude doublets and non-viable cells. Cells were then sequentially gated on CD3+ CD19− cells, followed by CD4+ CD8− cells. Activated cytokine producing cells were gated on using CD69 combined with IFNγ, IL-2, or TNFα, with gates established based upon the staining present in positive and FMO control samples.
    Figure Legend Snippet: Gating schematic utilized for analysis of intracellular cytokine staining data. Using FlowJo version 10, sequential gating was applied to multiparameter flow cytometric data. Debris was first excluded, followed by gating to exclude doublets and non-viable cells. Cells were then sequentially gated on CD3+ CD19− cells, followed by CD4+ CD8− cells. Activated cytokine producing cells were gated on using CD69 combined with IFNγ, IL-2, or TNFα, with gates established based upon the staining present in positive and FMO control samples.

    Techniques Used: Staining, Flow Cytometry

    When summed across specificities, CD4 T cells from pediatric subjects are less likely to produce combinations of cytokines that include IFNγ and are more likely to produce TNFα or IL-2 alone or together. To better illustrate differences in cytokine production between the pediatric and young adult cohorts, subject-level data across each stimulation condition was summed for each cytokine combination and then was divided by the total number of cytokine-producing cells across all conditions. Data are presented as box plots with each point representing a given subject. Statistical testing was performed using the Wilcoxon rank sum test with the Benjamini-Hochberg procedure to correct for multiple comparisons.
    Figure Legend Snippet: When summed across specificities, CD4 T cells from pediatric subjects are less likely to produce combinations of cytokines that include IFNγ and are more likely to produce TNFα or IL-2 alone or together. To better illustrate differences in cytokine production between the pediatric and young adult cohorts, subject-level data across each stimulation condition was summed for each cytokine combination and then was divided by the total number of cytokine-producing cells across all conditions. Data are presented as box plots with each point representing a given subject. Statistical testing was performed using the Wilcoxon rank sum test with the Benjamini-Hochberg procedure to correct for multiple comparisons.

    Techniques Used:

    16) Product Images from "IL‐12 and IL‐15 induce the expression of CXCR6 and CD49a on peripheral natural killer cells"

    Article Title: IL‐12 and IL‐15 induce the expression of CXCR6 and CD49a on peripheral natural killer cells

    Journal: Immunity, Inflammation and Disease

    doi: 10.1002/iid3.190

    (a) Representative flow cytometry plots showing gating strategy and individual frequencies of CD49a+ and CXCR6+ NK cell populations within the peripheral blood and hepatic perfusate. (b) A comparison of the frequency of CD49a+ ( n = 20) and CXCR6+ ( n = 22) NK cells within the peripheral blood and hepatic perfusate (paired samples). Dot plots display individual values. (Wilcoxon matched pairs test). (c) Distribution of frequencies of CD49a+ ( n = 35) and CD49a + CXCR6+ ( n = 27) NK cells within the hepatic lymphocyte population. Dot plot displays individual values and median. Individuals with high frequencies of CD49a+ NK cells are plotted with a cross. Representative flow cytometry plots gated on NK cells showing examples of individuals with average and high frequencies of CD49a + CXCR6+ NK cells. (d) Frequencies of CD49a + CXCR6+, CD49a + CXCR6−, CD49a‐CXCR6+, and CD49a‐CXCR6− NK cell subsets in the human liver ( n = 27). Dot plots display individual values and median. (e) Comparison of frequency of CD16 ( n = 12), CD57 ( n = 12), CD69 ( n = 11), NKG2C ( n = 22), and KIR+ ( n = 9) NK cells between liver‐resident subpopulations CD49a + CXCR6+, CD49a + CXCR6−, CD49a‐CXCR6+, and CD49a‐CXCR6− (Wilcoxon matched pairs test). Representative flow cytometry plots gated on CD49a± and CXCR6± NK cells showing expression of CD16, CD57, CD69, NKG2C, and KIR. (f) Percentage of IFNγ+ ( n = 7) and TNFα+ ( n = 6) NK cells within the hepatic CD49a + CXCR6+, CD49a + CXCR6−, CD49a‐CXCR6+, and CD49a‐CXCR6− NK cell populations following stimulation with IL‐12 10 ng/ml and IL‐15 1 ng/ml for 12 h, respectively. Dot plots display individual values and median. (Wilcoxon matched pairs test). Representative flow cytometry plots gated on CD49a± and CXCR6± NK cells showing IFNγ and TNFα production. p
    Figure Legend Snippet: (a) Representative flow cytometry plots showing gating strategy and individual frequencies of CD49a+ and CXCR6+ NK cell populations within the peripheral blood and hepatic perfusate. (b) A comparison of the frequency of CD49a+ ( n = 20) and CXCR6+ ( n = 22) NK cells within the peripheral blood and hepatic perfusate (paired samples). Dot plots display individual values. (Wilcoxon matched pairs test). (c) Distribution of frequencies of CD49a+ ( n = 35) and CD49a + CXCR6+ ( n = 27) NK cells within the hepatic lymphocyte population. Dot plot displays individual values and median. Individuals with high frequencies of CD49a+ NK cells are plotted with a cross. Representative flow cytometry plots gated on NK cells showing examples of individuals with average and high frequencies of CD49a + CXCR6+ NK cells. (d) Frequencies of CD49a + CXCR6+, CD49a + CXCR6−, CD49a‐CXCR6+, and CD49a‐CXCR6− NK cell subsets in the human liver ( n = 27). Dot plots display individual values and median. (e) Comparison of frequency of CD16 ( n = 12), CD57 ( n = 12), CD69 ( n = 11), NKG2C ( n = 22), and KIR+ ( n = 9) NK cells between liver‐resident subpopulations CD49a + CXCR6+, CD49a + CXCR6−, CD49a‐CXCR6+, and CD49a‐CXCR6− (Wilcoxon matched pairs test). Representative flow cytometry plots gated on CD49a± and CXCR6± NK cells showing expression of CD16, CD57, CD69, NKG2C, and KIR. (f) Percentage of IFNγ+ ( n = 7) and TNFα+ ( n = 6) NK cells within the hepatic CD49a + CXCR6+, CD49a + CXCR6−, CD49a‐CXCR6+, and CD49a‐CXCR6− NK cell populations following stimulation with IL‐12 10 ng/ml and IL‐15 1 ng/ml for 12 h, respectively. Dot plots display individual values and median. (Wilcoxon matched pairs test). Representative flow cytometry plots gated on CD49a± and CXCR6± NK cells showing IFNγ and TNFα production. p

    Techniques Used: Flow Cytometry, Cytometry, Expressing

    17) Product Images from "Functionality of Dengue Virus Specific Memory T Cell Responses in Individuals Who Were Hospitalized or Who Had Mild or Subclinical Dengue Infection"

    Article Title: Functionality of Dengue Virus Specific Memory T Cell Responses in Individuals Who Were Hospitalized or Who Had Mild or Subclinical Dengue Infection

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0003673

    Cross reactive dengue-specific immune responses and disease severity. A: Circulating NS3-specific IFNγ ex vivo ELISpot responses were measured in individuals who were hospitalized due to dengue and in those with past mild/sub clinical dengue infection. B: Granzyme B production by PBMCs from individuals who were hospitalized due to dengue and who had a past mild/sub clinical dengue infection, following stimulation with DENV-NS3 overlapping peptides. C: TNFα production by PBMCs from individuals who were hospitalized due to dengue and those with past mild/sub clinical dengue infection following stimulation with DENV-NS3 overlapping peptides.
    Figure Legend Snippet: Cross reactive dengue-specific immune responses and disease severity. A: Circulating NS3-specific IFNγ ex vivo ELISpot responses were measured in individuals who were hospitalized due to dengue and in those with past mild/sub clinical dengue infection. B: Granzyme B production by PBMCs from individuals who were hospitalized due to dengue and who had a past mild/sub clinical dengue infection, following stimulation with DENV-NS3 overlapping peptides. C: TNFα production by PBMCs from individuals who were hospitalized due to dengue and those with past mild/sub clinical dengue infection following stimulation with DENV-NS3 overlapping peptides.

    Techniques Used: Ex Vivo, Enzyme-linked Immunospot, Infection

    18) Product Images from "Deletion of the Mineralocorticoid Receptor in Myeloid Cells Attenuates Central Nervous System Autoimmunity"

    Article Title: Deletion of the Mineralocorticoid Receptor in Myeloid Cells Attenuates Central Nervous System Autoimmunity

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01319

    T cell proliferation and cytokine production in peripheral lymphoid organs and regulatory T cell (Treg) abundance after experimental autoimmune encephalomyelitis induction in MR lysM mice. (A,B) MR flox and MR lysM mice were adoptively transferred with CFSE-labeled RFP + 2D2 T cells and subsequently immunized with MOG 35–55 in CFA. Proliferation of MOG-specific T cells was investigated at days 3 and 5 p.i. in spleen (A) and draining lymph nodes (B) by FACS analysis. T cells with diluted CFSE were considered as proliferative regardless of their round of cell division. n = 3. (C–F) Production of the pro-inflammatory cytokines IL-17A (C) , GM-CSF (D) , IFNγ (E) , and TNFα (F) in vitro by splenocytes and lymph node cells at day 10 p.i. and after an additional culture for 72 h in the presence of the cognate antigen. n = 5–10. (G) Frequency of CD25 + FoxP3 + Treg in spleen, draining lymph nodes, blood and spinal cord [central nervous system (CNS)] as measured by FACS analysis at day 10 p.i. for peripheral organs and at the peak of disease for spinal cord. n = 14–17 (peripheral organs), n = 11 (spinal cord). Black bars/symbols refer to values from MR flox mice and white bars/symbols to values from MR lysM mice. All data are presented as the mean ± SEM (n.s., not significant, * p
    Figure Legend Snippet: T cell proliferation and cytokine production in peripheral lymphoid organs and regulatory T cell (Treg) abundance after experimental autoimmune encephalomyelitis induction in MR lysM mice. (A,B) MR flox and MR lysM mice were adoptively transferred with CFSE-labeled RFP + 2D2 T cells and subsequently immunized with MOG 35–55 in CFA. Proliferation of MOG-specific T cells was investigated at days 3 and 5 p.i. in spleen (A) and draining lymph nodes (B) by FACS analysis. T cells with diluted CFSE were considered as proliferative regardless of their round of cell division. n = 3. (C–F) Production of the pro-inflammatory cytokines IL-17A (C) , GM-CSF (D) , IFNγ (E) , and TNFα (F) in vitro by splenocytes and lymph node cells at day 10 p.i. and after an additional culture for 72 h in the presence of the cognate antigen. n = 5–10. (G) Frequency of CD25 + FoxP3 + Treg in spleen, draining lymph nodes, blood and spinal cord [central nervous system (CNS)] as measured by FACS analysis at day 10 p.i. for peripheral organs and at the peak of disease for spinal cord. n = 14–17 (peripheral organs), n = 11 (spinal cord). Black bars/symbols refer to values from MR flox mice and white bars/symbols to values from MR lysM mice. All data are presented as the mean ± SEM (n.s., not significant, * p

    Techniques Used: Mouse Assay, Labeling, FACS, In Vitro

    Functional characterization of bone marrow-derived macrophages (BMDM) from MR lysM mice. (A) Production of NO by unstimulated (w/o) or LPS/IFNγ-stimulated BMDM from MR lysM or MR flox mice after 24 h in vitro culture. n = 9–11. (B,C) Production of IL-6 or TNFα by LPS/IFNγ-stimulated BMDM from MR lysM or MR flox mice after 24 h in vitro culture. n = 16–24. (D) In vitro proliferation of CFSE-labeled RFP + 2D2 T cells stimulated with their cognate antigen in the presence of BMDM from MR lysM or MR flox mice after 48 and 72 h. T cells with diluted CFSE were considered as proliferative regardless of their round of cell division. n = 9. (E) IL-17A production by Th17-differentiated 2D2 cells cultured with BMDM from MR lysM or MR flox mice after 72 h. n = 11. (F) Analysis of the phagocytic activity of BMDM by incubating them with CFSE-labeled irradiated lymph node cells from C57BL/6 wt mice at a 2:1 ratio. The frequency of CFSE + BMDM was measured by flow cytometry after 2 h and 20 h. n = 10–12. Black bars/symbols refer to BMDM from MR flox mice and white bars/symbols to BMDM from MR lysM mice. All data are presented as the mean ± SEM (n.s., not significant, * p
    Figure Legend Snippet: Functional characterization of bone marrow-derived macrophages (BMDM) from MR lysM mice. (A) Production of NO by unstimulated (w/o) or LPS/IFNγ-stimulated BMDM from MR lysM or MR flox mice after 24 h in vitro culture. n = 9–11. (B,C) Production of IL-6 or TNFα by LPS/IFNγ-stimulated BMDM from MR lysM or MR flox mice after 24 h in vitro culture. n = 16–24. (D) In vitro proliferation of CFSE-labeled RFP + 2D2 T cells stimulated with their cognate antigen in the presence of BMDM from MR lysM or MR flox mice after 48 and 72 h. T cells with diluted CFSE were considered as proliferative regardless of their round of cell division. n = 9. (E) IL-17A production by Th17-differentiated 2D2 cells cultured with BMDM from MR lysM or MR flox mice after 72 h. n = 11. (F) Analysis of the phagocytic activity of BMDM by incubating them with CFSE-labeled irradiated lymph node cells from C57BL/6 wt mice at a 2:1 ratio. The frequency of CFSE + BMDM was measured by flow cytometry after 2 h and 20 h. n = 10–12. Black bars/symbols refer to BMDM from MR flox mice and white bars/symbols to BMDM from MR lysM mice. All data are presented as the mean ± SEM (n.s., not significant, * p

    Techniques Used: Functional Assay, Derivative Assay, Mouse Assay, In Vitro, Labeling, Cell Culture, Activity Assay, Irradiation, Flow Cytometry, Cytometry

    19) Product Images from "Accelerated immune senescence and reduced response to vaccination in ovariectomized female rhesus macaques"

    Article Title: Accelerated immune senescence and reduced response to vaccination in ovariectomized female rhesus macaques

    Journal:

    doi: 10.1007/s11357-010-9178-0

    Effect of ovariectomy on IFNγ and TNFα production by T cells. a Frequency of CM and EM T cells that secrete IFNγ and TNFα in response to CD3 stimulation was determined by intracellular cytokine staining and flow cytometry.
    Figure Legend Snippet: Effect of ovariectomy on IFNγ and TNFα production by T cells. a Frequency of CM and EM T cells that secrete IFNγ and TNFα in response to CD3 stimulation was determined by intracellular cytokine staining and flow cytometry.

    Techniques Used: Electron Microscopy, Staining, Flow Cytometry, Cytometry

    20) Product Images from "Natural killer T cells contribute to the control of acute retroviral infection"

    Article Title: Natural killer T cells contribute to the control of acute retroviral infection

    Journal: Retrovirology

    doi: 10.1186/s12977-017-0327-8

    Cytokine production by NKT cells after FV infection. Bone marrow cells ( white bars ) and splenocytes ( grey bars ) were isolated from naïve or FV-infected mice (3 dpi). Cells were stimulated and stained for the pro-inflammatory cytokines IFNγ ( a ) and TNFα ( b ) and the anti-inflammatory cytokines IL-10 ( c ) and IL-13 ( d ). Mean (±SEM) values are indicated by bars . At least nine animals per group out of at least six experiments were used for analysis. Differences between naïve and FV-infected mice were analyzed using the Mann–Whitney test and are indicated by single asterisk for p
    Figure Legend Snippet: Cytokine production by NKT cells after FV infection. Bone marrow cells ( white bars ) and splenocytes ( grey bars ) were isolated from naïve or FV-infected mice (3 dpi). Cells were stimulated and stained for the pro-inflammatory cytokines IFNγ ( a ) and TNFα ( b ) and the anti-inflammatory cytokines IL-10 ( c ) and IL-13 ( d ). Mean (±SEM) values are indicated by bars . At least nine animals per group out of at least six experiments were used for analysis. Differences between naïve and FV-infected mice were analyzed using the Mann–Whitney test and are indicated by single asterisk for p

    Techniques Used: Infection, Isolation, Mouse Assay, Staining, MANN-WHITNEY

    Related Articles

    Clone Assay:

    Article Title: CXCR3+ Regulatory T Cells Control TH1 Responses in Crescentic GN
    Article Snippet: Previously described methods for leukocyte isolation from murine kidneys, spleens, and lymph nodes were used., Measurements were performed on a BD FACS LSR II, and data were analyzed with the FlowJo software (TreeStar, Inc.). .. The following anti-mouse or anti-human antibodies were used: CD45, CD3, CD4, CD8, CD25, CD40L, CD44, CD62L, CD69, CXCR3, CCR4, CCR5, CCR6, CCR7, CCR3, γ d-TCR, NK1.1, IL-17A, IFN γ , TNF α , Foxp3, CD45RA, HLA-DR, and CD127 (Biolegend, San Diego, CA; BD Biosciences; eBioscience, San Diego, CA; and R & D Systems; clones are available on request). .. LIVE/DEAD Staining (near infrared; Invitrogen Molecular Probes, Eugene, OR) was used to exclude dead cells during flow cytometry and ensure viability of the cells after the stimulation procedure.

    Cell Isolation:

    Article Title: CD4 T cells control development and maintenance of brain-resident CD8 T cells during polyomavirus infection
    Article Snippet: Paragraph title: T cell isolation and flow cytometry ... Intracellular staining included anti-IFN-γ (clone XMG1.2; Biolegend), anti-TNF-α (clone XMG1.2; Biolegend), anti- IL-2 (clone JES6-5H4, Biolegend), anti- CD107a (clone 1D4B, BD Biosciences), and anti-CD107b (clone ABL-93, BD Biosciences).

    Amplification:

    Article Title: CD4 T cells control development and maintenance of brain-resident CD8 T cells during polyomavirus infection
    Article Snippet: Intracellular staining included anti-IFN-γ (clone XMG1.2; Biolegend), anti-TNF-α (clone XMG1.2; Biolegend), anti- IL-2 (clone JES6-5H4, Biolegend), anti- CD107a (clone 1D4B, BD Biosciences), and anti-CD107b (clone ABL-93, BD Biosciences). .. Lymphocytes isolated from IFN-γ eYFP reporter mice were surface stained with anti-CD8α and anti-CD44.

    Neutralization:

    Article Title: IMMUNOMODULATION OF ACTIVATED HEPATIC STELLATE CELLS BY MESENCHYMAL STEM CELLS
    Article Snippet: Paragraph title: Cytokine treatment, neutralization and protein quantification ... Anti-human IL-10 (BioLegend, San Diego, CA), TNF-α, (BioLegend, San Diego, CA), or HGF and anti-rat IL-6 (Cell Sciences, Canton, MA) were diluted in SC medium based on the half maximal inhibition concentrations given by the manufacturer.

    Cytometry:

    Article Title: CXCR3+ Regulatory T Cells Control TH1 Responses in Crescentic GN
    Article Snippet: Paragraph title: Flow Cytometry ... The following anti-mouse or anti-human antibodies were used: CD45, CD3, CD4, CD8, CD25, CD40L, CD44, CD62L, CD69, CXCR3, CCR4, CCR5, CCR6, CCR7, CCR3, γ d-TCR, NK1.1, IL-17A, IFN γ , TNF α , Foxp3, CD45RA, HLA-DR, and CD127 (Biolegend, San Diego, CA; BD Biosciences; eBioscience, San Diego, CA; and R & D Systems; clones are available on request).

    Article Title: STIM1 controls T cell–mediated immune regulation and inflammation in chronic infection
    Article Snippet: Paragraph title: Flow cytometry. ... The following anti-murine fluorophore-conjugated antibodies were used for staining: CD4 (RM4-5, BioLegend), CD8α (53-6.7, BD Biosciences), CD11c (HL3, BD Biosciences), CD11b (M1/70, BD Biosciences), Gr-1 (RB6-8C5, BD Biosciences), FasL (MFL3, eBioscience), Fas (15A7, eBioscience), IFN-γ (XMG1.2, BioLegend), TNF-α (MP6-XT22, BioLegend), CD62L (MEL-14, BD Biosciences), CD44 (IM7, BioLegend), CD25 (PC61, BD Biosciences), FOXP3 (FJK-16s, eBioscience), Helios (22F6, BioLegend), and NRP-1 (biotinylated goat polyclonal antibody, catalog BAF566, R & D Systems) followed by fluorophore-conjugated streptavidin (eBioscience).

    Article Title: CD4 T cells control development and maintenance of brain-resident CD8 T cells during polyomavirus infection
    Article Snippet: Paragraph title: T cell isolation and flow cytometry ... Intracellular staining included anti-IFN-γ (clone XMG1.2; Biolegend), anti-TNF-α (clone XMG1.2; Biolegend), anti- IL-2 (clone JES6-5H4, Biolegend), anti- CD107a (clone 1D4B, BD Biosciences), and anti-CD107b (clone ABL-93, BD Biosciences).

    Blocking Assay:

    Article Title: Resistance to cytotoxicity and sustained release of interleukin-6 and interleukin-8 in the presence of decreased interferon-γ after differentiation of glioblastoma by human natural killer cells
    Article Snippet: Human recombinant tumor necrosis factor alpha (hrTNF-α) and interferon gamma (hrIFN-γ) were purchased from Biolegend. .. Specificity of the antibodies was determined with ELISA and functional assays against hrTNF-α, hrIFN-γ, IL-6 and IL-8.

    Article Title: Interaction Between Macrophage Migration Inhibitory Factor and CD74 in Human Immunodeficiency Virus Type I Infected Primary Monocyte-Derived Macrophages Triggers the Production of Proinflammatory Mediators and Enhances Infection of Unactivated CD4+ T Cells
    Article Snippet: The neutralizing anti-MIF monoclonal antibody (clone NIHlllD.9) was obtained from ascites after purification using protein A/G spin column and resuspended at 5.15 mg/ml ( , ). .. A CD74 blocking antibody (BD Pharmingen, clone LN2), the recombinant human cytokines IL-6, IL-8, IL-1β (BioLegend), and TNFα (MiltenyiBiotec), and the cytokine neutralizing antibodies anti-IL-8 (R & D Systems), anti-IL-6, anti-IL-1β, and anti-TNFα (BioLegend) were obtained. .. Monocyte-derived macrophages were infected with the R5-tropic HIV and the infection was left to progress.

    Article Title: Taenia solium: Development of an Experimental Model of Porcine Neurocysticercosis
    Article Snippet: Endogenous peroxidase was inhibited by incubation with 0.3% (v/v) H2 O2 in PBS for 10 min. After washing twice with PBS, heat-mediated antigen retrieval method was performed by microwave treatment with 0.1 M sodium citrate solution (pH 6.0) for 5 min. Then, slides were rinsed three times in PBS buffer and sections were preincubated in a blocking solution consisting of 2% BSA (bovine serum albumin; Sigma-Aldrich) for 30 min. After two washes with Tris-EDTA buffer, sections were incubated with the primary antibody (described below) diluted in PBS buffer overnight at 4°C. .. The primary antibodies used in this study recognized: glial fibrillary acidic protein (GFAP; polyclonal rabbit DAKO, Glostrup, Denmark 1:100 dilution), vimentin (mouse clone V9, DAKO; 1:100 dilution), neuronal nuclear protein (NeuN; mouse clone MAB377, IgG; Chemicon, Temecula CA, USA; 1:1000), nestin (mouse anti-nestin monoclonal antibody, 1:100; Chemicon, Millipore Billerica, MA, USA), IL-4 (anti-human monoclonal antibody, 1:200 dilution, Biolegend), IL-6 (anti-human monoclonal antibody, 1:250 dilution Biolegend), IL-10 (anti-human monoclonal antibody, 1:100, Biolegend), IL-17A (anti-human monoclonal antibody, 1:150 Boise’s), TNF-α (anti-human monoclonal antibody, 1:500, Biolegend), CD54 (anti-human monoclonal antibody, 1:200 Biolegend), CD69 (anti-human monoclonal antibody, 1:200 Biolegend), CD80 (anti-human monoclonal antibody, 1:300 Biolegend), CD106 (anti-human monoclonal antibody, 1:250, Biolegend).

    Article Title: Endothelial CD47 promotes Vascular Endothelial-cadherin tyrosine phosphorylation and participates in T-cell recruitment at sites of inflammation in vivo
    Article Snippet: Recombinant murine TNF-α was purchased from BioLegend (San Diego, CA). .. Recombinant murine TNF-α was purchased from BioLegend (San Diego, CA).

    Enzyme-linked Immunosorbent Assay:

    Article Title: Induction of Dendritic Cell Maturation and Activation by a Potential Adjuvant, 2-Hydroxypropyl-β-Cyclodextrin
    Article Snippet: Recombinant murine GM-CSF was obtained from CreaGene. .. 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate and enzyme-linked immunosorbent assay (ELISA) kits for the quantification of human TNF-α, IL-6, IL-12p70, and IL-10, and mouse TNF-α and IL-6 were purchased from BioLegend (San Diego, CA, USA). .. 5,6-Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and the Vybrant® Alexa Fluor® 594 lipid raft-labeling kit were obtained from Molecular Probes (Eugene, OR, USA).

    Article Title: Resistance to cytotoxicity and sustained release of interleukin-6 and interleukin-8 in the presence of decreased interferon-γ after differentiation of glioblastoma by human natural killer cells
    Article Snippet: Human recombinant tumor necrosis factor alpha (hrTNF-α) and interferon gamma (hrIFN-γ) were purchased from Biolegend. .. Polyclonal IFN-γ, IL-6 and IL-8 antibodies were prepared in rabbits and purified.

    Article Title: Macrophage polarization in response to wear particles in vitro
    Article Snippet: Cytokine production, reflective of the pro-inflammatory and anti-inflammatory functions of M1 and M2 macrophages respectively, was assessed using ELISA. .. ELISA kits for TNF-α were purchased from Biolegend, and ELISA kits from IL-1ra were purchased from R & D Systems. .. ELISAs were run in duplicate.

    Article Title: Dab2, a negative regulator of DC immunogenicity, is an attractive molecular target for DC-based immunotherapy
    Article Snippet: FITC- or PE-conjugated anti-CD4, CD8, CD25, CD11c, CD40, CD80, CD86, MHCII, MHCI, and Foxp3 antibodies were purchased from BD Pharmingen and BioLegend. .. Cytokine ELISA kits for murine IL-6, IL-12p70, IL-10, IL-17A, IL-1β, TNF-α, TGF-β and γIFN were purchased from BioLegend and the kit for IL-13 was from Abcam. .. BMDCs were generated from mouse BM progenitor cells as described previously.

    Article Title: IMMUNOMODULATION OF ACTIVATED HEPATIC STELLATE CELLS BY MESENCHYMAL STEM CELLS
    Article Snippet: Quantification of human TNF-α, IL-10 and rat IL-6 and HGF was determined using an ELISA as per vendor instructions (Endogen, Rockford, IL). .. Anti-human IL-10 (BioLegend, San Diego, CA), TNF-α, (BioLegend, San Diego, CA), or HGF and anti-rat IL-6 (Cell Sciences, Canton, MA) were diluted in SC medium based on the half maximal inhibition concentrations given by the manufacturer.

    Article Title: Mincle-mediated translational regulation is required for strong nitric oxide production and inflammation resolution
    Article Snippet: GC7 (125 μM, 259545) was from EMD Millipore. .. TNFα and IL-1β (BioLegend) in culture supernatants were measured by ELISA. .. For immunoblot analysis, cells and tissues were lysed for 10 m at 4 °C in RIPA buffer (100 mM Tris–HCl (pH 8.0), 50 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, 50 mM β-glycerophosphate, 50 mM NaF, 0.1 mM Na3 VO4 , 0.5% sodium deoxycholate, with a protease inhibitor ‘cocktail' (1 mM PMSF, 10 μg ml–1 aprotinin, 5 μg ml–1 pepstatin and 5 μg ml–1 leupeptin), followed by centrifugation at 13,000g for 10 m at 4 °C for the removal of debris.

    Article Title: Snake Venom Disintegrin Inhibits the Activation of Toll-Like Receptors and Alleviates Sepsis through Integrin alphaVbeta3 Blockade
    Article Snippet: However, the detail mechanism regarding how Rn interferes with the TLRs-elicited signaling is under exploration. .. Enzyme-linked immunosorbent assays (ELISA) kit for IL-1β, IL-6, IL-8 and TNFα were purchased from BioLegend (San Diego, USA). .. Anti-phosphotyrosine mAbs, including anti-phosphorlated p38, ERK, JNK, FAK, PI3K, Akt, c-Src and Syk and IκB, MyD88 and anti-α-tubulin and FITC conjugated anti-mouse IgG were purchased from Santa Cruz (CA, USA).

    Article Title: Increased frequency of IL-6-producing non-classical monocytes in neuromyelitis optica spectrum disorder
    Article Snippet: Cytokine measurement was performed within 4 weeks after the collection of monocytes cultured media and aliquoted to appropriate amounts to avoid repeated freeze/thaw cycles. .. For measurement of cytokines, standard ELISA kits for IL-10, IL-6, IL-1β, TNFα (all from BioLegend, San Diego, CA, USA), and IL-23 (eBioscience, Vienna, Austria) were purchased and used according to the manufacturer’s instructions. .. Purified CD14+ monocytes cultured for 24 h, with or without stimulation, were labeled with primary antibodies directed against human CD80, CD86, HLA-DR, ICAM-1, or CD16 (all from BD Biosciences, San Jose, CA, USA).

    Article Title: Oligomeric procyanidins stimulate innate antiviral immunity in dengue virus infected human PBMCs
    Article Snippet: Cells were cultured for 48 hours before supernatant fluid and cells were collected. .. The supernatant fluids were used to measure DENV titers in endpoint titration assays as well as TNFα by ELISA (Biolegend). .. When DENV was not detected after 48 hour infection (n=1, a seronegative donor), samples were excluded from analyses.

    Incubation:

    Article Title: Taenia solium: Development of an Experimental Model of Porcine Neurocysticercosis
    Article Snippet: Endogenous peroxidase was inhibited by incubation with 0.3% (v/v) H2 O2 in PBS for 10 min. After washing twice with PBS, heat-mediated antigen retrieval method was performed by microwave treatment with 0.1 M sodium citrate solution (pH 6.0) for 5 min. Then, slides were rinsed three times in PBS buffer and sections were preincubated in a blocking solution consisting of 2% BSA (bovine serum albumin; Sigma-Aldrich) for 30 min. After two washes with Tris-EDTA buffer, sections were incubated with the primary antibody (described below) diluted in PBS buffer overnight at 4°C. .. The primary antibodies used in this study recognized: glial fibrillary acidic protein (GFAP; polyclonal rabbit DAKO, Glostrup, Denmark 1:100 dilution), vimentin (mouse clone V9, DAKO; 1:100 dilution), neuronal nuclear protein (NeuN; mouse clone MAB377, IgG; Chemicon, Temecula CA, USA; 1:1000), nestin (mouse anti-nestin monoclonal antibody, 1:100; Chemicon, Millipore Billerica, MA, USA), IL-4 (anti-human monoclonal antibody, 1:200 dilution, Biolegend), IL-6 (anti-human monoclonal antibody, 1:250 dilution Biolegend), IL-10 (anti-human monoclonal antibody, 1:100, Biolegend), IL-17A (anti-human monoclonal antibody, 1:150 Boise’s), TNF-α (anti-human monoclonal antibody, 1:500, Biolegend), CD54 (anti-human monoclonal antibody, 1:200 Biolegend), CD69 (anti-human monoclonal antibody, 1:200 Biolegend), CD80 (anti-human monoclonal antibody, 1:300 Biolegend), CD106 (anti-human monoclonal antibody, 1:250, Biolegend).

    Activity Assay:

    Article Title: Taenia solium: Development of an Experimental Model of Porcine Neurocysticercosis
    Article Snippet: After washing three times in PBS/A-T (1% BSA in PBS, plus 0.1% Triton X-100), 5 min each, slides were covered with secondary antibody conjugated with horseradish peroxidase (Dako-Kit) for 30 min at 37°C and rinsed with PBS/A-T. Peroxidase activity was visualized by incubating the samples for 2 min with 3-diaminobenzidine tetrahydrochloride (DAB, DAKO). .. The primary antibodies used in this study recognized: glial fibrillary acidic protein (GFAP; polyclonal rabbit DAKO, Glostrup, Denmark 1:100 dilution), vimentin (mouse clone V9, DAKO; 1:100 dilution), neuronal nuclear protein (NeuN; mouse clone MAB377, IgG; Chemicon, Temecula CA, USA; 1:1000), nestin (mouse anti-nestin monoclonal antibody, 1:100; Chemicon, Millipore Billerica, MA, USA), IL-4 (anti-human monoclonal antibody, 1:200 dilution, Biolegend), IL-6 (anti-human monoclonal antibody, 1:250 dilution Biolegend), IL-10 (anti-human monoclonal antibody, 1:100, Biolegend), IL-17A (anti-human monoclonal antibody, 1:150 Boise’s), TNF-α (anti-human monoclonal antibody, 1:500, Biolegend), CD54 (anti-human monoclonal antibody, 1:200 Biolegend), CD69 (anti-human monoclonal antibody, 1:200 Biolegend), CD80 (anti-human monoclonal antibody, 1:300 Biolegend), CD106 (anti-human monoclonal antibody, 1:250, Biolegend).

    Infection:

    Article Title: The sterol regulatory element binding proteins are essential for the metabolic programming of effector T cells and adaptive immunity
    Article Snippet: Paragraph title: LCMV infection, MHC I tetramer and intracellular cytokine staining ... Cells were stained for surface expression of CD8, then fixed, permeabilized and stained with antibodies to TNF, IFN-γ and IL-2 (BioLegend).

    Expressing:

    Article Title: IMMUNOMODULATION OF ACTIVATED HEPATIC STELLATE CELLS BY MESENCHYMAL STEM CELLS
    Article Snippet: Anti-human IL-10 (BioLegend, San Diego, CA), TNF-α, (BioLegend, San Diego, CA), or HGF and anti-rat IL-6 (Cell Sciences, Canton, MA) were diluted in SC medium based on the half maximal inhibition concentrations given by the manufacturer. .. Anti-human IL-10 (BioLegend, San Diego, CA), TNF-α, (BioLegend, San Diego, CA), or HGF and anti-rat IL-6 (Cell Sciences, Canton, MA) were diluted in SC medium based on the half maximal inhibition concentrations given by the manufacturer.

    Article Title: Taenia solium: Development of an Experimental Model of Porcine Neurocysticercosis
    Article Snippet: The primary antibodies used in this study recognized: glial fibrillary acidic protein (GFAP; polyclonal rabbit DAKO, Glostrup, Denmark 1:100 dilution), vimentin (mouse clone V9, DAKO; 1:100 dilution), neuronal nuclear protein (NeuN; mouse clone MAB377, IgG; Chemicon, Temecula CA, USA; 1:1000), nestin (mouse anti-nestin monoclonal antibody, 1:100; Chemicon, Millipore Billerica, MA, USA), IL-4 (anti-human monoclonal antibody, 1:200 dilution, Biolegend), IL-6 (anti-human monoclonal antibody, 1:250 dilution Biolegend), IL-10 (anti-human monoclonal antibody, 1:100, Biolegend), IL-17A (anti-human monoclonal antibody, 1:150 Boise’s), TNF-α (anti-human monoclonal antibody, 1:500, Biolegend), CD54 (anti-human monoclonal antibody, 1:200 Biolegend), CD69 (anti-human monoclonal antibody, 1:200 Biolegend), CD80 (anti-human monoclonal antibody, 1:300 Biolegend), CD106 (anti-human monoclonal antibody, 1:250, Biolegend). .. The primary antibodies used in this study recognized: glial fibrillary acidic protein (GFAP; polyclonal rabbit DAKO, Glostrup, Denmark 1:100 dilution), vimentin (mouse clone V9, DAKO; 1:100 dilution), neuronal nuclear protein (NeuN; mouse clone MAB377, IgG; Chemicon, Temecula CA, USA; 1:1000), nestin (mouse anti-nestin monoclonal antibody, 1:100; Chemicon, Millipore Billerica, MA, USA), IL-4 (anti-human monoclonal antibody, 1:200 dilution, Biolegend), IL-6 (anti-human monoclonal antibody, 1:250 dilution Biolegend), IL-10 (anti-human monoclonal antibody, 1:100, Biolegend), IL-17A (anti-human monoclonal antibody, 1:150 Boise’s), TNF-α (anti-human monoclonal antibody, 1:500, Biolegend), CD54 (anti-human monoclonal antibody, 1:200 Biolegend), CD69 (anti-human monoclonal antibody, 1:200 Biolegend), CD80 (anti-human monoclonal antibody, 1:300 Biolegend), CD106 (anti-human monoclonal antibody, 1:250, Biolegend).

    Article Title: The sterol regulatory element binding proteins are essential for the metabolic programming of effector T cells and adaptive immunity
    Article Snippet: To analyze cytokine expression, splenocytes were stimulated for 5 hours with 2 μg/ml of the MHC class I restricted LCMV-GP33-41 or NP396-404 peptide in the presence of 50 U/ml recombinant murine IL-2 (R & D Systems) and 1mg/ml brefeldin A (Sigma-Aldrich). .. Cells were stained for surface expression of CD8, then fixed, permeabilized and stained with antibodies to TNF, IFN-γ and IL-2 (BioLegend). .. Flow cytometric analysis was performed using an LSR II or the FACSVerse (Becton Dickinson) and analyzed using FlowJo software (Treestar).

    Article Title: Oligomeric procyanidins stimulate innate antiviral immunity in dengue virus infected human PBMCs
    Article Snippet: The supernatant fluids were used to measure DENV titers in endpoint titration assays as well as TNFα by ELISA (Biolegend). .. When DENV was not detected after 48 hour infection (n=1, a seronegative donor), samples were excluded from analyses.

    Ex Vivo:

    Article Title: The sterol regulatory element binding proteins are essential for the metabolic programming of effector T cells and adaptive immunity
    Article Snippet: Splenocytes were stained directly ex vivo with LCMV-Db GP33-41 or LCMV-Db GP396-404 specific tetramers and for surface expression of CD8. .. Cells were stained for surface expression of CD8, then fixed, permeabilized and stained with antibodies to TNF, IFN-γ and IL-2 (BioLegend).

    Western Blot:

    Article Title: GM-CSF Signalling Boosts Dramatically IL-1Production
    Article Snippet: Following antibodies were used for western blotting: anti-tubulin, anti-caspase-1 p20, anti-c-Rel and anti-USF-2 (Santa Cruz Biotechnlogy, Santa Cruz, CA, USA), anti-IL-1β antibody (R & D), anti-NLRP3 (Alexis), anti-caspase-11 (Biolegend, San Diego, CA, USA). .. Recombinant GM-CSF, TNF-α, IL-6 and IFN-γ were obtained from Biolegend, M-CSF from Milteny Biotech (BergischGladbach, Germany).

    Recombinase Polymerase Amplification:

    Article Title: Induction of Dendritic Cell Maturation and Activation by a Potential Adjuvant, 2-Hydroxypropyl-β-Cyclodextrin
    Article Snippet: 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate and enzyme-linked immunosorbent assay (ELISA) kits for the quantification of human TNF-α, IL-6, IL-12p70, and IL-10, and mouse TNF-α and IL-6 were purchased from BioLegend (San Diego, CA, USA). .. 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate and enzyme-linked immunosorbent assay (ELISA) kits for the quantification of human TNF-α, IL-6, IL-12p70, and IL-10, and mouse TNF-α and IL-6 were purchased from BioLegend (San Diego, CA, USA).

    High Performance Liquid Chromatography:

    Article Title: Oligomeric procyanidins stimulate innate antiviral immunity in dengue virus infected human PBMCs
    Article Snippet: Crude extract of Applepoly® , the putative trimer or tetramer procyanidins [10µg/ml, isolated from Applepoly® by normal phase HPLC as previously described ( )], monomeric procyanidin [catechin (Fisher) or epicatechin (Sigma), also 10µg/ml] or vehicle only was then added to the tissue culture media. .. The supernatant fluids were used to measure DENV titers in endpoint titration assays as well as TNFα by ELISA (Biolegend).

    Flow Cytometry:

    Article Title: CXCR3+ Regulatory T Cells Control TH1 Responses in Crescentic GN
    Article Snippet: Paragraph title: Flow Cytometry ... The following anti-mouse or anti-human antibodies were used: CD45, CD3, CD4, CD8, CD25, CD40L, CD44, CD62L, CD69, CXCR3, CCR4, CCR5, CCR6, CCR7, CCR3, γ d-TCR, NK1.1, IL-17A, IFN γ , TNF α , Foxp3, CD45RA, HLA-DR, and CD127 (Biolegend, San Diego, CA; BD Biosciences; eBioscience, San Diego, CA; and R & D Systems; clones are available on request).

    Article Title: STIM1 controls T cell–mediated immune regulation and inflammation in chronic infection
    Article Snippet: Paragraph title: Flow cytometry. ... The following anti-murine fluorophore-conjugated antibodies were used for staining: CD4 (RM4-5, BioLegend), CD8α (53-6.7, BD Biosciences), CD11c (HL3, BD Biosciences), CD11b (M1/70, BD Biosciences), Gr-1 (RB6-8C5, BD Biosciences), FasL (MFL3, eBioscience), Fas (15A7, eBioscience), IFN-γ (XMG1.2, BioLegend), TNF-α (MP6-XT22, BioLegend), CD62L (MEL-14, BD Biosciences), CD44 (IM7, BioLegend), CD25 (PC61, BD Biosciences), FOXP3 (FJK-16s, eBioscience), Helios (22F6, BioLegend), and NRP-1 (biotinylated goat polyclonal antibody, catalog BAF566, R & D Systems) followed by fluorophore-conjugated streptavidin (eBioscience).

    Article Title: CD4 T cells control development and maintenance of brain-resident CD8 T cells during polyomavirus infection
    Article Snippet: Paragraph title: T cell isolation and flow cytometry ... Intracellular staining included anti-IFN-γ (clone XMG1.2; Biolegend), anti-TNF-α (clone XMG1.2; Biolegend), anti- IL-2 (clone JES6-5H4, Biolegend), anti- CD107a (clone 1D4B, BD Biosciences), and anti-CD107b (clone ABL-93, BD Biosciences).

    Concentration Assay:

    Article Title: Resistance to cytotoxicity and sustained release of interleukin-6 and interleukin-8 in the presence of decreased interferon-γ after differentiation of glioblastoma by human natural killer cells
    Article Snippet: Human recombinant tumor necrosis factor alpha (hrTNF-α) and interferon gamma (hrIFN-γ) were purchased from Biolegend. .. Specificity of the antibodies was determined with ELISA and functional assays against hrTNF-α, hrIFN-γ, IL-6 and IL-8.

    Article Title: Interaction Between Macrophage Migration Inhibitory Factor and CD74 in Human Immunodeficiency Virus Type I Infected Primary Monocyte-Derived Macrophages Triggers the Production of Proinflammatory Mediators and Enhances Infection of Unactivated CD4+ T Cells
    Article Snippet: MIF antagonist MIF098 [3-(3-hydroxybenzyl)-5-methylbenzooxazol-2-one] was dissolved in DMSO at a concentration of 149 µM ( ). .. A CD74 blocking antibody (BD Pharmingen, clone LN2), the recombinant human cytokines IL-6, IL-8, IL-1β (BioLegend), and TNFα (MiltenyiBiotec), and the cytokine neutralizing antibodies anti-IL-8 (R & D Systems), anti-IL-6, anti-IL-1β, and anti-TNFα (BioLegend) were obtained.

    Cell Culture:

    Article Title: Resistance to cytotoxicity and sustained release of interleukin-6 and interleukin-8 in the presence of decreased interferon-γ after differentiation of glioblastoma by human natural killer cells
    Article Snippet: X01GB and X02GB GBM CSCs were isolated from freshly resected human tumor tissues [ , ] and were cultured under conditions that allow propagation of brain CSCs as described previously [ , ]. .. Human recombinant tumor necrosis factor alpha (hrTNF-α) and interferon gamma (hrIFN-γ) were purchased from Biolegend.

    Article Title: Mincle-mediated translational regulation is required for strong nitric oxide production and inflammation resolution
    Article Snippet: TNFα and IL-1β (BioLegend) in culture supernatants were measured by ELISA. .. For immunoblot analysis, cells and tissues were lysed for 10 m at 4 °C in RIPA buffer (100 mM Tris–HCl (pH 8.0), 50 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, 50 mM β-glycerophosphate, 50 mM NaF, 0.1 mM Na3 VO4 , 0.5% sodium deoxycholate, with a protease inhibitor ‘cocktail' (1 mM PMSF, 10 μg ml–1 aprotinin, 5 μg ml–1 pepstatin and 5 μg ml–1 leupeptin), followed by centrifugation at 13,000g for 10 m at 4 °C for the removal of debris.

    Article Title: Increased frequency of IL-6-producing non-classical monocytes in neuromyelitis optica spectrum disorder
    Article Snippet: Paragraph title: Cytokine detection in monocyte cultured media ... For measurement of cytokines, standard ELISA kits for IL-10, IL-6, IL-1β, TNFα (all from BioLegend, San Diego, CA, USA), and IL-23 (eBioscience, Vienna, Austria) were purchased and used according to the manufacturer’s instructions.

    Article Title: Oligomeric procyanidins stimulate innate antiviral immunity in dengue virus infected human PBMCs
    Article Snippet: The supernatant fluids were used to measure DENV titers in endpoint titration assays as well as TNFα by ELISA (Biolegend). .. The supernatant fluids were used to measure DENV titers in endpoint titration assays as well as TNFα by ELISA (Biolegend).

    Article Title: CD4 T cells control development and maintenance of brain-resident CD8 T cells during polyomavirus infection
    Article Snippet: For intracellular cytokine stimulation assays, lymphocytes were isolated from brain and spleen, cultured in DMEM/10% FBS for 5 h at 37°C with or without 1 μM LT359 peptide [ ], stained with Fixable Viability Dye, anti-CD8α, and anti-CD44, then permeabilized and fixed in FoxP3 buffer fixation and permeabilization solutions. .. Intracellular staining included anti-IFN-γ (clone XMG1.2; Biolegend), anti-TNF-α (clone XMG1.2; Biolegend), anti- IL-2 (clone JES6-5H4, Biolegend), anti- CD107a (clone 1D4B, BD Biosciences), and anti-CD107b (clone ABL-93, BD Biosciences).

    Light Microscopy:

    Article Title: Taenia solium: Development of an Experimental Model of Porcine Neurocysticercosis
    Article Snippet: The single labeled sections were examined by light microscopy Leica Galen III, and digital color video camera (SSC-DC14), on a Pentium IV, Windows 2000 computer. .. The primary antibodies used in this study recognized: glial fibrillary acidic protein (GFAP; polyclonal rabbit DAKO, Glostrup, Denmark 1:100 dilution), vimentin (mouse clone V9, DAKO; 1:100 dilution), neuronal nuclear protein (NeuN; mouse clone MAB377, IgG; Chemicon, Temecula CA, USA; 1:1000), nestin (mouse anti-nestin monoclonal antibody, 1:100; Chemicon, Millipore Billerica, MA, USA), IL-4 (anti-human monoclonal antibody, 1:200 dilution, Biolegend), IL-6 (anti-human monoclonal antibody, 1:250 dilution Biolegend), IL-10 (anti-human monoclonal antibody, 1:100, Biolegend), IL-17A (anti-human monoclonal antibody, 1:150 Boise’s), TNF-α (anti-human monoclonal antibody, 1:500, Biolegend), CD54 (anti-human monoclonal antibody, 1:200 Biolegend), CD69 (anti-human monoclonal antibody, 1:200 Biolegend), CD80 (anti-human monoclonal antibody, 1:300 Biolegend), CD106 (anti-human monoclonal antibody, 1:250, Biolegend).

    Purification:

    Article Title: Dichotomy between RIP1- and RIP3-Mediated Necroptosis in Tumor Necrosis Factor-?-Induced Shock
    Article Snippet: The zVAD, the ApoAlert annexin V-FITC antibody α-FADD, α-RIP1 and the monoclonal α-crmA antibody were purchased from BD Biosciences (Heidel-berg, Germany). .. Recombinant mouse TNFα (carrier-free) and purified α-human TNFα were purchased from BioLegend (Uithoorn, Netherlands). .. The cyclophilin A antibody and the caspase-8 antibody were obtained from Cell Signaling (Frankfurt/Main, Germany).

    Article Title: Resistance to cytotoxicity and sustained release of interleukin-6 and interleukin-8 in the presence of decreased interferon-γ after differentiation of glioblastoma by human natural killer cells
    Article Snippet: Human recombinant tumor necrosis factor alpha (hrTNF-α) and interferon gamma (hrIFN-γ) were purchased from Biolegend. .. Monoclonal antibodies to TNF-α were purified in our laboratory from ascites of mice injected with TNF-α hybridomas.

    Article Title: Interaction Between Macrophage Migration Inhibitory Factor and CD74 in Human Immunodeficiency Virus Type I Infected Primary Monocyte-Derived Macrophages Triggers the Production of Proinflammatory Mediators and Enhances Infection of Unactivated CD4+ T Cells
    Article Snippet: The neutralizing anti-MIF monoclonal antibody (clone NIHlllD.9) was obtained from ascites after purification using protein A/G spin column and resuspended at 5.15 mg/ml ( , ). .. A CD74 blocking antibody (BD Pharmingen, clone LN2), the recombinant human cytokines IL-6, IL-8, IL-1β (BioLegend), and TNFα (MiltenyiBiotec), and the cytokine neutralizing antibodies anti-IL-8 (R & D Systems), anti-IL-6, anti-IL-1β, and anti-TNFα (BioLegend) were obtained.

    Article Title: Endothelial CD47 promotes Vascular Endothelial-cadherin tyrosine phosphorylation and participates in T-cell recruitment at sites of inflammation in vivo
    Article Snippet: Recombinant murine TNF-α was purchased from BioLegend (San Diego, CA). .. Recombinant murine TNF-α was purchased from BioLegend (San Diego, CA).

    Inhibition:

    Article Title: IMMUNOMODULATION OF ACTIVATED HEPATIC STELLATE CELLS BY MESENCHYMAL STEM CELLS
    Article Snippet: For all neutralization experiments, the ratio of MSCs to SCs was 1:1. .. Anti-human IL-10 (BioLegend, San Diego, CA), TNF-α, (BioLegend, San Diego, CA), or HGF and anti-rat IL-6 (Cell Sciences, Canton, MA) were diluted in SC medium based on the half maximal inhibition concentrations given by the manufacturer. .. Fresh medium with neutralizing antibodies was added after 48 hours of coculture.

    Injection:

    Article Title: CD4 T cells control development and maintenance of brain-resident CD8 T cells during polyomavirus infection
    Article Snippet: For intravascular staining, animals were injected i.v. with FITC-conjugated anti-CD45 (clone 30-F11, BD Biosciences) through the tail vein three minutes before the brains were excised as described [ ]. .. Intracellular staining included anti-IFN-γ (clone XMG1.2; Biolegend), anti-TNF-α (clone XMG1.2; Biolegend), anti- IL-2 (clone JES6-5H4, Biolegend), anti- CD107a (clone 1D4B, BD Biosciences), and anti-CD107b (clone ABL-93, BD Biosciences).

    Recombinant:

    Article Title: Dichotomy between RIP1- and RIP3-Mediated Necroptosis in Tumor Necrosis Factor-?-Induced Shock
    Article Snippet: The zVAD, the ApoAlert annexin V-FITC antibody α-FADD, α-RIP1 and the monoclonal α-crmA antibody were purchased from BD Biosciences (Heidel-berg, Germany). .. Recombinant mouse TNFα (carrier-free) and purified α-human TNFα were purchased from BioLegend (Uithoorn, Netherlands). .. The cyclophilin A antibody and the caspase-8 antibody were obtained from Cell Signaling (Frankfurt/Main, Germany).

    Article Title: Induction of Dendritic Cell Maturation and Activation by a Potential Adjuvant, 2-Hydroxypropyl-β-Cyclodextrin
    Article Snippet: Recombinant murine GM-CSF was obtained from CreaGene. .. 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate and enzyme-linked immunosorbent assay (ELISA) kits for the quantification of human TNF-α, IL-6, IL-12p70, and IL-10, and mouse TNF-α and IL-6 were purchased from BioLegend (San Diego, CA, USA).

    Article Title: Resistance to cytotoxicity and sustained release of interleukin-6 and interleukin-8 in the presence of decreased interferon-γ after differentiation of glioblastoma by human natural killer cells
    Article Snippet: Human recombinant IL-2 was obtained from NIH-BRB. .. Human recombinant tumor necrosis factor alpha (hrTNF-α) and interferon gamma (hrIFN-γ) were purchased from Biolegend. .. Monoclonal antibodies to TNF-α were purified in our laboratory from ascites of mice injected with TNF-α hybridomas.

    Article Title: Interaction Between Macrophage Migration Inhibitory Factor and CD74 in Human Immunodeficiency Virus Type I Infected Primary Monocyte-Derived Macrophages Triggers the Production of Proinflammatory Mediators and Enhances Infection of Unactivated CD4+ T Cells
    Article Snippet: The neutralizing anti-MIF monoclonal antibody (clone NIHlllD.9) was obtained from ascites after purification using protein A/G spin column and resuspended at 5.15 mg/ml ( , ). .. A CD74 blocking antibody (BD Pharmingen, clone LN2), the recombinant human cytokines IL-6, IL-8, IL-1β (BioLegend), and TNFα (MiltenyiBiotec), and the cytokine neutralizing antibodies anti-IL-8 (R & D Systems), anti-IL-6, anti-IL-1β, and anti-TNFα (BioLegend) were obtained. .. Monocyte-derived macrophages were infected with the R5-tropic HIV and the infection was left to progress.

    Article Title: GM-CSF Signalling Boosts Dramatically IL-1Production
    Article Snippet: Following antibodies were used for western blotting: anti-tubulin, anti-caspase-1 p20, anti-c-Rel and anti-USF-2 (Santa Cruz Biotechnlogy, Santa Cruz, CA, USA), anti-IL-1β antibody (R & D), anti-NLRP3 (Alexis), anti-caspase-11 (Biolegend, San Diego, CA, USA). .. Recombinant GM-CSF, TNF-α, IL-6 and IFN-γ were obtained from Biolegend, M-CSF from Milteny Biotech (BergischGladbach, Germany). .. GM-CSF and FLT3L-derived BM DCs were generated by incubating freshly prepared BM cells for 8 days in IMDM medium supplemented with 20 ng/ml GM-CSF or 100 ng/ml FLT3L, respectively.

    Article Title: Dab2, a negative regulator of DC immunogenicity, is an attractive molecular target for DC-based immunotherapy
    Article Snippet: Anti-phospho-tyrosine-STAT5, anti-phospho-Smad2, anti-pan-STAT5, anti-pan-Akt and anti-phospho-serine-Akt antibodies were obtained from Cell Signaling Inc. Anti-β-actin , HRP-conjugated anti-rabbit and anti-mouse IgGs were purchased from Sigma, anti-Smad2 and anti-phospho-serine antibodies from Abcam, murine GM-CSF from Creagene Inc., recombinant human TGF-β1 from R & D system and lipopolysaccharide (LPS, from Escherichia coli O111:B4) from Sigma–Aldrich. .. Cytokine ELISA kits for murine IL-6, IL-12p70, IL-10, IL-17A, IL-1β, TNF-α, TGF-β and γIFN were purchased from BioLegend and the kit for IL-13 was from Abcam.

    Article Title: The sterol regulatory element binding proteins are essential for the metabolic programming of effector T cells and adaptive immunity
    Article Snippet: To analyze cytokine expression, splenocytes were stimulated for 5 hours with 2 μg/ml of the MHC class I restricted LCMV-GP33-41 or NP396-404 peptide in the presence of 50 U/ml recombinant murine IL-2 (R & D Systems) and 1mg/ml brefeldin A (Sigma-Aldrich). .. Cells were stained for surface expression of CD8, then fixed, permeabilized and stained with antibodies to TNF, IFN-γ and IL-2 (BioLegend).

    Article Title: Endothelial CD47 promotes Vascular Endothelial-cadherin tyrosine phosphorylation and participates in T-cell recruitment at sites of inflammation in vivo
    Article Snippet: Recombinant human TNF-α and CXCL12 were from PeproTech (Rocky Hill, NJ). .. Recombinant murine TNF-α was purchased from BioLegend (San Diego, CA). .. The Src kinase inhibitor PP2 and the p38 MAP kinase inhibitor SB203580 were from Calbiochem (San Diego, CA).

    Magnetic Beads:

    Article Title: Induction of Dendritic Cell Maturation and Activation by a Potential Adjuvant, 2-Hydroxypropyl-β-Cyclodextrin
    Article Snippet: Anti-human CD14 magnetic beads (clone: MΦP9) and anti-human CD3 magnetic beads (clone: HIT3a) were purchased from BD Biosciences (San Diego, CA, USA). .. 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate and enzyme-linked immunosorbent assay (ELISA) kits for the quantification of human TNF-α, IL-6, IL-12p70, and IL-10, and mouse TNF-α and IL-6 were purchased from BioLegend (San Diego, CA, USA).

    Isolation:

    Article Title: CXCR3+ Regulatory T Cells Control TH1 Responses in Crescentic GN
    Article Snippet: Previously described methods for leukocyte isolation from murine kidneys, spleens, and lymph nodes were used., Measurements were performed on a BD FACS LSR II, and data were analyzed with the FlowJo software (TreeStar, Inc.). .. The following anti-mouse or anti-human antibodies were used: CD45, CD3, CD4, CD8, CD25, CD40L, CD44, CD62L, CD69, CXCR3, CCR4, CCR5, CCR6, CCR7, CCR3, γ d-TCR, NK1.1, IL-17A, IFN γ , TNF α , Foxp3, CD45RA, HLA-DR, and CD127 (Biolegend, San Diego, CA; BD Biosciences; eBioscience, San Diego, CA; and R & D Systems; clones are available on request).

    Article Title: Resistance to cytotoxicity and sustained release of interleukin-6 and interleukin-8 in the presence of decreased interferon-γ after differentiation of glioblastoma by human natural killer cells
    Article Snippet: X01GB and X02GB GBM CSCs were isolated from freshly resected human tumor tissues [ , ] and were cultured under conditions that allow propagation of brain CSCs as described previously [ , ]. .. Human recombinant tumor necrosis factor alpha (hrTNF-α) and interferon gamma (hrIFN-γ) were purchased from Biolegend.

    Article Title: Oligomeric procyanidins stimulate innate antiviral immunity in dengue virus infected human PBMCs
    Article Snippet: Crude extract of Applepoly® , the putative trimer or tetramer procyanidins [10µg/ml, isolated from Applepoly® by normal phase HPLC as previously described ( )], monomeric procyanidin [catechin (Fisher) or epicatechin (Sigma), also 10µg/ml] or vehicle only was then added to the tissue culture media. .. The supernatant fluids were used to measure DENV titers in endpoint titration assays as well as TNFα by ELISA (Biolegend).

    Article Title: CD4 T cells control development and maintenance of brain-resident CD8 T cells during polyomavirus infection
    Article Snippet: For intracellular cytokine stimulation assays, lymphocytes were isolated from brain and spleen, cultured in DMEM/10% FBS for 5 h at 37°C with or without 1 μM LT359 peptide [ ], stained with Fixable Viability Dye, anti-CD8α, and anti-CD44, then permeabilized and fixed in FoxP3 buffer fixation and permeabilization solutions. .. Intracellular staining included anti-IFN-γ (clone XMG1.2; Biolegend), anti-TNF-α (clone XMG1.2; Biolegend), anti- IL-2 (clone JES6-5H4, Biolegend), anti- CD107a (clone 1D4B, BD Biosciences), and anti-CD107b (clone ABL-93, BD Biosciences).

    Immunohistochemistry:

    Article Title: Taenia solium: Development of an Experimental Model of Porcine Neurocysticercosis
    Article Snippet: Paragraph title: Immunohistochemical Analysis ... The primary antibodies used in this study recognized: glial fibrillary acidic protein (GFAP; polyclonal rabbit DAKO, Glostrup, Denmark 1:100 dilution), vimentin (mouse clone V9, DAKO; 1:100 dilution), neuronal nuclear protein (NeuN; mouse clone MAB377, IgG; Chemicon, Temecula CA, USA; 1:1000), nestin (mouse anti-nestin monoclonal antibody, 1:100; Chemicon, Millipore Billerica, MA, USA), IL-4 (anti-human monoclonal antibody, 1:200 dilution, Biolegend), IL-6 (anti-human monoclonal antibody, 1:250 dilution Biolegend), IL-10 (anti-human monoclonal antibody, 1:100, Biolegend), IL-17A (anti-human monoclonal antibody, 1:150 Boise’s), TNF-α (anti-human monoclonal antibody, 1:500, Biolegend), CD54 (anti-human monoclonal antibody, 1:200 Biolegend), CD69 (anti-human monoclonal antibody, 1:200 Biolegend), CD80 (anti-human monoclonal antibody, 1:300 Biolegend), CD106 (anti-human monoclonal antibody, 1:250, Biolegend).

    Labeling:

    Article Title: Taenia solium: Development of an Experimental Model of Porcine Neurocysticercosis
    Article Snippet: The single labeled sections were examined by light microscopy Leica Galen III, and digital color video camera (SSC-DC14), on a Pentium IV, Windows 2000 computer. .. The primary antibodies used in this study recognized: glial fibrillary acidic protein (GFAP; polyclonal rabbit DAKO, Glostrup, Denmark 1:100 dilution), vimentin (mouse clone V9, DAKO; 1:100 dilution), neuronal nuclear protein (NeuN; mouse clone MAB377, IgG; Chemicon, Temecula CA, USA; 1:1000), nestin (mouse anti-nestin monoclonal antibody, 1:100; Chemicon, Millipore Billerica, MA, USA), IL-4 (anti-human monoclonal antibody, 1:200 dilution, Biolegend), IL-6 (anti-human monoclonal antibody, 1:250 dilution Biolegend), IL-10 (anti-human monoclonal antibody, 1:100, Biolegend), IL-17A (anti-human monoclonal antibody, 1:150 Boise’s), TNF-α (anti-human monoclonal antibody, 1:500, Biolegend), CD54 (anti-human monoclonal antibody, 1:200 Biolegend), CD69 (anti-human monoclonal antibody, 1:200 Biolegend), CD80 (anti-human monoclonal antibody, 1:300 Biolegend), CD106 (anti-human monoclonal antibody, 1:250, Biolegend).

    Titration:

    Article Title: Oligomeric procyanidins stimulate innate antiviral immunity in dengue virus infected human PBMCs
    Article Snippet: Cells were cultured for 48 hours before supernatant fluid and cells were collected. .. The supernatant fluids were used to measure DENV titers in endpoint titration assays as well as TNFα by ELISA (Biolegend). .. When DENV was not detected after 48 hour infection (n=1, a seronegative donor), samples were excluded from analyses.

    Staining:

    Article Title: STIM1 controls T cell–mediated immune regulation and inflammation in chronic infection
    Article Snippet: For analysis of immune cell populations, mouse lungs were processed for flow cytometry as previously described ( ). .. The following anti-murine fluorophore-conjugated antibodies were used for staining: CD4 (RM4-5, BioLegend), CD8α (53-6.7, BD Biosciences), CD11c (HL3, BD Biosciences), CD11b (M1/70, BD Biosciences), Gr-1 (RB6-8C5, BD Biosciences), FasL (MFL3, eBioscience), Fas (15A7, eBioscience), IFN-γ (XMG1.2, BioLegend), TNF-α (MP6-XT22, BioLegend), CD62L (MEL-14, BD Biosciences), CD44 (IM7, BioLegend), CD25 (PC61, BD Biosciences), FOXP3 (FJK-16s, eBioscience), Helios (22F6, BioLegend), and NRP-1 (biotinylated goat polyclonal antibody, catalog BAF566, R & D Systems) followed by fluorophore-conjugated streptavidin (eBioscience). .. For analysis of human T cells, the following fluorophore-conjugated antibodies were used for flow cytometry: CD4 (OKT4, BioLegend), CD8α (HIT8a, BioLegend), IFN-γ (4S.B3, BioLegend), TNF-α (MAb11, BioLegend), Helios (22F6, BioLegend), and FOXP3 (259D, BioLegend).

    Article Title: The sterol regulatory element binding proteins are essential for the metabolic programming of effector T cells and adaptive immunity
    Article Snippet: To analyze cytokine expression, splenocytes were stimulated for 5 hours with 2 μg/ml of the MHC class I restricted LCMV-GP33-41 or NP396-404 peptide in the presence of 50 U/ml recombinant murine IL-2 (R & D Systems) and 1mg/ml brefeldin A (Sigma-Aldrich). .. Cells were stained for surface expression of CD8, then fixed, permeabilized and stained with antibodies to TNF, IFN-γ and IL-2 (BioLegend). .. Flow cytometric analysis was performed using an LSR II or the FACSVerse (Becton Dickinson) and analyzed using FlowJo software (Treestar).

    Article Title: CD4 T cells control development and maintenance of brain-resident CD8 T cells during polyomavirus infection
    Article Snippet: For intracellular cytokine stimulation assays, lymphocytes were isolated from brain and spleen, cultured in DMEM/10% FBS for 5 h at 37°C with or without 1 μM LT359 peptide [ ], stained with Fixable Viability Dye, anti-CD8α, and anti-CD44, then permeabilized and fixed in FoxP3 buffer fixation and permeabilization solutions. .. Intracellular staining included anti-IFN-γ (clone XMG1.2; Biolegend), anti-TNF-α (clone XMG1.2; Biolegend), anti- IL-2 (clone JES6-5H4, Biolegend), anti- CD107a (clone 1D4B, BD Biosciences), and anti-CD107b (clone ABL-93, BD Biosciences). .. CD4 T cells were stained with anti-CD4 and anti-CD44, permeabilized as described, and then stained with FoxP3 (clone FJK-16s, Invitrogen).

    Mouse Assay:

    Article Title: Dab2, a negative regulator of DC immunogenicity, is an attractive molecular target for DC-based immunotherapy
    Article Snippet: Paragraph title: Mice, cell lines, and reagents ... Cytokine ELISA kits for murine IL-6, IL-12p70, IL-10, IL-17A, IL-1β, TNF-α, TGF-β and γIFN were purchased from BioLegend and the kit for IL-13 was from Abcam.

    Article Title: The sterol regulatory element binding proteins are essential for the metabolic programming of effector T cells and adaptive immunity
    Article Snippet: Mice were infected intraperitoneally (i.p.) with 2 × 104 plaque forming units (PFU) of LCMV-Armstrong. .. Cells were stained for surface expression of CD8, then fixed, permeabilized and stained with antibodies to TNF, IFN-γ and IL-2 (BioLegend).

    Article Title: CD4 T cells control development and maintenance of brain-resident CD8 T cells during polyomavirus infection
    Article Snippet: After isolation from perfused or intravascularly stained mice, cells were stained with Fixable Viability Dye (eBioscience, San Diego, CA), APC-Db LT359 tetramers (NIH Tetramer Core Facility, Atlanta, GA), and the following surface antibodies: CD8α (clone 53–6.7, eBioscience), CD44 (clone IM7, eBioscience), PD-1 (clone RMPI-30, Biolegend), Tim-3 (clone RMT3-23, Biolegend), 2B4 (clone m2B4(B6)4581, Biolegend), CD103 (clone M290, BD Horizon), CD69 (clone HI.2F3, Biolegend), CD49d (clone MRF4.8, Biolegend), CD162 (clone 2PH1, BD Biosciences), CD11a (clone 2D7, BD Biosciences), CD127 (clone A7R34, Biolegend), KLRG1 (clone 2F1, BD Biosciences), CD25 (clone PC61.5.3, Invitrogen), CD44 (clone IM7, BD Biosciences) and CD4 (clone RM4-5, BD Biosciences). .. Intracellular staining included anti-IFN-γ (clone XMG1.2; Biolegend), anti-TNF-α (clone XMG1.2; Biolegend), anti- IL-2 (clone JES6-5H4, Biolegend), anti- CD107a (clone 1D4B, BD Biosciences), and anti-CD107b (clone ABL-93, BD Biosciences).

    Software:

    Article Title: CXCR3+ Regulatory T Cells Control TH1 Responses in Crescentic GN
    Article Snippet: Previously described methods for leukocyte isolation from murine kidneys, spleens, and lymph nodes were used., Measurements were performed on a BD FACS LSR II, and data were analyzed with the FlowJo software (TreeStar, Inc.). .. The following anti-mouse or anti-human antibodies were used: CD45, CD3, CD4, CD8, CD25, CD40L, CD44, CD62L, CD69, CXCR3, CCR4, CCR5, CCR6, CCR7, CCR3, γ d-TCR, NK1.1, IL-17A, IFN γ , TNF α , Foxp3, CD45RA, HLA-DR, and CD127 (Biolegend, San Diego, CA; BD Biosciences; eBioscience, San Diego, CA; and R & D Systems; clones are available on request).

    Article Title: STIM1 controls T cell–mediated immune regulation and inflammation in chronic infection
    Article Snippet: The following anti-murine fluorophore-conjugated antibodies were used for staining: CD4 (RM4-5, BioLegend), CD8α (53-6.7, BD Biosciences), CD11c (HL3, BD Biosciences), CD11b (M1/70, BD Biosciences), Gr-1 (RB6-8C5, BD Biosciences), FasL (MFL3, eBioscience), Fas (15A7, eBioscience), IFN-γ (XMG1.2, BioLegend), TNF-α (MP6-XT22, BioLegend), CD62L (MEL-14, BD Biosciences), CD44 (IM7, BioLegend), CD25 (PC61, BD Biosciences), FOXP3 (FJK-16s, eBioscience), Helios (22F6, BioLegend), and NRP-1 (biotinylated goat polyclonal antibody, catalog BAF566, R & D Systems) followed by fluorophore-conjugated streptavidin (eBioscience). .. For apoptotic cell staining, fluorophore-conjugated annexin V probe (BD Biosciences) and propidium iodide (PI) (BD Biosciences) were used.

    Article Title: CD4 T cells control development and maintenance of brain-resident CD8 T cells during polyomavirus infection
    Article Snippet: Intracellular staining included anti-IFN-γ (clone XMG1.2; Biolegend), anti-TNF-α (clone XMG1.2; Biolegend), anti- IL-2 (clone JES6-5H4, Biolegend), anti- CD107a (clone 1D4B, BD Biosciences), and anti-CD107b (clone ABL-93, BD Biosciences). .. The intracellular signal of YFP was amplified by staining with anti-GFP (clone FM264G, Biolegend).

    Real-time Polymerase Chain Reaction:

    Article Title: Oligomeric procyanidins stimulate innate antiviral immunity in dengue virus infected human PBMCs
    Article Snippet: The supernatant fluids were used to measure DENV titers in endpoint titration assays as well as TNFα by ELISA (Biolegend). .. When DENV was not detected after 48 hour infection (n=1, a seronegative donor), samples were excluded from analyses.

    Functional Assay:

    Article Title: Resistance to cytotoxicity and sustained release of interleukin-6 and interleukin-8 in the presence of decreased interferon-γ after differentiation of glioblastoma by human natural killer cells
    Article Snippet: Human recombinant tumor necrosis factor alpha (hrTNF-α) and interferon gamma (hrIFN-γ) were purchased from Biolegend. .. Polyclonal IFN-γ, IL-6 and IL-8 antibodies were prepared in rabbits and purified.

    Activation Assay:

    Article Title: Taenia solium: Development of an Experimental Model of Porcine Neurocysticercosis
    Article Snippet: The primary antibodies used in this study recognized: glial fibrillary acidic protein (GFAP; polyclonal rabbit DAKO, Glostrup, Denmark 1:100 dilution), vimentin (mouse clone V9, DAKO; 1:100 dilution), neuronal nuclear protein (NeuN; mouse clone MAB377, IgG; Chemicon, Temecula CA, USA; 1:1000), nestin (mouse anti-nestin monoclonal antibody, 1:100; Chemicon, Millipore Billerica, MA, USA), IL-4 (anti-human monoclonal antibody, 1:200 dilution, Biolegend), IL-6 (anti-human monoclonal antibody, 1:250 dilution Biolegend), IL-10 (anti-human monoclonal antibody, 1:100, Biolegend), IL-17A (anti-human monoclonal antibody, 1:150 Boise’s), TNF-α (anti-human monoclonal antibody, 1:500, Biolegend), CD54 (anti-human monoclonal antibody, 1:200 Biolegend), CD69 (anti-human monoclonal antibody, 1:200 Biolegend), CD80 (anti-human monoclonal antibody, 1:300 Biolegend), CD106 (anti-human monoclonal antibody, 1:250, Biolegend). .. The primary antibodies used in this study recognized: glial fibrillary acidic protein (GFAP; polyclonal rabbit DAKO, Glostrup, Denmark 1:100 dilution), vimentin (mouse clone V9, DAKO; 1:100 dilution), neuronal nuclear protein (NeuN; mouse clone MAB377, IgG; Chemicon, Temecula CA, USA; 1:1000), nestin (mouse anti-nestin monoclonal antibody, 1:100; Chemicon, Millipore Billerica, MA, USA), IL-4 (anti-human monoclonal antibody, 1:200 dilution, Biolegend), IL-6 (anti-human monoclonal antibody, 1:250 dilution Biolegend), IL-10 (anti-human monoclonal antibody, 1:100, Biolegend), IL-17A (anti-human monoclonal antibody, 1:150 Boise’s), TNF-α (anti-human monoclonal antibody, 1:500, Biolegend), CD54 (anti-human monoclonal antibody, 1:200 Biolegend), CD69 (anti-human monoclonal antibody, 1:200 Biolegend), CD80 (anti-human monoclonal antibody, 1:300 Biolegend), CD106 (anti-human monoclonal antibody, 1:250, Biolegend).

    Article Title: The anti-tumor efficacy of IL-2/IL-21-cultured polyfunctional neu-specific T-cells is TNF-alpha/IL-17 dependent
    Article Snippet: Cancer Research. .. Griffin GK, Newton G, Tarrio ML, Bu DX, Maganto-Garcia E, Azcutia V, et al. IL-17 and TNF-alpha sustain neutrophil recruitment during inflammation through synergistic effects on endothelial activation. .. Kryczek I, Banerjee M, Cheng P, Vatan L, Szeliga W, Wei S, et al. Phenotype, distribution, generation, and functional and clinical relevance of Th17 cells in the human tumor environments.

    FACS:

    Article Title: CXCR3+ Regulatory T Cells Control TH1 Responses in Crescentic GN
    Article Snippet: Previously described methods for leukocyte isolation from murine kidneys, spleens, and lymph nodes were used., Measurements were performed on a BD FACS LSR II, and data were analyzed with the FlowJo software (TreeStar, Inc.). .. The following anti-mouse or anti-human antibodies were used: CD45, CD3, CD4, CD8, CD25, CD40L, CD44, CD62L, CD69, CXCR3, CCR4, CCR5, CCR6, CCR7, CCR3, γ d-TCR, NK1.1, IL-17A, IFN γ , TNF α , Foxp3, CD45RA, HLA-DR, and CD127 (Biolegend, San Diego, CA; BD Biosciences; eBioscience, San Diego, CA; and R & D Systems; clones are available on request).

    other:

    Article Title: The role of microglial P2X7: modulation of cell death and cytokine release
    Article Snippet: Interferon γ (IFNγ), TNFα, IL6, and IL1β were purchased from Biolegend.

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    BioLegend rat anti mouse tnfα
    Blocking antibodies to <t>TNFα</t> and TNFRs prevents cholangiocyte killing.
    Rat Anti Mouse Tnfα, supplied by BioLegend, 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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    99
    BioLegend rat anti mouse tnfα mab
    <t>TNFα</t> instruct the neutrophils to migrate into the lymphatic system upon antigen sensitisation. Neutrophil migration into the lymphatic system of the cremaster muscle following antigen sensitisation with complete Freund’s adjuvant (CFA+Ag) was induced in WT and TNFRdbKO animals as well as in chimeric animals exhibiting neutrophils deficient in TNFRs. ( a ) Time course of TNFα release in the cremaster muscles of WT mice following intra-scrotal injection of CFA+Ag and as quantified by ELISA. ( b ) TNFα release in mice subjected to clodronate liposome-induced macrophage depletion. ( c ) Number of extravasated neutrophils in cremaster muscles of WT and TNFRdbKO mice at 16 hrs post-CFA+Ag-stimulation as quantified by confocal microscopy. ( d ) Number of neutrophils within cremaster lymphatic vessels of WT and TNFRdbKO mice at 16 hrs post-CFA+Ag-stimulation as quantified by confocal microscopy. ( e ) Percentage of neutrophils in dLNs of WT and TNFRdbKO mice at 16 hrs post-CFA+Ag-stimulation as quantified by flow cytometry. ( f ) Number of extravasated neutrophils in cremaster muscles at 16 hrs post-CFA+Ag-stimulation from chimeric animals receiving bone marrow transplant from WT or TNFRdbKO donor mice and as quantified by confocal microscopy. ( g ) Number of neutrophils within cremaster lymphatic vessels at 16 hrs post-CFA+Ag-stimulation from chimeric animals receiving bone marrow transplant from WT or TNFRdbKO donor mice and as quantified by confocal microscopy. ( h ) Number of neutrophils found in the dLNs of chimeric animals receiving bone marrow transplant from WT or TNFRdbKO donor mice as quantified by confocal microscopy 16 hrs post-CFA+Ag-stimulation. Data are expressed as mean ± SEM of N = 5–12 animals per group from at least 5–10 experiments. Statistically significant differences between stimulated and control groups or between WT and TNFRdbKO mice are indicated by asterisks: *P
    Rat Anti Mouse Tnfα Mab, supplied by BioLegend, 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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    83
    BioLegend murine tnfα
    P2X 1 deficiency and P2X 1 inhibition in E. coli -induced sepsis . ARD6 was injected into anesthetized P 2 X 1 + / + and P 2 X 1 − / − mice at a concentration of either low (61 million) or high (248 million) number of bacteria. (A) Kaplan-Meier plot shows survival (high dose), n = 6 for control and n = 12 for both genotypes. (B) Survival in the presence or absence of the P2X 1 antagonist NF449 (100 mg kg −1 , iv ) in balb/c mice. The mice were exposed to the high concentration of bacteria, n = 6 for control with or without NF449 and n = 10–12 for ARD6 with or without NF449. (C) Absorbance of plasma as an indication of plasma haemolysis after the low dose, n = 8 for both genotypes (D) Levels of thrombin-antithrombin (TAT) complexes in plasma 2.5 h after the low dose, n = 8 for both genotypes. (E) Levels of <t>TNFα,</t> KC, IL-1β, and IL-6 2.5 h after the low dose of ARD6, n = 13 both genotypes. * p
    Murine Tnfα, supplied by BioLegend, used in various techniques. Bioz Stars score: 83/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    BioLegend anti tnfα mab
    BV6 does not induce NF-κB nuclear translocation SW620 cells were either untreated (a), treated with <t>TNFα</t> (b, 100 U/ml), BV6 (c, 5 μM) or both TNFα and BV6 (d) for 60 min. Cells were fixed, permeabilized, and stained with p65-specific antibody, followed by fluorescent dye-conjugated 2 nd antibody. The images were obtained with a confocal microscope. a1-d1 are low amplification images and a2-d2 are high amplification images.
    Anti Tnfα Mab, supplied by BioLegend, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Blocking antibodies to TNFα and TNFRs prevents cholangiocyte killing.

    Journal:

    Article Title: Preferential TNF α signaling via TNFR2 regulates epithelial injury and duct obstruction in experimental biliary atresia

    doi: 10.1172/jci.insight.88747

    Figure Lengend Snippet: Blocking antibodies to TNFα and TNFRs prevents cholangiocyte killing.

    Article Snippet: Neutralizing rat anti-mouse TNFα (clone MP6-XT22) and isotype rat IgG1 antibodies were obtained from BioLegend, while isotype Armenian hamster IgG antibodies were from BioXCell.

    Techniques: Blocking Assay

    Blocking of TNFα/TNFR prevents experimental biliary atresia.

    Journal:

    Article Title: Preferential TNF α signaling via TNFR2 regulates epithelial injury and duct obstruction in experimental biliary atresia

    doi: 10.1172/jci.insight.88747

    Figure Lengend Snippet: Blocking of TNFα/TNFR prevents experimental biliary atresia.

    Article Snippet: Neutralizing rat anti-mouse TNFα (clone MP6-XT22) and isotype rat IgG1 antibodies were obtained from BioLegend, while isotype Armenian hamster IgG antibodies were from BioXCell.

    Techniques: Blocking Assay

    Hepatic NK and DCs express TNFα.

    Journal:

    Article Title: Preferential TNF α signaling via TNFR2 regulates epithelial injury and duct obstruction in experimental biliary atresia

    doi: 10.1172/jci.insight.88747

    Figure Lengend Snippet: Hepatic NK and DCs express TNFα.

    Article Snippet: Neutralizing rat anti-mouse TNFα (clone MP6-XT22) and isotype rat IgG1 antibodies were obtained from BioLegend, while isotype Armenian hamster IgG antibodies were from BioXCell.

    Techniques:

    Activation markers and cytokine/chemokine expression following blockade of TNFα or TNFR1/2.

    Journal:

    Article Title: Preferential TNF α signaling via TNFR2 regulates epithelial injury and duct obstruction in experimental biliary atresia

    doi: 10.1172/jci.insight.88747

    Figure Lengend Snippet: Activation markers and cytokine/chemokine expression following blockade of TNFα or TNFR1/2.

    Article Snippet: Neutralizing rat anti-mouse TNFα (clone MP6-XT22) and isotype rat IgG1 antibodies were obtained from BioLegend, while isotype Armenian hamster IgG antibodies were from BioXCell.

    Techniques: Activation Assay, Expressing

    Expression of TNFα and TNFR1/2 in cholangiocytes and NK-mediated cytotoxicity.

    Journal:

    Article Title: Preferential TNF α signaling via TNFR2 regulates epithelial injury and duct obstruction in experimental biliary atresia

    doi: 10.1172/jci.insight.88747

    Figure Lengend Snippet: Expression of TNFα and TNFR1/2 in cholangiocytes and NK-mediated cytotoxicity.

    Article Snippet: Neutralizing rat anti-mouse TNFα (clone MP6-XT22) and isotype rat IgG1 antibodies were obtained from BioLegend, while isotype Armenian hamster IgG antibodies were from BioXCell.

    Techniques: Expressing

    Anti-TNFα antibodies suppress the phenotype and bile duct injury.

    Journal:

    Article Title: Preferential TNF α signaling via TNFR2 regulates epithelial injury and duct obstruction in experimental biliary atresia

    doi: 10.1172/jci.insight.88747

    Figure Lengend Snippet: Anti-TNFα antibodies suppress the phenotype and bile duct injury.

    Article Snippet: Neutralizing rat anti-mouse TNFα (clone MP6-XT22) and isotype rat IgG1 antibodies were obtained from BioLegend, while isotype Armenian hamster IgG antibodies were from BioXCell.

    Techniques:

    Expression of TNFα and TNFRs in humans and mice with biliary atresia.

    Journal:

    Article Title: Preferential TNF α signaling via TNFR2 regulates epithelial injury and duct obstruction in experimental biliary atresia

    doi: 10.1172/jci.insight.88747

    Figure Lengend Snippet: Expression of TNFα and TNFRs in humans and mice with biliary atresia.

    Article Snippet: Neutralizing rat anti-mouse TNFα (clone MP6-XT22) and isotype rat IgG1 antibodies were obtained from BioLegend, while isotype Armenian hamster IgG antibodies were from BioXCell.

    Techniques: Expressing, Mouse Assay

    TNFα instruct the neutrophils to migrate into the lymphatic system upon antigen sensitisation. Neutrophil migration into the lymphatic system of the cremaster muscle following antigen sensitisation with complete Freund’s adjuvant (CFA+Ag) was induced in WT and TNFRdbKO animals as well as in chimeric animals exhibiting neutrophils deficient in TNFRs. ( a ) Time course of TNFα release in the cremaster muscles of WT mice following intra-scrotal injection of CFA+Ag and as quantified by ELISA. ( b ) TNFα release in mice subjected to clodronate liposome-induced macrophage depletion. ( c ) Number of extravasated neutrophils in cremaster muscles of WT and TNFRdbKO mice at 16 hrs post-CFA+Ag-stimulation as quantified by confocal microscopy. ( d ) Number of neutrophils within cremaster lymphatic vessels of WT and TNFRdbKO mice at 16 hrs post-CFA+Ag-stimulation as quantified by confocal microscopy. ( e ) Percentage of neutrophils in dLNs of WT and TNFRdbKO mice at 16 hrs post-CFA+Ag-stimulation as quantified by flow cytometry. ( f ) Number of extravasated neutrophils in cremaster muscles at 16 hrs post-CFA+Ag-stimulation from chimeric animals receiving bone marrow transplant from WT or TNFRdbKO donor mice and as quantified by confocal microscopy. ( g ) Number of neutrophils within cremaster lymphatic vessels at 16 hrs post-CFA+Ag-stimulation from chimeric animals receiving bone marrow transplant from WT or TNFRdbKO donor mice and as quantified by confocal microscopy. ( h ) Number of neutrophils found in the dLNs of chimeric animals receiving bone marrow transplant from WT or TNFRdbKO donor mice as quantified by confocal microscopy 16 hrs post-CFA+Ag-stimulation. Data are expressed as mean ± SEM of N = 5–12 animals per group from at least 5–10 experiments. Statistically significant differences between stimulated and control groups or between WT and TNFRdbKO mice are indicated by asterisks: *P

    Journal: Scientific Reports

    Article Title: Endogenous TNFα orchestrates the trafficking of neutrophils into and within lymphatic vessels during acute inflammation

    doi: 10.1038/srep44189

    Figure Lengend Snippet: TNFα instruct the neutrophils to migrate into the lymphatic system upon antigen sensitisation. Neutrophil migration into the lymphatic system of the cremaster muscle following antigen sensitisation with complete Freund’s adjuvant (CFA+Ag) was induced in WT and TNFRdbKO animals as well as in chimeric animals exhibiting neutrophils deficient in TNFRs. ( a ) Time course of TNFα release in the cremaster muscles of WT mice following intra-scrotal injection of CFA+Ag and as quantified by ELISA. ( b ) TNFα release in mice subjected to clodronate liposome-induced macrophage depletion. ( c ) Number of extravasated neutrophils in cremaster muscles of WT and TNFRdbKO mice at 16 hrs post-CFA+Ag-stimulation as quantified by confocal microscopy. ( d ) Number of neutrophils within cremaster lymphatic vessels of WT and TNFRdbKO mice at 16 hrs post-CFA+Ag-stimulation as quantified by confocal microscopy. ( e ) Percentage of neutrophils in dLNs of WT and TNFRdbKO mice at 16 hrs post-CFA+Ag-stimulation as quantified by flow cytometry. ( f ) Number of extravasated neutrophils in cremaster muscles at 16 hrs post-CFA+Ag-stimulation from chimeric animals receiving bone marrow transplant from WT or TNFRdbKO donor mice and as quantified by confocal microscopy. ( g ) Number of neutrophils within cremaster lymphatic vessels at 16 hrs post-CFA+Ag-stimulation from chimeric animals receiving bone marrow transplant from WT or TNFRdbKO donor mice and as quantified by confocal microscopy. ( h ) Number of neutrophils found in the dLNs of chimeric animals receiving bone marrow transplant from WT or TNFRdbKO donor mice as quantified by confocal microscopy 16 hrs post-CFA+Ag-stimulation. Data are expressed as mean ± SEM of N = 5–12 animals per group from at least 5–10 experiments. Statistically significant differences between stimulated and control groups or between WT and TNFRdbKO mice are indicated by asterisks: *P

    Article Snippet: The following primary antibodies were used for immunofluorescence labelling for confocal imaging and confocal IVM: rat anti–mouse LYVE-1 mAb (clone ALY7; eBioscience); non-blocking rat anti–mouse PECAM-1 Ab (clone C390, eBioscience); rat anti–mouse ICAM-1 mAb (clone YN1/1.4.7; purified and Alexa 488-conjugated, eBioscience); rat anti–mouse MAC-1 mAb (clone M1/70, BioLegend); monoclonal rat anti–mouse MRP14 mAb (clone 2B10; a gift from N. Hogg, Cancer Research UK, London, UK); rat anti-mouse Ly6G mAb (clone 1A8, Alexa 647-conjugated, Biolegend); rat anti-mouse CXCR4 mAb (clone 2B11, PE-conjugated, eBioscience), rat anti-mouse/human High Endothelial Venule mAb (MECA-79, Alexa 488-conjugated eBioscience), rat anti-mouse CCR7 mAb (clone 4B12, Alexa 488- or biotin-conjugated, eBioscience); rat anti-mouse CD45.2 mAb (clone 104, PE/Cy7-conjugated, Biolegend), rat anti-mouse TNFα mAb (clone MP6-XT22, Biolegend), anti-mouse CCL21, anti-mouse CXCL1 and anti-mouse CXCL12 mAbs (R & D systems), polyclonal goat anti–mouse TNFR p55 and p75 Abs (R & D Systems).

    Techniques: Migration, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Flow Cytometry, Cytometry

    Schematic diagram illustrating the dual mechanisms of action of TNFα leading to the trafficking of neutrophils into and within the lymphatic vasculature upon acute inflammation in vivo . During the acute inflammatory response of the tissue following antigen sensitisation, endogenous TNFα release primed the freshly recruited neutrophils. This cytokine allow these leukocytes to be attracted to the lymphatic vessels in a CCR7 dependent manner (intravasation). Furthermore, endogenous TNFα also stimulate the lymphatic endothelium to express ICAM-1 on their surface, allowing the neutrophils present in the lymphatic vessels to adhere and crawl along the luminal side in the correct direction toward the flow of the vessel.

    Journal: Scientific Reports

    Article Title: Endogenous TNFα orchestrates the trafficking of neutrophils into and within lymphatic vessels during acute inflammation

    doi: 10.1038/srep44189

    Figure Lengend Snippet: Schematic diagram illustrating the dual mechanisms of action of TNFα leading to the trafficking of neutrophils into and within the lymphatic vasculature upon acute inflammation in vivo . During the acute inflammatory response of the tissue following antigen sensitisation, endogenous TNFα release primed the freshly recruited neutrophils. This cytokine allow these leukocytes to be attracted to the lymphatic vessels in a CCR7 dependent manner (intravasation). Furthermore, endogenous TNFα also stimulate the lymphatic endothelium to express ICAM-1 on their surface, allowing the neutrophils present in the lymphatic vessels to adhere and crawl along the luminal side in the correct direction toward the flow of the vessel.

    Article Snippet: The following primary antibodies were used for immunofluorescence labelling for confocal imaging and confocal IVM: rat anti–mouse LYVE-1 mAb (clone ALY7; eBioscience); non-blocking rat anti–mouse PECAM-1 Ab (clone C390, eBioscience); rat anti–mouse ICAM-1 mAb (clone YN1/1.4.7; purified and Alexa 488-conjugated, eBioscience); rat anti–mouse MAC-1 mAb (clone M1/70, BioLegend); monoclonal rat anti–mouse MRP14 mAb (clone 2B10; a gift from N. Hogg, Cancer Research UK, London, UK); rat anti-mouse Ly6G mAb (clone 1A8, Alexa 647-conjugated, Biolegend); rat anti-mouse CXCR4 mAb (clone 2B11, PE-conjugated, eBioscience), rat anti-mouse/human High Endothelial Venule mAb (MECA-79, Alexa 488-conjugated eBioscience), rat anti-mouse CCR7 mAb (clone 4B12, Alexa 488- or biotin-conjugated, eBioscience); rat anti-mouse CD45.2 mAb (clone 104, PE/Cy7-conjugated, Biolegend), rat anti-mouse TNFα mAb (clone MP6-XT22, Biolegend), anti-mouse CCL21, anti-mouse CXCL1 and anti-mouse CXCL12 mAbs (R & D systems), polyclonal goat anti–mouse TNFR p55 and p75 Abs (R & D Systems).

    Techniques: In Vivo, Flow Cytometry

    TNFα controls the crawling of neutrophils into the lymphatic vessels in vivo . The effect of anti- TNFα blocking mAb on neutrophil crawling along the luminal side of the lymphatic endothelium was analysed by intravital confocal microscopy (IVM) using LysM-GFP mice subjected to CFA+Ag-induced cremaster inflammation and immunostained in vivo with a non-blocking dose of Alexa555-conjugated anti-LYVE-1 mAb. Isotype control or anti- TNFα blocking mAbs were injected i.s. 4 hrs post-inflammation. ( a ) The pictures are representative still images at one time point of the IVM recording showing lymphatic-infiltrated neutrophils (green) and their associated crawling path (time-coloured mapped line) and/or directionality (arrow) as analysed by IMARIS software (LYVE-1 with an opacity filter of 5% to see the intravasated leukocytes) from CTL (left panel) or anti- TNFα (right panel) mAb-treated groups. ( b ) The graphs show the crawling paths of lymphatic-infiltrated neutrophils in the X Y planes of the lymphatic vessels from CTL mAb (left panel) and anti- TNFα mAb (right panel) treated groups. ( c ) Quantification (in percentage) of neutrophils crawling in the afferent (flow) or opposite direction (anti-flow) of the lymphatic vessel. Mean speed ( d ), directionality ( e ) and straightness ( f ) of neutrophils crawling in CTL mAb and anti- TNFα treated groups. A total of 280 cells were analysed. Results are expressed as mean ± SEM of N = 4–9 mice (each mouse representing one independent experiment). Significant differences between flow and anti-flow crawling cells are indicated by *P

    Journal: Scientific Reports

    Article Title: Endogenous TNFα orchestrates the trafficking of neutrophils into and within lymphatic vessels during acute inflammation

    doi: 10.1038/srep44189

    Figure Lengend Snippet: TNFα controls the crawling of neutrophils into the lymphatic vessels in vivo . The effect of anti- TNFα blocking mAb on neutrophil crawling along the luminal side of the lymphatic endothelium was analysed by intravital confocal microscopy (IVM) using LysM-GFP mice subjected to CFA+Ag-induced cremaster inflammation and immunostained in vivo with a non-blocking dose of Alexa555-conjugated anti-LYVE-1 mAb. Isotype control or anti- TNFα blocking mAbs were injected i.s. 4 hrs post-inflammation. ( a ) The pictures are representative still images at one time point of the IVM recording showing lymphatic-infiltrated neutrophils (green) and their associated crawling path (time-coloured mapped line) and/or directionality (arrow) as analysed by IMARIS software (LYVE-1 with an opacity filter of 5% to see the intravasated leukocytes) from CTL (left panel) or anti- TNFα (right panel) mAb-treated groups. ( b ) The graphs show the crawling paths of lymphatic-infiltrated neutrophils in the X Y planes of the lymphatic vessels from CTL mAb (left panel) and anti- TNFα mAb (right panel) treated groups. ( c ) Quantification (in percentage) of neutrophils crawling in the afferent (flow) or opposite direction (anti-flow) of the lymphatic vessel. Mean speed ( d ), directionality ( e ) and straightness ( f ) of neutrophils crawling in CTL mAb and anti- TNFα treated groups. A total of 280 cells were analysed. Results are expressed as mean ± SEM of N = 4–9 mice (each mouse representing one independent experiment). Significant differences between flow and anti-flow crawling cells are indicated by *P

    Article Snippet: The following primary antibodies were used for immunofluorescence labelling for confocal imaging and confocal IVM: rat anti–mouse LYVE-1 mAb (clone ALY7; eBioscience); non-blocking rat anti–mouse PECAM-1 Ab (clone C390, eBioscience); rat anti–mouse ICAM-1 mAb (clone YN1/1.4.7; purified and Alexa 488-conjugated, eBioscience); rat anti–mouse MAC-1 mAb (clone M1/70, BioLegend); monoclonal rat anti–mouse MRP14 mAb (clone 2B10; a gift from N. Hogg, Cancer Research UK, London, UK); rat anti-mouse Ly6G mAb (clone 1A8, Alexa 647-conjugated, Biolegend); rat anti-mouse CXCR4 mAb (clone 2B11, PE-conjugated, eBioscience), rat anti-mouse/human High Endothelial Venule mAb (MECA-79, Alexa 488-conjugated eBioscience), rat anti-mouse CCR7 mAb (clone 4B12, Alexa 488- or biotin-conjugated, eBioscience); rat anti-mouse CD45.2 mAb (clone 104, PE/Cy7-conjugated, Biolegend), rat anti-mouse TNFα mAb (clone MP6-XT22, Biolegend), anti-mouse CCL21, anti-mouse CXCL1 and anti-mouse CXCL12 mAbs (R & D systems), polyclonal goat anti–mouse TNFR p55 and p75 Abs (R & D Systems).

    Techniques: In Vivo, Blocking Assay, Confocal Microscopy, Mouse Assay, Injection, Software, CTL Assay, Flow Cytometry

    TNFα promotes CCR7-dependent migration of neutrophils into lymphatic vessels in vivo . ( a ) Analysis by flow cytometry of CCR7 expression (intracellular and cell-surface) on neutrophils isolated from the blood circulation, CFA+Ag-stimulated-cremaster muscles and dLNs of WT and CCR7KO animals. ( b ) CCR7 surface expression on tissue-infiltrated neutrophils from WT and TNFRdbKO mice subjected to CFA+Ag-induced inflammation. ( c–e ) WT and CCR7KO mice were subjected to TNFα-induced cremaster muscle inflammation and neutrophil responses in the tissue and dLNs was assessed by confocal microscopy 16 hrs post-inflammation. ( c ) Number of extravasated neutrophils in of WT and CCR7KO mice. ( d ) Number of intravasated neutrophils in lymphatic vessels of cremaster muscles from WT and CCR7KO mice. ( e ) Neutrophil number in the cremaster dLNs of WT and CCR7KO animals. ( f–k ) WT, CCR7KO mice or CCR7KO-neutrophil chimeric animals were subjected to antigen sensitisation (CFA+Ag) and neutrophil responses in the cremaster muscle and dLNs (16 hrs post-inflammation) was assessed by confocal microscopy. ( f ) Number of extravasated neutrophils in inflamed cremaster muscles of WT and CCR7KO mice. ( g ) Number of intravasated neutrophils in cremaster lymphatic vessels of WT and CCR7KO mice. ( h ) Neutrophil number in the cremaster dLNs of WT and CCR7KO mice. ( i ) Number of neutrophils recruited to the cremaster muscles from lethally irradiated WT animals receiving bone marrow transplant from either WT or CCR7KO donor mice. ( j ) Number of neutrophils within cremaster lymphatic vessels post-CFA+Ag-stimulation from lethally irradiated WT animals receiving bone marrow transplant from either WT or CCR7KO donor mice. ( k ) Neutrophil number in the cremaster dLNs from lethally irradiated WT animals receiving bone marrow transplant from either WT or CCR7KO donor mice. Data are expressed as mean ± SEM of N = 7–12 animals per group (from at least 5 independent experiments). Statistically significant differences between stimulated/specific mAb and unstimulated treatment/isotype control groups are indicated by asterisks: *P

    Journal: Scientific Reports

    Article Title: Endogenous TNFα orchestrates the trafficking of neutrophils into and within lymphatic vessels during acute inflammation

    doi: 10.1038/srep44189

    Figure Lengend Snippet: TNFα promotes CCR7-dependent migration of neutrophils into lymphatic vessels in vivo . ( a ) Analysis by flow cytometry of CCR7 expression (intracellular and cell-surface) on neutrophils isolated from the blood circulation, CFA+Ag-stimulated-cremaster muscles and dLNs of WT and CCR7KO animals. ( b ) CCR7 surface expression on tissue-infiltrated neutrophils from WT and TNFRdbKO mice subjected to CFA+Ag-induced inflammation. ( c–e ) WT and CCR7KO mice were subjected to TNFα-induced cremaster muscle inflammation and neutrophil responses in the tissue and dLNs was assessed by confocal microscopy 16 hrs post-inflammation. ( c ) Number of extravasated neutrophils in of WT and CCR7KO mice. ( d ) Number of intravasated neutrophils in lymphatic vessels of cremaster muscles from WT and CCR7KO mice. ( e ) Neutrophil number in the cremaster dLNs of WT and CCR7KO animals. ( f–k ) WT, CCR7KO mice or CCR7KO-neutrophil chimeric animals were subjected to antigen sensitisation (CFA+Ag) and neutrophil responses in the cremaster muscle and dLNs (16 hrs post-inflammation) was assessed by confocal microscopy. ( f ) Number of extravasated neutrophils in inflamed cremaster muscles of WT and CCR7KO mice. ( g ) Number of intravasated neutrophils in cremaster lymphatic vessels of WT and CCR7KO mice. ( h ) Neutrophil number in the cremaster dLNs of WT and CCR7KO mice. ( i ) Number of neutrophils recruited to the cremaster muscles from lethally irradiated WT animals receiving bone marrow transplant from either WT or CCR7KO donor mice. ( j ) Number of neutrophils within cremaster lymphatic vessels post-CFA+Ag-stimulation from lethally irradiated WT animals receiving bone marrow transplant from either WT or CCR7KO donor mice. ( k ) Neutrophil number in the cremaster dLNs from lethally irradiated WT animals receiving bone marrow transplant from either WT or CCR7KO donor mice. Data are expressed as mean ± SEM of N = 7–12 animals per group (from at least 5 independent experiments). Statistically significant differences between stimulated/specific mAb and unstimulated treatment/isotype control groups are indicated by asterisks: *P

    Article Snippet: The following primary antibodies were used for immunofluorescence labelling for confocal imaging and confocal IVM: rat anti–mouse LYVE-1 mAb (clone ALY7; eBioscience); non-blocking rat anti–mouse PECAM-1 Ab (clone C390, eBioscience); rat anti–mouse ICAM-1 mAb (clone YN1/1.4.7; purified and Alexa 488-conjugated, eBioscience); rat anti–mouse MAC-1 mAb (clone M1/70, BioLegend); monoclonal rat anti–mouse MRP14 mAb (clone 2B10; a gift from N. Hogg, Cancer Research UK, London, UK); rat anti-mouse Ly6G mAb (clone 1A8, Alexa 647-conjugated, Biolegend); rat anti-mouse CXCR4 mAb (clone 2B11, PE-conjugated, eBioscience), rat anti-mouse/human High Endothelial Venule mAb (MECA-79, Alexa 488-conjugated eBioscience), rat anti-mouse CCR7 mAb (clone 4B12, Alexa 488- or biotin-conjugated, eBioscience); rat anti-mouse CD45.2 mAb (clone 104, PE/Cy7-conjugated, Biolegend), rat anti-mouse TNFα mAb (clone MP6-XT22, Biolegend), anti-mouse CCL21, anti-mouse CXCL1 and anti-mouse CXCL12 mAbs (R & D systems), polyclonal goat anti–mouse TNFR p55 and p75 Abs (R & D Systems).

    Techniques: Migration, In Vivo, Flow Cytometry, Cytometry, Expressing, Isolation, Mouse Assay, Confocal Microscopy, Irradiation

    TNFα controls ICAM-1 expression on lymphatic endothelial cells in vivo . Cremaster muscles of WT mice were stimulated with TNFα or CFA+Ag (6–8 hrs) and immunostained with Alexa555-conjugated anti-LYVE-1 and Alexa488-conjugated anti-ICAM-1 (or an isotype control) mAbs to label the lymphatic vasculature and ICAM-1, respectively. ( a ) The pictures are representative confocal images of cremaster lymphatic vessels showing the expression of ICAM-1 on selected lymphatic vessels from a PBS-treated control (left panels), TNFα-stimulated (middle panels) and CFA+Ag-stimulated (right panels) animals. ( b ) ICAM-1 expression (mean fluorescent intensity or MFI) on vessels from PBS-treated control, TNFα-stimulated and CFA+Ag-stimulated cremaster muscles as quantified by IMARIS software. ( c ) ICAM-1 expression on lymphatic vessels of CFA+Ag-stimulated cremaster muscles from animals pre-treated with an anti- TNFα blocking mAb or isotype CTL mAb injected 4 hrs post-inflammation. Data are expressed as mean ± SEM of N = 8–12 vessels/animals from 4 animals per group (3 independent experiments). Statistically significant differences between the staining of isotype control and anti-ICAM-1 Abs treated groups are indicated by asterisks: *P

    Journal: Scientific Reports

    Article Title: Endogenous TNFα orchestrates the trafficking of neutrophils into and within lymphatic vessels during acute inflammation

    doi: 10.1038/srep44189

    Figure Lengend Snippet: TNFα controls ICAM-1 expression on lymphatic endothelial cells in vivo . Cremaster muscles of WT mice were stimulated with TNFα or CFA+Ag (6–8 hrs) and immunostained with Alexa555-conjugated anti-LYVE-1 and Alexa488-conjugated anti-ICAM-1 (or an isotype control) mAbs to label the lymphatic vasculature and ICAM-1, respectively. ( a ) The pictures are representative confocal images of cremaster lymphatic vessels showing the expression of ICAM-1 on selected lymphatic vessels from a PBS-treated control (left panels), TNFα-stimulated (middle panels) and CFA+Ag-stimulated (right panels) animals. ( b ) ICAM-1 expression (mean fluorescent intensity or MFI) on vessels from PBS-treated control, TNFα-stimulated and CFA+Ag-stimulated cremaster muscles as quantified by IMARIS software. ( c ) ICAM-1 expression on lymphatic vessels of CFA+Ag-stimulated cremaster muscles from animals pre-treated with an anti- TNFα blocking mAb or isotype CTL mAb injected 4 hrs post-inflammation. Data are expressed as mean ± SEM of N = 8–12 vessels/animals from 4 animals per group (3 independent experiments). Statistically significant differences between the staining of isotype control and anti-ICAM-1 Abs treated groups are indicated by asterisks: *P

    Article Snippet: The following primary antibodies were used for immunofluorescence labelling for confocal imaging and confocal IVM: rat anti–mouse LYVE-1 mAb (clone ALY7; eBioscience); non-blocking rat anti–mouse PECAM-1 Ab (clone C390, eBioscience); rat anti–mouse ICAM-1 mAb (clone YN1/1.4.7; purified and Alexa 488-conjugated, eBioscience); rat anti–mouse MAC-1 mAb (clone M1/70, BioLegend); monoclonal rat anti–mouse MRP14 mAb (clone 2B10; a gift from N. Hogg, Cancer Research UK, London, UK); rat anti-mouse Ly6G mAb (clone 1A8, Alexa 647-conjugated, Biolegend); rat anti-mouse CXCR4 mAb (clone 2B11, PE-conjugated, eBioscience), rat anti-mouse/human High Endothelial Venule mAb (MECA-79, Alexa 488-conjugated eBioscience), rat anti-mouse CCR7 mAb (clone 4B12, Alexa 488- or biotin-conjugated, eBioscience); rat anti-mouse CD45.2 mAb (clone 104, PE/Cy7-conjugated, Biolegend), rat anti-mouse TNFα mAb (clone MP6-XT22, Biolegend), anti-mouse CCL21, anti-mouse CXCL1 and anti-mouse CXCL12 mAbs (R & D systems), polyclonal goat anti–mouse TNFR p55 and p75 Abs (R & D Systems).

    Techniques: Expressing, In Vivo, Mouse Assay, Software, Blocking Assay, CTL Assay, Injection, Staining

    Dynamics of neutrophil migration into cremaster muscle lymphatics upon TNFα-stimulation. The dynamics of neutrophil migration into the tissue and lymphatic vessels was analysed by intravital confocal microscopy in TNFα-stimulated mouse cremaster muscles. ( a ) Representative 3D-reconstructed still image (2 μm cross-section) from a LysM-GFP × αSMA-CherryRFP mouse [exhibiting both endogenous GFP-fluorescent neutrophils (green) and RFP-fluorescent pericytes/smooth muscle cells (red) and immunostained with a non-blocking anti-PECAM-1 mAb (blue)] cremaster tissue showing a neutrophil within the lymphatic vessel (yellow arrow) post TNFα-stimulation. ( b ) Time-course of neutrophil extravasation in TNFα-stimulated cremaster muscles. ( c ) Time-course of neutrophil migration into lymphatic vessels upon TNFα-stimulation. ( d ) Total neutrophil-infiltrate in dLNs upon TNFα-stimulation. ( e ) Representative 3D-reconstructed still image of a post-capillary venule and an adjacent lymphatic vessel from a LysM-GFP mouse (immunostained with non-blocking anti-PECAM-1 mAb (blue)]. The right panel images illustrate a time-lapse series of 2 μm-thick cross-sections along the z-plane (dotted-yellow arrow) showing the migration of two neutrophils (Cell-1 Cell-2) into the lymphatic vessel. ( f ) Representative 3D-reconstructed still image of a lymphatic vessel from a TNFα-stimulated cremaster tissue of a LysM-GFP mouse and immunostained with an anti-LYVE-1 mAb (red) in vivo . Neutrophil crawling path (colour-coded line) and directionality (white arrow) is shown on the image. The bottom panel images are a series of high magnification cross-sections of the main image at indicated time-points illustrating the continuous attachment of the neutrophil to the lymphatic endothelium. ( g ) Percentage of neutrophils crawling in the afferent direction (flow) or in the opposite direction (anti-flow). Speed ( h ), directionality ( i ) and straightness ( j ) of neutrophils crawling in the afferent (flow) or opposite direction (anti-flow) of the cremaster lymphatic vessels. Data are expressed as mean ± SEM from 5–12 animals per group (at least 5 independent experiments). For the crawling parameter analysis, a total of 63 cells were quantified from 8 mice. Statistically significant differences between stimulated and unstimulated treatment groups are indicated by asterisks: *P

    Journal: Scientific Reports

    Article Title: Endogenous TNFα orchestrates the trafficking of neutrophils into and within lymphatic vessels during acute inflammation

    doi: 10.1038/srep44189

    Figure Lengend Snippet: Dynamics of neutrophil migration into cremaster muscle lymphatics upon TNFα-stimulation. The dynamics of neutrophil migration into the tissue and lymphatic vessels was analysed by intravital confocal microscopy in TNFα-stimulated mouse cremaster muscles. ( a ) Representative 3D-reconstructed still image (2 μm cross-section) from a LysM-GFP × αSMA-CherryRFP mouse [exhibiting both endogenous GFP-fluorescent neutrophils (green) and RFP-fluorescent pericytes/smooth muscle cells (red) and immunostained with a non-blocking anti-PECAM-1 mAb (blue)] cremaster tissue showing a neutrophil within the lymphatic vessel (yellow arrow) post TNFα-stimulation. ( b ) Time-course of neutrophil extravasation in TNFα-stimulated cremaster muscles. ( c ) Time-course of neutrophil migration into lymphatic vessels upon TNFα-stimulation. ( d ) Total neutrophil-infiltrate in dLNs upon TNFα-stimulation. ( e ) Representative 3D-reconstructed still image of a post-capillary venule and an adjacent lymphatic vessel from a LysM-GFP mouse (immunostained with non-blocking anti-PECAM-1 mAb (blue)]. The right panel images illustrate a time-lapse series of 2 μm-thick cross-sections along the z-plane (dotted-yellow arrow) showing the migration of two neutrophils (Cell-1 Cell-2) into the lymphatic vessel. ( f ) Representative 3D-reconstructed still image of a lymphatic vessel from a TNFα-stimulated cremaster tissue of a LysM-GFP mouse and immunostained with an anti-LYVE-1 mAb (red) in vivo . Neutrophil crawling path (colour-coded line) and directionality (white arrow) is shown on the image. The bottom panel images are a series of high magnification cross-sections of the main image at indicated time-points illustrating the continuous attachment of the neutrophil to the lymphatic endothelium. ( g ) Percentage of neutrophils crawling in the afferent direction (flow) or in the opposite direction (anti-flow). Speed ( h ), directionality ( i ) and straightness ( j ) of neutrophils crawling in the afferent (flow) or opposite direction (anti-flow) of the cremaster lymphatic vessels. Data are expressed as mean ± SEM from 5–12 animals per group (at least 5 independent experiments). For the crawling parameter analysis, a total of 63 cells were quantified from 8 mice. Statistically significant differences between stimulated and unstimulated treatment groups are indicated by asterisks: *P

    Article Snippet: The following primary antibodies were used for immunofluorescence labelling for confocal imaging and confocal IVM: rat anti–mouse LYVE-1 mAb (clone ALY7; eBioscience); non-blocking rat anti–mouse PECAM-1 Ab (clone C390, eBioscience); rat anti–mouse ICAM-1 mAb (clone YN1/1.4.7; purified and Alexa 488-conjugated, eBioscience); rat anti–mouse MAC-1 mAb (clone M1/70, BioLegend); monoclonal rat anti–mouse MRP14 mAb (clone 2B10; a gift from N. Hogg, Cancer Research UK, London, UK); rat anti-mouse Ly6G mAb (clone 1A8, Alexa 647-conjugated, Biolegend); rat anti-mouse CXCR4 mAb (clone 2B11, PE-conjugated, eBioscience), rat anti-mouse/human High Endothelial Venule mAb (MECA-79, Alexa 488-conjugated eBioscience), rat anti-mouse CCR7 mAb (clone 4B12, Alexa 488- or biotin-conjugated, eBioscience); rat anti-mouse CD45.2 mAb (clone 104, PE/Cy7-conjugated, Biolegend), rat anti-mouse TNFα mAb (clone MP6-XT22, Biolegend), anti-mouse CCL21, anti-mouse CXCL1 and anti-mouse CXCL12 mAbs (R & D systems), polyclonal goat anti–mouse TNFR p55 and p75 Abs (R & D Systems).

    Techniques: Migration, Confocal Microscopy, Blocking Assay, In Vivo, Flow Cytometry, Mouse Assay

    P2X 1 deficiency and P2X 1 inhibition in E. coli -induced sepsis . ARD6 was injected into anesthetized P 2 X 1 + / + and P 2 X 1 − / − mice at a concentration of either low (61 million) or high (248 million) number of bacteria. (A) Kaplan-Meier plot shows survival (high dose), n = 6 for control and n = 12 for both genotypes. (B) Survival in the presence or absence of the P2X 1 antagonist NF449 (100 mg kg −1 , iv ) in balb/c mice. The mice were exposed to the high concentration of bacteria, n = 6 for control with or without NF449 and n = 10–12 for ARD6 with or without NF449. (C) Absorbance of plasma as an indication of plasma haemolysis after the low dose, n = 8 for both genotypes (D) Levels of thrombin-antithrombin (TAT) complexes in plasma 2.5 h after the low dose, n = 8 for both genotypes. (E) Levels of TNFα, KC, IL-1β, and IL-6 2.5 h after the low dose of ARD6, n = 13 both genotypes. * p

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: P2X1, P2X4, and P2X7 Receptor Knock Out Mice Expose Differential Outcome of Sepsis Induced by α-Haemolysin Producing Escherichia coli

    doi: 10.3389/fcimb.2017.00113

    Figure Lengend Snippet: P2X 1 deficiency and P2X 1 inhibition in E. coli -induced sepsis . ARD6 was injected into anesthetized P 2 X 1 + / + and P 2 X 1 − / − mice at a concentration of either low (61 million) or high (248 million) number of bacteria. (A) Kaplan-Meier plot shows survival (high dose), n = 6 for control and n = 12 for both genotypes. (B) Survival in the presence or absence of the P2X 1 antagonist NF449 (100 mg kg −1 , iv ) in balb/c mice. The mice were exposed to the high concentration of bacteria, n = 6 for control with or without NF449 and n = 10–12 for ARD6 with or without NF449. (C) Absorbance of plasma as an indication of plasma haemolysis after the low dose, n = 8 for both genotypes (D) Levels of thrombin-antithrombin (TAT) complexes in plasma 2.5 h after the low dose, n = 8 for both genotypes. (E) Levels of TNFα, KC, IL-1β, and IL-6 2.5 h after the low dose of ARD6, n = 13 both genotypes. * p

    Article Snippet: Purified murine TNFα was purchased from BioLegend (Uithoorn, Netherlands).

    Techniques: Inhibition, Injection, Mouse Assay, Concentration Assay

    Casp8 and RIPK3 deficiency in E. coli -induced sepsis and TNFα-induced shock . ARD6 was injected into anesthetized wild type and casp8/RIPK3 DKO mice at a low (61 million) or high concentration (248 million). (A) Kaplan-Meier plot shows survival after the high dose, n = 6 for wild type and 8 for casp8/RIPK3 DKO . (B) Levels of TNFα, KC, IL-1β and IL-6 2.5 h after the low dose of ARD6, n = 7 for wild type and 6 for casp8/RIPK3 DKO . (C) Kaplan-Meier plot shows survival after TNFα shock (25 mg kg −1 ) was induced in wildtype, RIPK3 deficient and casp8/RIPK3 DKO , n = 8 in all three groups. * p

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: P2X1, P2X4, and P2X7 Receptor Knock Out Mice Expose Differential Outcome of Sepsis Induced by α-Haemolysin Producing Escherichia coli

    doi: 10.3389/fcimb.2017.00113

    Figure Lengend Snippet: Casp8 and RIPK3 deficiency in E. coli -induced sepsis and TNFα-induced shock . ARD6 was injected into anesthetized wild type and casp8/RIPK3 DKO mice at a low (61 million) or high concentration (248 million). (A) Kaplan-Meier plot shows survival after the high dose, n = 6 for wild type and 8 for casp8/RIPK3 DKO . (B) Levels of TNFα, KC, IL-1β and IL-6 2.5 h after the low dose of ARD6, n = 7 for wild type and 6 for casp8/RIPK3 DKO . (C) Kaplan-Meier plot shows survival after TNFα shock (25 mg kg −1 ) was induced in wildtype, RIPK3 deficient and casp8/RIPK3 DKO , n = 8 in all three groups. * p

    Article Snippet: Purified murine TNFα was purchased from BioLegend (Uithoorn, Netherlands).

    Techniques: Injection, Mouse Assay, Concentration Assay

    P2X 7 deficiency and E. coli -induced sepsis . ARD6 was injected into anesthetized P 2 X 7 + / + and P 2 X 7 − / − mice (low dose—41 million). (A) plasma TNFα, KC, IL-1β, and IL-6 measured 2.5 h after ARD6 injection, n = 7–8 for controls and 8–9 for each genotype. (B) Plasma thrombin-antithrombin (TAT) complexes—measured 2.5 h after ARD6 injection, n = 6–7 for controls and 10–11 for both genotypes. * p

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: P2X1, P2X4, and P2X7 Receptor Knock Out Mice Expose Differential Outcome of Sepsis Induced by α-Haemolysin Producing Escherichia coli

    doi: 10.3389/fcimb.2017.00113

    Figure Lengend Snippet: P2X 7 deficiency and E. coli -induced sepsis . ARD6 was injected into anesthetized P 2 X 7 + / + and P 2 X 7 − / − mice (low dose—41 million). (A) plasma TNFα, KC, IL-1β, and IL-6 measured 2.5 h after ARD6 injection, n = 7–8 for controls and 8–9 for each genotype. (B) Plasma thrombin-antithrombin (TAT) complexes—measured 2.5 h after ARD6 injection, n = 6–7 for controls and 10–11 for both genotypes. * p

    Article Snippet: Purified murine TNFα was purchased from BioLegend (Uithoorn, Netherlands).

    Techniques: Injection, Mouse Assay

    The effect of P2X 7 receptor inhibition in E. coli -induced sepsis . ARD6 was injected iv into anesthetized balb/cj mice (low dose—41 million). Two hours prior to ARD6 injection the animals were administered the P2X 7 antagonist BBG or vehicle (50 mg kg −1 , subcutaneously). (A) Kaplan-Meier plot shows survival, n = 6 for controls and 10–12 for ARD6 with or without BBG. (B) Absorbance of plasma as an indication of plasma haemolysis 2.5 h after ARD6 injection, n = 13 for all four groups. (C) Colony forming units from blood samples 2.5 h after a low number of bacteria (D) Levels of the cytokines TNFα, KC, IL-1β, and IL-6 2.5 h after ARD6 injection, n = 14 for ARD6 and 13 for ARD6 + BBG. * p

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: P2X1, P2X4, and P2X7 Receptor Knock Out Mice Expose Differential Outcome of Sepsis Induced by α-Haemolysin Producing Escherichia coli

    doi: 10.3389/fcimb.2017.00113

    Figure Lengend Snippet: The effect of P2X 7 receptor inhibition in E. coli -induced sepsis . ARD6 was injected iv into anesthetized balb/cj mice (low dose—41 million). Two hours prior to ARD6 injection the animals were administered the P2X 7 antagonist BBG or vehicle (50 mg kg −1 , subcutaneously). (A) Kaplan-Meier plot shows survival, n = 6 for controls and 10–12 for ARD6 with or without BBG. (B) Absorbance of plasma as an indication of plasma haemolysis 2.5 h after ARD6 injection, n = 13 for all four groups. (C) Colony forming units from blood samples 2.5 h after a low number of bacteria (D) Levels of the cytokines TNFα, KC, IL-1β, and IL-6 2.5 h after ARD6 injection, n = 14 for ARD6 and 13 for ARD6 + BBG. * p

    Article Snippet: Purified murine TNFα was purchased from BioLegend (Uithoorn, Netherlands).

    Techniques: Inhibition, Injection, Mouse Assay

    P2X 4 deficiency in E. coli -induced sepsis . ARD6 was injected into anesthetized P 2 X 4 + / + and P 2 X 4 − / − mice at a concentration of either low (61 million) or high (248 million) bacteria. (A) Kaplan-Meier plot shows survival after the high dose, n = 5 for each genotypes (B) Level of haemolysis after the low dose, n = 8 for P 2 X 4 + / + and 6 for P 2 X 4 − / − . (C) Levels of thrombin-antithrombin (TAT) complexes in plasma 2.5 h after the low dose of bacteria, n = 8 for each genotype. (D) Levels of TNFα, KC, IL-1β, and IL-6 2.5 h after the low dose of ARD6, n = 11 both genotypes. * p

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: P2X1, P2X4, and P2X7 Receptor Knock Out Mice Expose Differential Outcome of Sepsis Induced by α-Haemolysin Producing Escherichia coli

    doi: 10.3389/fcimb.2017.00113

    Figure Lengend Snippet: P2X 4 deficiency in E. coli -induced sepsis . ARD6 was injected into anesthetized P 2 X 4 + / + and P 2 X 4 − / − mice at a concentration of either low (61 million) or high (248 million) bacteria. (A) Kaplan-Meier plot shows survival after the high dose, n = 5 for each genotypes (B) Level of haemolysis after the low dose, n = 8 for P 2 X 4 + / + and 6 for P 2 X 4 − / − . (C) Levels of thrombin-antithrombin (TAT) complexes in plasma 2.5 h after the low dose of bacteria, n = 8 for each genotype. (D) Levels of TNFα, KC, IL-1β, and IL-6 2.5 h after the low dose of ARD6, n = 11 both genotypes. * p

    Article Snippet: Purified murine TNFα was purchased from BioLegend (Uithoorn, Netherlands).

    Techniques: Injection, Mouse Assay, Concentration Assay

    BV6 does not induce NF-κB nuclear translocation SW620 cells were either untreated (a), treated with TNFα (b, 100 U/ml), BV6 (c, 5 μM) or both TNFα and BV6 (d) for 60 min. Cells were fixed, permeabilized, and stained with p65-specific antibody, followed by fluorescent dye-conjugated 2 nd antibody. The images were obtained with a confocal microscope. a1-d1 are low amplification images and a2-d2 are high amplification images.

    Journal: Oncotarget

    Article Title: NF-κB functions as a molecular link between tumor cells and Th1/Tc1 T cells in the tumor microenvironment to exert radiation-mediated tumor suppression

    doi: 10.18632/oncotarget.8246

    Figure Lengend Snippet: BV6 does not induce NF-κB nuclear translocation SW620 cells were either untreated (a), treated with TNFα (b, 100 U/ml), BV6 (c, 5 μM) or both TNFα and BV6 (d) for 60 min. Cells were fixed, permeabilized, and stained with p65-specific antibody, followed by fluorescent dye-conjugated 2 nd antibody. The images were obtained with a confocal microscope. a1-d1 are low amplification images and a2-d2 are high amplification images.

    Article Snippet: Tumor cells were irradiated and then cultured in the presence of BV6 plus either IgG (20 μg/ml) or anti-TNFα mAb (Biolegend, 20 μg/ml) for 24h.

    Techniques: Translocation Assay, Staining, Microscopy, Amplification

    Radiation-induced NF-κB directly regulates TNFα transcription that acts in concert with BV6 to induce tumor cell death A. The human TNFα promoter structure. The three NF-κB-binding consensus sequence elements are indicated at the bottom of the bar. B. Tumor cells were irradiated at a dose of 50 Gy and then cultured for 90 min. Nuclear extracts were prepared and analyzed for NF-κB activation using EMSA with the three DNA probes of the human TNFα promoter as indicated in A. C. Tumor cells were irradiated with the indicated dose and cultured for 24h. Cells were then analyzed for human TNFα mRNA level using quantitative PCR. D. Tumor cells were treated as indicated for 24h and analyzed for cell death using PI staining and flow cytometry analysis. E. Tumor cells were irradiated and then cultured in the presence of BV6 (5 μM). IgG or anti-TNFα neutralization antibody (200 μg/ml) was added to the culture, respectively, for 24h. Cell death was analyzed by PI staining and flow cytometry analysis. * p

    Journal: Oncotarget

    Article Title: NF-κB functions as a molecular link between tumor cells and Th1/Tc1 T cells in the tumor microenvironment to exert radiation-mediated tumor suppression

    doi: 10.18632/oncotarget.8246

    Figure Lengend Snippet: Radiation-induced NF-κB directly regulates TNFα transcription that acts in concert with BV6 to induce tumor cell death A. The human TNFα promoter structure. The three NF-κB-binding consensus sequence elements are indicated at the bottom of the bar. B. Tumor cells were irradiated at a dose of 50 Gy and then cultured for 90 min. Nuclear extracts were prepared and analyzed for NF-κB activation using EMSA with the three DNA probes of the human TNFα promoter as indicated in A. C. Tumor cells were irradiated with the indicated dose and cultured for 24h. Cells were then analyzed for human TNFα mRNA level using quantitative PCR. D. Tumor cells were treated as indicated for 24h and analyzed for cell death using PI staining and flow cytometry analysis. E. Tumor cells were irradiated and then cultured in the presence of BV6 (5 μM). IgG or anti-TNFα neutralization antibody (200 μg/ml) was added to the culture, respectively, for 24h. Cell death was analyzed by PI staining and flow cytometry analysis. * p

    Article Snippet: Tumor cells were irradiated and then cultured in the presence of BV6 plus either IgG (20 μg/ml) or anti-TNFα mAb (Biolegend, 20 μg/ml) for 24h.

    Techniques: Binding Assay, Sequencing, Irradiation, Cell Culture, Activation Assay, Real-time Polymerase Chain Reaction, Staining, Flow Cytometry, Cytometry, Neutralization

    Radiation and TNFα induce apoptosis and necroptosis A. Tumor cells were irradiated (R) at a dose of 50 Gy and then cultured in the presence of BV6 (B, 5 μM) plus Z-VAD (Z, 10 μM), Nec-1 (10 μM), and NSA (2 μM), either alone or in combinations, for 24 hours. Cell death was determined by PI staining and flow cytometry analysis. Column: mean, bar: SD. B. Tumor cells were cultured in the presence of TNFα (T, 100 U/ml) and BV6 (B, 5 μM) plus Z-VAD (Z, 10 μM), Nec-1 (10 μM), and NSA (2 μM), either alone or in combinations, for 24h, and analyzed for cell death as in A. Column: mean; Bar: SD.

    Journal: Oncotarget

    Article Title: NF-κB functions as a molecular link between tumor cells and Th1/Tc1 T cells in the tumor microenvironment to exert radiation-mediated tumor suppression

    doi: 10.18632/oncotarget.8246

    Figure Lengend Snippet: Radiation and TNFα induce apoptosis and necroptosis A. Tumor cells were irradiated (R) at a dose of 50 Gy and then cultured in the presence of BV6 (B, 5 μM) plus Z-VAD (Z, 10 μM), Nec-1 (10 μM), and NSA (2 μM), either alone or in combinations, for 24 hours. Cell death was determined by PI staining and flow cytometry analysis. Column: mean, bar: SD. B. Tumor cells were cultured in the presence of TNFα (T, 100 U/ml) and BV6 (B, 5 μM) plus Z-VAD (Z, 10 μM), Nec-1 (10 μM), and NSA (2 μM), either alone or in combinations, for 24h, and analyzed for cell death as in A. Column: mean; Bar: SD.

    Article Snippet: Tumor cells were irradiated and then cultured in the presence of BV6 plus either IgG (20 μg/ml) or anti-TNFα mAb (Biolegend, 20 μg/ml) for 24h.

    Techniques: Irradiation, Cell Culture, Staining, Flow Cytometry, Cytometry

    BV6 activates the alternate but not the canonical NF-κB A. The indicated human sarcoma cells were treated with BV6 (5 μM) for the indicated time. Cells were then analyzed by Western blotting analysis for p100/p52, pIκBα, and IκBα. B. The indicated human colon carcinoma cells were treated with BV6 (5 μM) for the indicated time and then analyzed for p100/p52, pIκBα, and IκBα as in A. C. The indicated human sarcoma cells were treated with BV6 (5μM, 1 and 2h), TNFα (100 U/ml, 1h), or both as indicated. Nuclear extracts were prepared from the tumor cells and analyzed for canonical NF-κB activity using EMSA with NF-κB consensus sequence-containing DNA probes (Santa Cruz Biotech). Anti-p65 and anti-p50 antibodies were used to identify the canonical NF-κB-DNA complexes. D. SW620 cells were treated with BV6 (5 μM, for 0.5, 1, and 2h) and TNFα (1 h) and analyzed for NF-κB activation as in C.

    Journal: Oncotarget

    Article Title: NF-κB functions as a molecular link between tumor cells and Th1/Tc1 T cells in the tumor microenvironment to exert radiation-mediated tumor suppression

    doi: 10.18632/oncotarget.8246

    Figure Lengend Snippet: BV6 activates the alternate but not the canonical NF-κB A. The indicated human sarcoma cells were treated with BV6 (5 μM) for the indicated time. Cells were then analyzed by Western blotting analysis for p100/p52, pIκBα, and IκBα. B. The indicated human colon carcinoma cells were treated with BV6 (5 μM) for the indicated time and then analyzed for p100/p52, pIκBα, and IκBα as in A. C. The indicated human sarcoma cells were treated with BV6 (5μM, 1 and 2h), TNFα (100 U/ml, 1h), or both as indicated. Nuclear extracts were prepared from the tumor cells and analyzed for canonical NF-κB activity using EMSA with NF-κB consensus sequence-containing DNA probes (Santa Cruz Biotech). Anti-p65 and anti-p50 antibodies were used to identify the canonical NF-κB-DNA complexes. D. SW620 cells were treated with BV6 (5 μM, for 0.5, 1, and 2h) and TNFα (1 h) and analyzed for NF-κB activation as in C.

    Article Snippet: Tumor cells were irradiated and then cultured in the presence of BV6 plus either IgG (20 μg/ml) or anti-TNFα mAb (Biolegend, 20 μg/ml) for 24h.

    Techniques: Western Blot, Activity Assay, Sequencing, Activation Assay

    Model of radiation modulation of NF-κB and CTL anti-tumor immune response in the tumor microenvironment A. Radiation initiates at least four molecular effects: 1) radiation activates the canonical NF-κB p65/p50 and p50/p50 complexes that directly bind to the TNFα promoter to activate its transcription; 2) radiation-activated canonical NF-κB p65/p50 and p50/p50 complexes also directly bind to the FAS promoter to activate its transcription to increase tumor cell sensitivity to apoptosis induction by FasL of tumor-infiltrating Tc1 cells; 3) radiation-activated NF-κB also up-regulates the T cell chemokines CCL2 and CCL5 to attract T cell infiltration to the tumor microenvironment; and 4) radiation-generated dsDNA fragments activate the STING pathway, as characterized by increased IRF3 and IFNβ expression, to induce T cell activation. B. TNFα in the tumor microenvironment induces tumor apoptosis and necroptosis in an autocrine manner, and RIP1 is a key molecular switch that determines whether TNFα induces cell death or survival signaling pathways. cIAP1 and cIAP2 ubiquitylates RIP1 in the TNFR cytoplasmic complex (complex I) and the ubiquitylated RIP1 promotes cell survival by preventing the formation of the death complex IIb. BV6 induces cIAP1 and cIAP2 degradation. Degradation of cIAP1 and cIAP2 leads to activation of caspase 9. Degradation of cIAP1 and cIAP2 also leads to deubiquitylation of RIP1. Deubiquitylated RIP1 dissociates from the membrane-bound TNFR complex I to form cytosolic complex IIb that mediates both caspase-dependent apoptosis and RIP3-dependent necroptosis.

    Journal: Oncotarget

    Article Title: NF-κB functions as a molecular link between tumor cells and Th1/Tc1 T cells in the tumor microenvironment to exert radiation-mediated tumor suppression

    doi: 10.18632/oncotarget.8246

    Figure Lengend Snippet: Model of radiation modulation of NF-κB and CTL anti-tumor immune response in the tumor microenvironment A. Radiation initiates at least four molecular effects: 1) radiation activates the canonical NF-κB p65/p50 and p50/p50 complexes that directly bind to the TNFα promoter to activate its transcription; 2) radiation-activated canonical NF-κB p65/p50 and p50/p50 complexes also directly bind to the FAS promoter to activate its transcription to increase tumor cell sensitivity to apoptosis induction by FasL of tumor-infiltrating Tc1 cells; 3) radiation-activated NF-κB also up-regulates the T cell chemokines CCL2 and CCL5 to attract T cell infiltration to the tumor microenvironment; and 4) radiation-generated dsDNA fragments activate the STING pathway, as characterized by increased IRF3 and IFNβ expression, to induce T cell activation. B. TNFα in the tumor microenvironment induces tumor apoptosis and necroptosis in an autocrine manner, and RIP1 is a key molecular switch that determines whether TNFα induces cell death or survival signaling pathways. cIAP1 and cIAP2 ubiquitylates RIP1 in the TNFR cytoplasmic complex (complex I) and the ubiquitylated RIP1 promotes cell survival by preventing the formation of the death complex IIb. BV6 induces cIAP1 and cIAP2 degradation. Degradation of cIAP1 and cIAP2 leads to activation of caspase 9. Degradation of cIAP1 and cIAP2 also leads to deubiquitylation of RIP1. Deubiquitylated RIP1 dissociates from the membrane-bound TNFR complex I to form cytosolic complex IIb that mediates both caspase-dependent apoptosis and RIP3-dependent necroptosis.

    Article Snippet: Tumor cells were irradiated and then cultured in the presence of BV6 plus either IgG (20 μg/ml) or anti-TNFα mAb (Biolegend, 20 μg/ml) for 24h.

    Techniques: CTL Assay, Generated, Expressing, Activation Assay

    TNFα and BV6 cooperate to activate sequential necroptosis and apoptosis A. Tumor cells were cultured in the presence of TNFα (100 U/ml), BV6 (5 μM), or both for the indicated time and analyzed by Western blotting analysis for the indicated caspases. PARP was used as an apoptosis indicator. B. Tumor cells were cultured in the presence of TNFα (100 U/ml), BV6 (5 μM), plus Z-VAD (10 μM), Nec-1 (10 μM), or NSA (2 μM), respectively for 4 h, and analyzed by Western blotting for the indicated caspases and PARP.

    Journal: Oncotarget

    Article Title: NF-κB functions as a molecular link between tumor cells and Th1/Tc1 T cells in the tumor microenvironment to exert radiation-mediated tumor suppression

    doi: 10.18632/oncotarget.8246

    Figure Lengend Snippet: TNFα and BV6 cooperate to activate sequential necroptosis and apoptosis A. Tumor cells were cultured in the presence of TNFα (100 U/ml), BV6 (5 μM), or both for the indicated time and analyzed by Western blotting analysis for the indicated caspases. PARP was used as an apoptosis indicator. B. Tumor cells were cultured in the presence of TNFα (100 U/ml), BV6 (5 μM), plus Z-VAD (10 μM), Nec-1 (10 μM), or NSA (2 μM), respectively for 4 h, and analyzed by Western blotting for the indicated caspases and PARP.

    Article Snippet: Tumor cells were irradiated and then cultured in the presence of BV6 plus either IgG (20 μg/ml) or anti-TNFα mAb (Biolegend, 20 μg/ml) for 24h.

    Techniques: Cell Culture, Western Blot