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In the absence of IFNγ frequencies and numbers of IL17A producing colonic CD4 T cells are dramatically increased in colitic mice. Cytokine expression analysis of colonic lamina propria (LP) CD4 T cells, PMA/ionomycin-stimulated in the presence or absence of IFNγ. Colonic LP cells were isolated during active colitis (24–26 days post transfer of colitogenic Ifng +/+ , and Ifng −/− CD4 CD45RB hi T cells into Rag1 −/− , and Rag1 −/− Ifng −/− recipient mice, respectively). (A,B) Representative FACS blots showing IFNγ, IL17A, or <t>TNFα</t> expression of CD4 T cells. (C) Relative cell frequencies and (D) absolute cell numbers of cytokine expressing CD4 T cells. Bars indicate mean ± SD from three independent experiments with n = 5–9 mice per group. P -value was determined using the two-tailed Mann–Whitney test with * P
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1) Product Images from "Divergent Roles of Interferon-γ and Innate Lymphoid Cells in Innate and Adaptive Immune Cell-Mediated Intestinal Inflammation"

Article Title: Divergent Roles of Interferon-γ and Innate Lymphoid Cells in Innate and Adaptive Immune Cell-Mediated Intestinal Inflammation

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00023

In the absence of IFNγ frequencies and numbers of IL17A producing colonic CD4 T cells are dramatically increased in colitic mice. Cytokine expression analysis of colonic lamina propria (LP) CD4 T cells, PMA/ionomycin-stimulated in the presence or absence of IFNγ. Colonic LP cells were isolated during active colitis (24–26 days post transfer of colitogenic Ifng +/+ , and Ifng −/− CD4 CD45RB hi T cells into Rag1 −/− , and Rag1 −/− Ifng −/− recipient mice, respectively). (A,B) Representative FACS blots showing IFNγ, IL17A, or TNFα expression of CD4 T cells. (C) Relative cell frequencies and (D) absolute cell numbers of cytokine expressing CD4 T cells. Bars indicate mean ± SD from three independent experiments with n = 5–9 mice per group. P -value was determined using the two-tailed Mann–Whitney test with * P
Figure Legend Snippet: In the absence of IFNγ frequencies and numbers of IL17A producing colonic CD4 T cells are dramatically increased in colitic mice. Cytokine expression analysis of colonic lamina propria (LP) CD4 T cells, PMA/ionomycin-stimulated in the presence or absence of IFNγ. Colonic LP cells were isolated during active colitis (24–26 days post transfer of colitogenic Ifng +/+ , and Ifng −/− CD4 CD45RB hi T cells into Rag1 −/− , and Rag1 −/− Ifng −/− recipient mice, respectively). (A,B) Representative FACS blots showing IFNγ, IL17A, or TNFα expression of CD4 T cells. (C) Relative cell frequencies and (D) absolute cell numbers of cytokine expressing CD4 T cells. Bars indicate mean ± SD from three independent experiments with n = 5–9 mice per group. P -value was determined using the two-tailed Mann–Whitney test with * P

Techniques Used: Mouse Assay, Expressing, Isolation, FACS, Two Tailed Test, MANN-WHITNEY

Neutralization of IL17A and IL 17F attenuates exacerbating CD4 T cell-induced colitis in the absence of IFNγ. Rag1 −/− Ifng −/− mice were transferred with Ifng −/− CD4 CD45RB hi T cells and treated with anti-IL17A and anti-IL17F neutralizing antibodies, or isotype control (250 μg/mouse) twice a week. (A) Body weight during colitis induction and (B) histopathological scores. (C) Representative hematoxylin and eosin staining of colonic tissue sections from mice during active phase of colitis. Scale bars indicate 100 µm. (D) Serum level for TNFα, IL6, and IL1β. Cells were isolated from colitic mice from the colonic lamina propria (col LP), mesenteric lymph nodes (MLN), caudal lymph node (CLN) and spleen, restimulated with PMA/ionomycin for subsequent intracellular FACS analysis. Relative cell frequencies of (E) CD4 T cells and (F) cytokine expressing CD4 T cells are shown in experimental mice treated, with, and without, anti-IL17A/IL17F mAb’s. CD4 T cells were identified as single, live, autofluorescent negative, CD45 leukocytes, positive for CD4 and CD3. P -values were determined using the two-tailed Mann–Whitney test with * P
Figure Legend Snippet: Neutralization of IL17A and IL 17F attenuates exacerbating CD4 T cell-induced colitis in the absence of IFNγ. Rag1 −/− Ifng −/− mice were transferred with Ifng −/− CD4 CD45RB hi T cells and treated with anti-IL17A and anti-IL17F neutralizing antibodies, or isotype control (250 μg/mouse) twice a week. (A) Body weight during colitis induction and (B) histopathological scores. (C) Representative hematoxylin and eosin staining of colonic tissue sections from mice during active phase of colitis. Scale bars indicate 100 µm. (D) Serum level for TNFα, IL6, and IL1β. Cells were isolated from colitic mice from the colonic lamina propria (col LP), mesenteric lymph nodes (MLN), caudal lymph node (CLN) and spleen, restimulated with PMA/ionomycin for subsequent intracellular FACS analysis. Relative cell frequencies of (E) CD4 T cells and (F) cytokine expressing CD4 T cells are shown in experimental mice treated, with, and without, anti-IL17A/IL17F mAb’s. CD4 T cells were identified as single, live, autofluorescent negative, CD45 leukocytes, positive for CD4 and CD3. P -values were determined using the two-tailed Mann–Whitney test with * P

Techniques Used: Neutralization, Mouse Assay, Staining, Isolation, FACS, Expressing, Two Tailed Test, MANN-WHITNEY

2) Product Images from "Stearic acid induces CD11c expression in proinflammatory macrophages via epidermal fatty acid binding protein"

Article Title: Stearic acid induces CD11c expression in proinflammatory macrophages via epidermal fatty acid binding protein

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

doi: 10.4049/jimmunol.1701416

E-FABP deficiency protects high saturated fat-induced skin lesions (A) Weight of WT mice and E-FABP−/− mice before and after cocoa butter diet (45% fat) (n=16 for WT mice, and n=15/E-FABP−/− mice) and safflower diet (45% fat) (n=10/WT mice and n=15/E-FABP−/−) for 6 months. ( B ) The incidence of skin lesions in WT and E-FABP−/− mice fed with cocoa butter diet or safflower oil diet for 9 months. (C-D) Confocal analysis of CD11c+ macrophage infiltration (green color) in the skin tissue of WT and E-FABP−/− mice fed with cocoa butter for 9 months. Relative CD11c fluorescent intensity is shown in panel D . (E-G) Flow cytometric analysis of CD80 ( E ), CD86 ( F ) and MHCII ( G ) expression on CD11c+ macrophages in the skin of WT and E-FABP−/− mice fed with the cocoa butter diet for 9 months. (H-I) Measurement of serum levels of IL-6 ( H ), IL-1β ( I ) and TNFα ( J .
Figure Legend Snippet: E-FABP deficiency protects high saturated fat-induced skin lesions (A) Weight of WT mice and E-FABP−/− mice before and after cocoa butter diet (45% fat) (n=16 for WT mice, and n=15/E-FABP−/− mice) and safflower diet (45% fat) (n=10/WT mice and n=15/E-FABP−/−) for 6 months. ( B ) The incidence of skin lesions in WT and E-FABP−/− mice fed with cocoa butter diet or safflower oil diet for 9 months. (C-D) Confocal analysis of CD11c+ macrophage infiltration (green color) in the skin tissue of WT and E-FABP−/− mice fed with cocoa butter for 9 months. Relative CD11c fluorescent intensity is shown in panel D . (E-G) Flow cytometric analysis of CD80 ( E ), CD86 ( F ) and MHCII ( G ) expression on CD11c+ macrophages in the skin of WT and E-FABP−/− mice fed with the cocoa butter diet for 9 months. (H-I) Measurement of serum levels of IL-6 ( H ), IL-1β ( I ) and TNFα ( J .

Techniques Used: Mouse Assay, Flow Cytometry, Expressing

SA promotes expression of costimulatory molecules and production of inflammatory cytokines in M-CSF-induced bone marrow cells (A-B) Flow cytometric analyses of CD11c expression on bone marrow cells stimulated with M-CSF (20ng/ml) and indicated concentrations of SA for 48h. Average percentage of CD11c+ cells was shown in Panel B . (C-D) Realtime PCR analysis of mRNA levels of CD11c ( C ) and CD11b ( D ) in bone marrow cells after stimulation with M-CSF and SA for 48h. (E-H) Flow cytometric analysis of the mean fluorescent intensity (MFI) of MHCII ( E ), CD86 ( F ), CD80 ( G ), and CD54 ( H ) on bone marrow cells after stimulation with M-CSF and indicated SA for 48h. (J-O) Realtime PCR analysis of mRNA levels of inflammatory cytokines, including IL-6 ( J ), TNFα ( K ), IL-1β ( L ), IL-10 ( M ), IFNα ( N ) and IFNβ ( O ) in M-CSF-stimulated bone marrow cells in the presence or absence SA treatment (100μM) for 48h. (P-S) Measurement of protein levels of IL-6 ( P ), TNFα ( Q ) IL-1β ( R ) and IL-10 ( S ) in the supernatants of M-CSF-differentiated macrophages in the presence or absence SA treatment (100μM) for 48h. Data are shown as mean ± SEM (* p
Figure Legend Snippet: SA promotes expression of costimulatory molecules and production of inflammatory cytokines in M-CSF-induced bone marrow cells (A-B) Flow cytometric analyses of CD11c expression on bone marrow cells stimulated with M-CSF (20ng/ml) and indicated concentrations of SA for 48h. Average percentage of CD11c+ cells was shown in Panel B . (C-D) Realtime PCR analysis of mRNA levels of CD11c ( C ) and CD11b ( D ) in bone marrow cells after stimulation with M-CSF and SA for 48h. (E-H) Flow cytometric analysis of the mean fluorescent intensity (MFI) of MHCII ( E ), CD86 ( F ), CD80 ( G ), and CD54 ( H ) on bone marrow cells after stimulation with M-CSF and indicated SA for 48h. (J-O) Realtime PCR analysis of mRNA levels of inflammatory cytokines, including IL-6 ( J ), TNFα ( K ), IL-1β ( L ), IL-10 ( M ), IFNα ( N ) and IFNβ ( O ) in M-CSF-stimulated bone marrow cells in the presence or absence SA treatment (100μM) for 48h. (P-S) Measurement of protein levels of IL-6 ( P ), TNFα ( Q ) IL-1β ( R ) and IL-10 ( S ) in the supernatants of M-CSF-differentiated macrophages in the presence or absence SA treatment (100μM) for 48h. Data are shown as mean ± SEM (* p

Techniques Used: Expressing, Flow Cytometry, Polymerase Chain Reaction

E-FABP deficiency reduces CD11c+ macrophages in obese mice WT and E-FABP−/− mice were fed on HFD (60% fat) for 20 weeks. Different tissues or organs were collected, respectively from the obese WT and E-FABP−/− mice (n=5) for analysis of the presence of CD11b+CD11c+ macrophages. ( A-D ) Flow cytometric surface staining for analysis of CD11b+CD11c+ macrophages in bone marrow ( A ), liver ( B ), lung ( C ) and skin ( D ). Average percentage of CD11c+ cells is shown in the right panel. (E-G) Measurement of cytokine levels of IL-6 ( E ), IL-1β ( F ) and TNFα ( G .
Figure Legend Snippet: E-FABP deficiency reduces CD11c+ macrophages in obese mice WT and E-FABP−/− mice were fed on HFD (60% fat) for 20 weeks. Different tissues or organs were collected, respectively from the obese WT and E-FABP−/− mice (n=5) for analysis of the presence of CD11b+CD11c+ macrophages. ( A-D ) Flow cytometric surface staining for analysis of CD11b+CD11c+ macrophages in bone marrow ( A ), liver ( B ), lung ( C ) and skin ( D ). Average percentage of CD11c+ cells is shown in the right panel. (E-G) Measurement of cytokine levels of IL-6 ( E ), IL-1β ( F ) and TNFα ( G .

Techniques Used: Mouse Assay, Flow Cytometry, Staining

3) Product Images from "Inflammation-associated upregulation of the sulfated steroid transporter Slc10a6 in mouse liver and macrophage cell lines"

Article Title: Inflammation-associated upregulation of the sulfated steroid transporter Slc10a6 in mouse liver and macrophage cell lines

Journal: Hepatology research : the official journal of the Japan Society of Hepatology

doi: 10.1111/hepr.12609

LPS-induction of Slc10a6mRNA expression by in the mouse macrophage cell line RAW264.7 A) Time dependent LPS (1 μg/mL) induction of Slc10a6 mRNA and TNFα in mouse macrophages (RAW264.7) incubated in the presence of vehicle (DMSO), or the NFκB and JNK pathway inhibitors Sulfasalazine (60 min pretreatment) and SP600125 (30 min pretreatment), respectively. Data shown is the average of 2 to 4 experiments (+SD) with each time point in triplicate. B) Effect of NR ligands 9cisRA and Dex on Slc10a6 mRNA levels in RAW264.7cells (24h). Ligands were added directly prior LPS treatment. C) Effect of the RXRα ligand 9cisRA on the LPS-mediated induction of Slc10a6 mRNA. Ligands were added directly prior LPS treatment. Data expressed as mean + SD. *P
Figure Legend Snippet: LPS-induction of Slc10a6mRNA expression by in the mouse macrophage cell line RAW264.7 A) Time dependent LPS (1 μg/mL) induction of Slc10a6 mRNA and TNFα in mouse macrophages (RAW264.7) incubated in the presence of vehicle (DMSO), or the NFκB and JNK pathway inhibitors Sulfasalazine (60 min pretreatment) and SP600125 (30 min pretreatment), respectively. Data shown is the average of 2 to 4 experiments (+SD) with each time point in triplicate. B) Effect of NR ligands 9cisRA and Dex on Slc10a6 mRNA levels in RAW264.7cells (24h). Ligands were added directly prior LPS treatment. C) Effect of the RXRα ligand 9cisRA on the LPS-mediated induction of Slc10a6 mRNA. Ligands were added directly prior LPS treatment. Data expressed as mean + SD. *P

Techniques Used: Expressing, Incubation

4) Product Images from "Extracellular poly(ADP-ribose) is a pro-inflammatory signal for macrophages"

Article Title: Extracellular poly(ADP-ribose) is a pro-inflammatory signal for macrophages

Journal: Chemistry & biology

doi: 10.1016/j.chembiol.2015.03.007

A dimer of PAR is sufficient to induce TNFα secretion
Figure Legend Snippet: A dimer of PAR is sufficient to induce TNFα secretion

Techniques Used:

5) Product Images from "Developmental induction of human T-cell responses against Candida albicans and Aspergillus fumigatus"

Article Title: Developmental induction of human T-cell responses against Candida albicans and Aspergillus fumigatus

Journal: Scientific Reports

doi: 10.1038/s41598-018-35161-5

Fungi specific Th1 cytokine expression by T cells of different age groups. CD4 + CD45RA + T cells from neonates, infants, children, and adults were stimulated with C . albicans (orange) or A . fumigatus (blue) (as in Fig. 1 ) for 3 and 6 days respectively ( A ) Frequency of T cells expressing intracellular IL-2 (left panel), TNFα (middle panel) or IFNγ (right panel) was determined by flow cytometry. ( B ) Determination of IL-2 (left panel), TNFα (middle panel) or IFNγ (right panel) cytokine release of CD4 + CD45RA + T cells of neonates, infants and children or adults by LegendPlex which were either stimulated or not for 3 days. ( C , D ) CD4 + CD45RA + T cells were stimulated with C . albicans ( C ) or A . fumigatus ( D ) as in A, and the cells expressing single or multiple cytokines IL-2, TNFα, and IFNγ were determined by flow cytometry and analysed by Boolean gating and shown as fraction of all CD4 + T cells in a pie chart. The subsets that simultaneously express no (grey), one (blue), two (yellow) or three (red) different cytokines are grouped by colour. The data are representative of at least 5 donors. Cumulative results are shown and each dot in ( A ) and ( B ) represent a different donor. The error bars in figures denote ± SD. * p
Figure Legend Snippet: Fungi specific Th1 cytokine expression by T cells of different age groups. CD4 + CD45RA + T cells from neonates, infants, children, and adults were stimulated with C . albicans (orange) or A . fumigatus (blue) (as in Fig. 1 ) for 3 and 6 days respectively ( A ) Frequency of T cells expressing intracellular IL-2 (left panel), TNFα (middle panel) or IFNγ (right panel) was determined by flow cytometry. ( B ) Determination of IL-2 (left panel), TNFα (middle panel) or IFNγ (right panel) cytokine release of CD4 + CD45RA + T cells of neonates, infants and children or adults by LegendPlex which were either stimulated or not for 3 days. ( C , D ) CD4 + CD45RA + T cells were stimulated with C . albicans ( C ) or A . fumigatus ( D ) as in A, and the cells expressing single or multiple cytokines IL-2, TNFα, and IFNγ were determined by flow cytometry and analysed by Boolean gating and shown as fraction of all CD4 + T cells in a pie chart. The subsets that simultaneously express no (grey), one (blue), two (yellow) or three (red) different cytokines are grouped by colour. The data are representative of at least 5 donors. Cumulative results are shown and each dot in ( A ) and ( B ) represent a different donor. The error bars in figures denote ± SD. * p

Techniques Used: Expressing, Flow Cytometry, Cytometry

6) Product Images from "Human immunodeficiency virus type-1 induces a regulatory B cell-like phenotype in vitro"

Article Title: Human immunodeficiency virus type-1 induces a regulatory B cell-like phenotype in vitro

Journal: Cellular and Molecular Immunology

doi: 10.1038/cmi.2017.48

Reduction of TNFα expression in CD4+ or CD8+ T cells after co-culture with HIV-1-exposed B cells. Intracellular expression of TNFα by CD4+ and CD8+ T cells was analyzed after co-culture of effector cells (EC) and non-treated (NT) or treated B cells (ratio Breg:EC=2:1). Correlation between the ratio of CD4/CD8 and the percentage of reduction in TNFα expression in co-culture experiments when ( a ) HIV-1 NL4-3 -exposed, ( b ) CD40L/IL-4-exposed, ( c ) CpG/CD40L/LPS-exposed and ( d ) CpG/CD40L/LPS/HIV-1 NL4-3 -exposed B cells were calculated considering the mock-treated B-cell co-culture as the baseline reference level. Black squares and solid lines represent the results from CD4+ T cells, and open squares and dotted black lines represent the results from CD8+ T cells. Each square represents one experiment.
Figure Legend Snippet: Reduction of TNFα expression in CD4+ or CD8+ T cells after co-culture with HIV-1-exposed B cells. Intracellular expression of TNFα by CD4+ and CD8+ T cells was analyzed after co-culture of effector cells (EC) and non-treated (NT) or treated B cells (ratio Breg:EC=2:1). Correlation between the ratio of CD4/CD8 and the percentage of reduction in TNFα expression in co-culture experiments when ( a ) HIV-1 NL4-3 -exposed, ( b ) CD40L/IL-4-exposed, ( c ) CpG/CD40L/LPS-exposed and ( d ) CpG/CD40L/LPS/HIV-1 NL4-3 -exposed B cells were calculated considering the mock-treated B-cell co-culture as the baseline reference level. Black squares and solid lines represent the results from CD4+ T cells, and open squares and dotted black lines represent the results from CD8+ T cells. Each square represents one experiment.

Techniques Used: Expressing, Co-Culture Assay

7) 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

8) Product Images from "Immunologic response to vaccine challenge in pregnant PTPN22 R620W carriers and non-carriers"

Article Title: Immunologic response to vaccine challenge in pregnant PTPN22 R620W carriers and non-carriers

Journal: PLoS ONE

doi: 10.1371/journal.pone.0181338

Influenza vaccination induces vaccine-reactive CD4 T cells in pregnant women 9–15 days after vaccination. (A) Frequency, among total CD4 T cells, of CD4 cells producing any of the cytokines IFNγ, IL-2 or TNFα after Fluzone stimulation (6 hours). (B-D) Frequency of CD4 T cells producing IL-2 (B), TNFα (C) or IFNγ (D) among total CD4 T cells after Fluzone stimulation. Horizontal lines indicate median values. P values are determined by Wilcoxon matched-pairs signed rank and Mann-Whitney tests.
Figure Legend Snippet: Influenza vaccination induces vaccine-reactive CD4 T cells in pregnant women 9–15 days after vaccination. (A) Frequency, among total CD4 T cells, of CD4 cells producing any of the cytokines IFNγ, IL-2 or TNFα after Fluzone stimulation (6 hours). (B-D) Frequency of CD4 T cells producing IL-2 (B), TNFα (C) or IFNγ (D) among total CD4 T cells after Fluzone stimulation. Horizontal lines indicate median values. P values are determined by Wilcoxon matched-pairs signed rank and Mann-Whitney tests.

Techniques Used: MANN-WHITNEY

9) 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

10) Product Images from "Deficiency of iPLA2β Primes Immune Cells for Proinflammation: Potential Involvement in Age-Related Mesenteric Lymph Node Lymphoma"

Article Title: Deficiency of iPLA2β Primes Immune Cells for Proinflammation: Potential Involvement in Age-Related Mesenteric Lymph Node Lymphoma

Journal: Cancers

doi: 10.3390/cancers7040901

Deficiency of iPLA 2 β increased apoptosis in spleen associated with the sensitized Th1/Th17 release by splenocytes. ( A ) Representative cleaved caspase 3 IHC-staining of a spleen of WT and KO ( left panel ) and its quantification of positive cells ( right panel ). Male mice at 19–24-months old were used ( N = 8–14 per group); ( B ) ELISA determination of spontaneous and ConA-stimulated release of IFNγ, IL-17, and TNFα (pg/mL) by splenocytes isolated from 3-month old WT and mutant male mice ( N = 4–6 per group). Saline or 10 µg/mL ConA was used to treat WT and KO splenocytes for 48 h, and a fold-increase of cytokine release by ConA treatment was calculated. * p
Figure Legend Snippet: Deficiency of iPLA 2 β increased apoptosis in spleen associated with the sensitized Th1/Th17 release by splenocytes. ( A ) Representative cleaved caspase 3 IHC-staining of a spleen of WT and KO ( left panel ) and its quantification of positive cells ( right panel ). Male mice at 19–24-months old were used ( N = 8–14 per group); ( B ) ELISA determination of spontaneous and ConA-stimulated release of IFNγ, IL-17, and TNFα (pg/mL) by splenocytes isolated from 3-month old WT and mutant male mice ( N = 4–6 per group). Saline or 10 µg/mL ConA was used to treat WT and KO splenocytes for 48 h, and a fold-increase of cytokine release by ConA treatment was calculated. * p

Techniques Used: Immunohistochemistry, Staining, Mouse Assay, Enzyme-linked Immunosorbent Assay, Isolation, Mutagenesis

iPLA 2 β deficiency leads to increased hepatic apoptosis associated with suppressed M1 cytokine release by KC either spontaneously or during LPS-stimulation. Male mice at 3 months old were used. Liver lymphocytes were treated with 10 µg/mL ConA for 48 h. KC were treated with 1 µg/mL LPS for 7 h. ( A ) iPLA 2 β IHC of human liver showed positive brown staining. IgG was used as (-) control; ( B ) In left panel, iPLA 2 β mRNA expression in livers of young mice was determined by qRT-PCR. In right panel, caspase 3/7 activity measured by luminescence normalized to the WT levels was obtained in liver homogenates of WT and KO mice ( N = 3 per group for PCR and N = 4–5 per group for luminescence); ( C ) Spontaneous or ConA-stimulated release of IFNγ, IL-17 and IL-10 measured by ELISA was determined in liver lymphocytes of 3-month old WT and KO ( N = 4–6 per group); Spontaneous or LPS-stimulated release of IL-6 and TNFα ( D ) as well as IL-10 and Il-4 ( E ) measured by ELISA was determined in KC isolated from WT and KO ( left panel ), and their fold increase by LPS was calculated ( right panel ) ( N = 6 per group). * p
Figure Legend Snippet: iPLA 2 β deficiency leads to increased hepatic apoptosis associated with suppressed M1 cytokine release by KC either spontaneously or during LPS-stimulation. Male mice at 3 months old were used. Liver lymphocytes were treated with 10 µg/mL ConA for 48 h. KC were treated with 1 µg/mL LPS for 7 h. ( A ) iPLA 2 β IHC of human liver showed positive brown staining. IgG was used as (-) control; ( B ) In left panel, iPLA 2 β mRNA expression in livers of young mice was determined by qRT-PCR. In right panel, caspase 3/7 activity measured by luminescence normalized to the WT levels was obtained in liver homogenates of WT and KO mice ( N = 3 per group for PCR and N = 4–5 per group for luminescence); ( C ) Spontaneous or ConA-stimulated release of IFNγ, IL-17 and IL-10 measured by ELISA was determined in liver lymphocytes of 3-month old WT and KO ( N = 4–6 per group); Spontaneous or LPS-stimulated release of IL-6 and TNFα ( D ) as well as IL-10 and Il-4 ( E ) measured by ELISA was determined in KC isolated from WT and KO ( left panel ), and their fold increase by LPS was calculated ( right panel ) ( N = 6 per group). * p

Techniques Used: Mouse Assay, Immunohistochemistry, Staining, Expressing, Quantitative RT-PCR, Activity Assay, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Isolation

Sublethal dose Jo2 treatment caused a very mild effect on liver injury and apoptosis but primes KC from mutant mice for a marked increase of IL-6 release either spontaneously or during LPS stimulation. Three-month old mice were treated with saline or 0.125 µg/g body weight Jo2 antibody for 6 h. KC were treated with saline or 1 µg/mL LPS for 7 h. ( A ) Activity of serum transaminases (AST and ALT) in U/L were determined in WT and KO mice ( N = 3–7 per group); ( B ) Caspase 8 and caspase 3/7 activities measured by luminescence were determined in liver homogenates of WT and KO ( N = 3–7 per group); Spontaneous ( C ) and LPS-stimulated ( D ) release of IL-6 and TNFα measured by ELISA was determined in KC isolated from WT and KO ( N = 6 per group), and the fold increase by Jo2 was calculated (right-hand panel). * p
Figure Legend Snippet: Sublethal dose Jo2 treatment caused a very mild effect on liver injury and apoptosis but primes KC from mutant mice for a marked increase of IL-6 release either spontaneously or during LPS stimulation. Three-month old mice were treated with saline or 0.125 µg/g body weight Jo2 antibody for 6 h. KC were treated with saline or 1 µg/mL LPS for 7 h. ( A ) Activity of serum transaminases (AST and ALT) in U/L were determined in WT and KO mice ( N = 3–7 per group); ( B ) Caspase 8 and caspase 3/7 activities measured by luminescence were determined in liver homogenates of WT and KO ( N = 3–7 per group); Spontaneous ( C ) and LPS-stimulated ( D ) release of IL-6 and TNFα measured by ELISA was determined in KC isolated from WT and KO ( N = 6 per group), and the fold increase by Jo2 was calculated (right-hand panel). * p

Techniques Used: Mutagenesis, Mouse Assay, Activity Assay, AST Assay, Enzyme-linked Immunosorbent Assay, Isolation

11) Product Images from "Glycogen Metabolism Supports Early Glycolytic Reprogramming and Activation in Dendritic Cells in Response to Both TLR and Syk-Dependent CLR Agonists"

Article Title: Glycogen Metabolism Supports Early Glycolytic Reprogramming and Activation in Dendritic Cells in Response to Both TLR and Syk-Dependent CLR Agonists

Journal: Cells

doi: 10.3390/cells9030715

Glycogen phosphorylase inhibition causes deficits in inflammatory cytokine expression and nitric oxide production in response to TLR and Syk-dependent CLR agonists: ( A and B ) DCs were stimulated for 6 h with the indicated ligands and GolgiPlug reagent (LPS = lipopolysaccharide, Pam = Pam2CSK4, Zy = Zymosan, and ZD = Zymosan depleted) in the presence or absence of PYG inhibitor (PYGib), and then assessed by flow cytometry for intracellular expression of IL-12p70 ( A ) and TNFα ( B ). The percent of cytokine-positive cells (from total CD11c+ cells) is depicted in the left panels, while the mean fluorescence intensities of cytokine-positive cells are depicted in the right panels. ( B ) Percent of CD40 and CD86 double-positive cells for each condition as depicted in ( A ). ( C ) DCs were stimulated for 18 h with the indicated conditions as above, and supernatants were then collected and analyzed for nitrite accumulation by Griess assay. For all graphs, statistical values are represented with an asterisk (*) as significant when p values are equal to or below 0.05, with mean +/− SD shown, n = 4.
Figure Legend Snippet: Glycogen phosphorylase inhibition causes deficits in inflammatory cytokine expression and nitric oxide production in response to TLR and Syk-dependent CLR agonists: ( A and B ) DCs were stimulated for 6 h with the indicated ligands and GolgiPlug reagent (LPS = lipopolysaccharide, Pam = Pam2CSK4, Zy = Zymosan, and ZD = Zymosan depleted) in the presence or absence of PYG inhibitor (PYGib), and then assessed by flow cytometry for intracellular expression of IL-12p70 ( A ) and TNFα ( B ). The percent of cytokine-positive cells (from total CD11c+ cells) is depicted in the left panels, while the mean fluorescence intensities of cytokine-positive cells are depicted in the right panels. ( B ) Percent of CD40 and CD86 double-positive cells for each condition as depicted in ( A ). ( C ) DCs were stimulated for 18 h with the indicated conditions as above, and supernatants were then collected and analyzed for nitrite accumulation by Griess assay. For all graphs, statistical values are represented with an asterisk (*) as significant when p values are equal to or below 0.05, with mean +/− SD shown, n = 4.

Techniques Used: Inhibition, Expressing, Flow Cytometry, Fluorescence, Griess Assay

12) Product Images from "The glucocorticoid receptor in recipient cells keeps cytokine secretion in acute graft-versus-host disease at bay"

Article Title: The glucocorticoid receptor in recipient cells keeps cytokine secretion in acute graft-versus-host disease at bay

Journal: Oncotarget

doi: 10.18632/oncotarget.24602

Cytokine serum levels in the early phase of aGvHD in the GR lysM model GR flox and GR lysM BALB/c mice were transplanted with BM and purified T cells from C57BL/6 wildtype mice; transfer of BM cells only served as a control. Mice were sacrificed and analyzed on day 4, 6 and 8 after aGvHD induction; analysis of BM controls was performed on day 6. Serum levels of IL-6, TNFα and IFNγ were determined by ELISA and are depicted as mean values ± SEM. N = 12 (BM), N = 5/5 (GR flox /GR lysM ; day 4), N = 27/28 (GR flox /GR lysM ; day 6), N = 6/7 (GR flox /GR lysM ; day 8); data pooled from multiple experiments. Statistical analyses were performed by Mann-Whitney U test ( * p
Figure Legend Snippet: Cytokine serum levels in the early phase of aGvHD in the GR lysM model GR flox and GR lysM BALB/c mice were transplanted with BM and purified T cells from C57BL/6 wildtype mice; transfer of BM cells only served as a control. Mice were sacrificed and analyzed on day 4, 6 and 8 after aGvHD induction; analysis of BM controls was performed on day 6. Serum levels of IL-6, TNFα and IFNγ were determined by ELISA and are depicted as mean values ± SEM. N = 12 (BM), N = 5/5 (GR flox /GR lysM ; day 4), N = 27/28 (GR flox /GR lysM ; day 6), N = 6/7 (GR flox /GR lysM ; day 8); data pooled from multiple experiments. Statistical analyses were performed by Mann-Whitney U test ( * p

Techniques Used: Mouse Assay, Purification, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Cytokine expression and secretion in the jejunum in the early phase of aGvHD in the GR lysM model GR flox and GR lysM BALB/c mice were transplanted with BM and purified T cells from C57BL/6 wildtype mice; transfer of BM cells only served as a control. Mice were sacrificed and analyzed on day 4, 6 and 8 after aGvHD induction; analysis of BM controls was performed on day 6. ( A ) Relative mRNA levels of IL-6, TNFα and IFNγ in jejunum biopsies were determined by quantitative RT-PCR using HPRT for normalization. Gene expression in BM control mice was arbitrarily set to 1. N = 4 (BM), N = 5/5 (GR flox /GR lysM ; day 4), N = 12/15 (GR flox /GR lysM ; day 6), N = 5/7 (GR flox /GR lysM ; day 8); data pooled from multiple experiments. ( B ) Jejunum biopsies were cultured for 24 hours in RPMI+ medium and IL-6, TNFα and IFNγ levels in the supernatant were determined by ELISA. N = 4 (BM), N = 5/5 (GR flox /GR lysM ; day 4), N = 14/15 (GR flox /GR lysM ; day 6), N = 6/8 (GR flox /GR lysM ; day 8); data pooled from multiple experiments. All values are depicted as mean ± SEM. Statistical analyses were performed by Mann-Whitney U test ( * p
Figure Legend Snippet: Cytokine expression and secretion in the jejunum in the early phase of aGvHD in the GR lysM model GR flox and GR lysM BALB/c mice were transplanted with BM and purified T cells from C57BL/6 wildtype mice; transfer of BM cells only served as a control. Mice were sacrificed and analyzed on day 4, 6 and 8 after aGvHD induction; analysis of BM controls was performed on day 6. ( A ) Relative mRNA levels of IL-6, TNFα and IFNγ in jejunum biopsies were determined by quantitative RT-PCR using HPRT for normalization. Gene expression in BM control mice was arbitrarily set to 1. N = 4 (BM), N = 5/5 (GR flox /GR lysM ; day 4), N = 12/15 (GR flox /GR lysM ; day 6), N = 5/7 (GR flox /GR lysM ; day 8); data pooled from multiple experiments. ( B ) Jejunum biopsies were cultured for 24 hours in RPMI+ medium and IL-6, TNFα and IFNγ levels in the supernatant were determined by ELISA. N = 4 (BM), N = 5/5 (GR flox /GR lysM ; day 4), N = 14/15 (GR flox /GR lysM ; day 6), N = 6/8 (GR flox /GR lysM ; day 8); data pooled from multiple experiments. All values are depicted as mean ± SEM. Statistical analyses were performed by Mann-Whitney U test ( * p

Techniques Used: Expressing, Mouse Assay, Purification, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

13) Product Images from "Inhibition of early T cell cytokine production by arsenic trioxide occurs independently of Nrf2"

Article Title: Inhibition of early T cell cytokine production by arsenic trioxide occurs independently of Nrf2

Journal: PLoS ONE

doi: 10.1371/journal.pone.0185579

ATO markedly inhibits protein secretion, but not gene expression, of TNFα by anti-CD3/anti-CD28-activated splenocytes from wild-type and Nrf2-null mice. Wild-type and Nrf2-null splenocytes were isolated and either left untreated (BKG) or treated with the vehicle (VH, PBS), 0 (activator alone), 1, or 2 μM ATO for 30 min. The treatment groups were then either left unactivated (BKG) or activated with anti-CD3/anti-CD28 for (A) 6 h prior to quantification of TNFα mRNA by real-time PCR, or (B) 24 h prior to quantification of TNFα protein in cell supernatants by ELISA. * denotes p
Figure Legend Snippet: ATO markedly inhibits protein secretion, but not gene expression, of TNFα by anti-CD3/anti-CD28-activated splenocytes from wild-type and Nrf2-null mice. Wild-type and Nrf2-null splenocytes were isolated and either left untreated (BKG) or treated with the vehicle (VH, PBS), 0 (activator alone), 1, or 2 μM ATO for 30 min. The treatment groups were then either left unactivated (BKG) or activated with anti-CD3/anti-CD28 for (A) 6 h prior to quantification of TNFα mRNA by real-time PCR, or (B) 24 h prior to quantification of TNFα protein in cell supernatants by ELISA. * denotes p

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

ATO inhibits cytokine production in isolated CD4 + T cells in a Nrf2-independent manner. Wild-type and Nrf2-null CD4 + T cells were isolated from splenocytes. They were left untreated (BKG) or treated with vehicle (VEH, PBS), 0.1 μM ATO, or 0.5 μM ATO for 30 min. The treatment groups were then either left unactivated (BKG) or activated with anti-CD3/anti-CD28 for 24 h prior to quantification of (A) IL-2, (B) IFNγ, (C) GM-CSF, and (D) TNFα protein in cell supernatants by ELISA. * denotes p
Figure Legend Snippet: ATO inhibits cytokine production in isolated CD4 + T cells in a Nrf2-independent manner. Wild-type and Nrf2-null CD4 + T cells were isolated from splenocytes. They were left untreated (BKG) or treated with vehicle (VEH, PBS), 0.1 μM ATO, or 0.5 μM ATO for 30 min. The treatment groups were then either left unactivated (BKG) or activated with anti-CD3/anti-CD28 for 24 h prior to quantification of (A) IL-2, (B) IFNγ, (C) GM-CSF, and (D) TNFα protein in cell supernatants by ELISA. * denotes p

Techniques Used: Isolation, Enzyme-linked Immunosorbent Assay

14) Product Images from "Eicosapentaenoic and docosahexaenoic acid ethyl esters differentially enhance B-cell activity in murine obesity [S]"

Article Title: Eicosapentaenoic and docosahexaenoic acid ethyl esters differentially enhance B-cell activity in murine obesity [S]

Journal: Journal of Lipid Research

doi: 10.1194/jlr.M049809

B-cell cytokine secretion is differentially enhanced with EPA and DHA. Splenic B220 + B cells were isolated from mice consuming control, HF, HF-OA, HF-EPA, or HF-DHA diets for 5 or 10 weeks and stimulated for 24 h with LPS. TNFα, IL-6, and IL-10
Figure Legend Snippet: B-cell cytokine secretion is differentially enhanced with EPA and DHA. Splenic B220 + B cells were isolated from mice consuming control, HF, HF-OA, HF-EPA, or HF-DHA diets for 5 or 10 weeks and stimulated for 24 h with LPS. TNFα, IL-6, and IL-10

Techniques Used: Isolation, Mouse Assay

15) Product Images from "AMPK and SIRT1 activation contribute to inhibition of neuroinflammation by thymoquinone in BV2 microglia"

Article Title: AMPK and SIRT1 activation contribute to inhibition of neuroinflammation by thymoquinone in BV2 microglia

Journal: Molecular and Cellular Biochemistry

doi: 10.1007/s11010-017-3064-3

Inhibition of neuroinflammation by thymoquinone was abolished in the presence of compound C. BV2 cells were treated with compound C (10 µM), followed by thymoquinone (10 µM) and LPS (100 ng/ml) for 24 h. Cells were analysed for TNFα ( a ), IL-6 ( b ), IL-1β ( c ), nitrite ( d ), PGE 2 ( e ) and ROS ( f ). In g nuclear extracts from cells were added to 96-well plates to which an oligonucleotide containing the NF-κB consensus site (5′-GGGACTTTCC-3′) has been immobilised, followed by addition of NF-κB and HRP-conjugated antibodies. Absorbance was read in a microplate reader. (Mean ± SEM; ** p
Figure Legend Snippet: Inhibition of neuroinflammation by thymoquinone was abolished in the presence of compound C. BV2 cells were treated with compound C (10 µM), followed by thymoquinone (10 µM) and LPS (100 ng/ml) for 24 h. Cells were analysed for TNFα ( a ), IL-6 ( b ), IL-1β ( c ), nitrite ( d ), PGE 2 ( e ) and ROS ( f ). In g nuclear extracts from cells were added to 96-well plates to which an oligonucleotide containing the NF-κB consensus site (5′-GGGACTTTCC-3′) has been immobilised, followed by addition of NF-κB and HRP-conjugated antibodies. Absorbance was read in a microplate reader. (Mean ± SEM; ** p

Techniques Used: Inhibition

Inhibition of neuroinflammation by thymoquinone was abolished in the presence of EX527. BV2 cells were treated with EX527 (1 µM), followed by thymoquinone (10 µM) and LPS (100 ng/ml) for 24 h. Culture supernatants were collected and analysed for TNFα ( a ), IL-6 ( b ), IL-1β ( c ), nitrite ( d ) and PGE 2 ( e ). In f nuclear extracts from cells were added to 96-well plates to which an oligonucleotide containing the NF-κB consensus site (5′-GGGACTTTCC-3′) has been immobilised, followed by addition of NF-κB and HRP-conjugated antibodies. Absorbance was read in a microplate reader. Reduction of cellular ROS production by thymoquinone was not abolished in the presence of EX527 (G). (Mean ± SEM; ** p
Figure Legend Snippet: Inhibition of neuroinflammation by thymoquinone was abolished in the presence of EX527. BV2 cells were treated with EX527 (1 µM), followed by thymoquinone (10 µM) and LPS (100 ng/ml) for 24 h. Culture supernatants were collected and analysed for TNFα ( a ), IL-6 ( b ), IL-1β ( c ), nitrite ( d ) and PGE 2 ( e ). In f nuclear extracts from cells were added to 96-well plates to which an oligonucleotide containing the NF-κB consensus site (5′-GGGACTTTCC-3′) has been immobilised, followed by addition of NF-κB and HRP-conjugated antibodies. Absorbance was read in a microplate reader. Reduction of cellular ROS production by thymoquinone was not abolished in the presence of EX527 (G). (Mean ± SEM; ** p

Techniques Used: Inhibition

Inhibition of neuroinflammation by thymoquinone is dependent on SIRT1. Control siRNA- and SIRT1 siRNA-transfected BV2 cells were pre-treated with thymoquinone (10 μM) prior to stimulation with LPS (100 ng/ml) for 24 h. Culture supernatants were analysed for TNFα ( a ), IL-6 ( b ), IL-1β ( c ), nitrite ( d ) and PGE 2 ( e ). In f nuclear extracts from cells were added to 96-well plates to which an oligonucleotide containing the NF-κB consensus site (5′-GGGACTTTCC-3′) has been immobilised, followed by addition of NF-κB and HRP-conjugated antibodies. Absorbance was read in a microplate reader. Reduction of cellular ROS production in LPS-stimulated microglia is not dependent on SIRT1 ( g ). (Mean ± SEM; ** p
Figure Legend Snippet: Inhibition of neuroinflammation by thymoquinone is dependent on SIRT1. Control siRNA- and SIRT1 siRNA-transfected BV2 cells were pre-treated with thymoquinone (10 μM) prior to stimulation with LPS (100 ng/ml) for 24 h. Culture supernatants were analysed for TNFα ( a ), IL-6 ( b ), IL-1β ( c ), nitrite ( d ) and PGE 2 ( e ). In f nuclear extracts from cells were added to 96-well plates to which an oligonucleotide containing the NF-κB consensus site (5′-GGGACTTTCC-3′) has been immobilised, followed by addition of NF-κB and HRP-conjugated antibodies. Absorbance was read in a microplate reader. Reduction of cellular ROS production in LPS-stimulated microglia is not dependent on SIRT1 ( g ). (Mean ± SEM; ** p

Techniques Used: Inhibition, Transfection

Inhibition of neuroinflammation by thymoquinone is dependent on AMPKα. Control siRNA- and AMPKα siRNA-transfected BV2 cells were pre-treated with thymoquinone (10 μM) prior to stimulation with LPS (100 ng/ml) for 24 h. Cells were analysed for TNFα ( a ), IL-6 ( b ), IL-1β ( c ), nitrite ( d ) PGE 2 ( e ) and ROS ( f ). In g nuclear extracts from cells were added to 96-well plates to which an oligonucleotide containing the NF-κB consensus site (5′-GGGACTTTCC-3′) has been immobilised, followed by addition of NF-κB and HRP-conjugated antibodies. Absorbance was read in a microplate reader. Western blot experiments on cell extracts to determine knockout efficiency ( h ). (Mean ± SEM; ** p
Figure Legend Snippet: Inhibition of neuroinflammation by thymoquinone is dependent on AMPKα. Control siRNA- and AMPKα siRNA-transfected BV2 cells were pre-treated with thymoquinone (10 μM) prior to stimulation with LPS (100 ng/ml) for 24 h. Cells were analysed for TNFα ( a ), IL-6 ( b ), IL-1β ( c ), nitrite ( d ) PGE 2 ( e ) and ROS ( f ). In g nuclear extracts from cells were added to 96-well plates to which an oligonucleotide containing the NF-κB consensus site (5′-GGGACTTTCC-3′) has been immobilised, followed by addition of NF-κB and HRP-conjugated antibodies. Absorbance was read in a microplate reader. Western blot experiments on cell extracts to determine knockout efficiency ( h ). (Mean ± SEM; ** p

Techniques Used: Inhibition, Transfection, Western Blot, Knock-Out

16) Product Images from "Complement and alcoholic liver disease: Role of C1q in the pathogenesis of ethanol-induced liver injury in mice"

Article Title: Complement and alcoholic liver disease: Role of C1q in the pathogenesis of ethanol-induced liver injury in mice

Journal: Gastroenterology

doi: 10.1053/j.gastro.2010.04.041

TUNEL, F4/80 and C1q staining and inflammatory cytokine protein concentration in mouse livers after short-term ethanol feeding Wild-type and C1qa-/- mice were allowed free access to ethanol-containing diets (4d/11% ethanol) or pair-fed control diets. (A) TUNEL-positive nuclei and F4/80-positive cells were visualized by immunohistochemistry in formalin-fixed liver sections. (B) F4/80 and C1q-positive cells were visualized by immunohistochemistry in frozen liver sections. Images are representative of at least 4 mice per group and are shown at 200X magnification. (C/D) Livers lysates were prepared and quantity of IL-6 (C) and TNFα (D) protein was measured by ELISA. Values represent means ± SEM. Pair-fed n=4, ethanol-fed n=6. Values with different superscripts are significantly different from each other, C) p
Figure Legend Snippet: TUNEL, F4/80 and C1q staining and inflammatory cytokine protein concentration in mouse livers after short-term ethanol feeding Wild-type and C1qa-/- mice were allowed free access to ethanol-containing diets (4d/11% ethanol) or pair-fed control diets. (A) TUNEL-positive nuclei and F4/80-positive cells were visualized by immunohistochemistry in formalin-fixed liver sections. (B) F4/80 and C1q-positive cells were visualized by immunohistochemistry in frozen liver sections. Images are representative of at least 4 mice per group and are shown at 200X magnification. (C/D) Livers lysates were prepared and quantity of IL-6 (C) and TNFα (D) protein was measured by ELISA. Values represent means ± SEM. Pair-fed n=4, ethanol-fed n=6. Values with different superscripts are significantly different from each other, C) p

Techniques Used: TUNEL Assay, Staining, Protein Concentration, Mouse Assay, Immunohistochemistry, Enzyme-linked Immunosorbent Assay

Markers of oxidative stress and inflammatory cytokine expression after chronic ethanol feeding Wild-type and C1qa-/- mice were allowed free access to ethanol-containing diets (25d/32% ethanol) or pair-fed control diets. (A) Immunoreactive CYP2E1 protein in liver lysates was assessed by Western blot analysis. Values under the images represent arbitrary units of density for CYP2E1 relative to hsc 70, used as a loading control. (B/C) Immunoreactive 4-HNE adducts were visualized by immunohistochemistry in formalin-fixed liver sections and fluorescence intensity per 200X field determined using Image Pro. (D) MDA was measured biochemically using a TBARS assay kit. (E/F) IL-6 (E) and TNFα (F) protein in liver lysates was measured by ELISA. Values represent means ± SEM. Pair-fed n=4, ethanol-fed n=6, except in panel D where pair-fed n=8, ethanol-fed n=12. Values with different superscripts are significantly different from each other, C) p
Figure Legend Snippet: Markers of oxidative stress and inflammatory cytokine expression after chronic ethanol feeding Wild-type and C1qa-/- mice were allowed free access to ethanol-containing diets (25d/32% ethanol) or pair-fed control diets. (A) Immunoreactive CYP2E1 protein in liver lysates was assessed by Western blot analysis. Values under the images represent arbitrary units of density for CYP2E1 relative to hsc 70, used as a loading control. (B/C) Immunoreactive 4-HNE adducts were visualized by immunohistochemistry in formalin-fixed liver sections and fluorescence intensity per 200X field determined using Image Pro. (D) MDA was measured biochemically using a TBARS assay kit. (E/F) IL-6 (E) and TNFα (F) protein in liver lysates was measured by ELISA. Values represent means ± SEM. Pair-fed n=4, ethanol-fed n=6, except in panel D where pair-fed n=8, ethanol-fed n=12. Values with different superscripts are significantly different from each other, C) p

Techniques Used: Expressing, Mouse Assay, Western Blot, Immunohistochemistry, Fluorescence, Multiple Displacement Amplification, TBARS Assay, Enzyme-linked Immunosorbent Assay

17) Product Images from "A commensal symbiotic factor derived from Bacteroides fragilis promotes human CD39+Foxp3+ T cells and Treg function"

Article Title: A commensal symbiotic factor derived from Bacteroides fragilis promotes human CD39+Foxp3+ T cells and Treg function

Journal: Gut Microbes

doi: 10.1080/19490976.2015.1056973

PSA increases the frequency of CD39-expressing T regs and T reg function. PSA exposure significantly enhanced the frequency of CD39 and HLA-DR-expressing circulating T regs and resulted in greater capacity to suppress monocyte secretion of TNFα.
Figure Legend Snippet: PSA increases the frequency of CD39-expressing T regs and T reg function. PSA exposure significantly enhanced the frequency of CD39 and HLA-DR-expressing circulating T regs and resulted in greater capacity to suppress monocyte secretion of TNFα.

Techniques Used: Expressing

18) Product Images from "TREM-1 links dyslipidemia to inflammation and lipid deposition in atherosclerosis"

Article Title: TREM-1 links dyslipidemia to inflammation and lipid deposition in atherosclerosis

Journal: Nature Communications

doi: 10.1038/ncomms13151

TREM-1 synergizes with dyslipidemic factors for pro-inflammatory cytokine secretion. ( a – c ) Human CD14 hi monocytes were FACS-purified from buffy coats obtained from different blood donors. ( a ) Representative histograms for TREM-1 surface expression (lines; filled histograms represent isotype control stained cells) on human peripheral blood CD14 hi monocytes after 20 h of stimulation with the indicated stimuli. ( b ) MFI values for TREM-1 surface expression after subtraction of MFI values for matched isotype control-stained cells. Bars show mean values of seven independent experiments with error bars indicating the s.d. ( c ) Production of IL-1α, IL-1β and TNFα by CD14 hi monocytes after 20 h of culture with the indicated factors. Symbols show individual values of 6–7 independent experiments and different blood donors. * P
Figure Legend Snippet: TREM-1 synergizes with dyslipidemic factors for pro-inflammatory cytokine secretion. ( a – c ) Human CD14 hi monocytes were FACS-purified from buffy coats obtained from different blood donors. ( a ) Representative histograms for TREM-1 surface expression (lines; filled histograms represent isotype control stained cells) on human peripheral blood CD14 hi monocytes after 20 h of stimulation with the indicated stimuli. ( b ) MFI values for TREM-1 surface expression after subtraction of MFI values for matched isotype control-stained cells. Bars show mean values of seven independent experiments with error bars indicating the s.d. ( c ) Production of IL-1α, IL-1β and TNFα by CD14 hi monocytes after 20 h of culture with the indicated factors. Symbols show individual values of 6–7 independent experiments and different blood donors. * P

Techniques Used: FACS, Purification, Expressing, Staining

19) Product Images from "The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response"

Article Title: The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response

Journal: Cell Research

doi: 10.1038/cr.2016.16

Mex3B is essential for TLR3-mediated signaling in MEFs, BMDMs and DCs. (A) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in MEFs. MEFs (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 3 h, or infected with SeV or VSV for 12 h. Total RNA was then extracted for qPCR analysis. (B) Effects of Mex3B deficiency on poly(I:C)-induced phosphorylation of IRF3 and IκBα in MEFs. MEFs (2 × 10 5 ) were left untreated, infected with SeV or treated with poly(I:C) for the indicated times. Cells were lysed and immunoblot was performed with the indicated antibodies. (C , D) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in BMDMs (C) and DCs (D) . The cells (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), R837 (20 μg/ml) or PGN (10 μg/ml) for 3 h or infected with SeV, VSV, EMCV or HSV for 12 h before qPCR analysis. Data are represented as mean ± SEM. * P
Figure Legend Snippet: Mex3B is essential for TLR3-mediated signaling in MEFs, BMDMs and DCs. (A) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in MEFs. MEFs (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 3 h, or infected with SeV or VSV for 12 h. Total RNA was then extracted for qPCR analysis. (B) Effects of Mex3B deficiency on poly(I:C)-induced phosphorylation of IRF3 and IκBα in MEFs. MEFs (2 × 10 5 ) were left untreated, infected with SeV or treated with poly(I:C) for the indicated times. Cells were lysed and immunoblot was performed with the indicated antibodies. (C , D) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in BMDMs (C) and DCs (D) . The cells (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), R837 (20 μg/ml) or PGN (10 μg/ml) for 3 h or infected with SeV, VSV, EMCV or HSV for 12 h before qPCR analysis. Data are represented as mean ± SEM. * P

Techniques Used: Infection, Real-time Polymerase Chain Reaction

20) Product Images from "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 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

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01494

Identification of cytokines as responsible for enhancing human immunodeficiency virus type I (HIV-1) infection in unactivated CD4 + T-cells. (A) Unactivated CD4 + T-cells w ere stimulated with different combinations of cytokines for 72 h. Then, cells were infected and p24 antigen production was evaluated at days 4 and 7 post-infection. Each condition was compared with the corresponding RPMI condition (negative control). As a positive control, PHA stimulation was used. Percentage of living CD4 + T-cells (B) and percentage of infected (GFP + ) CD4 + T-cells (C) after stimulation with the denoted treatments are shown. Data represent mean ± SD from four independent donors evaluated in duplicate. Concentrations of cytokines used in these experiments corresponded to the average concentrations found in monocyte-derived macrophage (MDM) supernatants stimulated with 25 ng/ml macrophage migration inhibitory factor (MIF) (peak effect) as follows: 250 pg/ml IL-6, 9,000 pg/ml IL-8, 1,400 pg/ml TNF-α, and 20 pg/ml IL-1β. (D) Neutralization of IL-8, IL-6, IL-1 β, and TNFα biological activity with monoclonal neutralizing antibodies. Primary CD4 + T-cells were incubated with supernatants derived from the 25 ng/ml MIF-treated HIV-infected MDM neutralized previously with 18 µg/ml anti-IL-8, 20 ng/ml anti-IL-6, 2 µg/ml anti-IL-1β, and 2 µg/ml anti-TNFα antibodies. Non-neutralized and isotype control antibody conditions were tested for comparison. Also, RPMI and PHA controls were included. Viral production was evaluated at day 4 post-infection. Data were analyzed by one-way ANOVA followed by Dunnett’s post-test (all conditions versus the corresponding RMPI control) in (A) and Tukey’s post-test in (D) . * p
Figure Legend Snippet: Identification of cytokines as responsible for enhancing human immunodeficiency virus type I (HIV-1) infection in unactivated CD4 + T-cells. (A) Unactivated CD4 + T-cells w ere stimulated with different combinations of cytokines for 72 h. Then, cells were infected and p24 antigen production was evaluated at days 4 and 7 post-infection. Each condition was compared with the corresponding RPMI condition (negative control). As a positive control, PHA stimulation was used. Percentage of living CD4 + T-cells (B) and percentage of infected (GFP + ) CD4 + T-cells (C) after stimulation with the denoted treatments are shown. Data represent mean ± SD from four independent donors evaluated in duplicate. Concentrations of cytokines used in these experiments corresponded to the average concentrations found in monocyte-derived macrophage (MDM) supernatants stimulated with 25 ng/ml macrophage migration inhibitory factor (MIF) (peak effect) as follows: 250 pg/ml IL-6, 9,000 pg/ml IL-8, 1,400 pg/ml TNF-α, and 20 pg/ml IL-1β. (D) Neutralization of IL-8, IL-6, IL-1 β, and TNFα biological activity with monoclonal neutralizing antibodies. Primary CD4 + T-cells were incubated with supernatants derived from the 25 ng/ml MIF-treated HIV-infected MDM neutralized previously with 18 µg/ml anti-IL-8, 20 ng/ml anti-IL-6, 2 µg/ml anti-IL-1β, and 2 µg/ml anti-TNFα antibodies. Non-neutralized and isotype control antibody conditions were tested for comparison. Also, RPMI and PHA controls were included. Viral production was evaluated at day 4 post-infection. Data were analyzed by one-way ANOVA followed by Dunnett’s post-test (all conditions versus the corresponding RMPI control) in (A) and Tukey’s post-test in (D) . * p

Techniques Used: Infection, Negative Control, Positive Control, Derivative Assay, Migration, Neutralization, Activity Assay, Incubation

21) Product Images from "Type I Interferon Receptor Deficiency in Dendritic Cells Facilitates Systemic Murine Norovirus Persistence Despite Enhanced Adaptive Immunity"

Article Title: Type I Interferon Receptor Deficiency in Dendritic Cells Facilitates Systemic Murine Norovirus Persistence Despite Enhanced Adaptive Immunity

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005684

CW3 persistence in CD11c- Ifnar1 -/- mice is associated with enhanced CD8 T cell expansion. Cells were collected from spleens, MLNs, or Peyer’s patches of CD11c- Ifnar1 -/- mice and controls eight days after infection with CW3. ( A ) Total CD8 T cells were analyzed for expression of IFNAR1. ( B ) The number or percentage of CW3-tetramer positive CD8 T cells was determined by flow cytometry from the indicated tissue. ( C ) Splenocytes were stimulated ex vivo with CW3 peptide and stained for cell surface CD107a and intracellular IFNγ and TNFα. ( D ) RNA extracted from spleens was used to quantitate transcripts for Ifng , Tnfa , and Gzmb . ( E ) The percentage of cells in (C) positive for one, two, or three of the markers of activation is shown. Data in (B) is combined from three experiments and data in (A), (C), (D) and (E) is combined from two experiments with seven mice per data point in (E). Statistical significance was determined by unpaired t test (B, C and D) or 2-way ANOVA (A and C). n.s = p > 0.05, * = p≤0.05, ** = p≤0.01, *** = p≤0.001, **** = p≤0.0001.
Figure Legend Snippet: CW3 persistence in CD11c- Ifnar1 -/- mice is associated with enhanced CD8 T cell expansion. Cells were collected from spleens, MLNs, or Peyer’s patches of CD11c- Ifnar1 -/- mice and controls eight days after infection with CW3. ( A ) Total CD8 T cells were analyzed for expression of IFNAR1. ( B ) The number or percentage of CW3-tetramer positive CD8 T cells was determined by flow cytometry from the indicated tissue. ( C ) Splenocytes were stimulated ex vivo with CW3 peptide and stained for cell surface CD107a and intracellular IFNγ and TNFα. ( D ) RNA extracted from spleens was used to quantitate transcripts for Ifng , Tnfa , and Gzmb . ( E ) The percentage of cells in (C) positive for one, two, or three of the markers of activation is shown. Data in (B) is combined from three experiments and data in (A), (C), (D) and (E) is combined from two experiments with seven mice per data point in (E). Statistical significance was determined by unpaired t test (B, C and D) or 2-way ANOVA (A and C). n.s = p > 0.05, * = p≤0.05, ** = p≤0.01, *** = p≤0.001, **** = p≤0.0001.

Techniques Used: Mouse Assay, Infection, Expressing, Flow Cytometry, Cytometry, Ex Vivo, Staining, Activation Assay

22) Product Images from "miR-466a Targeting of TGF-β2 Contributes to FoxP3+ Regulatory T Cell Differentiation in a Murine Model of Allogeneic Transplantation"

Article Title: miR-466a Targeting of TGF-β2 Contributes to FoxP3+ Regulatory T Cell Differentiation in a Murine Model of Allogeneic Transplantation

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00688

Locked nucleic acid (LNA) mitigates draining lymph node (dLN) effector cell and cytokines. Female C57BL/6 mice were given either syn (BL6) or allo (C 3 H) tail skin grafts. Mice receiving allografts were given either LNA-466 (10 mg/kg) or LNA-Ctrl (10 mg/kg) intraperitoneally 1 day before skin transplantation and every third day after that until termination of the study. (A) Survival curve of mice receiving skin transplants and indicated LNA or controls; n = 4 (syngeneic), 7 (allogeneic + Ctrl), or 8 (allogeneic + LNA). dLNs were harvested 12 days after skin transplantation from indicated groups imaged in (B) , and absolute cell counts were taken in (C) . dLN cells were plated overnight in complete media and culture supernatants were harvested and subjected to enzyme-linked immunosorbent assay (ELISA) for TNFα (D) and IFNγ (E) . Serum was taken upon sacrifice and subjected to ELISA for transforming growth factor (TGF)-β1 (F) and TGF-β2 (G) . Dot plots of circulating cells double positive for FoxP3 and CD62L, data are gated on CD4 + cells (H) , and quantified in (I) . (J) Hematoxylin eosin stains of grafts excised from mice upon sacrifice. Data are presented as mean ± SEM; n = at least 4 per group. * P
Figure Legend Snippet: Locked nucleic acid (LNA) mitigates draining lymph node (dLN) effector cell and cytokines. Female C57BL/6 mice were given either syn (BL6) or allo (C 3 H) tail skin grafts. Mice receiving allografts were given either LNA-466 (10 mg/kg) or LNA-Ctrl (10 mg/kg) intraperitoneally 1 day before skin transplantation and every third day after that until termination of the study. (A) Survival curve of mice receiving skin transplants and indicated LNA or controls; n = 4 (syngeneic), 7 (allogeneic + Ctrl), or 8 (allogeneic + LNA). dLNs were harvested 12 days after skin transplantation from indicated groups imaged in (B) , and absolute cell counts were taken in (C) . dLN cells were plated overnight in complete media and culture supernatants were harvested and subjected to enzyme-linked immunosorbent assay (ELISA) for TNFα (D) and IFNγ (E) . Serum was taken upon sacrifice and subjected to ELISA for transforming growth factor (TGF)-β1 (F) and TGF-β2 (G) . Dot plots of circulating cells double positive for FoxP3 and CD62L, data are gated on CD4 + cells (H) , and quantified in (I) . (J) Hematoxylin eosin stains of grafts excised from mice upon sacrifice. Data are presented as mean ± SEM; n = at least 4 per group. * P

Techniques Used: Mouse Assay, Transplantation Assay, Enzyme-linked Immunosorbent Assay

Locked nucleic acid (LNA) reduces intragraft effector cells and cytokines. Female C57BL/6 mice were given either syn (BL6) or allo (C 3 H) tail skin grafts. Mice receiving allografts were given either LNA-466 (10 mg/kg) or LNA-Ctrl (10 mg/kg) intraperitoneally 1 day before skin transplantation and every third day after that until termination of the study. Upon rejection, grafts were aseptically excised, minced, and enzymatically digested to dislodge graft-infiltrating cells (GICs). GICs were spun down, culture supernatants were collected, and live cells were used for flow cytometric analysis. Representative dot plots displaying naïve (CD62L Low , CD44 Neg ), memory (CD62L + , CD44 HI ), and effector (CD62L Low , CD44 HI ) cell types gated on CD8 + [ (A) , upper row] and CD4 + [ (A) , lower row] cells. Percentages for CD8 + and CD4 + are quantified in (B,C) , respectively. Dot plots of GICs double-positive for FoxP3 and CD62L, data are gated on CD4 + cells (D) , and quantified in (E) ; n = 7 (allogeneic + Ctrl) or 8 (allogeneic + LNA). GIC supernatants were collected and subjected to enzyme-linked immunosorbent assay for the interrogation of effector cytokines TNFα (F) and IFNγ (G) , as well as anti-inflammatory cytokines transforming growth factor (TGF)-β1 (H) and TGF-β2 (I) . Data are presented as mean ± SEM. * P
Figure Legend Snippet: Locked nucleic acid (LNA) reduces intragraft effector cells and cytokines. Female C57BL/6 mice were given either syn (BL6) or allo (C 3 H) tail skin grafts. Mice receiving allografts were given either LNA-466 (10 mg/kg) or LNA-Ctrl (10 mg/kg) intraperitoneally 1 day before skin transplantation and every third day after that until termination of the study. Upon rejection, grafts were aseptically excised, minced, and enzymatically digested to dislodge graft-infiltrating cells (GICs). GICs were spun down, culture supernatants were collected, and live cells were used for flow cytometric analysis. Representative dot plots displaying naïve (CD62L Low , CD44 Neg ), memory (CD62L + , CD44 HI ), and effector (CD62L Low , CD44 HI ) cell types gated on CD8 + [ (A) , upper row] and CD4 + [ (A) , lower row] cells. Percentages for CD8 + and CD4 + are quantified in (B,C) , respectively. Dot plots of GICs double-positive for FoxP3 and CD62L, data are gated on CD4 + cells (D) , and quantified in (E) ; n = 7 (allogeneic + Ctrl) or 8 (allogeneic + LNA). GIC supernatants were collected and subjected to enzyme-linked immunosorbent assay for the interrogation of effector cytokines TNFα (F) and IFNγ (G) , as well as anti-inflammatory cytokines transforming growth factor (TGF)-β1 (H) and TGF-β2 (I) . Data are presented as mean ± SEM. * P

Techniques Used: Mouse Assay, Transplantation Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

23) Product Images from "Immune checkpoint protein VISTA regulates antitumor immunity by controlling myeloid cell–mediated inflammation and immunosuppression"

Article Title: Immune checkpoint protein VISTA regulates antitumor immunity by controlling myeloid cell–mediated inflammation and immunosuppression

Journal: Cancer immunology research

doi: 10.1158/2326-6066.CIR-18-0489

TLR/MyD88-mediated inflammation promotes the therapeutic efficacy of VISTA blockade. (A) WT or MyD88 KO mice were inoculated with B16-BL6 melanoma cells (200,000) on the flank on day 0. On day +7 mice bearing established tumors (~5 mm in diameter) were treated with VISTA-blocking mAb or isotype control IgG (n=10/group). Three hours later, serum was harvested and examined by ELISA for cytokines. (B) WT and MyD88 KO mice (n=6–7/group) were inoculated with EG7 thymoma cells (150,000). Beginning on day 2 and repeated every 2–3 days, tumor-bearing mice were treated with VISTA-blocking mAb or control IgG. Tumor size was measured by a caliper. Shown are representative results from three independent repeats. (C-E) Naïve WT mice (n=10) were inoculated with B16-BL6 melanoma cells (30,000) on day 0. On day +3, tumor-bearing mice were treated with a peptide vaccine containing CpG, R848, and peptides (details in Methods). Mice were also treated with VISTA-blocking mAb or control IgG every 2–3 days. (C) Tumor size was recorded. In a separate group of mice (n=10), tumor tissues (~6–8 mm in diameter) were harvested. Tumor-infiltrating CD4 + and CD8 + T cells were stimulated with anti-CD3. Percentage and number of IFNγ-expressing (D) CD8 + T cells and (E) CD4 + T cells were examined by flow cytometry. (F,H) To examine the inflammatory TME, we treated B16-BL6 tumor-bearing mice (n= 5–7/group) with the peptide vaccine and VISTA-blocking mAb or control IgG. Tumor tissues were harvested after three hours and homogenized. Cytokines (IL12p40, IL12p70, IL23p19, IL6, TNFα, and IFNβ) and chemokines (CCL4, CCL5, CXCL9, CXCL10) in the homogenates were quantified by ELISA. (G,I) B16-BL6 tumors were harvested from WT and Vsir −/− mice (n=5–7/group) following treatment with the peptide vaccine as described above. Cytokines and chemokines in tumor tissue homogenates were examined by ELISA. Statistical significance of all results was determined by unpaired two-tailed t test. Error bars represent SEM. *p
Figure Legend Snippet: TLR/MyD88-mediated inflammation promotes the therapeutic efficacy of VISTA blockade. (A) WT or MyD88 KO mice were inoculated with B16-BL6 melanoma cells (200,000) on the flank on day 0. On day +7 mice bearing established tumors (~5 mm in diameter) were treated with VISTA-blocking mAb or isotype control IgG (n=10/group). Three hours later, serum was harvested and examined by ELISA for cytokines. (B) WT and MyD88 KO mice (n=6–7/group) were inoculated with EG7 thymoma cells (150,000). Beginning on day 2 and repeated every 2–3 days, tumor-bearing mice were treated with VISTA-blocking mAb or control IgG. Tumor size was measured by a caliper. Shown are representative results from three independent repeats. (C-E) Naïve WT mice (n=10) were inoculated with B16-BL6 melanoma cells (30,000) on day 0. On day +3, tumor-bearing mice were treated with a peptide vaccine containing CpG, R848, and peptides (details in Methods). Mice were also treated with VISTA-blocking mAb or control IgG every 2–3 days. (C) Tumor size was recorded. In a separate group of mice (n=10), tumor tissues (~6–8 mm in diameter) were harvested. Tumor-infiltrating CD4 + and CD8 + T cells were stimulated with anti-CD3. Percentage and number of IFNγ-expressing (D) CD8 + T cells and (E) CD4 + T cells were examined by flow cytometry. (F,H) To examine the inflammatory TME, we treated B16-BL6 tumor-bearing mice (n= 5–7/group) with the peptide vaccine and VISTA-blocking mAb or control IgG. Tumor tissues were harvested after three hours and homogenized. Cytokines (IL12p40, IL12p70, IL23p19, IL6, TNFα, and IFNβ) and chemokines (CCL4, CCL5, CXCL9, CXCL10) in the homogenates were quantified by ELISA. (G,I) B16-BL6 tumors were harvested from WT and Vsir −/− mice (n=5–7/group) following treatment with the peptide vaccine as described above. Cytokines and chemokines in tumor tissue homogenates were examined by ELISA. Statistical significance of all results was determined by unpaired two-tailed t test. Error bars represent SEM. *p

Techniques Used: Mouse Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Two Tailed Test

24) Product Images from "Neuron-Specific Vitamin D Signaling Attenuates Microglia Activation and CNS Autoimmunity"

Article Title: Neuron-Specific Vitamin D Signaling Attenuates Microglia Activation and CNS Autoimmunity

Journal: Frontiers in Neurology

doi: 10.3389/fneur.2020.00019

Vitamin D signaling in primary neurons reduces pro-inflammatory cytokine production by microglia. (A) Primary neurons were isolated from the hippocampus of post-natal day 1 mice. Red—β-tubulin; Blue—DAPI. (B) Primary microglia stained with Iba1 (green) and DAPI (blue). The primary neurons were cultured with calcitriol and the media was collected and transferred to the primary microglia. After 24 h, the microglia were washed, activated with LPS, supernatants collected, and IL-6 (C) , IL-1β (D) , and TNFα (E) were measured in the supernatants by ELISA. * p
Figure Legend Snippet: Vitamin D signaling in primary neurons reduces pro-inflammatory cytokine production by microglia. (A) Primary neurons were isolated from the hippocampus of post-natal day 1 mice. Red—β-tubulin; Blue—DAPI. (B) Primary microglia stained with Iba1 (green) and DAPI (blue). The primary neurons were cultured with calcitriol and the media was collected and transferred to the primary microglia. After 24 h, the microglia were washed, activated with LPS, supernatants collected, and IL-6 (C) , IL-1β (D) , and TNFα (E) were measured in the supernatants by ELISA. * p

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

Vitamin D induces IL-34 in neurons, and IL-34 induces an anti-inflammatory phenotype in microglia. (A) IL34 transcripts were measured by real-time PCR in primary neurons treated with calcitriol. (B) IL-34 was measured by ELISA in the supernatants of primary neurons treated with calcitriol. Hmox1 (C) and Arg1 (D) transcripts were measured by real-time PCR in primary microglia treated with IL-34 and activated with LPS. IL-6 (E) , IL-1β (F) , and TNFα (G) were measured in the supernatants of primary microglia treated with IL-34 and activated with LPS. (H) NCM from calcitriol-treated primary neurons was treated with anti-IL34, transferred to primary microglia, and IL-6 was measured by ELISA. * p
Figure Legend Snippet: Vitamin D induces IL-34 in neurons, and IL-34 induces an anti-inflammatory phenotype in microglia. (A) IL34 transcripts were measured by real-time PCR in primary neurons treated with calcitriol. (B) IL-34 was measured by ELISA in the supernatants of primary neurons treated with calcitriol. Hmox1 (C) and Arg1 (D) transcripts were measured by real-time PCR in primary microglia treated with IL-34 and activated with LPS. IL-6 (E) , IL-1β (F) , and TNFα (G) were measured in the supernatants of primary microglia treated with IL-34 and activated with LPS. (H) NCM from calcitriol-treated primary neurons was treated with anti-IL34, transferred to primary microglia, and IL-6 was measured by ELISA. * p

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

25) Product Images from "Exercise Increases MAIT Cell Cytokine Expression but not Activation or Homing Markers"

Article Title: Exercise Increases MAIT Cell Cytokine Expression but not Activation or Homing Markers

Journal: Medicine and science in sports and exercise

doi: 10.1249/MSS.0000000000001780

MAIT cells expressing intracellular A) TNFα, B) INFɣ, and C) IL-17 following 4 hours of stimulation with 2 ng/μL of phorbol 12-myristate 13-acetate and 1 ug/μL ionomycin at baseline, 0 h and 1 h after sub-maximal aerobic exercise for n=17. The proportion of MAIT cell expressing each cytokine are displayed in the left panel and the absolute cell counts are displayed in the right panel. * p
Figure Legend Snippet: MAIT cells expressing intracellular A) TNFα, B) INFɣ, and C) IL-17 following 4 hours of stimulation with 2 ng/μL of phorbol 12-myristate 13-acetate and 1 ug/μL ionomycin at baseline, 0 h and 1 h after sub-maximal aerobic exercise for n=17. The proportion of MAIT cell expressing each cytokine are displayed in the left panel and the absolute cell counts are displayed in the right panel. * p

Techniques Used: Expressing

26) Product Images from "Immunosuppression via adenosine receptor activation by adenosine monophosphate released from apoptotic cells"

Article Title: Immunosuppression via adenosine receptor activation by adenosine monophosphate released from apoptotic cells

Journal: eLife

doi: 10.7554/eLife.02172

Immunosuppressive function of Pannexin 1 and A2a adenosine receptor in a mouse peritonitis model. ( A ) Mouse resident peritoneal macrophages were incubated with 50 μg/ml zymosan A in the presence (+) or absence (−) of 100 μM AMP for 16 hr. The TNFα and MIP-2 levels in the culture supernatant were determined in triplicate by ELISA, and the average values are plotted with S.D. (bars). The experiments were performed at least twice, and representative data are shown. ( B ) Peritoneal cells were collected at 3 hr after the injection of zymosan A (250 mg/kg), and stained with anti-Ly6B.2 and anti-Ly6G antibodies (left panel). Some of the Ly6B.2 and Ly6G double-positive cells (circled in the left panel) were stained with Cy5-labeled Annexin V (right panel). ( C and D ) After zymosan injection, peritoneal lavage fluid was collected at 2 hr (n = 5) or 6 hr (n = 7) of Panx1 +/+ , and at 2 hr (n = 5) or 6 hr (n = 10) of Panx1 −/− littermates ( C ), or 2 hr (n = 10) or 6 hr (n = 6) of Adora2a +/+ or +/− , and at 2 hr (n = 8) or 6 hr (n = 6) of Adora2a −/− mice ( D ). The TNFα and MIP-2 levels in the fluids were determined by ELISA. The Student’s t test was used for the statistical analysis, and the p values are shown. DOI: http://dx.doi.org/10.7554/eLife.02172.011
Figure Legend Snippet: Immunosuppressive function of Pannexin 1 and A2a adenosine receptor in a mouse peritonitis model. ( A ) Mouse resident peritoneal macrophages were incubated with 50 μg/ml zymosan A in the presence (+) or absence (−) of 100 μM AMP for 16 hr. The TNFα and MIP-2 levels in the culture supernatant were determined in triplicate by ELISA, and the average values are plotted with S.D. (bars). The experiments were performed at least twice, and representative data are shown. ( B ) Peritoneal cells were collected at 3 hr after the injection of zymosan A (250 mg/kg), and stained with anti-Ly6B.2 and anti-Ly6G antibodies (left panel). Some of the Ly6B.2 and Ly6G double-positive cells (circled in the left panel) were stained with Cy5-labeled Annexin V (right panel). ( C and D ) After zymosan injection, peritoneal lavage fluid was collected at 2 hr (n = 5) or 6 hr (n = 7) of Panx1 +/+ , and at 2 hr (n = 5) or 6 hr (n = 10) of Panx1 −/− littermates ( C ), or 2 hr (n = 10) or 6 hr (n = 6) of Adora2a +/+ or +/− , and at 2 hr (n = 8) or 6 hr (n = 6) of Adora2a −/− mice ( D ). The TNFα and MIP-2 levels in the fluids were determined by ELISA. The Student’s t test was used for the statistical analysis, and the p values are shown. DOI: http://dx.doi.org/10.7554/eLife.02172.011

Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay, Injection, Staining, Labeling, Mouse Assay

27) 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

28) 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

29) Product Images from "A Novel Role of Numb as A Regulator of Pro-inflammatory Cytokine Production in Macrophages in Response to Toll-like Receptor 4"

Article Title: A Novel Role of Numb as A Regulator of Pro-inflammatory Cytokine Production in Macrophages in Response to Toll-like Receptor 4

Journal: Scientific Reports

doi: 10.1038/srep12784

Expression of cytokines in macrophages lacking Numb after LPS stimulation. ( A – D ) Secretion levels of TNFα ( A ), IL-6 ( B ), IL-12p70 ( C ), and IL-10 ( D ) from control- (open bars) or sh Numb -infected macrophages (closed bars) following LPS stimulation were measured by ELISA. ( E – H ) mRNA levels of Tnfα ( E ), Il6 ( F ), Il12p40 ( G ), and Il10 ( H ) from control- or sh Numb -infected macrophages following LPS stimulation were measured by qPCR. Data are the mean ± SEM from the representative experiment of two independent experiments performed in triplicates.
Figure Legend Snippet: Expression of cytokines in macrophages lacking Numb after LPS stimulation. ( A – D ) Secretion levels of TNFα ( A ), IL-6 ( B ), IL-12p70 ( C ), and IL-10 ( D ) from control- (open bars) or sh Numb -infected macrophages (closed bars) following LPS stimulation were measured by ELISA. ( E – H ) mRNA levels of Tnfα ( E ), Il6 ( F ), Il12p40 ( G ), and Il10 ( H ) from control- or sh Numb -infected macrophages following LPS stimulation were measured by qPCR. Data are the mean ± SEM from the representative experiment of two independent experiments performed in triplicates.

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

Effect of silencing Numb on activation of MAPK and NF-κB pathways in activated macrophages and the interaction of Numb and Itch. ( A ) After stimulation with LPS for indicated times, phosphorylation levels of NF-κB p65, p38, ERK1/2, and JNK MAPK from GFP + macrophages were analyzed. Data are representative of two independent experiments. ( B , C ) GFP + macrophages containing control (open bars) or sh Numb (closed bars) vectors were stimulated with LPS for 1 hr prior to treatment with DMSO or with actinomycin D to inhibit mRNA synthesis. The relative amount of remaining Tnfα mRNA was measured by qPCR. Half-life of Tnfα mRNA was calculated using linear regression line equations and shown in ( C ). Data are the mean ± SEM from representative of two independent experiments performed in triplicates. ( D ) RAW264.7 cell line was transiently transfected with the control plasmid or pCIneoHA-Numb and stimulated with LPS as indicated. p38 MAPK and NF-κB p65 were detected by immunoblotting. ( E ) RAW264.7 cell line transfected with the control plasmid or pCIneoHA-Numb were stimulated with LPS for 1 hr. The amount of TNFα was measured by ELISA. ( F ) Co-immunoprecipitation of endogenous Numb from unstimulated or LPS-stimulated macrophages was analyzed by immunoblotting. ( G ) Expression of Itch in macrophages containing control or sh Numb vector was detected by immunoblotting following LPS stimulation. ( H ) RAW264.7 cell line transfected with the control plasmid or pCIneoHA-Numb were stimulated with LPS for the times indicated and the level of Itch was detected by immunoblotting.
Figure Legend Snippet: Effect of silencing Numb on activation of MAPK and NF-κB pathways in activated macrophages and the interaction of Numb and Itch. ( A ) After stimulation with LPS for indicated times, phosphorylation levels of NF-κB p65, p38, ERK1/2, and JNK MAPK from GFP + macrophages were analyzed. Data are representative of two independent experiments. ( B , C ) GFP + macrophages containing control (open bars) or sh Numb (closed bars) vectors were stimulated with LPS for 1 hr prior to treatment with DMSO or with actinomycin D to inhibit mRNA synthesis. The relative amount of remaining Tnfα mRNA was measured by qPCR. Half-life of Tnfα mRNA was calculated using linear regression line equations and shown in ( C ). Data are the mean ± SEM from representative of two independent experiments performed in triplicates. ( D ) RAW264.7 cell line was transiently transfected with the control plasmid or pCIneoHA-Numb and stimulated with LPS as indicated. p38 MAPK and NF-κB p65 were detected by immunoblotting. ( E ) RAW264.7 cell line transfected with the control plasmid or pCIneoHA-Numb were stimulated with LPS for 1 hr. The amount of TNFα was measured by ELISA. ( F ) Co-immunoprecipitation of endogenous Numb from unstimulated or LPS-stimulated macrophages was analyzed by immunoblotting. ( G ) Expression of Itch in macrophages containing control or sh Numb vector was detected by immunoblotting following LPS stimulation. ( H ) RAW264.7 cell line transfected with the control plasmid or pCIneoHA-Numb were stimulated with LPS for the times indicated and the level of Itch was detected by immunoblotting.

Techniques Used: Activation Assay, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Expressing

Expression of pro-inflammatory cytokines in macrophages lacking both Numb and Notch signaling after LPS stimulation. ( A – F ) GFP + macrophages containing control (open bars) or sh Numb (closed bars) vectors were pretreated with DAPT for 1 hr prior to LPS stimulation. The levels of intracellular TNFα, IL-6, and IL-12 were determined by flow cytometry ( A – C ) or ELISA ( D – F ). N.D. = not detectable. ( G ) Expression level of Rbpj κ from macrophages transduced with si Rbpj κ, or scrambled siRNA as control, was measured by qPCR. ( H – J ) Expression levels of TNFα, IL-6, and IL-12p70 from GFP + macrophages containing control (open bars) or sh Numb (closed bars) vectors which were transfected with scramble siRNA or si Rbpjk were measured by ELISA. N.D. = not detectable.
Figure Legend Snippet: Expression of pro-inflammatory cytokines in macrophages lacking both Numb and Notch signaling after LPS stimulation. ( A – F ) GFP + macrophages containing control (open bars) or sh Numb (closed bars) vectors were pretreated with DAPT for 1 hr prior to LPS stimulation. The levels of intracellular TNFα, IL-6, and IL-12 were determined by flow cytometry ( A – C ) or ELISA ( D – F ). N.D. = not detectable. ( G ) Expression level of Rbpj κ from macrophages transduced with si Rbpj κ, or scrambled siRNA as control, was measured by qPCR. ( H – J ) Expression levels of TNFα, IL-6, and IL-12p70 from GFP + macrophages containing control (open bars) or sh Numb (closed bars) vectors which were transfected with scramble siRNA or si Rbpjk were measured by ELISA. N.D. = not detectable.

Techniques Used: Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Transduction, Real-time Polymerase Chain Reaction, Transfection

30) Product Images from "Reduced gut microbiome protects from alcohol-induced neuroinflammation and alters intestinal and brain inflammasome expression"

Article Title: Reduced gut microbiome protects from alcohol-induced neuroinflammation and alters intestinal and brain inflammasome expression

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-018-1328-9

Antibiotic treatment protects from alcohol-induced inflammatory cytokine expression in the cortex. a Serum TNFα and IL-6 were measured by ELISA. b Expression levels of proinflammatory cytokines Tnfα , Mcp1 , Hmgb1 , Il-17 , Il-23 , Il-6 , and Cox2 were measured from the cortex of pair-fed (PF) or alcohol-fed (EtOH) mice with or without daily antibiotic treatment (Abx). Data are mean ± SEM, n = 5–10 mice/group. * p
Figure Legend Snippet: Antibiotic treatment protects from alcohol-induced inflammatory cytokine expression in the cortex. a Serum TNFα and IL-6 were measured by ELISA. b Expression levels of proinflammatory cytokines Tnfα , Mcp1 , Hmgb1 , Il-17 , Il-23 , Il-6 , and Cox2 were measured from the cortex of pair-fed (PF) or alcohol-fed (EtOH) mice with or without daily antibiotic treatment (Abx). Data are mean ± SEM, n = 5–10 mice/group. * p

Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Mouse Assay

Alcohol-induced small intestinal inflammation is reduced with gut bacterial load reduction. a Expression of proinflammatory cytokines Tnfα , Mcp1 , Hmgb1 , Il-17 , and Il-23 was measured from the small intestine of pair-fed (PF) or alcohol-fed (EtOH) mice with or without daily antibiotic treatment (Abx). b Expression of inflammasome components Nlrp3 , Asc , and Casp1 as well as the cytokines Il-1β and Il-18 were measured by qPCR. Data are mean ± SEM, n = 5–10 mice/group. * p
Figure Legend Snippet: Alcohol-induced small intestinal inflammation is reduced with gut bacterial load reduction. a Expression of proinflammatory cytokines Tnfα , Mcp1 , Hmgb1 , Il-17 , and Il-23 was measured from the small intestine of pair-fed (PF) or alcohol-fed (EtOH) mice with or without daily antibiotic treatment (Abx). b Expression of inflammasome components Nlrp3 , Asc , and Casp1 as well as the cytokines Il-1β and Il-18 were measured by qPCR. Data are mean ± SEM, n = 5–10 mice/group. * p

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

31) 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

32) Product Images from "Immunometabolic Cross-Talk and Regulation of Endocrine and Metabolic Functions: Saturated fatty acid combined with lipopolysaccharide stimulates a strong inflammatory response in hepatocytes in vivo and in vitro"

Article Title: Immunometabolic Cross-Talk and Regulation of Endocrine and Metabolic Functions: Saturated fatty acid combined with lipopolysaccharide stimulates a strong inflammatory response in hepatocytes in vivo and in vitro

Journal: American Journal of Physiology - Endocrinology and Metabolism

doi: 10.1152/ajpendo.00015.2018

Hepatocyte and RAW264.7 macrophages were cultured separately ( A and B ; E and F ) or together ( C and G ) and exposed to LPS, PA, or LPS plus PA for 24 h. After the treatment, IL-6 ( A–C ) and TNFα ( E–G ) in culture medium were quantified using ELISA. The amount of IL-6 ( D ) and TNFα ( H ) secretion by hepatocytes, RAW264.7 macrophages or coculture of hepatocytes and RAW264.7 macrophages was compared. Hepatocytes cultured alone or cultured with RAW264.7 macrophages in the coculture system were exposed to LPS, PA, or LPS plus PA for 24 h. After the treatment, IL-6 mRNA was isolated and compared between hepatocytes cultured alone and those in the coculture ( I ) and between RAW264.7 macrophages cultured alone and those in the coculture ( J ). The data (mean ± SD) presented are representative of three experiments with similar results. PA, palmitic acid.
Figure Legend Snippet: Hepatocyte and RAW264.7 macrophages were cultured separately ( A and B ; E and F ) or together ( C and G ) and exposed to LPS, PA, or LPS plus PA for 24 h. After the treatment, IL-6 ( A–C ) and TNFα ( E–G ) in culture medium were quantified using ELISA. The amount of IL-6 ( D ) and TNFα ( H ) secretion by hepatocytes, RAW264.7 macrophages or coculture of hepatocytes and RAW264.7 macrophages was compared. Hepatocytes cultured alone or cultured with RAW264.7 macrophages in the coculture system were exposed to LPS, PA, or LPS plus PA for 24 h. After the treatment, IL-6 mRNA was isolated and compared between hepatocytes cultured alone and those in the coculture ( I ) and between RAW264.7 macrophages cultured alone and those in the coculture ( J ). The data (mean ± SD) presented are representative of three experiments with similar results. PA, palmitic acid.

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

33) 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

34) Product Images from "Prevention of the ?-amyloid peptide-induced inflammatory process by inhibition of double-stranded RNA-dependent protein kinase in primary murine mixed co-cultures"

Article Title: Prevention of the ?-amyloid peptide-induced inflammatory process by inhibition of double-stranded RNA-dependent protein kinase in primary murine mixed co-cultures

Journal: Journal of Neuroinflammation

doi: 10.1186/1742-2094-8-72

Production (A) and release (B) of cytokines in murine astrocyte/neuron/microglia co-cultures . These co-cultures were pretreated with 210 nM C16 or its DMSO vehicle and exposed or not to 20 μM Aβ42 for 72 h in serum-free medium. TNFα, IL-1β and IL-6 were assessed by ELISA. Data are expressed as mean ± SEM of pg/mg protein for production and pg/mL for release (n = 5 in duplicate). *p
Figure Legend Snippet: Production (A) and release (B) of cytokines in murine astrocyte/neuron/microglia co-cultures . These co-cultures were pretreated with 210 nM C16 or its DMSO vehicle and exposed or not to 20 μM Aβ42 for 72 h in serum-free medium. TNFα, IL-1β and IL-6 were assessed by ELISA. Data are expressed as mean ± SEM of pg/mg protein for production and pg/mL for release (n = 5 in duplicate). *p

Techniques Used: Enzyme-linked Immunosorbent Assay

35) 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

36) Product Images from "CXCR2-dependent accumulation of tumor-associated neutrophils regulates T-cell immunity in pancreatic ductal adenocarcinoma"

Article Title: CXCR2-dependent accumulation of tumor-associated neutrophils regulates T-cell immunity in pancreatic ductal adenocarcinoma

Journal: Cancer immunology research

doi: 10.1158/2326-6066.CIR-16-0188

TNFα and KRAS/MEK inhibition induce CXCL5 expression in a NF-κB dependent manner (A) Heatmap of relative CXCR2 ligand expression by YFP + pancreatic and YFP − stromal cells in 4–6 months old CY, PCY, and KCY mice ( n = 3 per group). (B) Fold change of Cxcl5 and Csf2 (GM-CSF) expression in 4662 PDA cells treated with 10µM U0126 (MEK inhibitor) compared to DMSO. Graphs show mean ± s.d. of 3 independent experiments. *, P ≤ 0.05; **, P ≤ 0.01 (unpaired t -test). (C) Fold change of Cxcl5 and Kras expression in Kras siRNA–treated compared to control siRNA–treated 4662 PDA cells. Graph shows mean ± s.d. of 3 independent experiments. ***, P ≤ 0.001; ****, P ≤ 0.0001 (unpaired t -test). (D) Fold change of Cxcl5 and Yap1 expression in si- Yap1 treated compared to si-control treated 4662 PDA cells. Graph shows mean ± s.d. of 3 independent experiments. ****, P ≤ 0.0001 (unpaired t -test). (E) CXCL5 protein level in the supernatant of 4662 PDA cells treated with the indicated combinations of DMSO control, 10ng/mL TNFα, 10µM U0126 (MEK inhibitor), or 20µM Wedeloactone (NF-κB inhibitor). Graph shows mean ± s.d. of 3 independent experiments. *, P ≤ 0.05; **, P ≤ 0.01 (1-way ANOVA, Holm-Sidak’s multiple comparison test).
Figure Legend Snippet: TNFα and KRAS/MEK inhibition induce CXCL5 expression in a NF-κB dependent manner (A) Heatmap of relative CXCR2 ligand expression by YFP + pancreatic and YFP − stromal cells in 4–6 months old CY, PCY, and KCY mice ( n = 3 per group). (B) Fold change of Cxcl5 and Csf2 (GM-CSF) expression in 4662 PDA cells treated with 10µM U0126 (MEK inhibitor) compared to DMSO. Graphs show mean ± s.d. of 3 independent experiments. *, P ≤ 0.05; **, P ≤ 0.01 (unpaired t -test). (C) Fold change of Cxcl5 and Kras expression in Kras siRNA–treated compared to control siRNA–treated 4662 PDA cells. Graph shows mean ± s.d. of 3 independent experiments. ***, P ≤ 0.001; ****, P ≤ 0.0001 (unpaired t -test). (D) Fold change of Cxcl5 and Yap1 expression in si- Yap1 treated compared to si-control treated 4662 PDA cells. Graph shows mean ± s.d. of 3 independent experiments. ****, P ≤ 0.0001 (unpaired t -test). (E) CXCL5 protein level in the supernatant of 4662 PDA cells treated with the indicated combinations of DMSO control, 10ng/mL TNFα, 10µM U0126 (MEK inhibitor), or 20µM Wedeloactone (NF-κB inhibitor). Graph shows mean ± s.d. of 3 independent experiments. *, P ≤ 0.05; **, P ≤ 0.01 (1-way ANOVA, Holm-Sidak’s multiple comparison test).

Techniques Used: Inhibition, Expressing, Mouse Assay

37) Product Images from "Autocrine TNF? Signaling Renders Human Cancer Cells Susceptible to Smac-Mimetic-Induced Apoptosis"

Article Title: Autocrine TNF? Signaling Renders Human Cancer Cells Susceptible to Smac-Mimetic-Induced Apoptosis

Journal:

doi: 10.1016/j.ccr.2007.08.029

Sirna Candidate Screen Implicates TNFα Signaling as a Requirement for Smac-Mimetic-Mediated Apoptosis
Figure Legend Snippet: Sirna Candidate Screen Implicates TNFα Signaling as a Requirement for Smac-Mimetic-Mediated Apoptosis

Techniques Used:

Autocrine TNFα Signaling Is Required for Smac-Mimetic-Mediated Apoptosis
Figure Legend Snippet: Autocrine TNFα Signaling Is Required for Smac-Mimetic-Mediated Apoptosis

Techniques Used:

38) Product Images from "G2019s LRRK2 promotes mitochondrial fission and increases TNFα-mediated neuroinflammation responses"

Article Title: G2019s LRRK2 promotes mitochondrial fission and increases TNFα-mediated neuroinflammation responses

Journal: Animal Cells and Systems

doi: 10.1080/19768354.2019.1585948

Treatment with LRRK2 kinase inhibitor suppresses the TNFα release in Myc-G2019S LRRK2-expressed BV2 cells. (A) After 6 h transfection, Vector or myc-GS cells were incubated with or without GSK2578215A in the fresh growth medium for 36 h. To validate the LRRK2 transfection and inhibition by GSK2578215A, cell lysates were analyzed using anti-myc and pS1292 antibody. (B) The culture medium was collected and concentrated using a 3 K filtration tube, and the medium was subjected to mouse TNFα ELISA. Two-way analysis of variance (ANOVA) using Tukey’s multiple comparison test was used for the statistical analysis; **** p
Figure Legend Snippet: Treatment with LRRK2 kinase inhibitor suppresses the TNFα release in Myc-G2019S LRRK2-expressed BV2 cells. (A) After 6 h transfection, Vector or myc-GS cells were incubated with or without GSK2578215A in the fresh growth medium for 36 h. To validate the LRRK2 transfection and inhibition by GSK2578215A, cell lysates were analyzed using anti-myc and pS1292 antibody. (B) The culture medium was collected and concentrated using a 3 K filtration tube, and the medium was subjected to mouse TNFα ELISA. Two-way analysis of variance (ANOVA) using Tukey’s multiple comparison test was used for the statistical analysis; **** p

Techniques Used: Transfection, Plasmid Preparation, Incubation, Inhibition, Filtration, Enzyme-linked Immunosorbent Assay

Protein levels of pro- or active-TNFα in brains of G2019S transgenic mice. (A) Brain lysates from littermates and GS TG mice were subjected to Western blot analysis. Arrowhead or empty-arrowhead denotes pro- or active-TNFα, respectively. (B-C) TNFα protein was normalized to the levels of β-actin. Student’s T-test with two-tailed analysis was used for the statistical analysis; * p
Figure Legend Snippet: Protein levels of pro- or active-TNFα in brains of G2019S transgenic mice. (A) Brain lysates from littermates and GS TG mice were subjected to Western blot analysis. Arrowhead or empty-arrowhead denotes pro- or active-TNFα, respectively. (B-C) TNFα protein was normalized to the levels of β-actin. Student’s T-test with two-tailed analysis was used for the statistical analysis; * p

Techniques Used: Transgenic Assay, Mouse Assay, Western Blot, Two Tailed Test

39) Product Images from "Early intervention with a small molecule inhibitor for tumor nefosis factor-? prevents cognitive deficits in a triple transgenic mouse model of Alzheimer's disease"

Article Title: Early intervention with a small molecule inhibitor for tumor nefosis factor-? prevents cognitive deficits in a triple transgenic mouse model of Alzheimer's disease

Journal: Journal of Neuroinflammation

doi: 10.1186/1742-2094-9-99

Tumor necrosis factor-α gene and protein expression in the 3 × Tg mouse. (A) Fold-change (2^(− δ δCt)) is the normalized gene expression (2^(−δ Ct)) in the test sample (3 × Tg treated with Thal or 3,6′-DT) divided by the normalized gene expression (2^(−δ Ct)) in the control sample (vehicle-treated 3 × Tg). Fold-change values of less than one indicate a negative- or downregulation. Both Thal and 3,6′-DT downregulated TNFα gene expression but the value was significant only in the 3,6′-DT group ( P = 0.033). (B) TNFα protein levels are doubled in the cortex of 3 × Tg mice compared with Non-Tg mice. 3,6′-DT, but not Thal, reduced TNFα protein levels near to Non-Tg levels in 3 × Tg mice. n = 5 to 8 per group. One-way analysis of variance ( P = 0.062, * P
Figure Legend Snippet: Tumor necrosis factor-α gene and protein expression in the 3 × Tg mouse. (A) Fold-change (2^(− δ δCt)) is the normalized gene expression (2^(−δ Ct)) in the test sample (3 × Tg treated with Thal or 3,6′-DT) divided by the normalized gene expression (2^(−δ Ct)) in the control sample (vehicle-treated 3 × Tg). Fold-change values of less than one indicate a negative- or downregulation. Both Thal and 3,6′-DT downregulated TNFα gene expression but the value was significant only in the 3,6′-DT group ( P = 0.033). (B) TNFα protein levels are doubled in the cortex of 3 × Tg mice compared with Non-Tg mice. 3,6′-DT, but not Thal, reduced TNFα protein levels near to Non-Tg levels in 3 × Tg mice. n = 5 to 8 per group. One-way analysis of variance ( P = 0.062, * P

Techniques Used: Expressing, Mouse Assay

Thalidomide and 3,6 ′ -dithiothalidomide attenuate lipopolysaccharide-induced increase in tumor necrosis factor-α. BV2 cells were treated with 1 ng/mL LPS ± Thal or 3,6′-DT for 24 h. Initial studies in BV2 cells demonstrate that both Thal and 3,6′-DT are effective at attenuating LPS-induced TNFα release into culture media. 3,6′-DT has an IC 50 value of approximately 1 μM whereas the IC 50 for Thal is > 10 μM. n = 6 per group. One-way analysis of variance: P
Figure Legend Snippet: Thalidomide and 3,6 ′ -dithiothalidomide attenuate lipopolysaccharide-induced increase in tumor necrosis factor-α. BV2 cells were treated with 1 ng/mL LPS ± Thal or 3,6′-DT for 24 h. Initial studies in BV2 cells demonstrate that both Thal and 3,6′-DT are effective at attenuating LPS-induced TNFα release into culture media. 3,6′-DT has an IC 50 value of approximately 1 μM whereas the IC 50 for Thal is > 10 μM. n = 6 per group. One-way analysis of variance: P

Techniques Used:

Tumor necrosis factor-α protein from splenocytes isolated from Non-Tg and 3 × Tg mice. Splenocytes isolated from Non-Tg and 3 × Tg mice were cultured for 24 hours and TNFα levels measured by ELISA. 3,6′-DT-treated 3 × Tg mice had significantly reduced TNFα secretion compared with vehicle-treated 3 × Tg mice. One-way analysis of variance: P = 0.0184. * P
Figure Legend Snippet: Tumor necrosis factor-α protein from splenocytes isolated from Non-Tg and 3 × Tg mice. Splenocytes isolated from Non-Tg and 3 × Tg mice were cultured for 24 hours and TNFα levels measured by ELISA. 3,6′-DT-treated 3 × Tg mice had significantly reduced TNFα secretion compared with vehicle-treated 3 × Tg mice. One-way analysis of variance: P = 0.0184. * P

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

3,6 ′ -dithiothalidomide reduces tumor necrosis factor-α in central nervous system-infiltrating myelomonocytic/granulocytic leukocytes. (A) Non-Tg mice; (B) 3 × Tg mice; (C) 3,6′-DT mice. CNS-infiltrating leukocytes from whole mouse brains (n = 3 to 4 per group) were isolated and evaluated for the presence of CD45 hi and myelomonocytes/granulocytes (CD45 hi /Gr1 + /Ly6G hi ) by cell surface staining and flow cytometric analyses. There was a trend towards an increased percentage of CD45 hi cells and CD45 hi /Gr1 + /Ly6G hi (not significant) in 3 × Tg mice (B) relative to Non-Tg (A) mice. 3,6′-DT (C) did not alter the percentages of these cell populations. TNFα expression in the total CD45 hi population and in the granulocyte population was increased in 3 × Tg mice relative to Non-Tg mice. 3,6′-DT treatment did not reduce TNFα expression in the total CD45 hi population but specifically reduced TNFα expression in the CD45 hi /Gr1 + /Ly6G hi population ( P = 0.031). Flow cytometry results were quantified and are presented in Table 1 .
Figure Legend Snippet: 3,6 ′ -dithiothalidomide reduces tumor necrosis factor-α in central nervous system-infiltrating myelomonocytic/granulocytic leukocytes. (A) Non-Tg mice; (B) 3 × Tg mice; (C) 3,6′-DT mice. CNS-infiltrating leukocytes from whole mouse brains (n = 3 to 4 per group) were isolated and evaluated for the presence of CD45 hi and myelomonocytes/granulocytes (CD45 hi /Gr1 + /Ly6G hi ) by cell surface staining and flow cytometric analyses. There was a trend towards an increased percentage of CD45 hi cells and CD45 hi /Gr1 + /Ly6G hi (not significant) in 3 × Tg mice (B) relative to Non-Tg (A) mice. 3,6′-DT (C) did not alter the percentages of these cell populations. TNFα expression in the total CD45 hi population and in the granulocyte population was increased in 3 × Tg mice relative to Non-Tg mice. 3,6′-DT treatment did not reduce TNFα expression in the total CD45 hi population but specifically reduced TNFα expression in the CD45 hi /Gr1 + /Ly6G hi population ( P = 0.031). Flow cytometry results were quantified and are presented in Table 1 .

Techniques Used: Mouse Assay, Isolation, Staining, Flow Cytometry, Expressing, Cytometry

40) Product Images from "The role of microglial P2X7: modulation of cell death and cytokine release"

Article Title: The role of microglial P2X7: modulation of cell death and cytokine release

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-017-0904-8

Secretome of microglia treated with P2X7 antagonist upon pro-inflammatory stimulation. Microglia were incubated with 1 μM P2X7 antagonist A-804598 for an hour before exposure to 100 ng/ml LPS or 100 ng/ml LPS plus 10 ng/ml IFNγ for 22 h in the presence or absence of 380 μM BzATP for additional 2 h. a Heat map and hierarchical clustering of detectable 30 proteins released from microglia measured by Luminex multiplex. b – e Histogram of IL1β ( b ), IL1α ( c ), TNFα ( d ), and IL6 ( e ) release obtained from a . Data are shown as mean + SD of triplicates. Experiment repeated twice independently. One-way ANOVA followed by Tukey’s post hoc test. **** P
Figure Legend Snippet: Secretome of microglia treated with P2X7 antagonist upon pro-inflammatory stimulation. Microglia were incubated with 1 μM P2X7 antagonist A-804598 for an hour before exposure to 100 ng/ml LPS or 100 ng/ml LPS plus 10 ng/ml IFNγ for 22 h in the presence or absence of 380 μM BzATP for additional 2 h. a Heat map and hierarchical clustering of detectable 30 proteins released from microglia measured by Luminex multiplex. b – e Histogram of IL1β ( b ), IL1α ( c ), TNFα ( d ), and IL6 ( e ) release obtained from a . Data are shown as mean + SD of triplicates. Experiment repeated twice independently. One-way ANOVA followed by Tukey’s post hoc test. **** P

Techniques Used: Incubation, Luminex, Multiplex Assay

Related Articles

Flow Cytometry:

Article Title: Amphiregulin activates regulatory T lymphocytes and suppresses CD8+ T cell-mediated anti-tumor response in hepatocellular carcinoma cells
Article Snippet: .. Then cells were stained at room temperature for 30 min with Alexa Fluor® 488 anti-IFN-γ (XMG1.2, Biolegend), Alexa Fluor® 647 anti-TNF-α (MP6-XT22, Biolegend), APC anti-perforin (eBioOMAK-D, eBioscience) and PE anti-granzyme B (NGZB, eBioscience) respectively before analysis on a BD LSRII flow cytometer. .. For Foxp3 staining, Foxp3 fix/perm buffer (Biolegend) set was used according to the manufacturer's instruction.

Luciferase:

Article Title: The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response
Article Snippet: .. Reagents and antibodies Poly(I:C), LPS, R837, PGN, chloroquine (Invivogen); TNFα, IL-1β (R & D Systems); D-galactosamine (Sigma); GM-CSF (peproTech); ELISA kit for murine IFN-β, TNFα, IL-6 (Biolegend); dual-specific luciferase assay kit (Promega); EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); lysosome isolation kit (sigma); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (OriGene), phospho-IBα (Cell Signaling Technology), TLR3 (IMGENEX); rabbit monoclonal antibodies against TLR3, phospho-IRF3 (Cell Signaling Technology); rabbit polyclonal antibodies against IRF3 (Santa Cruz Biotechnology), TRIF (Cell Signaling Technology), TBK1 and phospho-TBK1(EPITOMICS), Flag (MBL); Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody, Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Invitrogen) were purchased from the indicated manufacturers. .. Mouse antisera against human Mex3B were raised against recombinant human Mex3B fragments containing aa236-517 and aa1-517, respectively.

Isolation:

Article Title: The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response
Article Snippet: .. Reagents and antibodies Poly(I:C), LPS, R837, PGN, chloroquine (Invivogen); TNFα, IL-1β (R & D Systems); D-galactosamine (Sigma); GM-CSF (peproTech); ELISA kit for murine IFN-β, TNFα, IL-6 (Biolegend); dual-specific luciferase assay kit (Promega); EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); lysosome isolation kit (sigma); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (OriGene), phospho-IBα (Cell Signaling Technology), TLR3 (IMGENEX); rabbit monoclonal antibodies against TLR3, phospho-IRF3 (Cell Signaling Technology); rabbit polyclonal antibodies against IRF3 (Santa Cruz Biotechnology), TRIF (Cell Signaling Technology), TBK1 and phospho-TBK1(EPITOMICS), Flag (MBL); Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody, Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Invitrogen) were purchased from the indicated manufacturers. .. Mouse antisera against human Mex3B were raised against recombinant human Mex3B fragments containing aa236-517 and aa1-517, respectively.

Cytometry:

Article Title: Amphiregulin activates regulatory T lymphocytes and suppresses CD8+ T cell-mediated anti-tumor response in hepatocellular carcinoma cells
Article Snippet: .. Then cells were stained at room temperature for 30 min with Alexa Fluor® 488 anti-IFN-γ (XMG1.2, Biolegend), Alexa Fluor® 647 anti-TNF-α (MP6-XT22, Biolegend), APC anti-perforin (eBioOMAK-D, eBioscience) and PE anti-granzyme B (NGZB, eBioscience) respectively before analysis on a BD LSRII flow cytometer. .. For Foxp3 staining, Foxp3 fix/perm buffer (Biolegend) set was used according to the manufacturer's instruction.

Enzyme-linked Immunosorbent Assay:

Article Title: The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response
Article Snippet: .. Reagents and antibodies Poly(I:C), LPS, R837, PGN, chloroquine (Invivogen); TNFα, IL-1β (R & D Systems); D-galactosamine (Sigma); GM-CSF (peproTech); ELISA kit for murine IFN-β, TNFα, IL-6 (Biolegend); dual-specific luciferase assay kit (Promega); EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); lysosome isolation kit (sigma); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (OriGene), phospho-IBα (Cell Signaling Technology), TLR3 (IMGENEX); rabbit monoclonal antibodies against TLR3, phospho-IRF3 (Cell Signaling Technology); rabbit polyclonal antibodies against IRF3 (Santa Cruz Biotechnology), TRIF (Cell Signaling Technology), TBK1 and phospho-TBK1(EPITOMICS), Flag (MBL); Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody, Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Invitrogen) were purchased from the indicated manufacturers. .. Mouse antisera against human Mex3B were raised against recombinant human Mex3B fragments containing aa236-517 and aa1-517, respectively.

Staining:

Article Title: IL-17A+GM-CSF+ Neutrophils Are the Major Infiltrating Cells in Interstitial Lung Disease in an Autoimmune Arthritis Model
Article Snippet: .. After fixing and perminization, intracellular molecules, including cytokines and transcription factors, were stained with PE/Cy7-IL-17A (BioLegend, clone: TC11-18H10.1), PerCP/Cy5.5-conjugated anti-RORγt (BD, clone: Q31-378), Alexa Fluor 647-conjugated anti-FOXP3 (BioLegend, clone:150D), PerCP/Cy5.5-conjugated anti-GM-CSF (BioLegend, clone: MP1-22E9), and Alexa Fluor 647-conjugated anti-TNF-α (BioLegend, clone: MP6-XT22). ..

Article Title: Amphiregulin activates regulatory T lymphocytes and suppresses CD8+ T cell-mediated anti-tumor response in hepatocellular carcinoma cells
Article Snippet: .. Then cells were stained at room temperature for 30 min with Alexa Fluor® 488 anti-IFN-γ (XMG1.2, Biolegend), Alexa Fluor® 647 anti-TNF-α (MP6-XT22, Biolegend), APC anti-perforin (eBioOMAK-D, eBioscience) and PE anti-granzyme B (NGZB, eBioscience) respectively before analysis on a BD LSRII flow cytometer. .. For Foxp3 staining, Foxp3 fix/perm buffer (Biolegend) set was used according to the manufacturer's instruction.

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  • 93
    BioLegend tnfα elisa kit
    In vivo treatment with GSC839 does not induce inflammation. (A) Production of inflammatory cytokines. C57BL/6 mice were subcutaneously immunized with 2.5 µg ovalbumin together with indicated amounts of GSC839, CpG oligo, or alum. The levels of serum <t>TNFα</t> and IL-6 24 h after immunization were measured by <t>ELISA.</t> Data were analyzed by Kruskal–Wallis test and Steel analysis was applied as post hoc analysis. * P
    Tnfα Elisa Kit, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend tnfα
    In the absence of IFNγ frequencies and numbers of IL17A producing colonic CD4 T cells are dramatically increased in colitic mice. Cytokine expression analysis of colonic lamina propria (LP) CD4 T cells, PMA/ionomycin-stimulated in the presence or absence of IFNγ. Colonic LP cells were isolated during active colitis (24–26 days post transfer of colitogenic Ifng +/+ , and Ifng −/− CD4 CD45RB hi T cells into Rag1 −/− , and Rag1 −/− Ifng −/− recipient mice, respectively). (A,B) Representative FACS blots showing IFNγ, IL17A, or <t>TNFα</t> expression of CD4 T cells. (C) Relative cell frequencies and (D) absolute cell numbers of cytokine expressing CD4 T cells. Bars indicate mean ± SD from three independent experiments with n = 5–9 mice per group. P -value was determined using the two-tailed Mann–Whitney test with * P
    Tnfα, supplied by BioLegend, used in various techniques. Bioz Stars score: 94/100, based on 188 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    In vivo treatment with GSC839 does not induce inflammation. (A) Production of inflammatory cytokines. C57BL/6 mice were subcutaneously immunized with 2.5 µg ovalbumin together with indicated amounts of GSC839, CpG oligo, or alum. The levels of serum TNFα and IL-6 24 h after immunization were measured by ELISA. Data were analyzed by Kruskal–Wallis test and Steel analysis was applied as post hoc analysis. * P

    Journal: Frontiers in Immunology

    Article Title: CD22-Binding Synthetic Sialosides Regulate B Lymphocyte Proliferation Through CD22 Ligand-Dependent and Independent Pathways, and Enhance Antibody Production in Mice

    doi: 10.3389/fimmu.2018.00820

    Figure Lengend Snippet: In vivo treatment with GSC839 does not induce inflammation. (A) Production of inflammatory cytokines. C57BL/6 mice were subcutaneously immunized with 2.5 µg ovalbumin together with indicated amounts of GSC839, CpG oligo, or alum. The levels of serum TNFα and IL-6 24 h after immunization were measured by ELISA. Data were analyzed by Kruskal–Wallis test and Steel analysis was applied as post hoc analysis. * P

    Article Snippet: TNFα and IL-6 were measured by TNFα ELISA kit and IL-6 ELISA kit (BioLegend), respectively, according to the manufacture’s protocol.

    Techniques: In Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay

    In the absence of IFNγ frequencies and numbers of IL17A producing colonic CD4 T cells are dramatically increased in colitic mice. Cytokine expression analysis of colonic lamina propria (LP) CD4 T cells, PMA/ionomycin-stimulated in the presence or absence of IFNγ. Colonic LP cells were isolated during active colitis (24–26 days post transfer of colitogenic Ifng +/+ , and Ifng −/− CD4 CD45RB hi T cells into Rag1 −/− , and Rag1 −/− Ifng −/− recipient mice, respectively). (A,B) Representative FACS blots showing IFNγ, IL17A, or TNFα expression of CD4 T cells. (C) Relative cell frequencies and (D) absolute cell numbers of cytokine expressing CD4 T cells. Bars indicate mean ± SD from three independent experiments with n = 5–9 mice per group. P -value was determined using the two-tailed Mann–Whitney test with * P

    Journal: Frontiers in Immunology

    Article Title: Divergent Roles of Interferon-γ and Innate Lymphoid Cells in Innate and Adaptive Immune Cell-Mediated Intestinal Inflammation

    doi: 10.3389/fimmu.2018.00023

    Figure Lengend Snippet: In the absence of IFNγ frequencies and numbers of IL17A producing colonic CD4 T cells are dramatically increased in colitic mice. Cytokine expression analysis of colonic lamina propria (LP) CD4 T cells, PMA/ionomycin-stimulated in the presence or absence of IFNγ. Colonic LP cells were isolated during active colitis (24–26 days post transfer of colitogenic Ifng +/+ , and Ifng −/− CD4 CD45RB hi T cells into Rag1 −/− , and Rag1 −/− Ifng −/− recipient mice, respectively). (A,B) Representative FACS blots showing IFNγ, IL17A, or TNFα expression of CD4 T cells. (C) Relative cell frequencies and (D) absolute cell numbers of cytokine expressing CD4 T cells. Bars indicate mean ± SD from three independent experiments with n = 5–9 mice per group. P -value was determined using the two-tailed Mann–Whitney test with * P

    Article Snippet: Anti-mouse CD4 (RM4-5), CD3 (145-2C11), TCRαβ (H57-597), CD45 (30-F11), IFNγ (XMG1.2), IL17A (clone TC11-18H10), TNFα (MP6-XT22), Thy1.2 (53-2.1), and Thy1.1 (OX-7) were purchased from Biolegend.

    Techniques: Mouse Assay, Expressing, Isolation, FACS, Two Tailed Test, MANN-WHITNEY

    Neutralization of IL17A and IL 17F attenuates exacerbating CD4 T cell-induced colitis in the absence of IFNγ. Rag1 −/− Ifng −/− mice were transferred with Ifng −/− CD4 CD45RB hi T cells and treated with anti-IL17A and anti-IL17F neutralizing antibodies, or isotype control (250 μg/mouse) twice a week. (A) Body weight during colitis induction and (B) histopathological scores. (C) Representative hematoxylin and eosin staining of colonic tissue sections from mice during active phase of colitis. Scale bars indicate 100 µm. (D) Serum level for TNFα, IL6, and IL1β. Cells were isolated from colitic mice from the colonic lamina propria (col LP), mesenteric lymph nodes (MLN), caudal lymph node (CLN) and spleen, restimulated with PMA/ionomycin for subsequent intracellular FACS analysis. Relative cell frequencies of (E) CD4 T cells and (F) cytokine expressing CD4 T cells are shown in experimental mice treated, with, and without, anti-IL17A/IL17F mAb’s. CD4 T cells were identified as single, live, autofluorescent negative, CD45 leukocytes, positive for CD4 and CD3. P -values were determined using the two-tailed Mann–Whitney test with * P

    Journal: Frontiers in Immunology

    Article Title: Divergent Roles of Interferon-γ and Innate Lymphoid Cells in Innate and Adaptive Immune Cell-Mediated Intestinal Inflammation

    doi: 10.3389/fimmu.2018.00023

    Figure Lengend Snippet: Neutralization of IL17A and IL 17F attenuates exacerbating CD4 T cell-induced colitis in the absence of IFNγ. Rag1 −/− Ifng −/− mice were transferred with Ifng −/− CD4 CD45RB hi T cells and treated with anti-IL17A and anti-IL17F neutralizing antibodies, or isotype control (250 μg/mouse) twice a week. (A) Body weight during colitis induction and (B) histopathological scores. (C) Representative hematoxylin and eosin staining of colonic tissue sections from mice during active phase of colitis. Scale bars indicate 100 µm. (D) Serum level for TNFα, IL6, and IL1β. Cells were isolated from colitic mice from the colonic lamina propria (col LP), mesenteric lymph nodes (MLN), caudal lymph node (CLN) and spleen, restimulated with PMA/ionomycin for subsequent intracellular FACS analysis. Relative cell frequencies of (E) CD4 T cells and (F) cytokine expressing CD4 T cells are shown in experimental mice treated, with, and without, anti-IL17A/IL17F mAb’s. CD4 T cells were identified as single, live, autofluorescent negative, CD45 leukocytes, positive for CD4 and CD3. P -values were determined using the two-tailed Mann–Whitney test with * P

    Article Snippet: Anti-mouse CD4 (RM4-5), CD3 (145-2C11), TCRαβ (H57-597), CD45 (30-F11), IFNγ (XMG1.2), IL17A (clone TC11-18H10), TNFα (MP6-XT22), Thy1.2 (53-2.1), and Thy1.1 (OX-7) were purchased from Biolegend.

    Techniques: Neutralization, Mouse Assay, Staining, Isolation, FACS, Expressing, Two Tailed Test, MANN-WHITNEY

    E-FABP deficiency protects high saturated fat-induced skin lesions (A) Weight of WT mice and E-FABP−/− mice before and after cocoa butter diet (45% fat) (n=16 for WT mice, and n=15/E-FABP−/− mice) and safflower diet (45% fat) (n=10/WT mice and n=15/E-FABP−/−) for 6 months. ( B ) The incidence of skin lesions in WT and E-FABP−/− mice fed with cocoa butter diet or safflower oil diet for 9 months. (C-D) Confocal analysis of CD11c+ macrophage infiltration (green color) in the skin tissue of WT and E-FABP−/− mice fed with cocoa butter for 9 months. Relative CD11c fluorescent intensity is shown in panel D . (E-G) Flow cytometric analysis of CD80 ( E ), CD86 ( F ) and MHCII ( G ) expression on CD11c+ macrophages in the skin of WT and E-FABP−/− mice fed with the cocoa butter diet for 9 months. (H-I) Measurement of serum levels of IL-6 ( H ), IL-1β ( I ) and TNFα ( J .

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

    Article Title: Stearic acid induces CD11c expression in proinflammatory macrophages via epidermal fatty acid binding protein

    doi: 10.4049/jimmunol.1701416

    Figure Lengend Snippet: E-FABP deficiency protects high saturated fat-induced skin lesions (A) Weight of WT mice and E-FABP−/− mice before and after cocoa butter diet (45% fat) (n=16 for WT mice, and n=15/E-FABP−/− mice) and safflower diet (45% fat) (n=10/WT mice and n=15/E-FABP−/−) for 6 months. ( B ) The incidence of skin lesions in WT and E-FABP−/− mice fed with cocoa butter diet or safflower oil diet for 9 months. (C-D) Confocal analysis of CD11c+ macrophage infiltration (green color) in the skin tissue of WT and E-FABP−/− mice fed with cocoa butter for 9 months. Relative CD11c fluorescent intensity is shown in panel D . (E-G) Flow cytometric analysis of CD80 ( E ), CD86 ( F ) and MHCII ( G ) expression on CD11c+ macrophages in the skin of WT and E-FABP−/− mice fed with the cocoa butter diet for 9 months. (H-I) Measurement of serum levels of IL-6 ( H ), IL-1β ( I ) and TNFα ( J .

    Article Snippet: Mouse IL-6, TNFα, IL-1β, and IL-10 levels in cell cultural supernatants or serum from obese/lean mice were measured using ELISA MAX™ standard set from BioLegend.

    Techniques: Mouse Assay, Flow Cytometry, Expressing

    SA promotes expression of costimulatory molecules and production of inflammatory cytokines in M-CSF-induced bone marrow cells (A-B) Flow cytometric analyses of CD11c expression on bone marrow cells stimulated with M-CSF (20ng/ml) and indicated concentrations of SA for 48h. Average percentage of CD11c+ cells was shown in Panel B . (C-D) Realtime PCR analysis of mRNA levels of CD11c ( C ) and CD11b ( D ) in bone marrow cells after stimulation with M-CSF and SA for 48h. (E-H) Flow cytometric analysis of the mean fluorescent intensity (MFI) of MHCII ( E ), CD86 ( F ), CD80 ( G ), and CD54 ( H ) on bone marrow cells after stimulation with M-CSF and indicated SA for 48h. (J-O) Realtime PCR analysis of mRNA levels of inflammatory cytokines, including IL-6 ( J ), TNFα ( K ), IL-1β ( L ), IL-10 ( M ), IFNα ( N ) and IFNβ ( O ) in M-CSF-stimulated bone marrow cells in the presence or absence SA treatment (100μM) for 48h. (P-S) Measurement of protein levels of IL-6 ( P ), TNFα ( Q ) IL-1β ( R ) and IL-10 ( S ) in the supernatants of M-CSF-differentiated macrophages in the presence or absence SA treatment (100μM) for 48h. Data are shown as mean ± SEM (* p

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

    Article Title: Stearic acid induces CD11c expression in proinflammatory macrophages via epidermal fatty acid binding protein

    doi: 10.4049/jimmunol.1701416

    Figure Lengend Snippet: SA promotes expression of costimulatory molecules and production of inflammatory cytokines in M-CSF-induced bone marrow cells (A-B) Flow cytometric analyses of CD11c expression on bone marrow cells stimulated with M-CSF (20ng/ml) and indicated concentrations of SA for 48h. Average percentage of CD11c+ cells was shown in Panel B . (C-D) Realtime PCR analysis of mRNA levels of CD11c ( C ) and CD11b ( D ) in bone marrow cells after stimulation with M-CSF and SA for 48h. (E-H) Flow cytometric analysis of the mean fluorescent intensity (MFI) of MHCII ( E ), CD86 ( F ), CD80 ( G ), and CD54 ( H ) on bone marrow cells after stimulation with M-CSF and indicated SA for 48h. (J-O) Realtime PCR analysis of mRNA levels of inflammatory cytokines, including IL-6 ( J ), TNFα ( K ), IL-1β ( L ), IL-10 ( M ), IFNα ( N ) and IFNβ ( O ) in M-CSF-stimulated bone marrow cells in the presence or absence SA treatment (100μM) for 48h. (P-S) Measurement of protein levels of IL-6 ( P ), TNFα ( Q ) IL-1β ( R ) and IL-10 ( S ) in the supernatants of M-CSF-differentiated macrophages in the presence or absence SA treatment (100μM) for 48h. Data are shown as mean ± SEM (* p

    Article Snippet: Mouse IL-6, TNFα, IL-1β, and IL-10 levels in cell cultural supernatants or serum from obese/lean mice were measured using ELISA MAX™ standard set from BioLegend.

    Techniques: Expressing, Flow Cytometry, Polymerase Chain Reaction

    E-FABP deficiency reduces CD11c+ macrophages in obese mice WT and E-FABP−/− mice were fed on HFD (60% fat) for 20 weeks. Different tissues or organs were collected, respectively from the obese WT and E-FABP−/− mice (n=5) for analysis of the presence of CD11b+CD11c+ macrophages. ( A-D ) Flow cytometric surface staining for analysis of CD11b+CD11c+ macrophages in bone marrow ( A ), liver ( B ), lung ( C ) and skin ( D ). Average percentage of CD11c+ cells is shown in the right panel. (E-G) Measurement of cytokine levels of IL-6 ( E ), IL-1β ( F ) and TNFα ( G .

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

    Article Title: Stearic acid induces CD11c expression in proinflammatory macrophages via epidermal fatty acid binding protein

    doi: 10.4049/jimmunol.1701416

    Figure Lengend Snippet: E-FABP deficiency reduces CD11c+ macrophages in obese mice WT and E-FABP−/− mice were fed on HFD (60% fat) for 20 weeks. Different tissues or organs were collected, respectively from the obese WT and E-FABP−/− mice (n=5) for analysis of the presence of CD11b+CD11c+ macrophages. ( A-D ) Flow cytometric surface staining for analysis of CD11b+CD11c+ macrophages in bone marrow ( A ), liver ( B ), lung ( C ) and skin ( D ). Average percentage of CD11c+ cells is shown in the right panel. (E-G) Measurement of cytokine levels of IL-6 ( E ), IL-1β ( F ) and TNFα ( G .

    Article Snippet: Mouse IL-6, TNFα, IL-1β, and IL-10 levels in cell cultural supernatants or serum from obese/lean mice were measured using ELISA MAX™ standard set from BioLegend.

    Techniques: Mouse Assay, Flow Cytometry, Staining

    Blocking antibodies to TNFα and TNFRs prevents cholangiocyte killing.

    Journal: JCI Insight

    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: JCI Insight

    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: JCI Insight

    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: JCI Insight

    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: JCI Insight

    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: JCI Insight

    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: JCI Insight

    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