Journal: Oncotarget

Article Title: Overexpression of complement component C5a accelerates the development of atherosclerosis in ApoE-knockout mice

doi: 10.18632/oncotarget.11180

Figure Lengend Snippet: Effect of C5a overexpression on inflammatory cytokines expression Serum from blood samples was taken 14 days after Ad-C5a injection. ELISA of the serum level of interferon γ (IFN-γ) A. , interleukin 6 (IL-6) B. and tumor necrosis factor α (TNF-α) C. N = 5 mice per treatment group. Data are means ± SEM. ** P

Article Snippet: We used mouse interleukin 6 (IL-6), interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) ELISA kits from eBioscience (San Diego, CA) and a C5a ELISA kit from Cloud-Clone (Houston, TX).

Techniques: Over Expression, Expressing, Injection, Enzyme-linked Immunosorbent Assay, Mouse Assay

Journal: Immunology

Article Title: Proliferating ?? T cells manifest high and spatially confined caspase-3 activity

doi: 10.1111/j.1365-2567.2011.03540.x

Figure Lengend Snippet: Responsiveness of day 7 effector αβ and γδ T cells to T-cell receptor (TCR) restimulation. Day 7 effector T cells were restimulated with soluble biotinylated anti-CD3 cross-linked with streptavidin for the times indicated. (a) Level and kinetics of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and total ERK level were evaluated by immunoblot. (b) The early activation marker CD69 was quantified by flow cytometry before and following CD3 restimulation for 18 hr, and (c) cell culture supernatants were assessed for interferon-γ (IFN-γ) production by immunoassay. Mean and SD are shown for each cell type ( n = 2).

Article Snippet: The concentration of interferon-γ (IFN-γ) in cell culture supernatants, secreted either by day 7 effector or anti-CD3 restimulated T cells (1 × 106 per condition × 18 hr), was determined using the Milliplex Mouse Cytokine Immunoassay (Millipore, Billerica, MA).

Techniques: Activation Assay, Marker, Flow Cytometry, Cytometry, Cell Culture

Journal: Immunology

Article Title: Early delayed-type hypersensitivity eosinophil infiltrates depend on T helper 2 cytokines and interferon-? via CXCR3 chemokines

doi: 10.1111/j.0019-2805.2004.01818.x

Figure Lengend Snippet: Keyhole limpet haemocyanin (KLH) delayed-type hypersensitivity (DTH) is dependent on interferon-γ (IFN-γ) on day 3 and day 5 after immunization. Left column: DTH was elicited on day 3 and 5 in IFN-γ +/+ mice (Group C and E), but

Article Snippet: Quantitative sandwich enzyme-linked immunosorbent assay (ELISA) was performed on ear extracts for IL-4, eotaxin, IL-5, interferon-γ (IFN-γ) and IP-10, using two different specific mAbs [all BD PharMingen, San Diego, CA, except for eotaxin (R & D, Minneapolis, MN) and IP-10 (Leinco Technologies, St Louis, MO)], in 0·1 m NaHCO3 (pH 8–9) at 4°.

Techniques: Mouse Assay

Journal: Immunology

Article Title: Early delayed-type hypersensitivity eosinophil infiltrates depend on T helper 2 cytokines and interferon-? via CXCR3 chemokines

doi: 10.1111/j.0019-2805.2004.01818.x

Figure Lengend Snippet: Production of interferon-γ (IFN-γ) and IP-10 [IFN-γ-inducible protein chemokine of 10 000 molecular weight (CXCL10)] in 24-hr keyhole limpet haemocyanin (KLH) delayed-type hypersensitivity (DTH) ear extracts, and dependency of

Article Snippet: Quantitative sandwich enzyme-linked immunosorbent assay (ELISA) was performed on ear extracts for IL-4, eotaxin, IL-5, interferon-γ (IFN-γ) and IP-10, using two different specific mAbs [all BD PharMingen, San Diego, CA, except for eotaxin (R & D, Minneapolis, MN) and IP-10 (Leinco Technologies, St Louis, MO)], in 0·1 m NaHCO3 (pH 8–9) at 4°.

Techniques: Molecular Weight

Journal: Frontiers in Immunology

Article Title: Endothelial Cell Amplification of Regulatory T Cells Is Differentially Modified by Immunosuppressors and Intravenous Immunoglobulin

doi: 10.3389/fimmu.2017.01761

Figure Lengend Snippet: The effect of mycophenolic acid (MPA), cyclosporine A (CsA), or intravenous immunoglobulin (IVIg) on endothelial cell (EC)-mediated allogeneic CD4 + -T cell polarization is maintained when PBMC have been exposed to immunosuppressors or IVIg. Figure shows the proportion of interleukin-17 producing cells [ (A) , n > 5 donors], interferon γ (IFN-γ) producing cells [ (B) , n > 5 donors], T memory cells [ (C) , n > 6 donors], and their proliferation [ (D) , n > 6 donors], Treg [ (E) , n > 6 donors], and Treg proliferation [ (F) , n > 6 donors]. Like ECs, PBMC were pretreated with MPA, CSA, or IVIg prior to coculture. In all conditions, PBMC and ECs were treated with the same immunomodulators. The concentrations used for stimulation of ECs and PBMC were 3 µg/ml for MPA, 5 µg/ml for CsA, and 1 mg/ml for IVIg. Results are expressed as the relative percentage of the control values corresponding to ECs treated with vehicle and PBMC treated by immunomodulators (represented by dotted lines). In all figures, horizontal lines show median values (* p

Article Snippet: ECs Pretreatment with Immunosuppressors Endothelial cells were cultured with interferon γ (IFN-γ) (200 IU/ml for 3 days; R & D Systems, Minneapolis, MN, USA) in tissue culture flasks and incubated, where indicated, with immunosuppressors: MPA (Sigma-Aldrich), CsA (Sandimmun® , Novartis), or with IVIg (Privigen® , CSL Behring) at the indicated concentrations.

Techniques:

Journal: Frontiers in Immunology

Article Title: Endothelial Cell Amplification of Regulatory T Cells Is Differentially Modified by Immunosuppressors and Intravenous Immunoglobulin

doi: 10.3389/fimmu.2017.01761

Figure Lengend Snippet: Effect of endothelial cell (EC) incubation with mycophenolic acid (MPA), cyclosporine A (CsA), and intravenous immunoglobulin (IVIg) on the proinflammatory activity of ECs. IL-6 secretion was quantified in the supernatants of cocultures where ECs was treated with MPA [ (A) , n > 19 donors], CsA [ (B) , n = 24 donors] or IVIg [ (C) , n > 6 donors]. IL-6 production by ECs incubated with vehicle alone is represented as 0. (D–F) show the expansion of the Th17 subset after a 7 day coculture of PBMC with EC pretreated with interferon γ (IFN-γ) and the indicated dose of MPA [ (D) , n = 7 donors], CsA [ (E) , n = 9 donors], or IVIg [ (F) , n > 18 donors]. Expansion of the Th1 subset under the same conditions is shown in [ (G) , n > 7 donors], [ (H) , n = 9 donors], and [ (I) , n > 18 donors]. Results are expressed as the relative percentage of the control values (ECs treated with vehicle alone, represented by dotted lines). Thick, horizontal lines represent median values in all the cocultures (* p

Article Snippet: ECs Pretreatment with Immunosuppressors Endothelial cells were cultured with interferon γ (IFN-γ) (200 IU/ml for 3 days; R & D Systems, Minneapolis, MN, USA) in tissue culture flasks and incubated, where indicated, with immunosuppressors: MPA (Sigma-Aldrich), CsA (Sandimmun® , Novartis), or with IVIg (Privigen® , CSL Behring) at the indicated concentrations.

Techniques: Incubation, Activity Assay

Journal: Frontiers in Immunology

Article Title: Endothelial Cell Amplification of Regulatory T Cells Is Differentially Modified by Immunosuppressors and Intravenous Immunoglobulin

doi: 10.3389/fimmu.2017.01761

Figure Lengend Snippet: Intravenous immunoglobulin (IVIg) pretreatment of endothelial cells (ECs) induces selective amplification of regulatory T cells in contrast with the commonly used immunosupressors mycophenolic acid (MPA) and cyclosporine A (CsA). (A,C,E) show the proportion of Treg expanded in cocultures of PBMC with EC pretreated with interferon γ (IFN-γ) and the indicated concentrations of MPA [ (A) , n = 8 donors], CsA [ (B) , n = 12 donors], or IVIg [ (C) , n > 13 donors]. [ (D) , n = 7 donors], [ (E) , n = 10 donors], and [ (F) , n > 12 donors] show the proliferation of Treg after MPA, CsA, or IVIg treatment of ECs, respectively. Results are expressed as the relative percentage of the control values (ECs treated with vehicle alone and represented by dotted lines). Thick, horizontal lines show the median values in each data set (* p

Article Snippet: ECs Pretreatment with Immunosuppressors Endothelial cells were cultured with interferon γ (IFN-γ) (200 IU/ml for 3 days; R & D Systems, Minneapolis, MN, USA) in tissue culture flasks and incubated, where indicated, with immunosuppressors: MPA (Sigma-Aldrich), CsA (Sandimmun® , Novartis), or with IVIg (Privigen® , CSL Behring) at the indicated concentrations.

Techniques: Amplification

Journal: Immunology

Article Title: Cooperative action of CD8 T lymphocytes and natural killer cells controls tumour growth under conditions of restricted T-cell receptor diversity

doi: 10.1111/j.1365-2567.2009.03150.x

Figure Lengend Snippet: P1A peptide dose-dependent proliferation, activation and interferon-γ (IFN-γ) secretion by TCRP1A DBA/2 and B10.D2 CD8 + T cells. (A) CFSE-labelled naïve lymph node (LN) CD8 T cells from TCRP1A DBA/2 (left) and TCRP1A B10.D2 (right)

Article Snippet: Interferon-γ (IFN-γ) in T-cell culture supernatants harvested following activation on day 3 was determined by enzyme-linked immunosorbent assay (ELISA) using mAb AN18 with biotin-conjugated R46A2 (PharMingen, San Diego, CA).

Techniques: Activation Assay

Journal: Breast Cancer : Targets and Therapy

Article Title: A GM-CSF and CD40L bystander vaccine is effective in a murine breast cancer model

doi: 10.2147/BCTT.S89563

Figure Lengend Snippet: Immune response analysis. Notes: ( A ) Average ELISPOT counts for interferon-γ (IFN-γ) and interleukin-2 (IL-2) between the 4T1 alone (4T1) and 4T1 + BCG (BCG) harvested lymphocytes cocultured with 4T1 lysates (top). The ELISPOT images above each bar are representative of the spot counts for each of the four groups. ( B ) The IFN-γ average ELISPOT counts are shown in the LLC tumor antigen alone versus the BCG vaccinated LLC mice. All experiments were done in triplicate. Spot counts in the treatment groups were adjusted for background using the no-tumor mouse lymphocytes. Error bars are the SD’s for the groups. * P -value statistically significant. Abbreviations: ELISPOT, enzyme-linked immunospot; LLC, Lewis lung carcinoma; SD, standard deviation.

Article Snippet: IL-2 and IFN-γ ELISPOT Harvested lymph nodes from each mouse were subjected to ELISPOT analysis using cytokine ELISPOT kits for mouse interleukin-2 (IL-2) and interferon-γ (IFN-γ) from Mabtech (Cincinnati, OH, USA).

Techniques: Enzyme-linked Immunospot, Mouse Assay, Standard Deviation

Journal: Scientific Reports

Article Title: Nitric Oxide Inhibits Hetero-adhesion of Cancer Cells to Endothelial Cells: Restraining Circulating Tumor Cells from Initiating Metastatic Cascade

doi: 10.1038/srep04344

Figure Lengend Snippet: Flow cytometric analysis of cytokine-stimulated CD62E (E-selectin), CD54 (ICAM-1) and CD106 (VCAM-1) expression by HUVECs. HUVECs was cultivated with IL-1β (1 ng/mL), TNF-β (10 ng/mL), TNF-α (10 ng/mL), IL-6 (10 ng/mL), IFN-γ (10 ng/mL), ECGS (100 μg/mL) and fibronectin (100 ng/mL), respectively, for 4 h. Expression of CD62E, CD54 and CD106 were detected by flow cytometry using silver area as isotype controls. Bar graph shows the percentage of positive cells (right panels), contour polt analysis (left panels) shows CD62E + , CD54 + and CD106 + cells, in comparison with isotype controls. Bars represent the mean ± SEM (n = 3).

Article Snippet: Human interleukin-1 beta (IL-1β), human interferon gama (IFN-γ), human tumor necrosis factor alpha (TNF-α), human lymphotoxin (LT, TNF-β) and mouse anti-human beta-actin (β-actin) antibody were purchased from Cell Signaling Technology, Inc. Recombinant human interleukin-6 (IL-6) was purchased from Peprotech, Inc.

Techniques: Flow Cytometry, Expressing, Cytometry

Journal: Scientific Reports

Article Title: Nitric Oxide Inhibits Hetero-adhesion of Cancer Cells to Endothelial Cells: Restraining Circulating Tumor Cells from Initiating Metastatic Cascade

doi: 10.1038/srep04344

Figure Lengend Snippet: Flow cytometric and western blot analyses of effects of CAP-NO and CAP on CAMs. HUVECs were pretreated with 1 ng/mL IL-1β for 4 h followed by incubation with CAP-NO or CAP for another 12 h (unless otherwise stated). Flow cytometric analysis showed significant inhibition by CAP-NO on expression induced by IL-1β (1 ng/mL) of ICAM-1 (a), E-selectin (b), and VCAM-1 (c). Results are expressed as the mean fluorescence intensities and calculated as percentage of controls. (d) Effects of CAP-NO on E-selectin expression at indicated time points. (e) Expression of VCAM-1 determined by flow cytometry using green curve as a control (IL-1β only), and silver area is an isotype control. (f) Effects of CAP-NO on CD29 expression at indicated time points. (g and h) Western blot analysis of VCAM-1 expression by HUVECs pretreated with IFN-γ (10 ng/mL) or IL-1β (1 ng/mL) for 4 h followed by incubation with CAP-NO (0, 1, 10 and 100 μM) for another 12 h. Band intensity was quantified by using Image Lab analysis software, and calculated as percentage of controls (IL-1β only). Bars represent the mean ± SEM (n = 3); *, P

Article Snippet: Human interleukin-1 beta (IL-1β), human interferon gama (IFN-γ), human tumor necrosis factor alpha (TNF-α), human lymphotoxin (LT, TNF-β) and mouse anti-human beta-actin (β-actin) antibody were purchased from Cell Signaling Technology, Inc. Recombinant human interleukin-6 (IL-6) was purchased from Peprotech, Inc.

Techniques: Flow Cytometry, Western Blot, Incubation, Inhibition, Expressing, Fluorescence, Cytometry, Software

Journal: Journal of Veterinary Research

Article Title: Toxicological Evaluation of Flumequine in Pubertal Male Rats After Oral Administration for Six Weeks

doi: 10.1515/jvetres-2018-0012

Figure Lengend Snippet: Changes in plasma cytokines in Wistar male rats treated with corn oil (control) or flumequine (FLU) 53, 200, and 750 mg/kg for six weeks. IL-10 – interleukin-10; IL-6 – interleukin-6; TNF-α – tumour necrosis factor-α; IFN-γ – interferon-γ; GM-CSF – granulocyte-macrophage colony-stimulating factor; IL-1β – interleukin-1β; IL-2 – interleukin-2. Values are mean ±SD for seven rats in each group. ∗ P

Article Snippet: Serum concentrations of interleukin-10 (IL-10), interleukin-6 (IL-6), tumour necrosis factor-α (TNFα), interferon-γ (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1β (IL-1β), and interleukin-2 (IL-2) were determined with a Bio-plex rat cytokine-9-plex A panel (Bio-Rad, USA).

Techniques:

Journal: BMC Immunology

Article Title: Lupus-like oral mucosal lesions in mercury-induced autoimmune response in Brown Norway rats

doi: 10.1186/1471-2172-14-47

Figure Lengend Snippet: Tissue expression of interleukin-4 (IL-4) and interferon-γ (IFN-γ) mRNA in the tongue of the mercury-treated rats by real-time reverse transcription-polymerase chain reaction (qRT-PCR). A and B : qRT-PCR analyses examine expression levels of mRNA encoding IL-4 (A) and IFN-γ (B) normalized by those of glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Data represents mean ± standard error (SE) of pooled data derived from three to five independent experiments. *, significantly different at p

Article Snippet: Predesigned primers and probe reagents for rat interleukin-4 (IL-4), interferon-γ (IFN-γ), and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were commercially obtained from Roche Diagnostics.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Ultrasensitive Quantification of Cytokine Proteins in Single Lymphocytes From Human Blood Following ex-vivo Stimulation

doi: 10.3389/fimmu.2018.02462

Figure Lengend Snippet: Cytokine capture assay for IFN-γ and TNF-α in CD8 T cells. Examples of gating strategies for sorting cells that were either IFN-γ and TNF-α positive. Cells were labeled with CD8 and (A) IFN-γ using a bispecific CD45/IFN-γ capture reagent; (B) TNF-α using a bispecific CD45/TNF-α capture reagent; or (C) TNF-α using a TNF-α capture assay with TNF-α processing inhibitor (TAPI-0). Stained cells were acquired and sorted on BD-FACS-Aria sorter.

Article Snippet: Reagents The SiMoA HD-1 analyzer, SiMoA consumables, and IFN-γ (SiMoA™ IFN-γ,138 Kit) and TNF-α (SiMoA™ TNF-α 2.0, 208 Kit) were purchased from Quanterix, Lexington, MA.

Techniques: Labeling, Staining, FACS

Journal: Frontiers in Immunology

Article Title: Ultrasensitive Quantification of Cytokine Proteins in Single Lymphocytes From Human Blood Following ex-vivo Stimulation

doi: 10.3389/fimmu.2018.02462

Figure Lengend Snippet: IFN-γ protein quantification at different mean fluorescent intensities (MFI). (A) Flow cytometry dot plot showing gating strategy for sorting CD8+IFN-γ+ cells at different MFI's using bin gating. The gates were set as such that the MFI values fall in a range of 100,000 with 8 different intensities following a two-fold incremental order and covering from the brightest to the dimmest IFN-γ expression on CD8 cells (B) Measured amounts of IFN-γ from cells sorted from various levels of staining for cells surface IFN-γ expressed as mean ± SD fluorescence intensity channel numbers (MFI).

Article Snippet: Reagents The SiMoA HD-1 analyzer, SiMoA consumables, and IFN-γ (SiMoA™ IFN-γ,138 Kit) and TNF-α (SiMoA™ TNF-α 2.0, 208 Kit) were purchased from Quanterix, Lexington, MA.

Techniques: Flow Cytometry, Cytometry, Expressing, Staining, Fluorescence

Journal: Frontiers in Immunology

Article Title: Ultrasensitive Quantification of Cytokine Proteins in Single Lymphocytes From Human Blood Following ex-vivo Stimulation

doi: 10.3389/fimmu.2018.02462

Figure Lengend Snippet: Quantification of IFN-γ in single CD8 T cells. (A) Reproducibility of the SiMoA standard curves for IFN-γ. Standard concentrations of IFN-γ were used to generate curves to calculate the unknown concentrations in cells (B) Comparison of measured IFN-γ protein at 1, 2, 5, and10 cell levels in CD8 cells. The amount of IFN-γ measured in femtograms of lysates from cell surface IFN-γ-positive cells or cells that were cell surface IFN-γ-negative. Left are data from IFN-γ-positive cells, right are data from IFN-γ-negative cells. Horizontal axis is the number of cells sorted into each lysate. (C) Competitive inhibition assay for IFN-γ assay. Five and ten cells were sorted in 25 μl of lysis buffer and the lysates were incubated with or without anti-IFN-γ inhibition antibody for 45 mins at 4°C. Lysates were transferred to V bottom SiMoA plates and IFN-γ assay was performed. Data are represented as mean and mean ± SD and differences among means were calculated using Mann–Whitney test. P

Article Snippet: Reagents The SiMoA HD-1 analyzer, SiMoA consumables, and IFN-γ (SiMoA™ IFN-γ,138 Kit) and TNF-α (SiMoA™ TNF-α 2.0, 208 Kit) were purchased from Quanterix, Lexington, MA.

Techniques: Inhibition, Lysis, Incubation, MANN-WHITNEY

Journal: Stem Cells and Development

Article Title: Nuclear Receptors Nur77 and Nurr1 Modulate Mesenchymal Stromal Cell Migration

doi: 10.1089/scd.2011.0076

Figure Lengend Snippet: Increased cytokine production in Nur77- and Nurr1-transduced FBMSC does not influence T-cell proliferation. (A) Four days after transduction, IL-6 and IL-8 protein secreted in supernatant of unstimulated FBMSC cultures were measured by ELISA after 24 h incubation and mRNA levels of HGF , TGF-β1 , and IDO were analyzed by RQ-PCR. Basal protein levels of IL-6 and IL-8 and HGF mRNA expression were significantly increased in Nur77- or Nurr1-transduced FBMSC compared with mock-transduced FBMSC. Data (mean±SD) normalized to unstimulated mock-transduced MSC. (B) Transduced FBMSC were stimulated with TNF-α and IFN-γ for 24 h or left untreated. Again the highest levels of IL-6 and IL-8 protein and HGF mRNA were observed in Nur77- and Nurr1-tranduced FBMSC. Data (mean±SD) normalized to unstimulated mock-transduced MSC. (C, D) Transduced FBMSC were cocultured with CD3/CD28 bead-activated PBMC. FBMSC with enhanced expression of Nur77 (C) or Nurr1 (D) have at least a similar capacity to inhibit T-cell proliferation compared with mock-transduced FBMSC. Beads were added to PBMC as negative control (CCPM count 49.157). Data represent pooled data (mean±SD) of 3 individual FBMSC donors cocultured with the same allogeneic PBMC. IL-6, interleukin-6; IL-8, interleukin-8; HGF, hepatocyte growth factor; TGF-β1, transforming growth factor-β1; IDO, indoleamine-2,3-dioxygenase; TNF-α, tumor necrosis factor-α; IFN-γ, interferon-γ; PBMC, peripheral blood mononuclear cells; CCPM, radioactivity counts per minute. * P ≤0.05.

Article Snippet: After 24 h, the medium was refreshed and cells were stimulated with tumor necrosis factor-α (TNF-α) (1,000 U/mL; Peprotech) or interferon-γ (IFN-γ) (1,000 U/mL; Peprotech) or left untreated.

Techniques: Transduction, Enzyme-linked Immunosorbent Assay, Incubation, Polymerase Chain Reaction, Expressing, Negative Control, Radioactivity