Journal: Frontiers in Pharmacology

Article Title: The TLR4-Active Morphine Metabolite Morphine-3-Glucuronide Does Not Elicit Macrophage Classical Activation In Vitro

doi: 10.3389/fphar.2016.00441

Figure Lengend Snippet: Effect of M3G co-administered with IFN-γ on expression of M1 markers in RAW264.7 cells. RAW264.7 cells (2 × 10 5 cells/well) were treated with 1 ng/ml IFN-γ alone or together with M3G (1, 5, 10, or 20 μM) for 12 h. Expression of iNOS, TNF-α, IL-6, and CD86 mRNA was determined by qRT-PCR. Results are shown relative to control (untreated) RAW264.7 cells. Mean ± SEM is shown, n = 4 independent experiments. ∗ p < 0.05, M3G + IFN-γ vs. IFN-γ alone, one-way ANOVA analysis with Dunnett’s multiple comparisons. Results are shown as mean ± SEM, n = 4–7 independent experiments.

Article Snippet: Amplification and quantification of cDNA was assessed, using TaqMan TM Fast Universal PCR Master Mix (Life Technologies, VIC, Australia) with AmpliTaq Gold TM DNA Polymerase and TaqMan TM Gene Expression Assays including human primers : IL-6 (Hs00985639-m1), IL-12 (Hs01011518-m1), IL-23 (Hs00900828-g1), TNFα (Hs01113624-g1), CXCL10 (Hs01124252-g1), and CXCL11(Hs04187682-g1) or mouse primers: iNOS (Mm00440502-m1), IL-6 (Mm00446190-m1), TNFα (Mm00443258-m1), CD86 (Mm00444543-m1) in a StepOnePlus 7500 real time PCR system (Applied Biosystems, Carlsbad, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Frontiers in Pharmacology

Article Title: The TLR4-Active Morphine Metabolite Morphine-3-Glucuronide Does Not Elicit Macrophage Classical Activation In Vitro

doi: 10.3389/fphar.2016.00441

Figure Lengend Snippet: Effect of M3G on expression of M1 markers in RAW264.7 cells. RAW264.7 cells (2 × 10 5 cells/well) were exposed to 0.001 or 0.01 ng/ml LPS, or M3G at the indicated concentrations (1, 5, 10, and 20 μM). The mRNA levels of iNOS, CD86, IL-6, or TNF-α were determined by qRT-PCR. Results are shown relative to control (untreated) RAW264.7 cells. Results are shown as mean ± SEM, n = 4–7 independent experiments. ∗∗ p < 0.01, LPS vs. control cells.

Article Snippet: Amplification and quantification of cDNA was assessed, using TaqMan TM Fast Universal PCR Master Mix (Life Technologies, VIC, Australia) with AmpliTaq Gold TM DNA Polymerase and TaqMan TM Gene Expression Assays including human primers : IL-6 (Hs00985639-m1), IL-12 (Hs01011518-m1), IL-23 (Hs00900828-g1), TNFα (Hs01113624-g1), CXCL10 (Hs01124252-g1), and CXCL11(Hs04187682-g1) or mouse primers: iNOS (Mm00440502-m1), IL-6 (Mm00446190-m1), TNFα (Mm00443258-m1), CD86 (Mm00444543-m1) in a StepOnePlus 7500 real time PCR system (Applied Biosystems, Carlsbad, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Control

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

Article Title: Human cytomegalovirus differentially controls B cell and T cell responses through effects on plasmacytoid dendritic cells.

doi: 10.4049/jimmunol.179.11.7767

Figure Lengend Snippet: FIGURE 2. HCMV activates PDCs. A, HCMV alters PDC morphology. PDCs were incubated overnight with medium or TB40/E at an MOI of 5 and examined by light microscopy and Evans blue staining of cytospin preparations. At 24 h, mock-infected PDCs showed plasmacytoid morphology, whereas TB40E-infected PDCs formed clusters, developed dendrites, and had larger cytoplasm. B and C, HCMV induces partial maturation of PDCs. PDCs were mock-infected (mock inf), stimulated with CpG-A (3 g/ml), or infected with TB40/E or VR1814 (MOI of 5) overnight. The stimuli were removed, cells were washed, and fresh medium was added. After 24 h, expression of CD83, HLA-DR, BDCA-2, CD80, and CD86 was measured by FACS. B, Values are means SEM of seven donors in independent experiments. , p 0.05. Y-axis values indicate MFI. C, Open histograms depict cells stained with isotype-matched control Ab. Representative data from one of seven experiments are shown.

Article Snippet: For cell surface staining, we used Abs against CD1a, CD3, CD4, CD8, CD14, CD19, CD20, CD38, CD80, CD86, HLA-DR, and mouse isotype controls (BD Pharmingen); CD83 (Immunotech); and BDCA-2 and BDCA-4 (Miltenyi Biotec).

Techniques: Incubation, Light Microscopy, Staining, Infection, Expressing, Control

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

Article Title: Human cytomegalovirus differentially controls B cell and T cell responses through effects on plasmacytoid dendritic cells.

doi: 10.4049/jimmunol.179.11.7767

Figure Lengend Snippet: FIGURE 4. Soluble factors secreted by HCMV-infected DCs contribute to B cell activation. A and B, PDCs were mock infected or infected with TB40/E (MOI of 5) for 4 h, washed, and resuspended in complete medium for 18 h. Supernatants were collected and filtered through 0.1-m filters to eliminate viral particles. B cells (2.5 105 cells/well) were cultured in 200 l of supernatant from PDC cultures in 96-well round-bottom plates. B cells were unstimulated or stimulated by B cell-receptor ligation (anti-Ig, 10 g/ml). A, After 3 days, B cells were harvested, stained for the B cell markers CD19, CD20, CD38, and CD86, and analyzed by FACS. Values are means SD. , p 0.05 (n 4 donors in independent experiments). B, For proliferation assay, B cells were labeled with 1 M CFSE, incubated with PDC supernatant for 5 days, harvested, and analyzed by FACS. Data from one representative of three donors in independent experiments are shown. C, MDCs were mock infected or infected with TB40/E (MOI of 5) for 4 h, washed, and resuspended in complete medium for 18 h. Supernatants were collected and filtered. B cells (2.5 105 cells/well) were cultured in 200 l of supernatant from MDC cultures. B cells were unstimulated or stimulated by B cell-receptor ligation (anti-Ig, 10 g/ml). After 3 days, B cells were harvested, stained for the B cell markers CD19, CD20, CD38, and CD86, and analyzed by FACS. Values are means SD. , p 0.05 (n 4 donors in independent experiments).

Article Snippet: For cell surface staining, we used Abs against CD1a, CD3, CD4, CD8, CD14, CD19, CD20, CD38, CD80, CD86, HLA-DR, and mouse isotype controls (BD Pharmingen); CD83 (Immunotech); and BDCA-2 and BDCA-4 (Miltenyi Biotec).

Techniques: Infection, Activation Assay, Cell Culture, Ligation, Staining, Proliferation Assay, Labeling, Incubation

Journal: Cell metabolism

Article Title: Ejection of damaged mitochondria and their removal by macrophages ensure efficient thermogenesis in brown adipose tissue

doi: 10.1016/j.cmet.2022.02.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CD86 Antibody, anti-mouse, FITC, Miltenyi Biotec , Miltenyi Biotec , Cat# 130–123-672; RRID:AB_2889633.

Techniques: Immunofluorescence, Recombinant, Cytometry, Purification, shRNA, Cell Culture, Lysis, XF Assay, Staining, Isolation, Transfection, Live Cell Imaging, Mouse Assay, Software, Expressing

Journal: Journal of leukocyte biology

Article Title: Adipose tissue of human omentum is a major source of dendritic cells, which lose MHC Class II and stimulatory function in Crohn's disease.

doi: 10.1189/jlb.0905501

Figure Lengend Snippet: Fig. 5. Morphology of omental DC. (a) A typical DC from a control omentum is shown. (b) An example of immunogold staining of the veils of a DC. The original bars indicate 1 m. In controls, gold labeling was always 5 gold grains per cell; 10 grains was considered positive. (c) The types of myeloid cells identified in the omental cells from two control patients; the proportion positive for HLA-DR staining is shown in the solid portion of the histograms, and those negative are hatched. M , Macrophage. (d) The gold grains per cell for the surface DR-labeling in the two experiments performed are shown. No evidence of DR-labeling was seen in either of the two Crohn’s patients studied. (e) A light microscopy picture of a cluster of putative DC immunostained with CD11c on a frozen section of omentum from a Crohn’s patient showing the dark, positive-peroxidase staining, which was absent from control slides. Cell nuclei, which were stained with hematoxylin, were distinguishable, showing that the veils were associated with mononuclear cells in the section. (f) Control for e using serum instead of specific antibody and with only hematoxylin staining visible. (g) Frozen section of control omentum, showing two cells specifically labeled for CD83 (TRITC-labeled). (h) The same section as g with label specific for CD86 (FITC-labeled).

Article Snippet: To block nonspecific binding sites, sections were incubated with normal mouse serum for 1 h and then incubated with FITC-conjugated primary antibody to CD86, CD80, or CD40 (Serotec) for 4 h, washed in PBS, and mounted under cover-slips using 4 ,6-diamidino-2-phenylindole-conjugated mounting medium (Vector Laboratories).

Techniques: Control, Staining, Labeling, Light Microscopy

Journal: Journal of viral hepatitis

Article Title: A class C CpG toll-like receptor 9 agonist successfully induces robust interferon-alpha production by plasmacytoid dendritic cells from patients chronically infected with hepatitis C.

doi: 10.1111/j.1365-2893.2008.01011.x

Figure Lengend Snippet: Fig. 4 Immunophenotypic maturation of pDCs is not impaired in cHCV infection. After 20–24 h stimulation with TLR agonists, pDCs were processed for flow cytometry to identify immunophenotypic changes consistent with maturation. FACS histograms from a representative NHD and cHCV subject show (a) the rise in CD86 expression and (b) fall in BDCA-2 expression following stimulation with CpG-C (solid histogram) compared to unstimulated cells (open histogram overlay). The bar graphs show the average fold change in mean fluorescence intensity (MFI) ± SEM of (a) CD86 and (b) BDCA-2 on pDCs from 9 NHD (solid bars) and eight cHCV (open bars) subjects (seven NHD for TLR7 agonists) induced by the four TLR agonists. For each TLR agonist, the degree of (a) upregulation of CD86 and (b) downregulation of BDCA-2 did not differ significantly between NHD–pDC and cHCV–pDCs (n.s.). CpG-C was a significantly stronger stimulus for CD86 upregulation than CpG-A (NHD–pDCs, P < 0.001; cHCV–pDCs, P < 0.01) and for cHCV–pDCs loxoribine was also significantly more potent than CpG-A (P < 0.05). CpG-C caused significantly stronger downregulation of BDCA-2 than CpG-A (NHD–pDCs, P < 0.01; cHVC–pDCs, P < 0.01).

Article Snippet: Cells in PBS containing 0.1% NaN3, 1% BSA and 100 lg ⁄ mL human IgG (Jackson Immunoresearch, West Grove, PA, USA) were blocked for 15 min on ice then labelled with monoclonal antibodies against HLA-DR FITC (clone L243), CD86-FITC (clone FUN-1), CD83-APC (clone HB15e) (all from BD Biosciences) or CD303-APC (BDCA2, clone AC144; Miltenyi Biotec) or with isotype controls [murine IgG1 (clone MOPC 31, APC or FITC conjugated) or IgG2a FITC (clone G155-178) (all from BD Biosciences)].

Techniques: Infection, Cytometry, Expressing