Journal: The Journal of Biological Chemistry

Article Title: Human CD300C Delivers an Fc Receptor-?-dependent Activating Signal in Mast Cells and Monocytes and Differs from CD300A in Ligand Recognition *

doi: 10.1074/jbc.M112.434746

Figure Lengend Snippet: Cell surface expression of CD300A and CD300C in human hematopoietic cells. A, analysis of CD300A or CD300C expression in hematopoietic cells derived from human PB. FSChighSSChigh populations in neutrophils or eosinophils or FSClow/intSSClow/int populations in basophils, T cells, B cells, monocytes, NK cells, or PDCs were gated. The cells were stained with FITC-conjugated anti-CD3, CD19, CD56, BDCA-2, CD123, or CD16 mAb or allophycocyanin-conjugated anti-CD14 mAb and analyzed for CD300A or CD300C expression. B, human PB-derived Mφ-1 or Mφ-2 were stained with R-PE-conjugated anti-CD11b, CD14, or HLR-DR mAb or FITC-conjugated CD11c mAb and were analyzed for CD300A or CD300C expression. C, human monocyte-derived DCs were cultured for 24 h in the presence or absence of 1 ng/ml LPS or 10 ng/ml TNFα. The cells were stained with R-PE-conjugated anti-CD80, CD83, or CD86 mAb and were analyzed for CD300A or CD300C expression. D, human PB-derived mast cells were analyzed for CD300A or CD300C expression. The results of control staining are shown as filled histograms. All of the data are representative of three independent experiments.

Article Snippet: R-PE-conjugated anti-human blood dendritic cell antigen-2 mAb and FITC-conjugated CD16 or CD123 mAb were from Miltenyi Biotech.

Techniques: Expressing, Derivative Assay, Staining, Cell Culture, Control

Journal: Frontiers in Immunology

Article Title: Age-related increase of mitochondrial content in human memory CD4+ T cells contributes to ROS-mediated increased expression of proinflammatory cytokines

doi: 10.3389/fimmu.2022.911050

Figure Lengend Snippet: Capacity of cytokine production in memory CD4 + T cell. Cells were activated with PMA/ionomycin for 5 h, adding BFA for the last 3 hours for intracellular cytokine expression. (A) Representative example of cytokine expression in memory CD4 + T cells from young and aged donors after stimulation with PMA/ionomycin. (B) Frequencies in % and geometric mean fluorescence intensity (gMFI) of IL-2, IL-4, IL-10, IL-17, IFN-γ, and TNF-α producing memory CD4 + T cells (mean ± SEM; n = 7).

Article Snippet: Incubation was conducted by dissolving 1x 10 6 cells/ml in RPMI 1640 medium (Thermo Fisher Scientific) with 5% human AB serum (Sigma Aldrich) and 1% penicillin/streptomycin (Thermo Fisher Scientific) at 37˚C in a 5% CO 2 atmosphere Cells were collected, washed, fixed, permeabilized (Inside staining kit, Miltenyi Biotec) and stained with a combination of monoclonal antibodies including anti-human-IFNγ (PERCP-Cy5.5, Biolegend, clone 4SB3, Cat.# 502526), anti-human-IL-2 (APC-vio770, Miltenyi Biotec, clone N7.48 A, Cat.# 130-097-011), anti-human-IL-4 (PE, Miltenyi Biotec, clone 7A3-3, Cat.# 130-091-647), anti-human-IL-17A (FITC, Miltenyi Biotec, clone CZ8-23G1, Cat.# 130-094-520), and anti-human-TNF-α (PE-Vio770 (Miltenyi Biotec, clone cA2, Cat.# 130-096-755).

Techniques: Expressing, Fluorescence

Journal: Frontiers in Immunology

Article Title: Ultraviolet B Inhibits IL-17A/TNF-α-Stimulated Activation of Human Dermal Fibroblasts by Decreasing the Expression of IL-17RA and IL-17RC on Fibroblasts

doi: 10.3389/fimmu.2017.00091

Figure Lengend Snippet: Primers for the target genes in real-time quantitative PCR .

Article Snippet: Detection of IL-17RA and IL-17RC expression were performed using human IL-17RA-FITC and IL-17RC-FITC antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Ultraviolet B Inhibits IL-17A/TNF-α-Stimulated Activation of Human Dermal Fibroblasts by Decreasing the Expression of IL-17RA and IL-17RC on Fibroblasts

doi: 10.3389/fimmu.2017.00091

Figure Lengend Snippet: Real-time quantitative PCR analyses of HDFs stimulated with IL-17A (100 ng/ml) and TNF-α (10 ng/ml) alone or together for 24 h. The results demonstrate that both IL-17A and TNF-α induce significant increases in IL-6, IL-8, and CXCL-1 mRNA expression and IL-17A/TNF-α stimulation have additive or synergistic effect on IL-6, IL-8, and CXCL-1 mRNA expression . Additionally, IL-17A induces the mRNA expression of TNFR-2 and TNF-α induces IL-17RA and IL-17RC expression on HDFs. The data (fold change) are from one representative experiment performed in triplicate, repeated three times with similar results. HDFs, human dermal fibroblasts. Statistical significance indicated: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Detection of IL-17RA and IL-17RC expression were performed using human IL-17RA-FITC and IL-17RC-FITC antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: Frontiers in Immunology

Article Title: Ultraviolet B Inhibits IL-17A/TNF-α-Stimulated Activation of Human Dermal Fibroblasts by Decreasing the Expression of IL-17RA and IL-17RC on Fibroblasts

doi: 10.3389/fimmu.2017.00091

Figure Lengend Snippet: Real-time quantitative PCR analyses of HDFs treated with IL-17A (100 ng/ml)/TNF-α (10 ng/ml), UVB (30 mJ/cm 2 ), and TGF-β1 (2 ng/ml) for 24 h . (A) IL-17A/TNF-α-stimulated HDFs increase expression of IL-6, IL-8, and CXCL-1 mRNA, which are also seen in 30 mJ/cm 2 UVB-irradiated HDFs. But UVB irradiation inhibits IL-17A/TNF-α-induced IL-6, IL-8, and CXCL-1 mRNA expression of HDFs. IL-17A/TNF-α stimulation induce the expression of TNFR-2, IL-17RA, and IL-17RC mRNA. UVB irradiation upregulates the expression of TNFR-1 and TNFR-2 mRNA but downregulates IL-17RA and IL-17RC expression, and inhibits IL-17A/TNF-α-induced IL-17RA and IL-17RC mRNA expression. IL-17A/TNF-α and UVB treatment do not induce significant expression of TGF-β1 mRNA 24 h after culture, but Smad3 mRNA is upregulated in both UVB irradiation and IL-17A/TNF-α + UVB groups. (B) TGF-β1 significantly induces the Smad3 mRNA expression and downregulates the IL-17RA and IL-17RC expression in HDFs. The data (fold change) are from one representative experiment performed in triplicate, repeated three times with similar results. UVB, ultraviolet B; HDFs, human dermal fibroblasts. Statistical significance indicated: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Detection of IL-17RA and IL-17RC expression were performed using human IL-17RA-FITC and IL-17RC-FITC antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Irradiation

Journal: Frontiers in Immunology

Article Title: Ultraviolet B Inhibits IL-17A/TNF-α-Stimulated Activation of Human Dermal Fibroblasts by Decreasing the Expression of IL-17RA and IL-17RC on Fibroblasts

doi: 10.3389/fimmu.2017.00091

Figure Lengend Snippet: Protein detection by using Western blot, ELISA analysis, and flow cytometry . (A) Western blot of HDFs lysates confirms mRNA expression of IL-6, IL-8, and CXCL-1 at the protein level, although shows no significant increase in IL-8 protein of UVB group compared with Control group and no significant decrease in IL-6 and CXCL-1 protein of IL-17A/TNF-α + UVB group compared with IL-17A/TNF-α group. (B) ELISA analysis of the supernatant confirms mRNA expression of IL-6, IL-8, and CXCL-1 at the protein level. (C) The expression of IL-17RA and IL-17RC mRNA is confirmed by flow cytometry for detection of the surface expression of IL-17RA and IL-17RC on HDFs. The data (fold change) of Western blot are expressed as the mean ± SD of three independent experiments. Other data are from one representative experiment performed in triplicate, repeated three times with similar results. UVB, ultraviolet B; HDFs, human dermal fibroblasts. Statistical significance indicated: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Detection of IL-17RA and IL-17RC expression were performed using human IL-17RA-FITC and IL-17RC-FITC antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Control

Journal: Frontiers in Immunology

Article Title: Ultraviolet B Inhibits IL-17A/TNF-α-Stimulated Activation of Human Dermal Fibroblasts by Decreasing the Expression of IL-17RA and IL-17RC on Fibroblasts

doi: 10.3389/fimmu.2017.00091

Figure Lengend Snippet: Protein detection by using ELISA analysis, Western blot, and flow cytometry . (A) ELISA analysis shows significant TGF-β1 protein secretion in supernatant in both UVB and IL-17A/TNF-α + UVB groups. (B) Western blot shows the protein expression of Smad3 is significantly upregulated in both UVB and IL-17A/TNF-α + UVB groups. (C) Flow cytometry shows P144 (2 µg/ml) is able to block the inhibitory effect of UVB on the expression of IL-17RA and IL-17RC on HDFs. The data (fold change) of Western blot are expressed as the mean ± SD of three independent experiments. Other data are from one representative experiment performed in triplicate, repeated three times with similar results. UVB, ultraviolet B; HDFs, human dermal fibroblasts. Statistical significance indicated: * P < 0.05, ** P < 0.01.

Article Snippet: Detection of IL-17RA and IL-17RC expression were performed using human IL-17RA-FITC and IL-17RC-FITC antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Flow Cytometry, Expressing, Blocking Assay

Journal: Mediators of Inflammation

Article Title: Analysis of Th17 and Tc17 Frequencies and Antiviral Defenses in Gut-Associated Lymphoid Tissue of Chronic HIV-1 Positive Patients

doi: 10.1155/2015/395484

Figure Lengend Snippet: Activation markers, IFN- γ and IL-17A expression in CD4 + and CD8 + T cells subsets in blood and in gut. Panel (a): representative flow cytometry plots outlining CD38 and HLA-DR expression on naïve, effector, and central memory CD4 + and CD8 + lymphocytes derived from peripheral blood mononuclear cells (PBMCs) and gut-associated lymphoid tissue (GALT). Panel (b): representative flow cytometry plots outlining IFN- γ and IL-17A intracitoplasmatic expression on naïve, effector, and central memory CD4 + (Th1 and Th17) and CD8 + (Tc1 and Tc17) lymphocytes derived from PBMCs and GALT.

Article Snippet: Cultured cells were fixed, permeabilized (BD Cytofix/Cytoperm, Becton Dickinson, San Jose, CA, USA), and stained with combinations of fluorochrome-labeled monoclonal antibodies: CD3-PerCP, CD4-APC-Vio770, CD8-FITC, CD45RO-PE-Vio770, CD27-VioBlue, IFN- γ -APC, and IL-17A-PE (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Activation Assay, Expressing, Flow Cytometry, Derivative Assay