Journal: Redox Biology

Article Title: Regulation of type I interferon responses by mitochondria-derived reactive oxygen species in plasmacytoid dendritic cells

doi: 10.1016/j.redox.2017.07.016

Figure Lengend Snippet: Phenotypical analysis of AMA-exposed GEN2.2 cell. Cells were treated with AMA (0.5 μg/ml) and CpG-A (1 μM) separately and in combination or were left untreated. Following 6 h of stimulation the expression levels of CD40 (A), CD80 (B), CD86 (C) and HLA-DQ (D) cell surface proteins were analyzed by flow cytometry. Relative fluorescence intensity values were calculated using the respective isotype-matched control antibodies. The bars represent fold changes compared to the untreated control and data are expressed as the mean ± SD of four independent experiments. *p < 0.05 vs. control. AMA: Antimycin-A.

Article Snippet: After 24 h of treatments the phenotypical analysis of the cells was performed by flow cytometry using FITC-labeled monoclonal antibodies against CD40 and CD80 (Cat. no. 334306 and 305206 both from BioLegend, San Diego, CA, USA), PE-labeled CD86 (Cat. no. FAB141P from R&D System, Minneapolis, MN, USA), HLA-DQ (Cat. no. 318106 from BioLegend) and isotype-matched control antibodies (all from BioLegend).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: The American Journal of Pathology

Article Title: Antigen Presentation and MHC Class II Expression by Human Esophageal Epithelial Cells

doi: 10.1016/j.ajpath.2010.10.027

Figure Lengend Snippet: Reverse Transcription–PCR Conditions

Article Snippet: Slides were incubated overnight with goat anti-human HLA-DR (1:40; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human HLA-DP (1:40; Antigenix America, Huntington Station, NY), mouse anti-human HLA-DQ (1:40; Antigenix America), goat anti-human CD80 (15 μg/ml; R&D Systems, Minneapolis, MN), and goat anti-human CD86 (15 μg/ml, R&D Systems) or mouse or goat IgG as a negative control and then with biotinylated mouse anti-goat (Santa Cruz Biotechnology) or rabbit anti-mouse (Invitrogen, Carlsbad, CA) IgG secondary antibody as appropriate for 30 minutes.

Techniques:

Journal: The American Journal of Pathology

Article Title: Antigen Presentation and MHC Class II Expression by Human Esophageal Epithelial Cells

doi: 10.1016/j.ajpath.2010.10.027

Figure Lengend Snippet: Costimulatory molecules CD80 and CD86 were differentially expressed on esophageal mucosal biopsies. No staining was present in isotype control goat IgG (gIgG) for CD80 primary antibody (A and B). Human CD80 (hCD80) costimulatory molecule expression was found on biopsies from normal patients (C), but this expression was not present on EoE patient biopsies (D, n=3). No staining was present in isotype control goat IgG for CD86 primary antibody (E and F). Human CD86 (hCD86) was not expressed on normal patient biopsies (G), but was found on epithelial cells in biopsies from patients with EoE (H, n=3). Original magnification: ×400 for all images.

Article Snippet: Slides were incubated overnight with goat anti-human HLA-DR (1:40; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human HLA-DP (1:40; Antigenix America, Huntington Station, NY), mouse anti-human HLA-DQ (1:40; Antigenix America), goat anti-human CD80 (15 μg/ml; R&D Systems, Minneapolis, MN), and goat anti-human CD86 (15 μg/ml, R&D Systems) or mouse or goat IgG as a negative control and then with biotinylated mouse anti-goat (Santa Cruz Biotechnology) or rabbit anti-mouse (Invitrogen, Carlsbad, CA) IgG secondary antibody as appropriate for 30 minutes.

Techniques: Staining, Control, Expressing

Journal: The American Journal of Pathology

Article Title: Antigen Presentation and MHC Class II Expression by Human Esophageal Epithelial Cells

doi: 10.1016/j.ajpath.2010.10.027

Figure Lengend Snippet: RT-PCR demonstrated increased mRNA expression of HLA-DR, HLA-DP, HLA-DQ, and CIITA after IFNγ treatment (50 ng/ml) of HET-1A cells for 24, 48, and 72 hours. Costimulatory molecule CD80 and CD86 expression was not increased by IFNγ. GAPDH was used as a loading control and H2O as a negative control. Representative images are shown (n = 3). PCR product lengths are indicated at the left.

Article Snippet: Slides were incubated overnight with goat anti-human HLA-DR (1:40; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human HLA-DP (1:40; Antigenix America, Huntington Station, NY), mouse anti-human HLA-DQ (1:40; Antigenix America), goat anti-human CD80 (15 μg/ml; R&D Systems, Minneapolis, MN), and goat anti-human CD86 (15 μg/ml, R&D Systems) or mouse or goat IgG as a negative control and then with biotinylated mouse anti-goat (Santa Cruz Biotechnology) or rabbit anti-mouse (Invitrogen, Carlsbad, CA) IgG secondary antibody as appropriate for 30 minutes.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Negative Control

Journal: The American Journal of Pathology

Article Title: Antigen Presentation and MHC Class II Expression by Human Esophageal Epithelial Cells

doi: 10.1016/j.ajpath.2010.10.027

Figure Lengend Snippet: IFNγ induced HLA-DR expression on HET-1A cells. A: HET-1A cells expressed HLA-DR only after IFNγ treatment (50 ng/ml) for 16 to 48 hours as shown by flow cytometry (n = 3). Over 16 to 48 hours, IFNγ (50 ng/ml) treatment did not induce detectable CD80 (B) or CD86 (C) expression on HET-1A cell surface (n = 3).

Article Snippet: Slides were incubated overnight with goat anti-human HLA-DR (1:40; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human HLA-DP (1:40; Antigenix America, Huntington Station, NY), mouse anti-human HLA-DQ (1:40; Antigenix America), goat anti-human CD80 (15 μg/ml; R&D Systems, Minneapolis, MN), and goat anti-human CD86 (15 μg/ml, R&D Systems) or mouse or goat IgG as a negative control and then with biotinylated mouse anti-goat (Santa Cruz Biotechnology) or rabbit anti-mouse (Invitrogen, Carlsbad, CA) IgG secondary antibody as appropriate for 30 minutes.

Techniques: Expressing, Flow Cytometry

Journal: The American Journal of Pathology

Article Title: Antigen Presentation and MHC Class II Expression by Human Esophageal Epithelial Cells

doi: 10.1016/j.ajpath.2010.10.027

Figure Lengend Snippet: A: RT-PCR demonstrating that treatment of HET-1A cells with IL-4 (50 ng/ml) for 0, 24, 48, and 72 hours did not alter expression of HLA-DR or CD86. IL-4 did increase mRNA expression of CD80 after 24 hours. B: Treatment of HET-1A cells with IL-13 (50 ng/ml) for 0, 24, 48, and 72 hours also did not alter expression of HLA-DR or CD86. CD80 expression was markedly decreased after 24 hours of IL-13 treatment. GAPDH was used as a loading control and H2O as a negative control (n = 3). Representative images are shown. All bands were located at the expected base-pair lengths.

Article Snippet: Slides were incubated overnight with goat anti-human HLA-DR (1:40; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human HLA-DP (1:40; Antigenix America, Huntington Station, NY), mouse anti-human HLA-DQ (1:40; Antigenix America), goat anti-human CD80 (15 μg/ml; R&D Systems, Minneapolis, MN), and goat anti-human CD86 (15 μg/ml, R&D Systems) or mouse or goat IgG as a negative control and then with biotinylated mouse anti-goat (Santa Cruz Biotechnology) or rabbit anti-mouse (Invitrogen, Carlsbad, CA) IgG secondary antibody as appropriate for 30 minutes.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Negative Control

Journal: PLoS ONE

Article Title: Sulforaphane Epigenetically Regulates Innate Immune Responses of Porcine Monocyte-Derived Dendritic Cells Induced with Lipopolysaccharide

doi: 10.1371/journal.pone.0121574

Figure Lengend Snippet: List of primer sequences used in this study.

Article Snippet: Cells were stained with a mouse anti-human CD40 FITC Ab (clone G28.5, NB100-77786, Novus Biologicals), a mouse anti-human CD80 PE Ab (clone 37711, FAB140P, R&D systems) and a mouse anti-human CD86 APC Ab (clone 37301, FAB141A, R&D systems) antibodies.

Techniques: Amplification

Journal: PLoS ONE

Article Title: Sulforaphane Epigenetically Regulates Innate Immune Responses of Porcine Monocyte-Derived Dendritic Cells Induced with Lipopolysaccharide

doi: 10.1371/journal.pone.0121574

Figure Lengend Snippet: moDCs at day 7 in culture were used for cell phagocytosis and cell differentiation status analysis. moDCs were pre-incubated for 1 h with or without SFN (10 μM) before stimulation for 24 h LPS (1.0 μg/ml) or to the indicated concentrations. CD40, CD80, and CD86 cellular surface markers expression were analyzed by flow cytometry (A). The flow cytometry results shown were from one experiment of two independent experiments. CD40, CD80 and CD86 mean fluorescence intensity (MFI) determined by flow cytometry (B). The flow cytometry results were combined from two independent experiments and each experiment was performed from triplications. Data are mean ± standard deviations (SD) (the letters a and b P< 0.01). The phagocytic activity of moDCs was examined after stimulating with different concentration of LPS (0,5 μg/ml, 1,0 μg/ml, and 2,0 μg/ml) with or without 24 h pre-treatment with SFN (C). The mRNA expression of DCs surface markers CD40, CD80 and CD86 were quantified using qRT-PCR (D). The mRNA expression and phagocytosis results were combined from three independent experiments and each experiment was performed in four replications. The data represented as the mean ± standard deviations (SD) (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Cells were stained with a mouse anti-human CD40 FITC Ab (clone G28.5, NB100-77786, Novus Biologicals), a mouse anti-human CD80 PE Ab (clone 37711, FAB140P, R&D systems) and a mouse anti-human CD86 APC Ab (clone 37301, FAB141A, R&D systems) antibodies.

Techniques: Cell Differentiation, Incubation, Expressing, Flow Cytometry, Fluorescence, Activity Assay, Concentration Assay, Quantitative RT-PCR