Journal: PLoS ONE

Article Title: A Differential Concentration-Dependent Effect of IVIg on Neutrophil Functions: Relevance for Anti-Microbial and Anti-Inflammatory Mechanisms

doi: 10.1371/journal.pone.0026469

Figure Lengend Snippet: Panels A, B, C and D: Effect of IVIg and F(ab′) 2 fragments on adhesion molecule expression at the PMN surface. Whole-blood samples (500 µl) were incubated in a water bath with gentle horizontal agitation at 37°C for 45 min with PBS, HSA (1 or 25 mg/ml), or IVIg (1–25 mg/ml) (panel A, C) or equimolar concentrations of F(ab′) 2 fragments (panel B, D). LPS (10 ng/ml, 45 min) was used as a positive control. Samples were then stained with PE-anti-human CD11b or anti-L-selectin at 4°C for 30 min. Samples stained with anti-L-selectin were washed with ice-cold PBS and incubated at 4°C for 30 min with FITC-goat anti-mouse. Then, erythrocytes were lysed with FACS lysing solution and white blood cells were resuspended in 1% paraformaldehyde-PBS and analysed by flow cytometry. Nonspecific Ab binding was determined on cells incubated with the same concentrations of an irrelevant Ab of the same isotype. Results are expressed as mean fluorescence intensity (MFI). Values are means ± sem (n = 6). * Significantly different from sample incubated with PBS (p<0.05). Panels E and F: Effect of IVIg and F(ab′) 2 fragments on the PMN oxidative burst in response to formyl peptides. Whole-blood samples (500 µl) were pre-treated with hydroethidine (1500 ng/ml) for 15 min in a water bath with gentle horizontal agitation at 37°C. The samples were then incubated at 37°C for 45 min with PBS, HSA (1 or 25 mg/ml), or IVIg (1–25 mg/ml) (panel E) or equimolar concentrations of F(ab′) 2 (panel F) before stimulation with fMLP (10 −6 M) for 5 min. A sample incubated with LPS (10 ng/ml, 45 min) then with fMLP (10 −6 M, 5 min) was used as a positive control. Erythrocytes were lysed with FACS lysing solution and white blood cells were resuspended in 1% paraformaldehyde-PBS. Samples were analysed by flow cytometry. Results are expressed as mean fluorescence intensity (MFI). Values are means ± sem (n = 3). * Significantly different from sample incubated with fMLP (p<0.05).

Article Snippet: After incubation with PBS, HSA or IVIg as described above, samples were then stained at 4°C for 30 min with PE-anti-human CD11b (Dakopatts, Glostrup, Denmark) or purified anti-L-selectin (BD Biosciences, San Jose, CA).

Techniques: Expressing, Incubation, Positive Control, Staining, Flow Cytometry, Binding Assay, Fluorescence

Journal: PLoS ONE

Article Title: A Differential Concentration-Dependent Effect of IVIg on Neutrophil Functions: Relevance for Anti-Microbial and Anti-Inflammatory Mechanisms

doi: 10.1371/journal.pone.0026469

Figure Lengend Snippet: Panels A, B, C and D: Effect of IVIg and F(ab′)2 fragments on LPS modulation of adhesion molecule expression at the PMN surface. Whole-blood samples were incubated at 37°C for 15 min with PBS, IVIg (1–25 mg/ml) (panel A, C) or equimolar concentrations of F(ab′) 2 (panel B, D) and then with LPS (10 ng/ml) for 30 min. Samples were then stained with PE-anti-human CD11b or anti-L-selectin as described in the legend of . Results are expressed as mean fluorescence intensity (MFI). Values are means ± sem (n = 3). ° Significantly different from sample incubated with LPS (p<0.05). Panels E and F: Effect of IVIg and F(ab′) 2 fragments on LPS priming of the PMN oxidative burst in response to formyl peptides. Whole-blood samples (500 µl) were pre-treated with hydroethidine HE and incubated at 37°C for 15 min with PBS, IVIg (1–25 mg/ml) (panel E) or equimolar concentrations of F(ab′) 2 (panel F). Samples were then incubated with LPS and fMLP as described in the legend of . Results are expressed as mean fluorescence intensity (MFI). Values are means ± sem (n = 3). * Significantly different from sample incubated with LPS+fMLP (p<0.05).

Article Snippet: After incubation with PBS, HSA or IVIg as described above, samples were then stained at 4°C for 30 min with PE-anti-human CD11b (Dakopatts, Glostrup, Denmark) or purified anti-L-selectin (BD Biosciences, San Jose, CA).

Techniques: Expressing, Incubation, Staining, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: Critical role of the disintegrin metalloprotease ADAM17 for intestinal inflammation and regeneration in mice

doi: 10.1084/jem.20092366

Figure Lengend Snippet: Targeted generation of ADAM17 ex/ex mice. (A) Scheme for targeting the ADAM17 gene. Open arrowheads denote FLP recombinase sites, and LoxP sites are indicated by closed arrowheads. The altered allele contains an artificial exon starting with an in-frame translational stop codon placed downstream of exon 11. The artificial exon (E11a, yellow) containing the stop codon is flanked by noncanonical splice donor and acceptor sites (see Materials and methods). The black bar indicates the hybridization probe used for Southern blotting. (B) mRNA from liver and brain was analyzed by RT-PCR. (C) ADAM17 and ADAM10 Western blots of skin and heart tissues. (D and E) Shedding of L-selectin from B cells (D) and TNF from spleen cells (E) was analyzed by FACS and ELISA, respectively. (F) Serum levels of sTNF-R II were measured by ELISA. Data in D–F are shown as mean values ± SD. (G) Formation of milk ducts in mice at the age of 12 wk. Representative pictures from four animals are shown. Bars, 500 µm.

Article Snippet: All other reagents were obtained from the following sources: TGF-α (PeproTech); LPS, FITC-dextran, and myeloperoxidase (Sigma-Aldrich); PMA (EMD); anti-CD3, anti-CD28, anti–CD4-FITC, anti–B220-FITC, and anti–L-selectin–PE (BD); anti–phospho-STAT3 and anti–cyclin-D1 (Cell Signaling Technology); and horseradish peroxidase–coupled anti–goat antibody (Thermo Fisher Scientific).

Techniques: Hybridization, Southern Blot, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay