Journal: PLoS ONE

Article Title: Nod2 Activates NF-kB in CD4 + T Cells but Its Expression Is Dispensable for T Cell-Induced Colitis

doi: 10.1371/journal.pone.0082623

Figure Lengend Snippet: (A) Splenic CD4 + T cells isolated from C57BL/6 or NOD2 -/- mice were cultured alone or in presence of anti-CD3 (1-10µg/mL) and anti-CD28 (2µg/mL) monoclonal antibodies for 24 hours. Nod2 expression was assessed by western blot. (B) Naïve CD4 + CD45RB high , effector CD4 + CD45RB low Foxp3 - and regulatory CD4 + CD45RB low Foxp3 + splenic T cell subsets were isolated from Foxp3-GFP mice and Nod2 mRNA level was assessed by RT-qPCR. (C) Lamina propria lymphocytes were isolated from the cecum of NOD2-GFP mice and analyzed by flow cytometry. The histograms show the level of GFP expression. Cells were gated on total viable TCRβ + CD4 + cells (left panel), on TCRβ + CD4 + CD44 - cells (middle panel), or TCRβ + CD4 + CD44 + cells (right panel).

Article Snippet: The following antibodies were used for the experiments: anti-CD3ε (100331, BioLegend, San Diego, CA, USA), anti-CD3ε-FITC (11-0031-82, eBioscience, San Diego, CA, USA), anti-CD4-APC (17-0041-82, eBioscience), anti-CD4-A780 (47-0042-82, eBioscience), anti-CD8α (553027, BD Biosciences, San Jose, CA, USA), anti-CD8α-APC (17-0081-81, eBioscience), anti-TCRβ-APC-eFluor780 (47-5961-82, eBioscience), anti-CD44-PE (12-0441-82, eBioscience), anti-CD11b (553308, BD Biosciences), anti-CD25-APC (17-0251-82, eBioscience), anti-CD28 (16-0281-85, eBioscience), anti-CD45RB-PE (12-0455-82, eBioscience), anti-Foxp3-PE-Cy7 (25-5773-82, eBioscience), anti-B220 (553084, BD Biosciences), anti-Nod2 (14-5858, eBioscience), anti-c-Rel (sc-71, Santa Cruz Biotechnology, Heidelberg, Germany), anti-p65 (3034, Cell Signaling, Danvers, MA, USA), anti-β-actin (sc-130656, Santa Cruz Biotechnology), anti-RNA polymerase II (sc-899, Santa Cruz Biotechnology).

Techniques: Isolation, Cell Culture, Expressing, Western Blot, Quantitative RT-PCR, Flow Cytometry

Journal: Cell Death & Disease

Article Title: CD95 maintains stem cell-like and non-classical EMT programs in primary human glioblastoma cells

doi: 10.1038/cddis.2016.102

Figure Lengend Snippet: CD95 high GBM cells co-express known GSC markers. ( a ) CD95 expression in seven patient tumor samples analyzed by flow cytometry. ( b – e ) Co-expression of known GSC markers CD44 ( n =7), Integrin α6 (ITGA6; n =7), CD15 ( n =3) and CD133 ( n =7) within the patient samples (Friedman test). ( f – k ) Correlation of CD95 expression and known GCS markers in the TCGA GBM data set. ( n =519 patients, R : Pearson's correlation coefficient, P : probability for no or negative correlation)

Article Snippet: The following antibodies were used for flow cytometric stainings: Anti-human CD95 (APO1, Apogenix), labeled with LightningLink APC-Cy7 (Innova Biosciences Ltd., Cambridge, UK); Mouse IgG Isotype Control (Sigma Aldrich), labeled with LightningLink APC-Cy7; Anti-human APG101 (Apogenix), labeled with LightningLink APC (Innova); Rabbit IgG XP Isotype Control (Cell Signaling), labeled with LightningLink APC (Innova); Anti-human CD44 PE-Cy7 (eBioscience); Rat IgG2b K Isotype Control PE-Cy7 (eBioscience); Anti-human CD15 PerCP (BioLegend); Mouse IgG1 K Isotype Control PerCP (BioLegend, San Diego, CA, USA); Anti-human CD133 PE (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany); and Mouse IgG1 Isotype Control PE (Miltenyi).

Techniques: Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: A Functional Variant of PTPN22 Confers Risk for Vogt-Koyanagi-Harada Syndrome but Not for Ankylosing Spondylitis

doi: 10.1371/journal.pone.0096943

Figure Lengend Snippet: (A) The frequency of CD4 + CD44 + CD69 + T cells from normal controls carrying different genotypes of rs2488457 (CC = 9, CG = 18, GG = 4). (B) The frequency of CD4 + CD44 + CD25 + T cells from normal controls carrying different genotypes of rs2488457 (CC = 9, CG = 18, GG = 4). Data are represented as the mean.

Article Snippet: In order to determine the activation of CD4 + T cells, PBMCs were incubated with FITC-conjugated anti-human CD4, APC-conjugated anti-human CD44, PE-conjugated anti-human CD25, PE-cy7-conjugated anti-human CD69 or appropriate isotypes (eBioscience, San Diego, CA) for 30 minutes at 4°C.

Techniques:

Journal: Scientific Reports

Article Title: Virus infection drives IL-2 antibody complexes into pro-inflammatory agonists in mice

doi: 10.1038/srep37603

Figure Lengend Snippet: IL-2 Ab Cx or PBS was administered to CHIKV-infected mice on 3, 4 and 5 dpi. The popliteal lymph node (pLN) was isolated from these mice on 6 dpi. N.I mice were added as a control. Prophylactic treatment of IL-2 Ab Cx was included. (a) Representative scatterplots show Foxp3 and CD25 gated on CD4 + T cells. Foxp3 + Tregs and Foxp3 − Teffs are represented in red and blue respectively in the scatterplots. Numbers in scatterplots indicate percentage of Foxp3 + Tregs (in red) and Foxp3 − Teffs (in blue) in total CD4 + T cells. Histogram profiles of activation markers CD44 (left), CTLA-4 (middle) and CD25 (right) on (b) Foxp3 − Teffs and (c) Foxp3 + Tregs were also determined. The experiment was performed 3 times independently.

Article Snippet: Staining was performed using rat anti-CD45 (30-F11, BD Pharmingen), rat anti-CD4 (RM4–5, BD Pharmingen), rat anti-CD3 (17A2, Biolegend), rat anti-CD25 (PC61.5, eBioscience), hamster anti-CTLA-4 (UC10-4B9, eBioscience) and rat anti-CD44 (IM7, eBioscience) antibodies for 15 min. Stained cells were then washed with PBS and fixed using IC Fixation Buffer (eBioscience).

Techniques: Infection, Isolation, Activation Assay

Journal: Scientific Reports

Article Title: Virus infection drives IL-2 antibody complexes into pro-inflammatory agonists in mice

doi: 10.1038/srep37603

Figure Lengend Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated from 7 healthy donors and stimulated with increasing concentrations of human IL-2 (1 to 1000 ng/ml) stimulation. pSTAT5 signaling in CD4 + T cells were measured. (a) Representative histograms of pSTAT5 signaling in Teffs (top) and Tregs (bottom) in response to increasing concentration of IL-2. Line graph shows average percentage of pSTAT5 + Tregs and Teffs with respect to increasing concentration of IL-2 stimulation. (b) Representative histograms of pSTAT5 signaling in naive (CD45RA + ) (top) and memory (CD45RA − ) (bottom) CD4 + T cells in response to increasing concentration of IL-2. Line graph shows average percentage of pSTAT5 + naive and memory CD4 + T cells with respect to increasing concentration of IL-2 stimulation (n = 4). Data was presented as mean ± SD. (c) Blood was collected from 261 healthy donors and assessed for the percentage of memory population (CD45RA − ) in peripheral CD4 + T cells, CD4 + Teffs and CD4 + Tregs using flow cytometry. The percentage of peripheral memory CD4 + T cells population (CD44 + ) in mice was also assessed in comparison. (d) CD25 expression on naïve (CD45RA + ) and memory (CD45RA − ) population from (c) was also measured.

Article Snippet: Staining was performed using rat anti-CD45 (30-F11, BD Pharmingen), rat anti-CD4 (RM4–5, BD Pharmingen), rat anti-CD3 (17A2, Biolegend), rat anti-CD25 (PC61.5, eBioscience), hamster anti-CTLA-4 (UC10-4B9, eBioscience) and rat anti-CD44 (IM7, eBioscience) antibodies for 15 min. Stained cells were then washed with PBS and fixed using IC Fixation Buffer (eBioscience).

Techniques: Isolation, Concentration Assay, Flow Cytometry, Expressing