Journal: Blood Cancer Journal

Article Title: Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia

doi: 10.1038/bcj.2013.56

Figure Lengend Snippet: Flow cytometric gating strategy adopted to identify CD33+/CD34+ precursor cells. A representative JMML patient is showed. Mononuclear cells were initially gated to exclude debris and residual granulocytes by physical parameters ( a ); all myeloid cells were selected by their reactivity to anti-CD33 antibody ( b ); myeloid precursors were then identified as CD33+/CD34+ double positive cells ( c ). CD33+/CD34+ cells were further checked for their negativity to anti-CD14 antibody ( d ) and low expression of CD45 ( e ), as features of myeloid precursor cells. p-STAT5 response was then measured on these selected cells by dual SSC/STAT5 cytogram ( f ). In panel ( e ), only CD33+/CD34+/CD14− gated cells are shown. In panel ( f ), only CD33+/CD34+/CD14−/CD45low gated cells are shown.

Article Snippet: Samples were incubated with anti-phospho-STAT5 (p-STAT5) Alexa 488 (Y694, clone 47, BD Biosciences) or isotype IgG 1 k Alexa 488 (clone MOPC-21, BD Biosciences), anti-CD33 PE (clone P67.6, BD Biosciences), anti-CD34 APC (clone 8G12, BD Biosciences), anti-CD45 PerCP (clone 2D1, BD Biosciences) and anti-CD38 PE-Cy7 (clone HB7, BD Biosciences) antibodies.

Techniques: Expressing

Journal: Blood Cancer Journal

Article Title: Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia

doi: 10.1038/bcj.2013.56

Figure Lengend Snippet: Training set samples assessed the best threshold. The p-STAT5 responses were measured at each GM-CSF concentration in the training set of samples (11 JMMLs and 23 controls). The best dose to distinguish JMML from non-JMML samples was identified at 0.1 ng/ml of GM-CSF ( P <0.0001). p-STAT5-positive cells (%) were quantified by scaling the maximum % of p-STAT5+ cells at 100 and the unstimulated p-STAT5+ cells to 0.

Article Snippet: Samples were incubated with anti-phospho-STAT5 (p-STAT5) Alexa 488 (Y694, clone 47, BD Biosciences) or isotype IgG 1 k Alexa 488 (clone MOPC-21, BD Biosciences), anti-CD33 PE (clone P67.6, BD Biosciences), anti-CD34 APC (clone 8G12, BD Biosciences), anti-CD45 PerCP (clone 2D1, BD Biosciences) and anti-CD38 PE-Cy7 (clone HB7, BD Biosciences) antibodies.

Techniques: Concentration Assay

Journal: Blood Cancer Journal

Article Title: Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia

doi: 10.1038/bcj.2013.56

Figure Lengend Snippet: Representative flow cytometric contour plots of p-STAT5 response in CD33+/CD34+ cells. Dual SSC/STAT5 cytograms from a JMML (upper panels) and a control (lower panels) are shown. Contour plots are referred to CD33+/CD34+ cells identified by gating strategy described in . For each dose of GM-CSF, the raw percentage of responding p-STAT5-positive cells is shown. Response to stimulation at each GM-CSF dose was then quantified by scaling the maximum percentage of p-STAT5+ cells at 100 and the unstimulated p-STAT5+ cells to 0. According to this criteria, calculated p-STAT5 responses are indicated in parenthesis for each stimulation dose.

Article Snippet: Samples were incubated with anti-phospho-STAT5 (p-STAT5) Alexa 488 (Y694, clone 47, BD Biosciences) or isotype IgG 1 k Alexa 488 (clone MOPC-21, BD Biosciences), anti-CD33 PE (clone P67.6, BD Biosciences), anti-CD34 APC (clone 8G12, BD Biosciences), anti-CD45 PerCP (clone 2D1, BD Biosciences) and anti-CD38 PE-Cy7 (clone HB7, BD Biosciences) antibodies.

Techniques:

Journal: Blood Cancer Journal

Article Title: Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia

doi: 10.1038/bcj.2013.56

Figure Lengend Snippet: Comparison of p-STAT5-positive cells (%) induced by 0.1 ng/ml of GM-CSF in the validation series comprising 11 JMML samples (central box plot), 24 controls (left box plot) and 7 samples from patients with other diseases mimicking JMML at presentation (right box plot). The discriminating threshold (17.17% as assessed in the training set) is indicated. The bold line inside each box plot indicates the median level, while the upper and lower lines indicate the maximum and minimum observed values, respectively. There are no outliers.

Article Snippet: Samples were incubated with anti-phospho-STAT5 (p-STAT5) Alexa 488 (Y694, clone 47, BD Biosciences) or isotype IgG 1 k Alexa 488 (clone MOPC-21, BD Biosciences), anti-CD33 PE (clone P67.6, BD Biosciences), anti-CD34 APC (clone 8G12, BD Biosciences), anti-CD45 PerCP (clone 2D1, BD Biosciences) and anti-CD38 PE-Cy7 (clone HB7, BD Biosciences) antibodies.

Techniques:

Journal: Blood Cancer Journal

Article Title: Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia

doi: 10.1038/bcj.2013.56

Figure Lengend Snippet: Comparison of p-STAT5-positive cells (%) induced by 0.1 ng/ml of GM-CSF according to the different cell source. In all, 17 JMML BM samples (left box plot), 5 JMML PB samples (middle left box plot), 12 BM samples and 2 PB samples (middle right and right box plot, respectively) from patients with other diseases mimicking JMML are shown. The discriminating threshold (17.17% as assessed in the training set) is indicated. The bold line inside each box plot indicates the median level, while the upper and lower lines indicate the maximum and minimum observed values, respectively. There are no outliers.

Article Snippet: Samples were incubated with anti-phospho-STAT5 (p-STAT5) Alexa 488 (Y694, clone 47, BD Biosciences) or isotype IgG 1 k Alexa 488 (clone MOPC-21, BD Biosciences), anti-CD33 PE (clone P67.6, BD Biosciences), anti-CD34 APC (clone 8G12, BD Biosciences), anti-CD45 PerCP (clone 2D1, BD Biosciences) and anti-CD38 PE-Cy7 (clone HB7, BD Biosciences) antibodies.

Techniques:

Journal: Blood Cancer Journal

Article Title: Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia

doi: 10.1038/bcj.2013.56

Figure Lengend Snippet: Flow cytometric gating strategy adopted to identify CD33+/CD34+ precursor cells. A representative JMML patient is showed. Mononuclear cells were initially gated to exclude debris and residual granulocytes by physical parameters ( a ); all myeloid cells were selected by their reactivity to anti-CD33 antibody ( b ); myeloid precursors were then identified as CD33+/CD34+ double positive cells ( c ). CD33+/CD34+ cells were further checked for their negativity to anti-CD14 antibody ( d ) and low expression of CD45 ( e ), as features of myeloid precursor cells. p-STAT5 response was then measured on these selected cells by dual SSC/STAT5 cytogram ( f ). In panel ( e ), only CD33+/CD34+/CD14− gated cells are shown. In panel ( f ), only CD33+/CD34+/CD14−/CD45low gated cells are shown.

Article Snippet: Samples were incubated with anti-phospho-STAT5 (p-STAT5) Alexa 488 (Y694, clone 47, BD Biosciences) or isotype IgG 1 k Alexa 488 (clone MOPC-21, BD Biosciences), anti-CD33 PE (clone P67.6, BD Biosciences), anti-CD34 APC (clone 8G12, BD Biosciences), anti-CD45 PerCP (clone 2D1, BD Biosciences) and anti-CD38 PE-Cy7 (clone HB7, BD Biosciences) antibodies.

Techniques: Expressing

Journal: Blood Cancer Journal

Article Title: Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia

doi: 10.1038/bcj.2013.56

Figure Lengend Snippet: Representative flow cytometric contour plots of p-STAT5 response in CD33+/CD34+ cells. Dual SSC/STAT5 cytograms from a JMML (upper panels) and a control (lower panels) are shown. Contour plots are referred to CD33+/CD34+ cells identified by gating strategy described in . For each dose of GM-CSF, the raw percentage of responding p-STAT5-positive cells is shown. Response to stimulation at each GM-CSF dose was then quantified by scaling the maximum percentage of p-STAT5+ cells at 100 and the unstimulated p-STAT5+ cells to 0. According to this criteria, calculated p-STAT5 responses are indicated in parenthesis for each stimulation dose.

Article Snippet: Samples were incubated with anti-phospho-STAT5 (p-STAT5) Alexa 488 (Y694, clone 47, BD Biosciences) or isotype IgG 1 k Alexa 488 (clone MOPC-21, BD Biosciences), anti-CD33 PE (clone P67.6, BD Biosciences), anti-CD34 APC (clone 8G12, BD Biosciences), anti-CD45 PerCP (clone 2D1, BD Biosciences) and anti-CD38 PE-Cy7 (clone HB7, BD Biosciences) antibodies.

Techniques:

Journal: Frontiers in Immunology

Article Title: CREM Alpha Enhances IL-21 Production in T Cells In Vivo and In Vitro

doi: 10.3389/fimmu.2016.00618

Figure Lengend Snippet: Colitis in cAMP-responsive element modulator alpha (CREMα) transgenic (tg) mice is accompanied by higher local IL-21 expression . (A) Percentages of IL-21 expressing CD4 + T-cells in mesenterial lymph node (mLN) and (B) in spleen of age-matched CREMα tg mice and appropriate wild-type (WT) mice after 6 days of colitis induction measured by flow cytometry. Data show mean percentage ± SEM of IL-21 expressing CD4 + T-cells in at least four mice per group (* p < 0.05). Representative flow cytometric dot plots of IL-21-expressing CD4 + T-cells in a CD4 + -gate on the right. (C) mRNA levels of IL-21 in mLN cells ( n -fold compared to IL-21 mRNA in peripheral LN) of CREMα tg mice compared to their WT littermates after 6 days of colitis induction measured by real-time quantitative PCR ( n = 4, * p < 0.05). (D) mRNA levels of IL-21 in colon tissue of CREMα tg mice and appropriate WT mice after 6 days of dextran sodium sulfate (DSS) treatment compared to their untreated WT controls ( n -fold) measured by real-time quantitative PCR ( n = 4, * p < 0.05). (E) mRNA levels of IL-17, (F) IL-23, and (G) IFN-γ in colon tissue of CREMα tg mice and appropriate WT mice after 6 days of DSS treatment compared to their untreated WT controls ( n -fold) measured by real-time quantitative PCR ( n = 4–5). (H) Percentages of IL-17-expressing CD4 + T-cells in mLN and (I) in spleen of age-matched CREMα tg mice and appropriate WT mice after 6 days of colitis induction measured by flow cytometry. Data show mean percentage ± SEM of IL-17-expressing CD4 + T-cells in at least four mice per group (** p < 0.01). Representative flow cytometric dot plots of IL-17-expressing CD4 + T-cells in a CD4 + -gate on the right.

Article Snippet: Intracellular cytokines were stained with anti-IL-21-Alexa 647 (eBioscience) and anti IL-17-Alexa 488 (BD) antibodies.

Techniques: Transgenic Assay, Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: CREM Alpha Enhances IL-21 Production in T Cells In Vivo and In Vitro

doi: 10.3389/fimmu.2016.00618

Figure Lengend Snippet: Model of IL-21 induction by cAMP-responsive element modulator alpha (CREMα) . CREMα represses CD3ζ chain expression. CD3ζ chain, which normally transduces TCR signals into the cell, can be replaced by FcRγ. FcRγ signals much stronger than the CD3ζ chain. Therefore, higher amounts of Ca 2+ are released, which lead to an enhanced binding of NFATc2 to the IL-21 promoter and subsequently induces IL-21 expression. At the same time, CREMα also binds to the half-CRE site in the IL-21 promoter, which is only part due to the low CD3ζ expression as pathways downstream of the TCR complex signaling must be also involved. CREMα likewise binds to the IL-17 promoter and increases IL-17 expression while IL-21, to some extent, supports generation and stabilization of the IL-17 expression.

Article Snippet: Intracellular cytokines were stained with anti-IL-21-Alexa 647 (eBioscience) and anti IL-17-Alexa 488 (BD) antibodies.

Techniques: Expressing, Binding Assay