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    GE Healthcare peripheral blood mononuclear cells pbmcs
    FISH-Flow detection of antigen-specific T cell responses associated with latent tuberculosis infection. <t>PBMCs</t> from 65 LTBI+ and LTBI- donors were cultured +/- PPD stimulation, stained with FITC-labeled αCD3 antibody, hybridized with Cy5-labeled nucleic acid probes specific for IFNG, IL2, and no target control (GFP) and analyzed by flow cytometry, as detailed in the legend to Fig 1 . (a) Representative contour plots showing results with cells from an LTBI+ (top row) and an LTBI- (bottom row) donor, unstimulated and PPD-stimulated, as indicated. The rightmost panels in both rows show IFNG induction with PMA/Ionomycin stimulation as positive control. The upper right quadrant of each plot shows the frequencies of mRNA+CD3+ cells. (b) Frequencies of mRNA+CD3+ cells in the 65-donor population, by LTBI and stimulation status. The box plots show lower quartile, median, and upper quartile of the distribution. The lower whisker is the minimum, while the upper limit of the whisker represents the median + 1.5 x the interquartile range (values exceeding the upper limit of the whisker are shown as circles). Additionally, the mean is shown as (+) symbol. To confirm the reproducibility of the assay, selected donors were assayed twice with blood samples drawn > 4 months apart.
    Peripheral Blood Mononuclear Cells Pbmcs, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peripheral blood mononuclear cells pbmcs/product/GE Healthcare
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    peripheral blood mononuclear cells pbmcs - by Bioz Stars, 2021-04
    86/100 stars
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    86
    Thermo Fisher pbmcs
    Janus kinase inhibitors block the IL-12 and IFN-γ response in peripheral blood mononuclear cells <t>(PBMCs)</t> from STAT4 risk patients with systemic lupus erythematosus (SLE). (A) Healthy donor PBMCs were treated with serial dilutions of the JAK2 selective (JAK2i), the TYK2 selective (TYK2i) and the pan-JAK inhibitor (pan-JAKi) before being stimulated with 5 ng/mL IL-12, 0.1 ng/mL IFN-γ or 200 U/mL IFN-α for 20 min. IL-12-stimulated cells were <t>preactivated</t> with PHA/IL-2. Phosphorylation of STAT4 (pSTAT4) and STAT1 (pSTAT1) was determined by flow cytometry in IL-12-stimulated cells and IFN-γ or IFN-α-stimulated cells, respectively. Data (mean±SD of two individuals) from IL-12 and IFN-α-stimulated cells are shown for CD3 + T cells and from IFN-γ-stimulated cells for monocytes. (B) IC 50 values for each inhibitor. (C–E) PBMCs from healthy donors (HD), patients with SLE homozygous for the protective STAT4 allele (G/G) and patients with SLE carrying one or two STAT4 risk alleles (G/T+T/T) were incubated with 200 nM TYK2i (C, D), 70 nm JAK2i or 30 nM pan-JAKi (E). (C, D) Inhibition of IL-12-induced pSTAT4 in CD8 + T cells (C) and reduction in the frequency of IFN-γ + memory CD45RO + CD57 – CD8 + T cells (D). (E) Inhibition of IFN-γ-induced pSTAT1 in monocytes. (C–E) Open triangles and squares denote G/T and T/T patients with SLE, respectively.
    Pbmcs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbmcs/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pbmcs - by Bioz Stars, 2021-04
    86/100 stars
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    86
    Axis-Shield Diagnostics peripheral blood mononuclear cells pbmcs
    Lenalidomide maintenance therapy does not enhance MM-specific CD4 + T-cell responses Analysis of T-cell responses specific for the MAGE-C1 peptide SQSSPVSSFPSSTSS. ( A ) CD137 expression on CD4 + and CD8 + T cells from patient#02 at the pre <t>LEN</t> timepoint upon in vitro restimulation with the MAGE-C1 peptide. ( B ) <t>PBMCs</t> obtained at the pre LEN and the LEN timepoint were examined by IFN-γ ELISPOT to measure the T-cell response to the MAGE-C1 peptide. The bar graphs show the mean number of spots per million cells of four replicate wells and the error bars represent the standard deviation. A two-way ANOVA with Sidak’s multiple comparisons test was performed to evaluate statistical significant differences, ns: not significant, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001.
    Peripheral Blood Mononuclear Cells Pbmcs, supplied by Axis-Shield Diagnostics, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peripheral blood mononuclear cells pbmcs/product/Axis-Shield Diagnostics
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    peripheral blood mononuclear cells pbmcs - by Bioz Stars, 2021-04
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    96
    Millipore pbmcs
    The total number of circulating fibrocytes is unaffected by the leukocyte isolation strategy used. a Total number of circulating fibrocytes (defined as CD45 + lin − CD15 − CXCR4 + Col-1 + cells) per ml blood using two common leukocyte isolation techniques, e.g. the simple Pasteur pipette tube technique to isolate all white blood cells and <t>Ficoll</t> separation technique to isolate <t>PBMCs.</t> For this experiment we analyzed paired total white blood cells and PBMCs on the same day as blood withdrawal of 9 patients (4 IPF patients (black) and 5 PH patients (purple)) and 5 healthy controls (green). b Percentage of fibrocytes (of CD45 + cells) in the same patients after isolating all white blood cells (gray) and PBMCs (black). Data are depicted as median and interquartile
    Pbmcs, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbmcs/product/Millipore
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pbmcs - by Bioz Stars, 2021-04
    96/100 stars
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    N/A
    Mouse monoclonal antibody conjugated to APC which recognizes CD42b that also known as platelet glycoprotein GP1B CD42b is expressed by platelets and megakaryocytes CD42b participates in the process of coagulation
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    N/A
    Isotype Note IgG2b kappa Host Species Note Mouse BALB c Reactivity Note Human
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    Image Search Results


    FISH-Flow detection of antigen-specific T cell responses associated with latent tuberculosis infection. PBMCs from 65 LTBI+ and LTBI- donors were cultured +/- PPD stimulation, stained with FITC-labeled αCD3 antibody, hybridized with Cy5-labeled nucleic acid probes specific for IFNG, IL2, and no target control (GFP) and analyzed by flow cytometry, as detailed in the legend to Fig 1 . (a) Representative contour plots showing results with cells from an LTBI+ (top row) and an LTBI- (bottom row) donor, unstimulated and PPD-stimulated, as indicated. The rightmost panels in both rows show IFNG induction with PMA/Ionomycin stimulation as positive control. The upper right quadrant of each plot shows the frequencies of mRNA+CD3+ cells. (b) Frequencies of mRNA+CD3+ cells in the 65-donor population, by LTBI and stimulation status. The box plots show lower quartile, median, and upper quartile of the distribution. The lower whisker is the minimum, while the upper limit of the whisker represents the median + 1.5 x the interquartile range (values exceeding the upper limit of the whisker are shown as circles). Additionally, the mean is shown as (+) symbol. To confirm the reproducibility of the assay, selected donors were assayed twice with blood samples drawn > 4 months apart.

    Journal: PLoS ONE

    Article Title: Single-Cell Cytokine Gene Expression in Peripheral Blood Cells Correlates with Latent Tuberculosis Status

    doi: 10.1371/journal.pone.0144904

    Figure Lengend Snippet: FISH-Flow detection of antigen-specific T cell responses associated with latent tuberculosis infection. PBMCs from 65 LTBI+ and LTBI- donors were cultured +/- PPD stimulation, stained with FITC-labeled αCD3 antibody, hybridized with Cy5-labeled nucleic acid probes specific for IFNG, IL2, and no target control (GFP) and analyzed by flow cytometry, as detailed in the legend to Fig 1 . (a) Representative contour plots showing results with cells from an LTBI+ (top row) and an LTBI- (bottom row) donor, unstimulated and PPD-stimulated, as indicated. The rightmost panels in both rows show IFNG induction with PMA/Ionomycin stimulation as positive control. The upper right quadrant of each plot shows the frequencies of mRNA+CD3+ cells. (b) Frequencies of mRNA+CD3+ cells in the 65-donor population, by LTBI and stimulation status. The box plots show lower quartile, median, and upper quartile of the distribution. The lower whisker is the minimum, while the upper limit of the whisker represents the median + 1.5 x the interquartile range (values exceeding the upper limit of the whisker are shown as circles). Additionally, the mean is shown as (+) symbol. To confirm the reproducibility of the assay, selected donors were assayed twice with blood samples drawn > 4 months apart.

    Article Snippet: PBMC collection and cell culture Within two hours after blood collection, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque Plus (GE Healthcare, Waukesha, WI) density-gradient centrifugation and stored in liquid nitrogen.

    Techniques: Fluorescence In Situ Hybridization, Flow Cytometry, Infection, Cell Culture, Staining, Labeling, Cytometry, Positive Control, Whisker Assay

    Schematic representation of the dual-color FISH-Flow assay. (a) Human PBMCs were left unstimulated or stimulated with 10 μg/ml PPD for 6 hr, in the presence of costimulatory monoclonal antibodies (αCD28 and αCD49d, each at 0.1 μg/ml). The cells were then stained with FITC-labeled αCD3 antibody, fixed, permeabilized, and hybridized overnight with Cy5-labeled, gene-specific nucleic acid probes. After hybridization, 100,000 events were acquired on a BD LSRII flow cytometer and analyzed. (b) Gating strategy: Single cells (inside the diagonal gate) were selected in the forward scatter-height (FSC-H) versus forward scatter-area (FSC-A) plot. Lymphocytes were then identified in a FSC-A versus side scatter-area (SSC-A) plot. Unstimulated cells stained with FITC-labeled isotype-control antibody and Cy5-labeled nucleic acid probe were gated in a bivariate plot to identify background. These gates were applied to unstimulated and stimulated cells stained with FITC-labeled αCD3 antibody and Cy5-labeled nucleic acid probe to obtain the frequencies of events in each quadrant. The events in the upper right quadrant (double-positive cells) were used for subsequent analysis.

    Journal: PLoS ONE

    Article Title: Single-Cell Cytokine Gene Expression in Peripheral Blood Cells Correlates with Latent Tuberculosis Status

    doi: 10.1371/journal.pone.0144904

    Figure Lengend Snippet: Schematic representation of the dual-color FISH-Flow assay. (a) Human PBMCs were left unstimulated or stimulated with 10 μg/ml PPD for 6 hr, in the presence of costimulatory monoclonal antibodies (αCD28 and αCD49d, each at 0.1 μg/ml). The cells were then stained with FITC-labeled αCD3 antibody, fixed, permeabilized, and hybridized overnight with Cy5-labeled, gene-specific nucleic acid probes. After hybridization, 100,000 events were acquired on a BD LSRII flow cytometer and analyzed. (b) Gating strategy: Single cells (inside the diagonal gate) were selected in the forward scatter-height (FSC-H) versus forward scatter-area (FSC-A) plot. Lymphocytes were then identified in a FSC-A versus side scatter-area (SSC-A) plot. Unstimulated cells stained with FITC-labeled isotype-control antibody and Cy5-labeled nucleic acid probe were gated in a bivariate plot to identify background. These gates were applied to unstimulated and stimulated cells stained with FITC-labeled αCD3 antibody and Cy5-labeled nucleic acid probe to obtain the frequencies of events in each quadrant. The events in the upper right quadrant (double-positive cells) were used for subsequent analysis.

    Article Snippet: PBMC collection and cell culture Within two hours after blood collection, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque Plus (GE Healthcare, Waukesha, WI) density-gradient centrifugation and stored in liquid nitrogen.

    Techniques: Fluorescence In Situ Hybridization, Flow Cytometry, Staining, Labeling, Hybridization, Cytometry

    Analysis of the mechanism of PPD-induced IFNG expression in T cells. PBMCs from an LTBI+ donor were treated with CsA, αIL-12, or isotype control antibody for 1 hr and then stimulated with PPD for 6 hr. The frequency of IFNG+CD3+ cells is shown in the upper right quadrant of each bichromatic contour plot. The title above each plot indicates the conditions used for stimulation (top line) and pretreatment (bottom line).

    Journal: PLoS ONE

    Article Title: Single-Cell Cytokine Gene Expression in Peripheral Blood Cells Correlates with Latent Tuberculosis Status

    doi: 10.1371/journal.pone.0144904

    Figure Lengend Snippet: Analysis of the mechanism of PPD-induced IFNG expression in T cells. PBMCs from an LTBI+ donor were treated with CsA, αIL-12, or isotype control antibody for 1 hr and then stimulated with PPD for 6 hr. The frequency of IFNG+CD3+ cells is shown in the upper right quadrant of each bichromatic contour plot. The title above each plot indicates the conditions used for stimulation (top line) and pretreatment (bottom line).

    Article Snippet: PBMC collection and cell culture Within two hours after blood collection, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque Plus (GE Healthcare, Waukesha, WI) density-gradient centrifugation and stored in liquid nitrogen.

    Techniques: Expressing

    Expression of CD69 and CD154 activation markers in CD4+ T cells. PPD-stimulated PBMC from LTBI+ donors were co-cultured with αCD154 antibody, stained for CD69, CD4, and CD3, probed with Cy5-labeled IFNG and IL2 nucleic acid probes, and analyzed by flow cytometry. Gates were set based on unstimulated negative controls, SEB-stimulated positive controls, and FMO controls. The scatter plots show data from one donor and the pie charts from three donors. (a) Frequencies of cytokine mRNA+CD4+ T cells (left panels) and CD69 and CD154 expression in mRNA+CD4+ T cells (right panels). The upper right quadrant shows mRNA+CD69+CD154+ cells. Cytokine: IFNG (top row) and IL2 (bottom row). (b) Frequencies of all combinations of CD69 and CD154 expression in IFNG+ (top row) and IL2+ (bottom row) CD4+ T cells, one donor per column.

    Journal: PLoS ONE

    Article Title: Single-Cell Cytokine Gene Expression in Peripheral Blood Cells Correlates with Latent Tuberculosis Status

    doi: 10.1371/journal.pone.0144904

    Figure Lengend Snippet: Expression of CD69 and CD154 activation markers in CD4+ T cells. PPD-stimulated PBMC from LTBI+ donors were co-cultured with αCD154 antibody, stained for CD69, CD4, and CD3, probed with Cy5-labeled IFNG and IL2 nucleic acid probes, and analyzed by flow cytometry. Gates were set based on unstimulated negative controls, SEB-stimulated positive controls, and FMO controls. The scatter plots show data from one donor and the pie charts from three donors. (a) Frequencies of cytokine mRNA+CD4+ T cells (left panels) and CD69 and CD154 expression in mRNA+CD4+ T cells (right panels). The upper right quadrant shows mRNA+CD69+CD154+ cells. Cytokine: IFNG (top row) and IL2 (bottom row). (b) Frequencies of all combinations of CD69 and CD154 expression in IFNG+ (top row) and IL2+ (bottom row) CD4+ T cells, one donor per column.

    Article Snippet: PBMC collection and cell culture Within two hours after blood collection, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque Plus (GE Healthcare, Waukesha, WI) density-gradient centrifugation and stored in liquid nitrogen.

    Techniques: Expressing, Activation Assay, Cell Culture, Staining, Labeling, Flow Cytometry, Cytometry

    Comparison of FISH-Flow and intracellular cytokine staining (ICS) for detection of antigen-specific T cell responses. PBMC from an LTBI+ donor were stimulated with PPD, SEB, or left unstimulated. All cells were co-cultured with αCD154 antibody, stained for CD69, CD4, and CD3, probed with Cy5-labeled IFNG (FISH-Flow) or Alexa 647 IFN-γ antibody (ICS), and analyzed by flow cytometry. In the ICS assay, samples were incubated in the presence of 2 μM monensin. Gates were based on unstimulated negative controls and FMO controls. (a) Frequencies of IFN-γ protein+ (IFN-γ) or IFNG mRNA+ (IFNG) cells among CD4+ T cells expressing the CD154 T cell activation marker. (b) Frequencies of IFN-γ protein+ (IFN-γ) or IFNG mRNA+ (IFNG) cells among CD4+ T cells expressing the CD69 T cell activation marker.

    Journal: PLoS ONE

    Article Title: Single-Cell Cytokine Gene Expression in Peripheral Blood Cells Correlates with Latent Tuberculosis Status

    doi: 10.1371/journal.pone.0144904

    Figure Lengend Snippet: Comparison of FISH-Flow and intracellular cytokine staining (ICS) for detection of antigen-specific T cell responses. PBMC from an LTBI+ donor were stimulated with PPD, SEB, or left unstimulated. All cells were co-cultured with αCD154 antibody, stained for CD69, CD4, and CD3, probed with Cy5-labeled IFNG (FISH-Flow) or Alexa 647 IFN-γ antibody (ICS), and analyzed by flow cytometry. In the ICS assay, samples were incubated in the presence of 2 μM monensin. Gates were based on unstimulated negative controls and FMO controls. (a) Frequencies of IFN-γ protein+ (IFN-γ) or IFNG mRNA+ (IFNG) cells among CD4+ T cells expressing the CD154 T cell activation marker. (b) Frequencies of IFN-γ protein+ (IFN-γ) or IFNG mRNA+ (IFNG) cells among CD4+ T cells expressing the CD69 T cell activation marker.

    Article Snippet: PBMC collection and cell culture Within two hours after blood collection, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque Plus (GE Healthcare, Waukesha, WI) density-gradient centrifugation and stored in liquid nitrogen.

    Techniques: Fluorescence In Situ Hybridization, Flow Cytometry, Staining, Cell Culture, Labeling, Cytometry, Incubation, Expressing, Activation Assay, Marker

    Analysis of IFNG expression in CD3+ and CD3- cell subsets. PBMC from LTBI+ donors were stimulated with PPD or left unstimulated, stained with antibodies against surface markers as indicated, probed with Cy5-labeled IFNG probes, and analyzed by flow cytometry. Gates were set based on unstimulated samples and Fluorescence Minus One (FMO) controls. The scatter plots show data from one donor and the bar graphs from three donors. In each panel, frequencies were calculated relative to the total number of cells in the panel. (a) Frequencies of IFNG+CD3+ cells (left panel) and IFNG+ cells in the CD4+, CD8+ and γδ T cell subsets (right panel). (b) Frequency of CD4+, CD8+, and γδ subsets in IFNG+CD3+ cells. (c) Frequencies of IFNG+CD3- cells (left panel) and IFNG+CD56+ cells (right panel, upper right quadrants). (d) Frequency of CD56 in IFNG+CD3- cells.

    Journal: PLoS ONE

    Article Title: Single-Cell Cytokine Gene Expression in Peripheral Blood Cells Correlates with Latent Tuberculosis Status

    doi: 10.1371/journal.pone.0144904

    Figure Lengend Snippet: Analysis of IFNG expression in CD3+ and CD3- cell subsets. PBMC from LTBI+ donors were stimulated with PPD or left unstimulated, stained with antibodies against surface markers as indicated, probed with Cy5-labeled IFNG probes, and analyzed by flow cytometry. Gates were set based on unstimulated samples and Fluorescence Minus One (FMO) controls. The scatter plots show data from one donor and the bar graphs from three donors. In each panel, frequencies were calculated relative to the total number of cells in the panel. (a) Frequencies of IFNG+CD3+ cells (left panel) and IFNG+ cells in the CD4+, CD8+ and γδ T cell subsets (right panel). (b) Frequency of CD4+, CD8+, and γδ subsets in IFNG+CD3+ cells. (c) Frequencies of IFNG+CD3- cells (left panel) and IFNG+CD56+ cells (right panel, upper right quadrants). (d) Frequency of CD56 in IFNG+CD3- cells.

    Article Snippet: PBMC collection and cell culture Within two hours after blood collection, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque Plus (GE Healthcare, Waukesha, WI) density-gradient centrifugation and stored in liquid nitrogen.

    Techniques: Expressing, Staining, Labeling, Flow Cytometry, Cytometry, Fluorescence

    Janus kinase inhibitors block the IL-12 and IFN-γ response in peripheral blood mononuclear cells (PBMCs) from STAT4 risk patients with systemic lupus erythematosus (SLE). (A) Healthy donor PBMCs were treated with serial dilutions of the JAK2 selective (JAK2i), the TYK2 selective (TYK2i) and the pan-JAK inhibitor (pan-JAKi) before being stimulated with 5 ng/mL IL-12, 0.1 ng/mL IFN-γ or 200 U/mL IFN-α for 20 min. IL-12-stimulated cells were preactivated with PHA/IL-2. Phosphorylation of STAT4 (pSTAT4) and STAT1 (pSTAT1) was determined by flow cytometry in IL-12-stimulated cells and IFN-γ or IFN-α-stimulated cells, respectively. Data (mean±SD of two individuals) from IL-12 and IFN-α-stimulated cells are shown for CD3 + T cells and from IFN-γ-stimulated cells for monocytes. (B) IC 50 values for each inhibitor. (C–E) PBMCs from healthy donors (HD), patients with SLE homozygous for the protective STAT4 allele (G/G) and patients with SLE carrying one or two STAT4 risk alleles (G/T+T/T) were incubated with 200 nM TYK2i (C, D), 70 nm JAK2i or 30 nM pan-JAKi (E). (C, D) Inhibition of IL-12-induced pSTAT4 in CD8 + T cells (C) and reduction in the frequency of IFN-γ + memory CD45RO + CD57 – CD8 + T cells (D). (E) Inhibition of IFN-γ-induced pSTAT1 in monocytes. (C–E) Open triangles and squares denote G/T and T/T patients with SLE, respectively.

    Journal: Annals of the Rheumatic Diseases

    Article Title: The STAT4 SLE risk allele rs7574865[T] is associated with increased IL-12-induced IFN-γ production in T cells from patients with SLE

    doi: 10.1136/annrheumdis-2017-212794

    Figure Lengend Snippet: Janus kinase inhibitors block the IL-12 and IFN-γ response in peripheral blood mononuclear cells (PBMCs) from STAT4 risk patients with systemic lupus erythematosus (SLE). (A) Healthy donor PBMCs were treated with serial dilutions of the JAK2 selective (JAK2i), the TYK2 selective (TYK2i) and the pan-JAK inhibitor (pan-JAKi) before being stimulated with 5 ng/mL IL-12, 0.1 ng/mL IFN-γ or 200 U/mL IFN-α for 20 min. IL-12-stimulated cells were preactivated with PHA/IL-2. Phosphorylation of STAT4 (pSTAT4) and STAT1 (pSTAT1) was determined by flow cytometry in IL-12-stimulated cells and IFN-γ or IFN-α-stimulated cells, respectively. Data (mean±SD of two individuals) from IL-12 and IFN-α-stimulated cells are shown for CD3 + T cells and from IFN-γ-stimulated cells for monocytes. (B) IC 50 values for each inhibitor. (C–E) PBMCs from healthy donors (HD), patients with SLE homozygous for the protective STAT4 allele (G/G) and patients with SLE carrying one or two STAT4 risk alleles (G/T+T/T) were incubated with 200 nM TYK2i (C, D), 70 nm JAK2i or 30 nM pan-JAKi (E). (C, D) Inhibition of IL-12-induced pSTAT4 in CD8 + T cells (C) and reduction in the frequency of IFN-γ + memory CD45RO + CD57 – CD8 + T cells (D). (E) Inhibition of IFN-γ-induced pSTAT1 in monocytes. (C–E) Open triangles and squares denote G/T and T/T patients with SLE, respectively.

    Article Snippet: In some experiments, PBMCs were preactivated with 1.5% phytohaemagglutinin (PHA; Life Technologies) and 2.5 ng/mL IL-2 (Miltenyi) for 72 hours.

    Techniques: Blocking Assay, Flow Cytometry, Cytometry, Incubation, Inhibition

    PHA/IL-2 preactivated T cells from patients with systemic lupus erythematosus (SLE) carrying the STAT4 risk allele have an increased phosphorylation of STAT4 in response to IL-12. PHA/IL-2 pre-activated peripheral blood mononuclear cells were stimulated with 50 ng/mL IL-12 for 20 min. Phosphorylation of STAT4 (pSTAT4) was determined in indicated cell populations using flow cytometry. (A) Histograms from one representative donor. (B–D) Cumulative data from 19 homozygous protective (G/G, black circles), 20 heterozygous (G/T, open triangles) and 9 homozygous risk (T/T, open squares) STAT4 patients with SLE. Due to

    Journal: Annals of the Rheumatic Diseases

    Article Title: The STAT4 SLE risk allele rs7574865[T] is associated with increased IL-12-induced IFN-γ production in T cells from patients with SLE

    doi: 10.1136/annrheumdis-2017-212794

    Figure Lengend Snippet: PHA/IL-2 preactivated T cells from patients with systemic lupus erythematosus (SLE) carrying the STAT4 risk allele have an increased phosphorylation of STAT4 in response to IL-12. PHA/IL-2 pre-activated peripheral blood mononuclear cells were stimulated with 50 ng/mL IL-12 for 20 min. Phosphorylation of STAT4 (pSTAT4) was determined in indicated cell populations using flow cytometry. (A) Histograms from one representative donor. (B–D) Cumulative data from 19 homozygous protective (G/G, black circles), 20 heterozygous (G/T, open triangles) and 9 homozygous risk (T/T, open squares) STAT4 patients with SLE. Due to

    Article Snippet: In some experiments, PBMCs were preactivated with 1.5% phytohaemagglutinin (PHA; Life Technologies) and 2.5 ng/mL IL-2 (Miltenyi) for 72 hours.

    Techniques: Flow Cytometry, Cytometry

    PHA/IL-2 preactivated T cells from STAT4 risk patients with systemic lupus erythematosus (SLE) have an increased IFN-γ production in response to IL-12. (A, B) PHA/IL-2 activated peripheral blood mononuclear cells were re-stimulated with 5 ng/mL IL-12 for 15 hours (A) or 20 ng/mL PMA together with 1 ng/mL A23187 for 6 hours (B). The frequency of IFN-γ + cells was determined in CD8 + and CD4 + T cells and subsets thereof using flow cytometry. Data from 18 STAT4 homozygous protective (G/G, black circles), 21 heterozygous (G/T, open triangles) and 9 homozygous risk (T/T, open squares) patients with SLE. Data of CD8 + T cells from one G/T individual (indicated as a filled triangle in A) was considered an outlier and was excluded from the statistical analysis of CD8 + T cells and subsets thereof. Due to

    Journal: Annals of the Rheumatic Diseases

    Article Title: The STAT4 SLE risk allele rs7574865[T] is associated with increased IL-12-induced IFN-γ production in T cells from patients with SLE

    doi: 10.1136/annrheumdis-2017-212794

    Figure Lengend Snippet: PHA/IL-2 preactivated T cells from STAT4 risk patients with systemic lupus erythematosus (SLE) have an increased IFN-γ production in response to IL-12. (A, B) PHA/IL-2 activated peripheral blood mononuclear cells were re-stimulated with 5 ng/mL IL-12 for 15 hours (A) or 20 ng/mL PMA together with 1 ng/mL A23187 for 6 hours (B). The frequency of IFN-γ + cells was determined in CD8 + and CD4 + T cells and subsets thereof using flow cytometry. Data from 18 STAT4 homozygous protective (G/G, black circles), 21 heterozygous (G/T, open triangles) and 9 homozygous risk (T/T, open squares) patients with SLE. Data of CD8 + T cells from one G/T individual (indicated as a filled triangle in A) was considered an outlier and was excluded from the statistical analysis of CD8 + T cells and subsets thereof. Due to

    Article Snippet: In some experiments, PBMCs were preactivated with 1.5% phytohaemagglutinin (PHA; Life Technologies) and 2.5 ng/mL IL-2 (Miltenyi) for 72 hours.

    Techniques: Flow Cytometry, Cytometry

    Lenalidomide maintenance therapy does not enhance MM-specific CD4 + T-cell responses Analysis of T-cell responses specific for the MAGE-C1 peptide SQSSPVSSFPSSTSS. ( A ) CD137 expression on CD4 + and CD8 + T cells from patient#02 at the pre LEN timepoint upon in vitro restimulation with the MAGE-C1 peptide. ( B ) PBMCs obtained at the pre LEN and the LEN timepoint were examined by IFN-γ ELISPOT to measure the T-cell response to the MAGE-C1 peptide. The bar graphs show the mean number of spots per million cells of four replicate wells and the error bars represent the standard deviation. A two-way ANOVA with Sidak’s multiple comparisons test was performed to evaluate statistical significant differences, ns: not significant, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001.

    Journal: Oncotarget

    Article Title: Impact of lenalidomide maintenance on the immune environment of multiple myeloma patients with low tumor burden after autologous stem cell transplantation

    doi: 10.18632/oncotarget.24944

    Figure Lengend Snippet: Lenalidomide maintenance therapy does not enhance MM-specific CD4 + T-cell responses Analysis of T-cell responses specific for the MAGE-C1 peptide SQSSPVSSFPSSTSS. ( A ) CD137 expression on CD4 + and CD8 + T cells from patient#02 at the pre LEN timepoint upon in vitro restimulation with the MAGE-C1 peptide. ( B ) PBMCs obtained at the pre LEN and the LEN timepoint were examined by IFN-γ ELISPOT to measure the T-cell response to the MAGE-C1 peptide. The bar graphs show the mean number of spots per million cells of four replicate wells and the error bars represent the standard deviation. A two-way ANOVA with Sidak’s multiple comparisons test was performed to evaluate statistical significant differences, ns: not significant, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001.

    Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated from leukapheresis (pre-LEN timepoint) and whole blood (LEN timepoint) samples through lymphoprep gradient-centrifugation (Axis-Shield) following the manufacturer’s instructions before being cryopreserved.

    Techniques: Expressing, In Vitro, Enzyme-linked Immunospot, Standard Deviation

    Lenalidomide maintenance therapy results in an improved CD8 + T cell functionality, which is hampered by non-CD8 + T cells present in the periphery ( A ) PBMCs obtained at the pre LEN and the LEN timepoint were examined by IFN-γ ELISPOT to measure the CD8 + T-cell response to viral recall epitopes derived for CMV, EBV and influenza (CEF) ( n = 6). The number of spot forming units (SFU) as well as the activity in each well was determined. Bar graphs show the mean number of spots per million PBMCs or the mean activity of four replicate wells and the error bars show the standard deviation. Spot numbers and activity measured upon culture in the absence of peptide were subtracted. ( B – E ) CD8 + T cells purified from PBMC samples obtained at the pre LEN and LEN timepoint were examined by IFN-γ/IL-2/TNF-α FLUOROSPOT to characterize the CD8 + T-cell response to the CEF peptide pool. (B–C) Bar graphs show the mean number of spots per million CD8 + T cells of five replicate wells and the error bars show the standard deviation. Spot numbers and activity measured upon culture in the absence of peptide were subtracted. ( D ) Raw data showing three-color FLUOROSPOT results. One representative well is shown for each patient and timepoint. Cytokine-specific spots are shown in green (IFN-γ), red (TNF-α), blue (IL-2) or overlays of these colors. ( E ) The pie charts indicate the portion of mono- and polyfunctional CEF-specific CD8 + T cells. A two-way ANOVA with Sidak’s multiple comparisons test was performed to detect differences between the pre LEN and the LEN timepoint, ns: not significant, * p

    Journal: Oncotarget

    Article Title: Impact of lenalidomide maintenance on the immune environment of multiple myeloma patients with low tumor burden after autologous stem cell transplantation

    doi: 10.18632/oncotarget.24944

    Figure Lengend Snippet: Lenalidomide maintenance therapy results in an improved CD8 + T cell functionality, which is hampered by non-CD8 + T cells present in the periphery ( A ) PBMCs obtained at the pre LEN and the LEN timepoint were examined by IFN-γ ELISPOT to measure the CD8 + T-cell response to viral recall epitopes derived for CMV, EBV and influenza (CEF) ( n = 6). The number of spot forming units (SFU) as well as the activity in each well was determined. Bar graphs show the mean number of spots per million PBMCs or the mean activity of four replicate wells and the error bars show the standard deviation. Spot numbers and activity measured upon culture in the absence of peptide were subtracted. ( B – E ) CD8 + T cells purified from PBMC samples obtained at the pre LEN and LEN timepoint were examined by IFN-γ/IL-2/TNF-α FLUOROSPOT to characterize the CD8 + T-cell response to the CEF peptide pool. (B–C) Bar graphs show the mean number of spots per million CD8 + T cells of five replicate wells and the error bars show the standard deviation. Spot numbers and activity measured upon culture in the absence of peptide were subtracted. ( D ) Raw data showing three-color FLUOROSPOT results. One representative well is shown for each patient and timepoint. Cytokine-specific spots are shown in green (IFN-γ), red (TNF-α), blue (IL-2) or overlays of these colors. ( E ) The pie charts indicate the portion of mono- and polyfunctional CEF-specific CD8 + T cells. A two-way ANOVA with Sidak’s multiple comparisons test was performed to detect differences between the pre LEN and the LEN timepoint, ns: not significant, * p

    Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated from leukapheresis (pre-LEN timepoint) and whole blood (LEN timepoint) samples through lymphoprep gradient-centrifugation (Axis-Shield) following the manufacturer’s instructions before being cryopreserved.

    Techniques: Enzyme-linked Immunospot, Derivative Assay, Activity Assay, Standard Deviation, Purification

    The total number of circulating fibrocytes is unaffected by the leukocyte isolation strategy used. a Total number of circulating fibrocytes (defined as CD45 + lin − CD15 − CXCR4 + Col-1 + cells) per ml blood using two common leukocyte isolation techniques, e.g. the simple Pasteur pipette tube technique to isolate all white blood cells and Ficoll separation technique to isolate PBMCs. For this experiment we analyzed paired total white blood cells and PBMCs on the same day as blood withdrawal of 9 patients (4 IPF patients (black) and 5 PH patients (purple)) and 5 healthy controls (green). b Percentage of fibrocytes (of CD45 + cells) in the same patients after isolating all white blood cells (gray) and PBMCs (black). Data are depicted as median and interquartile

    Journal: Respiratory Research

    Article Title: Fibrocytes are increased in lung and peripheral blood of patients with idiopathic pulmonary fibrosis

    doi: 10.1186/s12931-018-0798-8

    Figure Lengend Snippet: The total number of circulating fibrocytes is unaffected by the leukocyte isolation strategy used. a Total number of circulating fibrocytes (defined as CD45 + lin − CD15 − CXCR4 + Col-1 + cells) per ml blood using two common leukocyte isolation techniques, e.g. the simple Pasteur pipette tube technique to isolate all white blood cells and Ficoll separation technique to isolate PBMCs. For this experiment we analyzed paired total white blood cells and PBMCs on the same day as blood withdrawal of 9 patients (4 IPF patients (black) and 5 PH patients (purple)) and 5 healthy controls (green). b Percentage of fibrocytes (of CD45 + cells) in the same patients after isolating all white blood cells (gray) and PBMCs (black). Data are depicted as median and interquartile

    Article Snippet: Briefly, following Ficoll density centrifugation, 2 × 10^5 PBMCs were plated into culture-slides (sigma, C7182, 0.8 cm2 /well) in complete culture medium (Dulbecco’s modified Eagle’s medium) (DMEM) supplemented with 20% fetal calf serum, 2 mM L-glutamine, 100 U/mL of penicillin, 100 mg/mL of streptomycin) (Life Technologies, Grand Island, NY) at 37 °C and 5% CO2.

    Techniques: Isolation, Transferring