peripheral blood mononuclear cells pbmc Search Results


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  • 99
    Millipore histopaque 1077
    γH2AX foci intensity in lymphocytes isolated before (Pre-PET + 0 Gy, Control) and after (Post-PET + 0 Gy, Exposed) a PET scan procedure, as measured by the H2AX Phosphorylation Assay Kit (Flow Cytometry). Mean channel values for quadruplicates were averaged and normalized to an antibody control tube specific to each sample and donor. Error bars represent the standard error of the mean. Lymphocytes were processed for measurement directly following isolation from whole blood using <t>Histopaque-1077</t> and resuspended at 5 × 10 5 cells/mL in complete Rosewell Park Memorial Institute medium. PET indicates positron emission tomography.
    Histopaque 1077, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13409 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore red blood cell lysis buffer
    PDK1 knockout in the haematopoietic system blocks the development of mature T and B cells. PDK1 fl/fl /Vav-Cre +ve mice were found to have an increased spleen size ( A ) and weight ( B ) relative to PDK1 +/+ /Vav-Cre +ve control mice. Post <t>lysis</t> of <t>red</t> <t>blood</t> cells however spleen <t>cell</t> numbers were similar between the PDK1 fl/fl /Vav-Cre +ve mice ( n =15) and PDK1 +/+ /Vav-Cre +ve ( n =18), PDK1 +/+ /Vav-Cre −ve ( n =6) and PDK1 fl/fl /Vav-Cre −ve ( n =6) control genotypes ( C ). Error bars represent standard deviation. H E staining of the spleens showed that PDK1 knockout resulted in a disruption of the white pulp ( D ). Analysis of T- and B-cell populations in the spleen by FACS ( E , F ) demonstrated that PDK1 fl/fl /Vav-Cre +ve mice ( n =7) had negligible numbers of mature T and B cells relative to PDK1 +/+ /Vav-Cre +ve ( n =6), PDK1 +/+ /Vav-Cre −ve ( n =6) and PDK1 fl/fl /Vav-Cre −ve ( n =4) control genotypes. Error bars represent the standard deviation of 4–7 mice per genotype. In ( B , C and E ) a P -value (Student's t -test) of
    Red Blood Cell Lysis Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 19303 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Axis-Shield Diagnostics peripheral blood mononuclear cells pbmcs
    Comparison of HBV-specific cell responses in the presence or absence of HBeAg. (A) C protein-specific T-cell responses elicited in ELISPOT assay; (B) Cytokine <t>IFN-Y</t> responses in cultured supernatants of <t>PBMCs</t> stimulated by C protein. HBeAg(−): HBeAg-negative subjects; HBeAg(+): HBeAg-positive subjects; HIV+: HIV co-infected; HIV-: not co-infected with HIV.
    Peripheral Blood Mononuclear Cells Pbmcs, supplied by Axis-Shield Diagnostics, used in various techniques. Bioz Stars score: 94/100, based on 1342 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher pbmc
    The distribution of fragments of mRNA, rRNA, tRNA, miRNA, and others in the small <t>RNA</t> libraries. a The <t>PBMC</t> of heat-stressed cattle. b The PBMC of non-heat-stressed cattle
    Pbmc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 14117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson peripheral blood mononuclear cells pbmcs
    Systemic immune response to <t>LCMV-GP33</t> peptide C57BL/6 mice (5 mice/group) were challenged subcutaneously with TC-1 tumor cells and subsequently treated with various combinations of cisplatin, CpG and GP33 peptide as shown. The <t>PBMCs</t> were analyzed 1 week after the last antigen delivery. The presence of GP33-specific CD8+ T cells in PBMCs was analyzed using intracellular cytokine staining for IFN-γ and CD8+ staining followed by flow cytometry analysis. A. Representative flow cytometry contour plot depicting the frequency of IFN-γ-secreting CD8+ T cells among PBMCs after being pulsed with GP33 peptide. B. Bar graph depicting the number of IFN-γ+ CD8+ T cells among PBMCs (mean ±S.E).
    Peripheral Blood Mononuclear Cells Pbmcs, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 950 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Nycomed peripheral blood mononuclear cells pbmc
    Dose–response curves for <t>ADCC.</t> The procedures for OxLDL incubated and Ab treatment were the same as described in the legend for Fig. . As effectors normal rat <t>PBMC</t> were added to the culture instead of complement. Note increased 51Cr release
    Peripheral Blood Mononuclear Cells Pbmc, supplied by Nycomed, used in various techniques. Bioz Stars score: 92/100, based on 502 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Biochrom peripheral blood mononuclear cells pbmc
    miR-20b expression in T cells and in <t>PBMC</t> from natalizumab-associated progressive multifocal leukoencephalopathy (PML). (A) PBMC were isolated using a <t>Ficoll</t> gradient and CD4+ T cells were sorted with magnetic beads ( n = 9; randomly selected patients of our initial longitudinal cohort consisting of 17 patients). miR-20b expression (qPCR) was measured in the CD4+ and CD4-negative fraction and compared using t- test. (B) CD4+ cells were activated by plate-bound anti-CD3-antibody and soluble anti-CD28-antibody without further cytokines, termed “Th0”, and with IL-1 (10 ng/mL), IL-6 (10 ng/mL), IL-23 (10 ng/mL) and TGF- β (5 ng/mL), termed “Th17”, as well as freshly isolated CD4+ T cells, termed “fresh”. miR-20b levels were measured by qPCR. (C) miR-20b levels in PBMC from natalizumab-treated patients ( n = 9, left bar) were compared to miR-20b levels in two PBMC samples from patients with natalizumab-associated PML (each patient one bar).
    Peripheral Blood Mononuclear Cells Pbmc, supplied by Biochrom, used in various techniques. Bioz Stars score: 93/100, based on 447 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore human peripheral blood mononuclear cells pbmc
    Detection of ApoE isoforms in differentiating hNP1 cells. Differentiating hNP1 cells were exposed to medium alone (Unt), mock-infected <t>PBMC</t> supernatants (Mock), and <t>HIV-infected</t> PBMC supernatants (HIV), and to rApoE3 or rApoE4 at 3 μg/ml, as described in Materials and Methods. At day 16 of differentiation, and 24 h after replenishment of rApoE in the cultures, cells were lysed for quantitative immunoblotting to detect ApoE. Lower blot depicts representative endogenous ApoE detected in cell culture lysates with molecular weight of 34 kDa, and rApoE with molecular weight of 36 kDa. Upper blot corresponds to beta-actin used as an internal control. For comparison, recombinant ApoE3 and ApoE4 were loaded directly on the gel at a 3 ng per lane, as shown. Bar graphs present ApoE signal normalized to beta-actin; each bar represents the mean of two experiments
    Human Peripheral Blood Mononuclear Cells Pbmc, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 145 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Ficoll-Paque Pharmacia peripheral blood mononuclear cell pbmc
    Semi-quantitative <t>PCR</t> analysis of BLV pol detects differences in proviral load between BLV SGV and BLV-rabbits. <t>PBMC</t> were harvested at month indicated after challenge with 1 × 10 8 FLK/BLV cells from indicated rabbits that had been infected with BLV SGV for 24 months or from age-matched naïve controls. DNA from 50,000 PMBC was subjected to nested PCR and gel electrophoresis and stained with ethidium bromide. The position of the 591-bp amplicon is designated by the labeled arrow. In parallel, DNA from 20,000 PBMC was subjected to PCR with primers complementary to β- actin . The position of the 594-bp amplicon is designated by the labeled arrow and the signals were quantified by densitometry.
    Peripheral Blood Mononuclear Cell Pbmc, supplied by Ficoll-Paque Pharmacia, used in various techniques. Bioz Stars score: 89/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore ceramide
    Exposure to C1P or <t>ceramide</t> induces P-glycoprotein transport activity at the blood-brain barrier. (A) Dose response of 20 minutes ceramide treatment, showing that ceramide increases specific P-glycoprotein activity in a concentration-dependent manner.
    Ceramide, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 483 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore peripheral blood mononuclear cells
    Generation, isolation, and characterization of epidermal growth factor receptor (EGFR)‐specific chimeric antigen receptor (CAR)‐engineered natural killer (NK) <t>cells</t> (EGFR‐CAR NK cells). (A) Flow cytometric analysis of phenotypic and subset composition of <t>peripheral</t> <t>blood</t> <t>mononuclear</t> cells (PBMCs) labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE. (B‐C) Flow cytometric analysis of phenotypic and subset composition of NK cells labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE, and anti‐CD69‐APC‐Cy7. (D) The percentage of CD3‐/CD56 + cells in day 0 PBMCs and day14 PBMC culture. (E) Real‐time PCR and (F) Western blotting analyses of the expression of exogenous CD3ζ in the non‐transduced NK cells, Con‐CAR NK cells, EGFR‐CAR‐1 NK cells, and EGFR‐CAR‐2 NK cells. β‐actin was used as an endogenous control. (G) The transduced NK cells stained with IgG‐FITC and EGFR‐FITC antibodies were detected by flow cytometry
    Peripheral Blood Mononuclear Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 187 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson human pbmcs
    <t>EAT-2</t> over-expression enhances human DC maturation. Human <t>PBMCs</t> were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-EAT2 or rAd5-Null. At 72 hpi, cells were stained for surface expression of CD86 (A), HLA-DR (B), CD80 (C) and CD83 (D) and FACS analysis was performed. Data are representative of at least three independent experiments from three different blood donors with similar results. Samples were plated in quadruplicates. Bars represent mean ± SD. Statistical analysis was completed using a one way ANOVA with a Student–Newman–Keuls post-hoc test. P
    Human Pbmcs, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 603 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson blood mononuclear cells pbmc
    Intracellular and plasma pharmacokinetic profiles of <t>imatinib</t> in five individual patients (solid lines, plasma concentrations; broken lines, peripheral blood mononuclear cell concentrations)
    Blood Mononuclear Cells Pbmc, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 177 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Valiant peripheral blood mononuclear cells pbmcs
    Isolation and molecular characterization of T cell subsets from <t>PNH</t> patients ( A ) Gating strategy for T cell subsets for cell sorting. <t>PBMCs</t> were stained with ViViD, anti-CD14-Pacific Blue, anti-CD19-Pacific Blue, anti-CD3-BV605, anti-CD4-V500, anti-CD8-APC-H7, anti-CD45RA-PE-Cy7, and anti-CD45RO-APC. Lymphocytes were gated based on their scatter characteristics or forward scatter height vs. forward scatter area. Live T cells were gated based on positive for CD3 and negative for ViViD, CD14, and CD19 to remove dead cells, monocytes, and B cells. CD4 + and CD8 + T cells were then gated based on the characteristic expression patterns of CD45RA and CD45RO. Four different T cell subsets [CD4 + naïve (CD45RA + CD45RO - ), CD4 + memory (CD45RA - CD45RO + ), CD8 + naïve (CD45RA + CD45RO - ), and CD8 + memory (CD45RA - CD45RO + ) T cells] were sorted for RNA-seq. ( B ) Transcriptional validation of the naïve and memory T cell identity by analyzing expression levels of CD4, CD8, CCR5, and EOMES. FPKM (fragments per kilobase of exon per million fragments mapped). * p
    Peripheral Blood Mononuclear Cells Pbmcs, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare pbmcs
    The systemic cellular immune response to sand fly saliva in volunteers in a cutaneous leishmaniasis-endemic area of Mali. Peripheral blood mononuclear cells <t>(PBMCs)</t> were obtained from 255 individuals aged 11–65 years from the villages of Kemena and Sougoula. IFN-γ, IL-12, IL-10, IL-13, and IL-5 levels were measured in the supernatants of PBMCs from exposed volunteers 96 hours after stimulation with Phlebotomus duboscqi salivary gland homogenate. ( a ) Overall <t>cytokine</t> production by tested individuals. ( b ) Pairwise correlation of expressed cytokines (log-transformed cytokine levels). Numbers in insets indicate the Spearman correlation coefficient and the 95% confidence intervals for each correlation. ( c ) Cytokine responses of individuals in ( a ) stratified by T H 1-predominant, T H 2-predominant, or T H 1/T H 2 mixed responses to sand fly salivary proteins.
    Pbmcs, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 10970 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    γH2AX foci intensity in lymphocytes isolated before (Pre-PET + 0 Gy, Control) and after (Post-PET + 0 Gy, Exposed) a PET scan procedure, as measured by the H2AX Phosphorylation Assay Kit (Flow Cytometry). Mean channel values for quadruplicates were averaged and normalized to an antibody control tube specific to each sample and donor. Error bars represent the standard error of the mean. Lymphocytes were processed for measurement directly following isolation from whole blood using Histopaque-1077 and resuspended at 5 × 10 5 cells/mL in complete Rosewell Park Memorial Institute medium. PET indicates positron emission tomography.

    Journal: Dose-Response

    Article Title: Biological Response of Positron Emission Tomography Scan Exposure and Adaptive Response in Humans

    doi: 10.1177/1559325815611904

    Figure Lengend Snippet: γH2AX foci intensity in lymphocytes isolated before (Pre-PET + 0 Gy, Control) and after (Post-PET + 0 Gy, Exposed) a PET scan procedure, as measured by the H2AX Phosphorylation Assay Kit (Flow Cytometry). Mean channel values for quadruplicates were averaged and normalized to an antibody control tube specific to each sample and donor. Error bars represent the standard error of the mean. Lymphocytes were processed for measurement directly following isolation from whole blood using Histopaque-1077 and resuspended at 5 × 10 5 cells/mL in complete Rosewell Park Memorial Institute medium. PET indicates positron emission tomography.

    Article Snippet: Lymphocytes were isolated from whole blood using Histopaque 1077 by centrifugation at room temperature for 30 minutes at 300g , according to the manufacturer’s instructions (Sigma-Aldrich, Oakville, Ontario).

    Techniques: Isolation, Positron Emission Tomography, Phosphorylation Assay, Flow Cytometry, Cytometry

    DGC induces TXNIP down-regulation in T cells. ( A ) Representative Western blot (lower panel) and quantification (upper panel) of TXNIP with CD3ζ as loading control from T cells isolated from blood samples incubated for 4 hours (4 h blood, Procedure II, Fig. 1B ) after no treatment (−) or centrifugation (+). ( B ) Representative Western blot (lower panel) and quantification (upper panel) of TXNIP with CD3ζ as loading control from T cells immediately isolated and lysed (0 h, Procedure I, Fig. 1B ), isolated and incubated for 4 hours before lysis (4 h T cells, Procedure I, Fig. 1B ), and isolated and lysed after incubation of PBMC for 4 hours (4 h PBMC, Procedure III, Fig. 1B ) after DGC on either Lymphoprep, Histopaque or Ficoll-Paque as indicated. ( C ) Representative Western blot (lower panel) and quantification (upper panel) of TXNIP with CD3ζ as loading control from T cells isolated from blood samples incubated for 4 hours (4 h blood, Procedure II, Fig. 1B ) with no addition (Control) or with Lymphoprep, Histopaque or Ficoll-Paque as indicated. ( D ) Overview of different T cell isolation and incubation procedures used. The text in the red frames gives the procedure used and the nomenclature for the T cell lysates analyzed and shown in ( E) lanes 4–9. ( E ) Representative Western blot (lower panel) and quantification (upper panel) of TXNIP with CD3ζ as loading control from T cells immediately isolated and lysed (lane 1: 0 h, Procedure I, Fig. 1B ; lane 4: monocyte depletion, 0 h, Procedure IV, ( D) ; lane 7: T cell enrichment, 0 h, Procedure VI, D ), isolated and incubated for 4 hours before lysis (lane 2: 4 h T cells, Procedure I, Fig. 1B ; lane 5: monocyte depletion, 4 h T cells, Procedure IV, ( D) ; lane 8: T cell enrichment, 4 h T cells, Procedure VI, D ), and isolated and lysed after incubation of PBMC for 4 hours (lane 3: 4 h PBMC, Procedure III, Fig. 1B ; lane 6: monocyte depletion, 4 h PBMC, Procedure V, D ) or T cells for 5 h (lane 9: T cell enrichment, 5 h T cells, Procedure VI, D ). ( A–C , E ) Each Western blot is representative for Western blots obtained from at least 3 different biological experiments and the quantification shows the mean + SEM of the band densities of TXNIP from Western blots obtained from at least 3 different biological experiments. The positions of the relevant molecular weight markers and their molecular weight in kDa are given to the right of each Western blot.

    Journal: Scientific Reports

    Article Title: Tumor necrosis factor induces rapid down-regulation of TXNIP in human T cells

    doi: 10.1038/s41598-019-53234-x

    Figure Lengend Snippet: DGC induces TXNIP down-regulation in T cells. ( A ) Representative Western blot (lower panel) and quantification (upper panel) of TXNIP with CD3ζ as loading control from T cells isolated from blood samples incubated for 4 hours (4 h blood, Procedure II, Fig. 1B ) after no treatment (−) or centrifugation (+). ( B ) Representative Western blot (lower panel) and quantification (upper panel) of TXNIP with CD3ζ as loading control from T cells immediately isolated and lysed (0 h, Procedure I, Fig. 1B ), isolated and incubated for 4 hours before lysis (4 h T cells, Procedure I, Fig. 1B ), and isolated and lysed after incubation of PBMC for 4 hours (4 h PBMC, Procedure III, Fig. 1B ) after DGC on either Lymphoprep, Histopaque or Ficoll-Paque as indicated. ( C ) Representative Western blot (lower panel) and quantification (upper panel) of TXNIP with CD3ζ as loading control from T cells isolated from blood samples incubated for 4 hours (4 h blood, Procedure II, Fig. 1B ) with no addition (Control) or with Lymphoprep, Histopaque or Ficoll-Paque as indicated. ( D ) Overview of different T cell isolation and incubation procedures used. The text in the red frames gives the procedure used and the nomenclature for the T cell lysates analyzed and shown in ( E) lanes 4–9. ( E ) Representative Western blot (lower panel) and quantification (upper panel) of TXNIP with CD3ζ as loading control from T cells immediately isolated and lysed (lane 1: 0 h, Procedure I, Fig. 1B ; lane 4: monocyte depletion, 0 h, Procedure IV, ( D) ; lane 7: T cell enrichment, 0 h, Procedure VI, D ), isolated and incubated for 4 hours before lysis (lane 2: 4 h T cells, Procedure I, Fig. 1B ; lane 5: monocyte depletion, 4 h T cells, Procedure IV, ( D) ; lane 8: T cell enrichment, 4 h T cells, Procedure VI, D ), and isolated and lysed after incubation of PBMC for 4 hours (lane 3: 4 h PBMC, Procedure III, Fig. 1B ; lane 6: monocyte depletion, 4 h PBMC, Procedure V, D ) or T cells for 5 h (lane 9: T cell enrichment, 5 h T cells, Procedure VI, D ). ( A–C , E ) Each Western blot is representative for Western blots obtained from at least 3 different biological experiments and the quantification shows the mean + SEM of the band densities of TXNIP from Western blots obtained from at least 3 different biological experiments. The positions of the relevant molecular weight markers and their molecular weight in kDa are given to the right of each Western blot.

    Article Snippet: Where indicated, Histopaque® –1077 (10771, Sigma-Aldrich) or Ficoll-PaqueTM (GE17-5442-02, Sigma-Aldrich) was used and centrifuged for 30 min at 400 g, 24 °C with the brake off as specified by the manufacturers’ instructions.

    Techniques: Western Blot, Isolation, Incubation, Centrifugation, Lysis, Cell Isolation, Molecular Weight

    PDK1 knockout in the haematopoietic system blocks the development of mature T and B cells. PDK1 fl/fl /Vav-Cre +ve mice were found to have an increased spleen size ( A ) and weight ( B ) relative to PDK1 +/+ /Vav-Cre +ve control mice. Post lysis of red blood cells however spleen cell numbers were similar between the PDK1 fl/fl /Vav-Cre +ve mice ( n =15) and PDK1 +/+ /Vav-Cre +ve ( n =18), PDK1 +/+ /Vav-Cre −ve ( n =6) and PDK1 fl/fl /Vav-Cre −ve ( n =6) control genotypes ( C ). Error bars represent standard deviation. H E staining of the spleens showed that PDK1 knockout resulted in a disruption of the white pulp ( D ). Analysis of T- and B-cell populations in the spleen by FACS ( E , F ) demonstrated that PDK1 fl/fl /Vav-Cre +ve mice ( n =7) had negligible numbers of mature T and B cells relative to PDK1 +/+ /Vav-Cre +ve ( n =6), PDK1 +/+ /Vav-Cre −ve ( n =6) and PDK1 fl/fl /Vav-Cre −ve ( n =4) control genotypes. Error bars represent the standard deviation of 4–7 mice per genotype. In ( B , C and E ) a P -value (Student's t -test) of

    Journal: The EMBO Journal

    Article Title: PDK1 regulates VDJ recombination, cell-cycle exit and survival during B-cell development

    doi: 10.1038/emboj.2013.40

    Figure Lengend Snippet: PDK1 knockout in the haematopoietic system blocks the development of mature T and B cells. PDK1 fl/fl /Vav-Cre +ve mice were found to have an increased spleen size ( A ) and weight ( B ) relative to PDK1 +/+ /Vav-Cre +ve control mice. Post lysis of red blood cells however spleen cell numbers were similar between the PDK1 fl/fl /Vav-Cre +ve mice ( n =15) and PDK1 +/+ /Vav-Cre +ve ( n =18), PDK1 +/+ /Vav-Cre −ve ( n =6) and PDK1 fl/fl /Vav-Cre −ve ( n =6) control genotypes ( C ). Error bars represent standard deviation. H E staining of the spleens showed that PDK1 knockout resulted in a disruption of the white pulp ( D ). Analysis of T- and B-cell populations in the spleen by FACS ( E , F ) demonstrated that PDK1 fl/fl /Vav-Cre +ve mice ( n =7) had negligible numbers of mature T and B cells relative to PDK1 +/+ /Vav-Cre +ve ( n =6), PDK1 +/+ /Vav-Cre −ve ( n =6) and PDK1 fl/fl /Vav-Cre −ve ( n =4) control genotypes. Error bars represent the standard deviation of 4–7 mice per genotype. In ( B , C and E ) a P -value (Student's t -test) of

    Article Snippet: Isolation of B-cell progenitors and cell culture Single cell bone marrow suspensions were treated with red blood cell lysis buffer (Sigma) and then Gr1, CD11b, Ter119 and CD3-positive cells were depleted using MACS columns.

    Techniques: Knock-Out, Mouse Assay, Lysis, Standard Deviation, Staining, FACS

    Pre-existing T. spiralis infection in mice attenuates RSV-induced pulmonary inflammation. Pro-inflammatory cytokine levels and inflammatory cell influx in the bronchoalveolar lavage fluid were quantified. Individual lung homogenates collected from the left lobe of 3 mice were used to assess IFN-γ ( a ) and IL-6 ( b ) levels. Bronchoalveolar lavage fluid was collected in 500 uL PBS from the right lobe of the remaining 3 mice by constricting the pulmonary blood vessels with a clamp ( c ). Cells were counted under the microscope after RBC lysis. Data are representative of three independent experiments presented as mean ± SEM, and statistical significance was determined using one-way ANOVA with Tukey’s post hoc analysis (* p

    Journal: Cells

    Article Title: Preliminary Trichinella spiralis Infection Ameliorates Subsequent RSV Infection-Induced Inflammatory Response

    doi: 10.3390/cells9051314

    Figure Lengend Snippet: Pre-existing T. spiralis infection in mice attenuates RSV-induced pulmonary inflammation. Pro-inflammatory cytokine levels and inflammatory cell influx in the bronchoalveolar lavage fluid were quantified. Individual lung homogenates collected from the left lobe of 3 mice were used to assess IFN-γ ( a ) and IL-6 ( b ) levels. Bronchoalveolar lavage fluid was collected in 500 uL PBS from the right lobe of the remaining 3 mice by constricting the pulmonary blood vessels with a clamp ( c ). Cells were counted under the microscope after RBC lysis. Data are representative of three independent experiments presented as mean ± SEM, and statistical significance was determined using one-way ANOVA with Tukey’s post hoc analysis (* p

    Article Snippet: Collected BALF samples from the murine lungs were centrifuged at 1200 RPM, 4 °C, 3 min, followed by red blood cell lysis using RBC lysis buffer (Sigma Aldrich, St. Louis, MO, USA).

    Techniques: Infection, Mouse Assay, Microscopy, Lysis

    Comparison of HBV-specific cell responses in the presence or absence of HBeAg. (A) C protein-specific T-cell responses elicited in ELISPOT assay; (B) Cytokine IFN-Y responses in cultured supernatants of PBMCs stimulated by C protein. HBeAg(−): HBeAg-negative subjects; HBeAg(+): HBeAg-positive subjects; HIV+: HIV co-infected; HIV-: not co-infected with HIV.

    Journal: Virology Journal

    Article Title: Hepatitis B virus (HBV)-specific T-cell responses to recombinant HBV core protein in patients with normal liver function and co-infected with chronic HBV and human immunodeficiency virus 1 (HIV-1)

    doi: 10.1186/1743-422X-10-232

    Figure Lengend Snippet: Comparison of HBV-specific cell responses in the presence or absence of HBeAg. (A) C protein-specific T-cell responses elicited in ELISPOT assay; (B) Cytokine IFN-Y responses in cultured supernatants of PBMCs stimulated by C protein. HBeAg(−): HBeAg-negative subjects; HBeAg(+): HBeAg-positive subjects; HIV+: HIV co-infected; HIV-: not co-infected with HIV.

    Article Snippet: Human interferon (IFN)-γ ELISPOT assay Peripheral whole blood was obtained from all subjects and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation with Ficoll Lymphoprep (Axis –Shield PoC AS, Oslo, Norway).

    Techniques: Enzyme-linked Immunospot, Cell Culture, Infection

    The distribution of fragments of mRNA, rRNA, tRNA, miRNA, and others in the small RNA libraries. a The PBMC of heat-stressed cattle. b The PBMC of non-heat-stressed cattle

    Journal: Cell Stress & Chaperones

    Article Title: Differential expression of microRNAs associated with thermal stress in Frieswal (Bos taurus x Bos indicus) crossbred dairy cattle

    doi: 10.1007/s12192-017-0833-6

    Figure Lengend Snippet: The distribution of fragments of mRNA, rRNA, tRNA, miRNA, and others in the small RNA libraries. a The PBMC of heat-stressed cattle. b The PBMC of non-heat-stressed cattle

    Article Snippet: To identify miRNAs in Frieswal cattle, two independent small RNA libraries were constructed from the PBMC of normal and heat-stressed animals through Ion Torrent deep sequencing.

    Techniques:

    Fold changes of HSP70 and HSP90 in the Frieswal PBMC collected 18 °C (normal) and 45 °C (heat stress) environmental temperatures. * P

    Journal: Cell Stress & Chaperones

    Article Title: Differential expression of microRNAs associated with thermal stress in Frieswal (Bos taurus x Bos indicus) crossbred dairy cattle

    doi: 10.1007/s12192-017-0833-6

    Figure Lengend Snippet: Fold changes of HSP70 and HSP90 in the Frieswal PBMC collected 18 °C (normal) and 45 °C (heat stress) environmental temperatures. * P

    Article Snippet: To identify miRNAs in Frieswal cattle, two independent small RNA libraries were constructed from the PBMC of normal and heat-stressed animals through Ion Torrent deep sequencing.

    Techniques:

    Systemic immune response to LCMV-GP33 peptide C57BL/6 mice (5 mice/group) were challenged subcutaneously with TC-1 tumor cells and subsequently treated with various combinations of cisplatin, CpG and GP33 peptide as shown. The PBMCs were analyzed 1 week after the last antigen delivery. The presence of GP33-specific CD8+ T cells in PBMCs was analyzed using intracellular cytokine staining for IFN-γ and CD8+ staining followed by flow cytometry analysis. A. Representative flow cytometry contour plot depicting the frequency of IFN-γ-secreting CD8+ T cells among PBMCs after being pulsed with GP33 peptide. B. Bar graph depicting the number of IFN-γ+ CD8+ T cells among PBMCs (mean ±S.E).

    Journal: Vaccine

    Article Title: Cancer immunotherapy using a potent immunodominant CTL epitope

    doi: 10.1016/j.vaccine.2014.09.021

    Figure Lengend Snippet: Systemic immune response to LCMV-GP33 peptide C57BL/6 mice (5 mice/group) were challenged subcutaneously with TC-1 tumor cells and subsequently treated with various combinations of cisplatin, CpG and GP33 peptide as shown. The PBMCs were analyzed 1 week after the last antigen delivery. The presence of GP33-specific CD8+ T cells in PBMCs was analyzed using intracellular cytokine staining for IFN-γ and CD8+ staining followed by flow cytometry analysis. A. Representative flow cytometry contour plot depicting the frequency of IFN-γ-secreting CD8+ T cells among PBMCs after being pulsed with GP33 peptide. B. Bar graph depicting the number of IFN-γ+ CD8+ T cells among PBMCs (mean ±S.E).

    Article Snippet: To detect HPV16 E7-specific and LCMV GP33-specific CD8+ T cell responses by IFN-γ intracellular staining, peripheral blood mononuclear cells (PBMCs), single-cell suspended splenocytes or tumor infiltrating lymphocytes were stimulated with either HPV E7aa49-57 or LCMV GP33 peptide (1μg/ml) in the presence of Golgiplug (BD Pharmingen, San Diego, CA) at 37°C overnight.

    Techniques: Mouse Assay, Staining, Flow Cytometry, Cytometry

    Proliferation of lymphocytes in LNs and PBMCs as measured by flow cytometry. (A) Ki-67 immunostaining of lymphocytes isolated from LNs of healthy animals on day −18 preexposure ( n = 2) and from LNs of surviving animals ( n = 4) on days 9 and 22 p.i. (B to D) Ki-67 immunostaining of CD4 + T cells (B), CD8 + T cells (C), and CD20 + B cells (D) in PBMCs in treated and untreated survivors versus moribund NHPs. Abbreviations: LN, lymph node; NHP, nonhuman primate; PBMCs, peripheral blood mononuclear cells; p.i., postinfection.

    Journal: Journal of Virology

    Article Title: [18F]-Fluorodeoxyglucose Uptake in Lymphoid Tissue Serves as a Predictor of Disease Outcome in the Nonhuman Primate Model of Monkeypox Virus Infection

    doi: 10.1128/JVI.00897-17

    Figure Lengend Snippet: Proliferation of lymphocytes in LNs and PBMCs as measured by flow cytometry. (A) Ki-67 immunostaining of lymphocytes isolated from LNs of healthy animals on day −18 preexposure ( n = 2) and from LNs of surviving animals ( n = 4) on days 9 and 22 p.i. (B to D) Ki-67 immunostaining of CD4 + T cells (B), CD8 + T cells (C), and CD20 + B cells (D) in PBMCs in treated and untreated survivors versus moribund NHPs. Abbreviations: LN, lymph node; NHP, nonhuman primate; PBMCs, peripheral blood mononuclear cells; p.i., postinfection.

    Article Snippet: PBMC samples and lymphocytes isolated from LNs were surface stained with a custom antibody cocktail (BD Biosciences) for CD3, CD4, CD8, CD14, CD16, CD20, CD45, and Ki-67.

    Techniques: Flow Cytometry, Cytometry, Immunostaining, Isolation

    Dose–response curves for ADCC. The procedures for OxLDL incubated and Ab treatment were the same as described in the legend for Fig. . As effectors normal rat PBMC were added to the culture instead of complement. Note increased 51Cr release

    Journal: Cell Stress & Chaperones

    Article Title: Evidence of a role for both anti-Hsp70 antibody and endothelial surface membrane Hsp70 in atherosclerosis

    doi: 10.1007/s12192-013-0404-4

    Figure Lengend Snippet: Dose–response curves for ADCC. The procedures for OxLDL incubated and Ab treatment were the same as described in the legend for Fig. . As effectors normal rat PBMC were added to the culture instead of complement. Note increased 51Cr release

    Article Snippet: To determine antibody-dependent cellular cytotoxicity (ADCC), peripheral blood mononuclear cells (PBMC) were isolated from healthy rats by density centrifugation (Lymphoprep, density 1,083; Nycomed Pharmaceuticals Oslo, Norway) as described previously (Jurgens et al. ).

    Techniques: Incubation

    miR-20b expression in T cells and in PBMC from natalizumab-associated progressive multifocal leukoencephalopathy (PML). (A) PBMC were isolated using a Ficoll gradient and CD4+ T cells were sorted with magnetic beads ( n = 9; randomly selected patients of our initial longitudinal cohort consisting of 17 patients). miR-20b expression (qPCR) was measured in the CD4+ and CD4-negative fraction and compared using t- test. (B) CD4+ cells were activated by plate-bound anti-CD3-antibody and soluble anti-CD28-antibody without further cytokines, termed “Th0”, and with IL-1 (10 ng/mL), IL-6 (10 ng/mL), IL-23 (10 ng/mL) and TGF- β (5 ng/mL), termed “Th17”, as well as freshly isolated CD4+ T cells, termed “fresh”. miR-20b levels were measured by qPCR. (C) miR-20b levels in PBMC from natalizumab-treated patients ( n = 9, left bar) were compared to miR-20b levels in two PBMC samples from patients with natalizumab-associated PML (each patient one bar).

    Journal: Annals of Clinical and Translational Neurology

    Article Title: Natalizumab restores aberrant miRNA expression profile in multiple sclerosis and reveals a critical role for miR-20b

    doi: 10.1002/acn3.152

    Figure Lengend Snippet: miR-20b expression in T cells and in PBMC from natalizumab-associated progressive multifocal leukoencephalopathy (PML). (A) PBMC were isolated using a Ficoll gradient and CD4+ T cells were sorted with magnetic beads ( n = 9; randomly selected patients of our initial longitudinal cohort consisting of 17 patients). miR-20b expression (qPCR) was measured in the CD4+ and CD4-negative fraction and compared using t- test. (B) CD4+ cells were activated by plate-bound anti-CD3-antibody and soluble anti-CD28-antibody without further cytokines, termed “Th0”, and with IL-1 (10 ng/mL), IL-6 (10 ng/mL), IL-23 (10 ng/mL) and TGF- β (5 ng/mL), termed “Th17”, as well as freshly isolated CD4+ T cells, termed “fresh”. miR-20b levels were measured by qPCR. (C) miR-20b levels in PBMC from natalizumab-treated patients ( n = 9, left bar) were compared to miR-20b levels in two PBMC samples from patients with natalizumab-associated PML (each patient one bar).

    Article Snippet: Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by Ficoll (Biochrom, Berlin, Germany) gradient and CD4+ cells were isolated by magnetic bead separation using STEMCELL EasySep Human CD4+ T Cell Enrichment Kit according to the manufacturer’s instructions.

    Techniques: Expressing, Isolation, Magnetic Beads, Real-time Polymerase Chain Reaction

    Detection of ApoE isoforms in differentiating hNP1 cells. Differentiating hNP1 cells were exposed to medium alone (Unt), mock-infected PBMC supernatants (Mock), and HIV-infected PBMC supernatants (HIV), and to rApoE3 or rApoE4 at 3 μg/ml, as described in Materials and Methods. At day 16 of differentiation, and 24 h after replenishment of rApoE in the cultures, cells were lysed for quantitative immunoblotting to detect ApoE. Lower blot depicts representative endogenous ApoE detected in cell culture lysates with molecular weight of 34 kDa, and rApoE with molecular weight of 36 kDa. Upper blot corresponds to beta-actin used as an internal control. For comparison, recombinant ApoE3 and ApoE4 were loaded directly on the gel at a 3 ng per lane, as shown. Bar graphs present ApoE signal normalized to beta-actin; each bar represents the mean of two experiments

    Journal: Journal of Neuroimmune Pharmacology

    Article Title: Apolipoprotein E4 Suppresses Neuronal-Specific Gene Expression in Maturing Neuronal Progenitor Cells Exposed to HIV

    doi: 10.1007/s11481-017-9734-9

    Figure Lengend Snippet: Detection of ApoE isoforms in differentiating hNP1 cells. Differentiating hNP1 cells were exposed to medium alone (Unt), mock-infected PBMC supernatants (Mock), and HIV-infected PBMC supernatants (HIV), and to rApoE3 or rApoE4 at 3 μg/ml, as described in Materials and Methods. At day 16 of differentiation, and 24 h after replenishment of rApoE in the cultures, cells were lysed for quantitative immunoblotting to detect ApoE. Lower blot depicts representative endogenous ApoE detected in cell culture lysates with molecular weight of 34 kDa, and rApoE with molecular weight of 36 kDa. Upper blot corresponds to beta-actin used as an internal control. For comparison, recombinant ApoE3 and ApoE4 were loaded directly on the gel at a 3 ng per lane, as shown. Bar graphs present ApoE signal normalized to beta-actin; each bar represents the mean of two experiments

    Article Snippet: “HIV-Exposed” HIV-1 containing supernatants were obtained from human peripheral blood mononuclear cells (PBMC) that were stimulated with mitogens phytohemagglutinin (PHA, Sigma-Aldrich, St Lois, MO) and recombinant human interleukin-2 (IL-2, Roche Diagnostics, Indianapolis, IN), then infected with HIV-1 as described previously (McCarthy et al. ).

    Techniques: Infection, Cell Culture, Molecular Weight, Recombinant

    Semi-quantitative PCR analysis of BLV pol detects differences in proviral load between BLV SGV and BLV-rabbits. PBMC were harvested at month indicated after challenge with 1 × 10 8 FLK/BLV cells from indicated rabbits that had been infected with BLV SGV for 24 months or from age-matched naïve controls. DNA from 50,000 PMBC was subjected to nested PCR and gel electrophoresis and stained with ethidium bromide. The position of the 591-bp amplicon is designated by the labeled arrow. In parallel, DNA from 20,000 PBMC was subjected to PCR with primers complementary to β- actin . The position of the 594-bp amplicon is designated by the labeled arrow and the signals were quantified by densitometry.

    Journal: Virology

    Article Title: Long-term infection with retroviral structural gene vector provides protection against bovine leukemia virus disease in rabbits

    doi: 10.1016/j.virol.2004.09.001

    Figure Lengend Snippet: Semi-quantitative PCR analysis of BLV pol detects differences in proviral load between BLV SGV and BLV-rabbits. PBMC were harvested at month indicated after challenge with 1 × 10 8 FLK/BLV cells from indicated rabbits that had been infected with BLV SGV for 24 months or from age-matched naïve controls. DNA from 50,000 PMBC was subjected to nested PCR and gel electrophoresis and stained with ethidium bromide. The position of the 591-bp amplicon is designated by the labeled arrow. In parallel, DNA from 20,000 PBMC was subjected to PCR with primers complementary to β- actin . The position of the 594-bp amplicon is designated by the labeled arrow and the signals were quantified by densitometry.

    Article Snippet: To prepare peripheral blood mononuclear cell (PBMC) samples for PCR, PBMCs were isolated by a Ficoll-Paque (Pharmacia) gradient centrifugation and washed three times with phosphate buffered saline.

    Techniques: Real-time Polymerase Chain Reaction, Infection, Nested PCR, Nucleic Acid Electrophoresis, Staining, Amplification, Labeling, Polymerase Chain Reaction

    Semiquantitative PCR analysis to evaluate differences in proviral load. (A) PCR to detect BLV pol in PBMC from treated rabbits. Rabbit PBMC were harvested at 1, 4, and 10 months postinoculation and subjected to PCR with BLV pol primers KB560 and KB561 (10 × 10 4 PBMC) or β- actin to control for sample variation (2 × 10 4 PBMC). Each panel is labeled with month of sample harvest. Lanes are labeled with the source of PBMC DNA by rabbit number and treatment, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). In lower panels, the corresponding rabbit samples are designated by the matching parallel black lines. Positions of BLV pol amplicon (591 bp) and β- actin amplicon (594 bp) are designated. *, not determined (ND) because sample was not available; †, not determined because the animal died before harvest. (B) PCR standard curve. PBMC lysate harvested at 1 month postinoculation from BLV rabbit 44-10 was serially diluted in a range of 5 × 10 2 to 5 × 10 4 PBMC and subjected to PCR with BLV pol primers KB560 and KB561. Each lane is labeled with the number of cells used for PCR amplification, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). The arrow on the left indicates the position of BLV pol amplicon (591 bp), and the lines on the right indicate positions of 676- and 571-bp DNA size markers.

    Journal: Journal of Virology

    Article Title: Bovine Leukemia Virus Structural Gene Vectors Are Immunogenic and Lack Pathogenicity in a Rabbit Model

    doi:

    Figure Lengend Snippet: Semiquantitative PCR analysis to evaluate differences in proviral load. (A) PCR to detect BLV pol in PBMC from treated rabbits. Rabbit PBMC were harvested at 1, 4, and 10 months postinoculation and subjected to PCR with BLV pol primers KB560 and KB561 (10 × 10 4 PBMC) or β- actin to control for sample variation (2 × 10 4 PBMC). Each panel is labeled with month of sample harvest. Lanes are labeled with the source of PBMC DNA by rabbit number and treatment, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). In lower panels, the corresponding rabbit samples are designated by the matching parallel black lines. Positions of BLV pol amplicon (591 bp) and β- actin amplicon (594 bp) are designated. *, not determined (ND) because sample was not available; †, not determined because the animal died before harvest. (B) PCR standard curve. PBMC lysate harvested at 1 month postinoculation from BLV rabbit 44-10 was serially diluted in a range of 5 × 10 2 to 5 × 10 4 PBMC and subjected to PCR with BLV pol primers KB560 and KB561. Each lane is labeled with the number of cells used for PCR amplification, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). The arrow on the left indicates the position of BLV pol amplicon (591 bp), and the lines on the right indicate positions of 676- and 571-bp DNA size markers.

    Article Snippet: To prepare PBMCs samples for PCR, PBMCs were isolated by Ficoll-Paque (Pharmacia) gradient centrifugation and washed three times with phosphate-buffered saline.

    Techniques: Polymerase Chain Reaction, Labeling, Positive Control, Marker, Amplification

    Exposure to C1P or ceramide induces P-glycoprotein transport activity at the blood-brain barrier. (A) Dose response of 20 minutes ceramide treatment, showing that ceramide increases specific P-glycoprotein activity in a concentration-dependent manner.

    Journal: Molecular Pharmacology

    Article Title:

    doi: 10.1124/mol.116.107169

    Figure Lengend Snippet: Exposure to C1P or ceramide induces P-glycoprotein transport activity at the blood-brain barrier. (A) Dose response of 20 minutes ceramide treatment, showing that ceramide increases specific P-glycoprotein activity in a concentration-dependent manner.

    Article Snippet: Ceramide from bovine spinal cord, mouse monoclonal β -actin antibody, chlorpromazine hydrochloride, Ficoll PM 400, cycloheximide, SC-51089 hydrate [3-chloro- N ʹ-(3-pyridin-4-ylpropanoyl)-6 H -benzo[ b ][1,4]benzoxazepine-5-carbohydrazide;hydrate;hydrochloride], PF-04418948 [1-(4-fluorobenzoyl)-3-[(6-methoxynaphthalen-2-yl)oxymethyl]azetidine-3-carboxylic acid], celecoxib, NVP-231 [ N -(2-benzamido-1,3-benzothiazol-6-yl)adamantane-1-carboxamide], and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

    Techniques: Activity Assay, Concentration Assay

    Proposed signaling cascade for the induction of P-glycoprotein activity by ceramide and C1P.

    Journal: Molecular Pharmacology

    Article Title:

    doi: 10.1124/mol.116.107169

    Figure Lengend Snippet: Proposed signaling cascade for the induction of P-glycoprotein activity by ceramide and C1P.

    Article Snippet: Ceramide from bovine spinal cord, mouse monoclonal β -actin antibody, chlorpromazine hydrochloride, Ficoll PM 400, cycloheximide, SC-51089 hydrate [3-chloro- N ʹ-(3-pyridin-4-ylpropanoyl)-6 H -benzo[ b ][1,4]benzoxazepine-5-carbohydrazide;hydrate;hydrochloride], PF-04418948 [1-(4-fluorobenzoyl)-3-[(6-methoxynaphthalen-2-yl)oxymethyl]azetidine-3-carboxylic acid], celecoxib, NVP-231 [ N -(2-benzamido-1,3-benzothiazol-6-yl)adamantane-1-carboxamide], and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

    Techniques: Activity Assay

    Generation, isolation, and characterization of epidermal growth factor receptor (EGFR)‐specific chimeric antigen receptor (CAR)‐engineered natural killer (NK) cells (EGFR‐CAR NK cells). (A) Flow cytometric analysis of phenotypic and subset composition of peripheral blood mononuclear cells (PBMCs) labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE. (B‐C) Flow cytometric analysis of phenotypic and subset composition of NK cells labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE, and anti‐CD69‐APC‐Cy7. (D) The percentage of CD3‐/CD56 + cells in day 0 PBMCs and day14 PBMC culture. (E) Real‐time PCR and (F) Western blotting analyses of the expression of exogenous CD3ζ in the non‐transduced NK cells, Con‐CAR NK cells, EGFR‐CAR‐1 NK cells, and EGFR‐CAR‐2 NK cells. β‐actin was used as an endogenous control. (G) The transduced NK cells stained with IgG‐FITC and EGFR‐FITC antibodies were detected by flow cytometry

    Journal: Cell Proliferation

    Article Title: Targeting epidermal growth factor‐overexpressing triple‐negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor, et al. Targeting epidermal growth factor‐overexpressing triple‐negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor

    doi: 10.1111/cpr.12858

    Figure Lengend Snippet: Generation, isolation, and characterization of epidermal growth factor receptor (EGFR)‐specific chimeric antigen receptor (CAR)‐engineered natural killer (NK) cells (EGFR‐CAR NK cells). (A) Flow cytometric analysis of phenotypic and subset composition of peripheral blood mononuclear cells (PBMCs) labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE. (B‐C) Flow cytometric analysis of phenotypic and subset composition of NK cells labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE, and anti‐CD69‐APC‐Cy7. (D) The percentage of CD3‐/CD56 + cells in day 0 PBMCs and day14 PBMC culture. (E) Real‐time PCR and (F) Western blotting analyses of the expression of exogenous CD3ζ in the non‐transduced NK cells, Con‐CAR NK cells, EGFR‐CAR‐1 NK cells, and EGFR‐CAR‐2 NK cells. β‐actin was used as an endogenous control. (G) The transduced NK cells stained with IgG‐FITC and EGFR‐FITC antibodies were detected by flow cytometry

    Article Snippet: Peripheral blood mononuclear cells were isolated from the whole blood of healthy donors by Ficoll density gradient centrifugation.

    Techniques: Isolation, Labeling, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Staining, Flow Cytometry

    EAT-2 over-expression enhances human DC maturation. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-EAT2 or rAd5-Null. At 72 hpi, cells were stained for surface expression of CD86 (A), HLA-DR (B), CD80 (C) and CD83 (D) and FACS analysis was performed. Data are representative of at least three independent experiments from three different blood donors with similar results. Samples were plated in quadruplicates. Bars represent mean ± SD. Statistical analysis was completed using a one way ANOVA with a Student–Newman–Keuls post-hoc test. P

    Journal: International Immunology

    Article Title: Manipulation of EAT-2 expression promotes induction of multiple beneficial regulatory and effector functions of the human innate immune system as a novel immunomodulatory strategy

    doi: 10.1093/intimm/dxt061

    Figure Lengend Snippet: EAT-2 over-expression enhances human DC maturation. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-EAT2 or rAd5-Null. At 72 hpi, cells were stained for surface expression of CD86 (A), HLA-DR (B), CD80 (C) and CD83 (D) and FACS analysis was performed. Data are representative of at least three independent experiments from three different blood donors with similar results. Samples were plated in quadruplicates. Bars represent mean ± SD. Statistical analysis was completed using a one way ANOVA with a Student–Newman–Keuls post-hoc test. P

    Article Snippet: To identify human immune cells transduced by Ad-mediated EAT-2 or GFP gene transduction, human PBMCs (2×106 ) were infected with multiplicity of infection (MOI) of 10000 of Ad5-EAT-2 or Ad5-GFP for 48h and were then stained with the following antibodies: APC-Cy7-CD14, PE-CD1a, Alexa Fluor 700-CD8a, V450-CD4, APC-CD65 and PerCpCy5.5-CD3 (all at 4 µg ml−1 ; BD Biosciences).

    Techniques: Over Expression, Isolation, Infection, Staining, Expressing, FACS

    Transduction of human innate and adaptive immune cells by GFP-expressing rAd5 vector. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-GFP. FACS analysis for GFP expression at 48h following rAd5-GFP infection of human NK cells (A), CD4 + and CD8 + T cells (B), CD14 + monocytes and CD1a + cells (C) is shown. Data were collected using a BD LSRII flow cytometer and analyzed using FlowJo software.

    Journal: International Immunology

    Article Title: Manipulation of EAT-2 expression promotes induction of multiple beneficial regulatory and effector functions of the human innate immune system as a novel immunomodulatory strategy

    doi: 10.1093/intimm/dxt061

    Figure Lengend Snippet: Transduction of human innate and adaptive immune cells by GFP-expressing rAd5 vector. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-GFP. FACS analysis for GFP expression at 48h following rAd5-GFP infection of human NK cells (A), CD4 + and CD8 + T cells (B), CD14 + monocytes and CD1a + cells (C) is shown. Data were collected using a BD LSRII flow cytometer and analyzed using FlowJo software.

    Article Snippet: To identify human immune cells transduced by Ad-mediated EAT-2 or GFP gene transduction, human PBMCs (2×106 ) were infected with multiplicity of infection (MOI) of 10000 of Ad5-EAT-2 or Ad5-GFP for 48h and were then stained with the following antibodies: APC-Cy7-CD14, PE-CD1a, Alexa Fluor 700-CD8a, V450-CD4, APC-CD65 and PerCpCy5.5-CD3 (all at 4 µg ml−1 ; BD Biosciences).

    Techniques: Transduction, Expressing, Plasmid Preparation, Isolation, Infection, FACS, Flow Cytometry, Cytometry, Software

    Intracellular staining analysis for EAT-2 expression in human immune cells following rAd5-EAT2 infection. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-EAT2 or rAd5-EAT2(R31Q). Intracellular staining analysis for EAT-2 expression at 48h following Ad infection of human NK cells (A), CD14 + monocytes (B) and CD1a + DCs (C) is shown. Data were collected using a BD LSRII flow cytometer and analyzed using FlowJo software.

    Journal: International Immunology

    Article Title: Manipulation of EAT-2 expression promotes induction of multiple beneficial regulatory and effector functions of the human innate immune system as a novel immunomodulatory strategy

    doi: 10.1093/intimm/dxt061

    Figure Lengend Snippet: Intracellular staining analysis for EAT-2 expression in human immune cells following rAd5-EAT2 infection. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-EAT2 or rAd5-EAT2(R31Q). Intracellular staining analysis for EAT-2 expression at 48h following Ad infection of human NK cells (A), CD14 + monocytes (B) and CD1a + DCs (C) is shown. Data were collected using a BD LSRII flow cytometer and analyzed using FlowJo software.

    Article Snippet: To identify human immune cells transduced by Ad-mediated EAT-2 or GFP gene transduction, human PBMCs (2×106 ) were infected with multiplicity of infection (MOI) of 10000 of Ad5-EAT-2 or Ad5-GFP for 48h and were then stained with the following antibodies: APC-Cy7-CD14, PE-CD1a, Alexa Fluor 700-CD8a, V450-CD4, APC-CD65 and PerCpCy5.5-CD3 (all at 4 µg ml−1 ; BD Biosciences).

    Techniques: Staining, Expressing, Infection, Isolation, Flow Cytometry, Cytometry, Software

    EAT-2 over-expression enhances the cytolytic activity of human NK cells. Human PBMCs isolated from fresh buffy coat material were either mock infected or infected with MOI of 10000 with rAd5-EAT2, rAd5-EAT2(R31Q) or rAd5-Null for 72h and then co-cultured with CFSE-labeled K562 cells for an additional 48h at an E:T ratio of 1:10. Viability of K562 cells was assessed by either PI (A and B) or ViViD (C). Data are representative of two (A) or three (B) independent experiments from three different blood donors with similar results. Samples were plated in quadruplicates. Bars represent mean ± SD. Statistical analysis was completed using a one way ANOVA with a Student–Newman–Keuls post-hoc test. P

    Journal: International Immunology

    Article Title: Manipulation of EAT-2 expression promotes induction of multiple beneficial regulatory and effector functions of the human innate immune system as a novel immunomodulatory strategy

    doi: 10.1093/intimm/dxt061

    Figure Lengend Snippet: EAT-2 over-expression enhances the cytolytic activity of human NK cells. Human PBMCs isolated from fresh buffy coat material were either mock infected or infected with MOI of 10000 with rAd5-EAT2, rAd5-EAT2(R31Q) or rAd5-Null for 72h and then co-cultured with CFSE-labeled K562 cells for an additional 48h at an E:T ratio of 1:10. Viability of K562 cells was assessed by either PI (A and B) or ViViD (C). Data are representative of two (A) or three (B) independent experiments from three different blood donors with similar results. Samples were plated in quadruplicates. Bars represent mean ± SD. Statistical analysis was completed using a one way ANOVA with a Student–Newman–Keuls post-hoc test. P

    Article Snippet: To identify human immune cells transduced by Ad-mediated EAT-2 or GFP gene transduction, human PBMCs (2×106 ) were infected with multiplicity of infection (MOI) of 10000 of Ad5-EAT-2 or Ad5-GFP for 48h and were then stained with the following antibodies: APC-Cy7-CD14, PE-CD1a, Alexa Fluor 700-CD8a, V450-CD4, APC-CD65 and PerCpCy5.5-CD3 (all at 4 µg ml−1 ; BD Biosciences).

    Techniques: Over Expression, Activity Assay, Isolation, Infection, Cell Culture, Labeling

    Intracellular and plasma pharmacokinetic profiles of imatinib in five individual patients (solid lines, plasma concentrations; broken lines, peripheral blood mononuclear cell concentrations)

    Journal: British Journal of Clinical Pharmacology

    Article Title: Population pharmacokinetics of imatinib and the role of ?1-acid glycoprotein

    doi: 10.1111/j.1365-2125.2006.02719.x

    Figure Lengend Snippet: Intracellular and plasma pharmacokinetic profiles of imatinib in five individual patients (solid lines, plasma concentrations; broken lines, peripheral blood mononuclear cell concentrations)

    Article Snippet: In these five patients, intracellular concentrations of imatinib were also measured in peripheral blood mononuclear cells (PBMC) obtained from four blood samples drawn before and 2, 4 and 6 h after drug intake using Vacutainer® CPT (Cell Preparation Tubes; Becton Dickinson, Allschwil, Switzerland), according to the manufacturer’s recommended procedure and the method previously developed in our laboratory for the intracellular measurement of anti-HIV drugs [ ].

    Techniques:

    Isolation and molecular characterization of T cell subsets from PNH patients ( A ) Gating strategy for T cell subsets for cell sorting. PBMCs were stained with ViViD, anti-CD14-Pacific Blue, anti-CD19-Pacific Blue, anti-CD3-BV605, anti-CD4-V500, anti-CD8-APC-H7, anti-CD45RA-PE-Cy7, and anti-CD45RO-APC. Lymphocytes were gated based on their scatter characteristics or forward scatter height vs. forward scatter area. Live T cells were gated based on positive for CD3 and negative for ViViD, CD14, and CD19 to remove dead cells, monocytes, and B cells. CD4 + and CD8 + T cells were then gated based on the characteristic expression patterns of CD45RA and CD45RO. Four different T cell subsets [CD4 + naïve (CD45RA + CD45RO - ), CD4 + memory (CD45RA - CD45RO + ), CD8 + naïve (CD45RA + CD45RO - ), and CD8 + memory (CD45RA - CD45RO + ) T cells] were sorted for RNA-seq. ( B ) Transcriptional validation of the naïve and memory T cell identity by analyzing expression levels of CD4, CD8, CCR5, and EOMES. FPKM (fragments per kilobase of exon per million fragments mapped). * p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: T Cell Transcriptomes from Paroxysmal Nocturnal Hemoglobinuria Patients Reveal Novel Signaling Pathways

    doi: 10.4049/jimmunol.1601299

    Figure Lengend Snippet: Isolation and molecular characterization of T cell subsets from PNH patients ( A ) Gating strategy for T cell subsets for cell sorting. PBMCs were stained with ViViD, anti-CD14-Pacific Blue, anti-CD19-Pacific Blue, anti-CD3-BV605, anti-CD4-V500, anti-CD8-APC-H7, anti-CD45RA-PE-Cy7, and anti-CD45RO-APC. Lymphocytes were gated based on their scatter characteristics or forward scatter height vs. forward scatter area. Live T cells were gated based on positive for CD3 and negative for ViViD, CD14, and CD19 to remove dead cells, monocytes, and B cells. CD4 + and CD8 + T cells were then gated based on the characteristic expression patterns of CD45RA and CD45RO. Four different T cell subsets [CD4 + naïve (CD45RA + CD45RO - ), CD4 + memory (CD45RA - CD45RO + ), CD8 + naïve (CD45RA + CD45RO - ), and CD8 + memory (CD45RA - CD45RO + ) T cells] were sorted for RNA-seq. ( B ) Transcriptional validation of the naïve and memory T cell identity by analyzing expression levels of CD4, CD8, CCR5, and EOMES. FPKM (fragments per kilobase of exon per million fragments mapped). * p

    Article Snippet: For cell sorting, peripheral blood mononuclear cells (PBMCs) from PNH patients and healthy controls were separated from PB samples using Lymphocyte Separation Medium (MP Biomedicals, Santa Ana, CA).

    Techniques: Isolation, FACS, Staining, Expressing, RNA Sequencing Assay

    The systemic cellular immune response to sand fly saliva in volunteers in a cutaneous leishmaniasis-endemic area of Mali. Peripheral blood mononuclear cells (PBMCs) were obtained from 255 individuals aged 11–65 years from the villages of Kemena and Sougoula. IFN-γ, IL-12, IL-10, IL-13, and IL-5 levels were measured in the supernatants of PBMCs from exposed volunteers 96 hours after stimulation with Phlebotomus duboscqi salivary gland homogenate. ( a ) Overall cytokine production by tested individuals. ( b ) Pairwise correlation of expressed cytokines (log-transformed cytokine levels). Numbers in insets indicate the Spearman correlation coefficient and the 95% confidence intervals for each correlation. ( c ) Cytokine responses of individuals in ( a ) stratified by T H 1-predominant, T H 2-predominant, or T H 1/T H 2 mixed responses to sand fly salivary proteins.

    Journal: The Journal of Investigative Dermatology

    Article Title: Delayed-Type Hypersensitivity to Sand Fly Saliva in Humans from a Leishmaniasis-Endemic Area of Mali Is TH1-Mediated and Persists to Midlife

    doi: 10.1038/jid.2012.315

    Figure Lengend Snippet: The systemic cellular immune response to sand fly saliva in volunteers in a cutaneous leishmaniasis-endemic area of Mali. Peripheral blood mononuclear cells (PBMCs) were obtained from 255 individuals aged 11–65 years from the villages of Kemena and Sougoula. IFN-γ, IL-12, IL-10, IL-13, and IL-5 levels were measured in the supernatants of PBMCs from exposed volunteers 96 hours after stimulation with Phlebotomus duboscqi salivary gland homogenate. ( a ) Overall cytokine production by tested individuals. ( b ) Pairwise correlation of expressed cytokines (log-transformed cytokine levels). Numbers in insets indicate the Spearman correlation coefficient and the 95% confidence intervals for each correlation. ( c ) Cytokine responses of individuals in ( a ) stratified by T H 1-predominant, T H 2-predominant, or T H 1/T H 2 mixed responses to sand fly salivary proteins.

    Article Snippet: Blood collection and cytokine measurements To evaluate cytokine levels, PBMCs isolated by density-gradient centrifugation (Ficoll-Paque PLUS; GE Healthcare, Pittsburgh, PA) from blood collected in heparinized Vacutainer tubes (BD Diagnostics, Hunt Valley, MD).

    Techniques: Transformation Assay