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  • 99
    Lonza peripheral blood mononuclear cells pbmcs
    ShK-186 inhibits antineutrophil cytoplasmic antibodies targeting proteinase 3 (PR3-ANCA) production in vitro . (A) IgG production after peripheral blood mononuclear cell <t>(PBMC)</t> stimulation with CpG, B cell-activating factor (BAFF), and interleukin (IL)-21 from 5 healthy controls (HCs) (open circles) and 13 granulomatosis with polyangiitis <t>(GPA)</t> patients (gray circles) in the presence and absence of 1 nM ShK-186. (B) PR3-ANCA production after PBMC stimulation with CpG, BAFF, and IL-21 from 13 GPA patients in the presence and absence of 1 nM ShK-186 (* p
    Peripheral Blood Mononuclear Cells Pbmcs, supplied by Lonza, used in various techniques. Bioz Stars score: 99/100, based on 126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC primary peripheral blood mononuclear cells pbmc normal human
    S. sonnei induces the secretion of IL-1β, IL-18, NLRP3, ASC, and active caspase-1 in macrophages. (A) J774A.1 macrophages, <t>THP-1</t> macrophages, <t>PBMCs</t> or BMDM were primed with 1 μg/ml LPS for 4 h and then infected with S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. (B–E) J774A.1 macrophages or THP-1 macrophages were primed with 1 μg/ml LPS for 4 h followed and then infected with S. sonnei for an additional 20 h or stimulated with 5 mM ATP for an additional 0.5 h. The levels of IL-1β (B) , IL-18 (C) , caspase-1 (D) , NLRP3, and ASC (E) in the supernatants were measured by Western blotting. The ELISA data are expressed as the mean ± SD of four separate experiments. The Western blotting results are representative of three different experiments. * and *** indicate significant differences at the levels of p
    Primary Peripheral Blood Mononuclear Cells Pbmc Normal Human, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore human peripheral blood mononuclear cells pbmc
    Fusion efficiency enhancement . The fusions were performed according to a standard protocol, where the culture media and PEG were reconstituted from powder forms with either NPD or DI water. For each fusion, <t>PBMC</t> from a single batch were divided into two equal fractures and used to prepare two parallel experiments, in NPD or DI based reagents. The figure presents percent of <t>hybridoma-positive</t> wells in each fusion experiment. The percent was calculated as the number of hybridoma-positive wells from 96-well plates where the cells were seeded and grown after the fusion process. The difference between the NPD- and DI-fusion results was found to be statistically significant by Chi-square analysis (p
    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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    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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    Axis-Shield Diagnostics human pbmcs
    Human PBMC subsets express receptors for p17. <t>PBMCs</t> were isolated from leukopaks from healthy donors (New York Blood Center) with <t>Lymphoprep.</t> After RBC lysis, cells were stained with p17-Alexa Fluor 430 and surface markers. Live cells were gated as follows:
    Human Pbmcs, supplied by Axis-Shield Diagnostics, used in various techniques. Bioz Stars score: 92/100, based on 140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Axis-Shield Diagnostics human peripheral blood mononuclear cells pbmcs
    Infection with Mtb HN878 triggers increased IL-22 production through a TLR2-dependent pathway Lung cells isolated from B6, Il1r-/- and Tlr2-/- mice were infected with either Mtb H37Rv (Rv) or Mtb HN878 (HN) at MOI of 0.1 for 7 days, and the protein levels of ( a ) IL-1β and ( b ) IL-22 were assessed in culture supernatants by ELISA and luminex assay, respectively. Lung cells isolated from B6 mice were treated with cell wall extracted from Mtb H37Rv (Rv) or Mtb HN878 (HN), (10μg/ml each). Protein levels of ( c ) IL-1β and ( d ) IL-22 were assessed by ELISA and luminex assay respectively. <t>PBMCs</t> were isolated from <t>ATB</t> patients and were stimulated with Mtb H37Rv (Rv) or Mtb HN878 (HN) cell wall extracts (20 μg/ml each) and the protein levels of ( e ) IL-22 were measured by luminex assays. Error bars represent means ± s.e.m. n = 3-5 for a-d. n = 17 for human ATB patients for e. ND, not detected; UI, uninfected; UN, untreated. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 by ( a,b ) one-way ANOVA, ( c,d ) Student's t test ( e ) two-way ANOVA
    Human Peripheral Blood Mononuclear Cells Pbmcs, supplied by Axis-Shield Diagnostics, used in various techniques. Bioz Stars score: 93/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    AllCells LLC human pbmcs
    Detection of SMN Protein in SMNA Type 1 <t>PBMCs.</t> SMA Type I patient samples (N = 4) were tested in the SMN ELISA at dilutions of 1∶4 with 1.25×10 6 and 2.5×10 6 PBMCs. SMN protein signal was detected in all samples, with an average of 8.32 pg SMN protein per 10 6 cells. Based on the average of 70.2 pg SMN protein per 10 6 cells calculated for adult normal donor PBMCs, the amount of SMN protein in PBMCs of Type I SMA patients in this patient cohort is 88% less than normal. Error bars represent standard deviations.
    Human Pbmcs, supplied by AllCells LLC, used in various techniques. Bioz Stars score: 92/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore peripheral blood mononuclear cells pbmcs
    Effects of PGRN on the differentiation of human OCs and PGRN as a predictor of osteoporosis. A , <t>HBMCs</t> and <t>PBMCs</t> were differentiated with RANKL in the absence or presence of PGRN. TRAP + MNCs were counted as described above. B , four sh-RNAs targeting human
    Peripheral Blood Mononuclear Cells Pbmcs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3440 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ActivX human pbmcs
    Lu AF58786 inhibits phosphorylation of <t>LRRK2,</t> Rab10 and Rab12 in cultured and immune stimulated human <t>PBMCs.</t> ( A ) LRRK2 (pSer910, pSer935, pSer955 and pSer973) phospho-site hits plotted with mean and individual replicate values of the quantified log 10 -transformed CMPD/DMSO ratio as well as the associated q-value of the performed mean rank (n = 5–8). ( B ) Rab10 (pThr73) and Rab12 (pSer106) switch II domain phospho-site hits plotted with mean and individual replicate values of the quantified log 10 -transformed CMPD/DMSO ratio as well as the associated q-value of the performed mean rank (n = 5–8). CMPD, compound (Lu AF58786). DMSO, dimethyl sulfoxide.
    Human Pbmcs, supplied by ActivX, used in various techniques. Bioz Stars score: 92/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biochrom human peripheral blood mononuclear cells pbmcs
    Lu AF58786 inhibits phosphorylation of <t>LRRK2,</t> Rab10 and Rab12 in cultured and immune stimulated human <t>PBMCs.</t> ( A ) LRRK2 (pSer910, pSer935, pSer955 and pSer973) phospho-site hits plotted with mean and individual replicate values of the quantified log 10 -transformed CMPD/DMSO ratio as well as the associated q-value of the performed mean rank (n = 5–8). ( B ) Rab10 (pThr73) and Rab12 (pSer106) switch II domain phospho-site hits plotted with mean and individual replicate values of the quantified log 10 -transformed CMPD/DMSO ratio as well as the associated q-value of the performed mean rank (n = 5–8). CMPD, compound (Lu AF58786). DMSO, dimethyl sulfoxide.
    Human Peripheral Blood Mononuclear Cells Pbmcs, supplied by Biochrom, used in various techniques. Bioz Stars score: 93/100, based on 109 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Lonza human pbmc human peripheral blood mononuclear cells cryopreserved
    Lu AF58786 inhibits phosphorylation of <t>LRRK2,</t> Rab10 and Rab12 in cultured and immune stimulated human <t>PBMCs.</t> ( A ) LRRK2 (pSer910, pSer935, pSer955 and pSer973) phospho-site hits plotted with mean and individual replicate values of the quantified log 10 -transformed CMPD/DMSO ratio as well as the associated q-value of the performed mean rank (n = 5–8). ( B ) Rab10 (pThr73) and Rab12 (pSer106) switch II domain phospho-site hits plotted with mean and individual replicate values of the quantified log 10 -transformed CMPD/DMSO ratio as well as the associated q-value of the performed mean rank (n = 5–8). CMPD, compound (Lu AF58786). DMSO, dimethyl sulfoxide.
    Human Pbmc Human Peripheral Blood Mononuclear Cells Cryopreserved, supplied by Lonza, used in various techniques. Bioz Stars score: 97/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Nycomed human peripheral blood mononuclear cells pbmcs
    Lu AF58786 inhibits phosphorylation of <t>LRRK2,</t> Rab10 and Rab12 in cultured and immune stimulated human <t>PBMCs.</t> ( A ) LRRK2 (pSer910, pSer935, pSer955 and pSer973) phospho-site hits plotted with mean and individual replicate values of the quantified log 10 -transformed CMPD/DMSO ratio as well as the associated q-value of the performed mean rank (n = 5–8). ( B ) Rab10 (pThr73) and Rab12 (pSer106) switch II domain phospho-site hits plotted with mean and individual replicate values of the quantified log 10 -transformed CMPD/DMSO ratio as well as the associated q-value of the performed mean rank (n = 5–8). CMPD, compound (Lu AF58786). DMSO, dimethyl sulfoxide.
    Human Peripheral Blood Mononuclear Cells Pbmcs, supplied by Nycomed, used in various techniques. Bioz Stars score: 92/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cellular Technology Ltd human pbmcs
    The evaluation of the immunostimulatory effects of <t>NIPAM-hemin.</t> Abbreviations: NIPAM, N -isopropylacrylamide; hemin, ferriprotoporphyrin IX chloride; IFN-γ, interferon-γ; IL, interleukin; NK, natural killer; <t>PBMCs,</t> peripheral blood mononuclear cells; ELISA, enzyme-linked immunosorbent assay; TLRs, toll-like receptors; SEAP, secreted embryonic alkaline phosphatase.
    Human Pbmcs, supplied by Cellular Technology Ltd, used in various techniques. Bioz Stars score: 93/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Sanquin human pbmcs
    Histone deacetylase (HDAC) inhibitors modulate proinflammatory immune response to Candida albicans. (a–c) Peripheral blood mononuclear cells <t>(PBMCs)</t> were isolated from buffy coats provided by <t>Sanquin</t> Blood Bank, Nijmegen, the Netherlands. Cells
    Human Pbmcs, supplied by Sanquin, used in various techniques. Bioz Stars score: 92/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson human peripheral blood mononuclear cells pbmcs
    CD40L expressed by recombinant ALVAC virus enhances specific antiviral CTL activity. CD4 + T cell-containing or -depleted <t>PBMCs</t> from four HIV-1-infected individuals and three HIV-1-uninfected individuals were cocultured with autologous MDDCs that were either pulsed or not with HLA-restricted epitopes of HIV-1 proteins (for HIV-1 infected individuals) or EBV proteins (for HIV-1-uninfected individuals). MDDCs were previously infected or not (medium) with parental ALVAC II or ALVAC recombinant vA3131-2 expressing human CD40L at an MOI of 10 for 48 h. On day 10, specific CTL activity was assessed by intracellular flow cytometric analysis of IFN-γproducing CD8 + T cells and 51 Cr release assay. (A) Representative intracellular IFN-γ flow cytometric data obtained from HIV-1-positive participant #1. (B) Summary data from intracellular IFN-γ flow <t>cytometry</t> of HIV-1-positive participant #2–#4 are graphically depicted. Open bars represent CD4 + T cell-containing condition and dark bar represent CD4 + T cell -depleted conditions. (C) A representative 51 Cr release assay result from HIV-1-positive participant #1 is shown. Similar results were obtained with HIV-1-positive participant #2 (data not shown). (D) Summary data from intracellular IFN-γ flow cytometric analysis of EBV-positive participant #5–#7 are graphically depicted. Open bars represent CD4 + T cell -containing condition and dark bars represent CD4 + T cell -depleted conditions. (E) A representative 51 Cr release assay result from EBV-positive participant #6 is shown. Similar results were obtained with EBV-positive participant #5 (data not shown). DC, MDDCs not pulsed with peptide; DCp, MDDCs pulsed with peptide; vA3131-2/DCp, vA3131-2-infected MDDCs pulsed with peptide; ALVAC/DCp, parental ALVAC II-infected MDDCs pulsed with peptide.
    Human Peripheral Blood Mononuclear Cells Pbmcs, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Ficoll-Paque Pharmacia human pbmc
    CD40L expressed by recombinant ALVAC virus enhances specific antiviral CTL activity. CD4 + T cell-containing or -depleted <t>PBMCs</t> from four HIV-1-infected individuals and three HIV-1-uninfected individuals were cocultured with autologous MDDCs that were either pulsed or not with HLA-restricted epitopes of HIV-1 proteins (for HIV-1 infected individuals) or EBV proteins (for HIV-1-uninfected individuals). MDDCs were previously infected or not (medium) with parental ALVAC II or ALVAC recombinant vA3131-2 expressing human CD40L at an MOI of 10 for 48 h. On day 10, specific CTL activity was assessed by intracellular flow cytometric analysis of IFN-γproducing CD8 + T cells and 51 Cr release assay. (A) Representative intracellular IFN-γ flow cytometric data obtained from HIV-1-positive participant #1. (B) Summary data from intracellular IFN-γ flow <t>cytometry</t> of HIV-1-positive participant #2–#4 are graphically depicted. Open bars represent CD4 + T cell-containing condition and dark bar represent CD4 + T cell -depleted conditions. (C) A representative 51 Cr release assay result from HIV-1-positive participant #1 is shown. Similar results were obtained with HIV-1-positive participant #2 (data not shown). (D) Summary data from intracellular IFN-γ flow cytometric analysis of EBV-positive participant #5–#7 are graphically depicted. Open bars represent CD4 + T cell -containing condition and dark bars represent CD4 + T cell -depleted conditions. (E) A representative 51 Cr release assay result from EBV-positive participant #6 is shown. Similar results were obtained with EBV-positive participant #5 (data not shown). DC, MDDCs not pulsed with peptide; DCp, MDDCs pulsed with peptide; vA3131-2/DCp, vA3131-2-infected MDDCs pulsed with peptide; ALVAC/DCp, parental ALVAC II-infected MDDCs pulsed with peptide.
    Human Pbmc, supplied by Ficoll-Paque Pharmacia, used in various techniques. Bioz Stars score: 92/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    AllCells LLC human peripheral blood mononuclear cells pbmc
    CD40L expressed by recombinant ALVAC virus enhances specific antiviral CTL activity. CD4 + T cell-containing or -depleted <t>PBMCs</t> from four HIV-1-infected individuals and three HIV-1-uninfected individuals were cocultured with autologous MDDCs that were either pulsed or not with HLA-restricted epitopes of HIV-1 proteins (for HIV-1 infected individuals) or EBV proteins (for HIV-1-uninfected individuals). MDDCs were previously infected or not (medium) with parental ALVAC II or ALVAC recombinant vA3131-2 expressing human CD40L at an MOI of 10 for 48 h. On day 10, specific CTL activity was assessed by intracellular flow cytometric analysis of IFN-γproducing CD8 + T cells and 51 Cr release assay. (A) Representative intracellular IFN-γ flow cytometric data obtained from HIV-1-positive participant #1. (B) Summary data from intracellular IFN-γ flow <t>cytometry</t> of HIV-1-positive participant #2–#4 are graphically depicted. Open bars represent CD4 + T cell-containing condition and dark bar represent CD4 + T cell -depleted conditions. (C) A representative 51 Cr release assay result from HIV-1-positive participant #1 is shown. Similar results were obtained with HIV-1-positive participant #2 (data not shown). (D) Summary data from intracellular IFN-γ flow cytometric analysis of EBV-positive participant #5–#7 are graphically depicted. Open bars represent CD4 + T cell -containing condition and dark bars represent CD4 + T cell -depleted conditions. (E) A representative 51 Cr release assay result from EBV-positive participant #6 is shown. Similar results were obtained with EBV-positive participant #5 (data not shown). DC, MDDCs not pulsed with peptide; DCp, MDDCs pulsed with peptide; vA3131-2/DCp, vA3131-2-infected MDDCs pulsed with peptide; ALVAC/DCp, parental ALVAC II-infected MDDCs pulsed with peptide.
    Human Peripheral Blood Mononuclear Cells Pbmc, supplied by AllCells LLC, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Astarte Biologics human peripheral blood mononuclear cells pbmcs
    CD40L expressed by recombinant ALVAC virus enhances specific antiviral CTL activity. CD4 + T cell-containing or -depleted <t>PBMCs</t> from four HIV-1-infected individuals and three HIV-1-uninfected individuals were cocultured with autologous MDDCs that were either pulsed or not with HLA-restricted epitopes of HIV-1 proteins (for HIV-1 infected individuals) or EBV proteins (for HIV-1-uninfected individuals). MDDCs were previously infected or not (medium) with parental ALVAC II or ALVAC recombinant vA3131-2 expressing human CD40L at an MOI of 10 for 48 h. On day 10, specific CTL activity was assessed by intracellular flow cytometric analysis of IFN-γproducing CD8 + T cells and 51 Cr release assay. (A) Representative intracellular IFN-γ flow cytometric data obtained from HIV-1-positive participant #1. (B) Summary data from intracellular IFN-γ flow <t>cytometry</t> of HIV-1-positive participant #2–#4 are graphically depicted. Open bars represent CD4 + T cell-containing condition and dark bar represent CD4 + T cell -depleted conditions. (C) A representative 51 Cr release assay result from HIV-1-positive participant #1 is shown. Similar results were obtained with HIV-1-positive participant #2 (data not shown). (D) Summary data from intracellular IFN-γ flow cytometric analysis of EBV-positive participant #5–#7 are graphically depicted. Open bars represent CD4 + T cell -containing condition and dark bars represent CD4 + T cell -depleted conditions. (E) A representative 51 Cr release assay result from EBV-positive participant #6 is shown. Similar results were obtained with EBV-positive participant #5 (data not shown). DC, MDDCs not pulsed with peptide; DCp, MDDCs pulsed with peptide; vA3131-2/DCp, vA3131-2-infected MDDCs pulsed with peptide; ALVAC/DCp, parental ALVAC II-infected MDDCs pulsed with peptide.
    Human Peripheral Blood Mononuclear Cells Pbmcs, supplied by Astarte Biologics, used in various techniques. Bioz Stars score: 92/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Ficoll-Paque Pharmacia human peripheral blood mononuclear cells pbmc
    CD40L expressed by recombinant ALVAC virus enhances specific antiviral CTL activity. CD4 + T cell-containing or -depleted <t>PBMCs</t> from four HIV-1-infected individuals and three HIV-1-uninfected individuals were cocultured with autologous MDDCs that were either pulsed or not with HLA-restricted epitopes of HIV-1 proteins (for HIV-1 infected individuals) or EBV proteins (for HIV-1-uninfected individuals). MDDCs were previously infected or not (medium) with parental ALVAC II or ALVAC recombinant vA3131-2 expressing human CD40L at an MOI of 10 for 48 h. On day 10, specific CTL activity was assessed by intracellular flow cytometric analysis of IFN-γproducing CD8 + T cells and 51 Cr release assay. (A) Representative intracellular IFN-γ flow cytometric data obtained from HIV-1-positive participant #1. (B) Summary data from intracellular IFN-γ flow <t>cytometry</t> of HIV-1-positive participant #2–#4 are graphically depicted. Open bars represent CD4 + T cell-containing condition and dark bar represent CD4 + T cell -depleted conditions. (C) A representative 51 Cr release assay result from HIV-1-positive participant #1 is shown. Similar results were obtained with HIV-1-positive participant #2 (data not shown). (D) Summary data from intracellular IFN-γ flow cytometric analysis of EBV-positive participant #5–#7 are graphically depicted. Open bars represent CD4 + T cell -containing condition and dark bars represent CD4 + T cell -depleted conditions. (E) A representative 51 Cr release assay result from EBV-positive participant #6 is shown. Similar results were obtained with EBV-positive participant #5 (data not shown). DC, MDDCs not pulsed with peptide; DCp, MDDCs pulsed with peptide; vA3131-2/DCp, vA3131-2-infected MDDCs pulsed with peptide; ALVAC/DCp, parental ALVAC II-infected MDDCs pulsed with peptide.
    Human Peripheral Blood Mononuclear Cells Pbmc, supplied by Ficoll-Paque Pharmacia, used in various techniques. Bioz Stars score: 91/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    10X Genomics human pbmc cite seq dataset
    The performance of BREM-SC with the public human <t>PBMC</t> <t>CITE-Seq</t> dataset (from 10X Genomics). The UMAP projection of cells are colored by the approximate ground truth ( A ) and BREM-SC clustering results ( B ).
    Human Pbmc Cite Seq Dataset, supplied by 10X Genomics, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Reachbio human pbmcs
    (A) Transcriptome analysis of <t>KSHV</t> during de novo infection of human <t>PBMCs.</t> Total RNAs extracted from de novo -infected PBMCs harvested at 4 h, 24 h, 48 h, 72 h, and 120 h postinfection were subjected to cDNA library preparation using a TrueSeq RNA-seq
    Human Pbmcs, supplied by Reachbio, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ShK-186 inhibits antineutrophil cytoplasmic antibodies targeting proteinase 3 (PR3-ANCA) production in vitro . (A) IgG production after peripheral blood mononuclear cell (PBMC) stimulation with CpG, B cell-activating factor (BAFF), and interleukin (IL)-21 from 5 healthy controls (HCs) (open circles) and 13 granulomatosis with polyangiitis (GPA) patients (gray circles) in the presence and absence of 1 nM ShK-186. (B) PR3-ANCA production after PBMC stimulation with CpG, BAFF, and IL-21 from 13 GPA patients in the presence and absence of 1 nM ShK-186 (* p

    Journal: Frontiers in Immunology

    Article Title: Kv1.3 Channel Blockade Modulates the Effector Function of B Cells in Granulomatosis with Polyangiitis

    doi: 10.3389/fimmu.2017.01205

    Figure Lengend Snippet: ShK-186 inhibits antineutrophil cytoplasmic antibodies targeting proteinase 3 (PR3-ANCA) production in vitro . (A) IgG production after peripheral blood mononuclear cell (PBMC) stimulation with CpG, B cell-activating factor (BAFF), and interleukin (IL)-21 from 5 healthy controls (HCs) (open circles) and 13 granulomatosis with polyangiitis (GPA) patients (gray circles) in the presence and absence of 1 nM ShK-186. (B) PR3-ANCA production after PBMC stimulation with CpG, BAFF, and IL-21 from 13 GPA patients in the presence and absence of 1 nM ShK-186 (* p

    Article Snippet: Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from GPA patients, who produce ANCA upon in vitro induction , and stored in RPMI 1640 (Lonza, Basel, Switzerland) supplemented with 50 µg/mL gentamycin (GIBCO, Life Technologies, Grand Island, NY, USA), 10% fetal calf serum (FCS, Lonza), and 10% dimethyl sulfoxide (DMSO).

    Techniques: In Vitro

    IWP-2 does not inhibit Wnt production from primary human CD8+ T cells. CD8+ T cells were isolated from PBMCs from healthy donors by negative selection and subsequently activated with 1 μg each anti-CD3/anti-CD28 then propagated in presence of 100 units/ml IL-2 for three days. On the third day supernatant was collected and 25 μl was analyzed by western blot for presence of Wnt 1 (A), 3 (B), 6 (C), 7a (D), 10a (E), and 16a (F).

    Journal: PLoS ONE

    Article Title: Porcupine Is Not Required for the Production of the Majority of Wnts from Primary Human Astrocytes and CD8+ T Cells

    doi: 10.1371/journal.pone.0092159

    Figure Lengend Snippet: IWP-2 does not inhibit Wnt production from primary human CD8+ T cells. CD8+ T cells were isolated from PBMCs from healthy donors by negative selection and subsequently activated with 1 μg each anti-CD3/anti-CD28 then propagated in presence of 100 units/ml IL-2 for three days. On the third day supernatant was collected and 25 μl was analyzed by western blot for presence of Wnt 1 (A), 3 (B), 6 (C), 7a (D), 10a (E), and 16a (F).

    Article Snippet: Culture and Activation of Human Peripheral Blood Mononuclear Cells (PBMCs) and Isolation of CD8+ T cells PBMCs were isolated from venous blood of healthy donors using lymphocyte separation medium (Lonza Biologics, Portsmouth, NH) and density centrifugation.

    Techniques: Isolation, Selection, Western Blot

    C. trachomatis infectivity from a cell culture of ECC1, human PBMCs and a co-culture of ECC1 and PBMCs, with or without azithromycin treatment. ECC1/PBMCs/co-culture of ECC1 and PBMCs were infected with C. trachomatis in MOI of 0.1. Cultures were either treated with azithromycin at 20 h PI, or not. The Chlamydia infected cells and culture supernatant were harvested a 44 h PI, sonicated and used to infect a new ECC1 monolayer for enumeration of recoverable IFUs. Data are presented as mean ± SD IFU/m ( n = 9) determinations

    Journal: BMC Infectious Diseases

    Article Title: High expression of IDO1 and TGF-β1 during recurrence and post infection clearance with Chlamydia trachomatis, are independent of host IFN-γ response

    doi: 10.1186/s12879-019-3843-4

    Figure Lengend Snippet: C. trachomatis infectivity from a cell culture of ECC1, human PBMCs and a co-culture of ECC1 and PBMCs, with or without azithromycin treatment. ECC1/PBMCs/co-culture of ECC1 and PBMCs were infected with C. trachomatis in MOI of 0.1. Cultures were either treated with azithromycin at 20 h PI, or not. The Chlamydia infected cells and culture supernatant were harvested a 44 h PI, sonicated and used to infect a new ECC1 monolayer for enumeration of recoverable IFUs. Data are presented as mean ± SD IFU/m ( n = 9) determinations

    Article Snippet: Experiments were conducted in 48-well plates, where co-culture of 40,000 ECC1 cells/well and 8 × 105 female human PBMCs (Lonza, Australia) cells/well were seeded with RPMI-1640 medium (Sigma-Aldrich, Australia) containing 10% heat inactivated FCS (Life Technologies, Australia), 120 μg/ml streptomycin (Sigma-Aldrich, Australia), 50 μg/ml Gentamycin (Gibco, Australia), 37 °C, 5% CO2 .

    Techniques: Infection, Cell Culture, Co-Culture Assay, Sonication

    Immunological, bacterial and biochemical factors that are associated with initial or repeated C. trachomatis infections in women. In the figure, the chlamydial developmental cycle is described. Infection is initiated with the chlamydial EBs that convert into RBs, multiply and convert back to EBs. Then, the infectious progeny is released from the host cell to initiate an additional cycle. Upon infection, immune cells are recruited to the infected area, among them CD4+ expressing Th1 cells that produce IFN-γ. IFN-γ induces the production of IDO1 that catabolizes tryptophan into kynurenine, depleting the host tryptophan pools. This triggers the tryptophan auxotroph Chlamydia to enter its persistence form, or in severe tryptophan starvation, to its death. Vaginal tract microbiota has an important role in health and disease. Among these bacterial communities, CST IV was associated with current or previous Chlamydia infection, low tryptophan levels and high kynurenine/tryptophan ratios. On the other hand, vaginal Lactobacillus crispatus was shown to inhibit chlamydial growth. Initial and repeated Chlamydia infections were associated with high kynurenine/tryptophan ratios. Repeated Chlamydia infection was shown to be associated with high kynurenine levels. Although low tryptophan levels were found to inhibit Chlamydia in vitro and were associated with natural clearance in vivo, tryptophan depletion is also related to the inhibition of Th1 immunity. Kynurenine and IDO1 are also known to inhibit T cells and local immunity. IDO1 and TGF-β1 are known to synergistically activate tolerogenic effect in pDCs. Azithromycin was shown to be effective in killing Chlamydia , however, also eliciting an anti-inflammatory response. Chlamydia infection clearance post azithromycin treatment in women was found to elicit IDO1, TGF-β1 and FoxP3 regulatory immune response. Repeated Chlamydia infection in women had similar effects on the expression levels of these genes, and might have been triggered by high kynurenine/tryptophan ratios. Chlamydia infection in in vitro ECC1 and PBMCs co-culture was found to elicit IDO1, TGF-β1 and FoxP3 as well

    Journal: BMC Infectious Diseases

    Article Title: High expression of IDO1 and TGF-β1 during recurrence and post infection clearance with Chlamydia trachomatis, are independent of host IFN-γ response

    doi: 10.1186/s12879-019-3843-4

    Figure Lengend Snippet: Immunological, bacterial and biochemical factors that are associated with initial or repeated C. trachomatis infections in women. In the figure, the chlamydial developmental cycle is described. Infection is initiated with the chlamydial EBs that convert into RBs, multiply and convert back to EBs. Then, the infectious progeny is released from the host cell to initiate an additional cycle. Upon infection, immune cells are recruited to the infected area, among them CD4+ expressing Th1 cells that produce IFN-γ. IFN-γ induces the production of IDO1 that catabolizes tryptophan into kynurenine, depleting the host tryptophan pools. This triggers the tryptophan auxotroph Chlamydia to enter its persistence form, or in severe tryptophan starvation, to its death. Vaginal tract microbiota has an important role in health and disease. Among these bacterial communities, CST IV was associated with current or previous Chlamydia infection, low tryptophan levels and high kynurenine/tryptophan ratios. On the other hand, vaginal Lactobacillus crispatus was shown to inhibit chlamydial growth. Initial and repeated Chlamydia infections were associated with high kynurenine/tryptophan ratios. Repeated Chlamydia infection was shown to be associated with high kynurenine levels. Although low tryptophan levels were found to inhibit Chlamydia in vitro and were associated with natural clearance in vivo, tryptophan depletion is also related to the inhibition of Th1 immunity. Kynurenine and IDO1 are also known to inhibit T cells and local immunity. IDO1 and TGF-β1 are known to synergistically activate tolerogenic effect in pDCs. Azithromycin was shown to be effective in killing Chlamydia , however, also eliciting an anti-inflammatory response. Chlamydia infection clearance post azithromycin treatment in women was found to elicit IDO1, TGF-β1 and FoxP3 regulatory immune response. Repeated Chlamydia infection in women had similar effects on the expression levels of these genes, and might have been triggered by high kynurenine/tryptophan ratios. Chlamydia infection in in vitro ECC1 and PBMCs co-culture was found to elicit IDO1, TGF-β1 and FoxP3 as well

    Article Snippet: Experiments were conducted in 48-well plates, where co-culture of 40,000 ECC1 cells/well and 8 × 105 female human PBMCs (Lonza, Australia) cells/well were seeded with RPMI-1640 medium (Sigma-Aldrich, Australia) containing 10% heat inactivated FCS (Life Technologies, Australia), 120 μg/ml streptomycin (Sigma-Aldrich, Australia), 50 μg/ml Gentamycin (Gibco, Australia), 37 °C, 5% CO2 .

    Techniques: Infection, Expressing, In Vitro, In Vivo, Inhibition, Co-Culture Assay

    IDO1, TGF-β1, FoxP3 and IFN-γ expression levels as a response to C. trachomatis infection, with or without azithromycin treatment. Transcript levels of IDO1, TGF-β1, FoxP3 and IFN-γ (2 - ΔCT ) were measured from cell cultures of ( a ) human endometrial cell line ECC1, ( b ) PBMCs of C. trachomatis negative female, and ( c ) co-culture of ECC1 and PBMCs. Transcript levels were compared between treatments; without Chlamydia infection or azithromycin treatment (−CT –AZ), no Chlamydia infection with azithromycin (−CT + AZ), with Chlamydia infection no azithromycin (+CT –AZ) and with Chlamydia infection and azithromycin treatment (+CT + AZ). Cells were infected with C. trachomatis at MOI of 0.1. Azithromycin was added to the culture at 20 h PI, or at 44 h post seeding in control treatments. Total RNA was isolated from cultures at 44 h PI. Data are presented as mean ± SD. Significant differences are indicated in the graph ( p

    Journal: BMC Infectious Diseases

    Article Title: High expression of IDO1 and TGF-β1 during recurrence and post infection clearance with Chlamydia trachomatis, are independent of host IFN-γ response

    doi: 10.1186/s12879-019-3843-4

    Figure Lengend Snippet: IDO1, TGF-β1, FoxP3 and IFN-γ expression levels as a response to C. trachomatis infection, with or without azithromycin treatment. Transcript levels of IDO1, TGF-β1, FoxP3 and IFN-γ (2 - ΔCT ) were measured from cell cultures of ( a ) human endometrial cell line ECC1, ( b ) PBMCs of C. trachomatis negative female, and ( c ) co-culture of ECC1 and PBMCs. Transcript levels were compared between treatments; without Chlamydia infection or azithromycin treatment (−CT –AZ), no Chlamydia infection with azithromycin (−CT + AZ), with Chlamydia infection no azithromycin (+CT –AZ) and with Chlamydia infection and azithromycin treatment (+CT + AZ). Cells were infected with C. trachomatis at MOI of 0.1. Azithromycin was added to the culture at 20 h PI, or at 44 h post seeding in control treatments. Total RNA was isolated from cultures at 44 h PI. Data are presented as mean ± SD. Significant differences are indicated in the graph ( p

    Article Snippet: Experiments were conducted in 48-well plates, where co-culture of 40,000 ECC1 cells/well and 8 × 105 female human PBMCs (Lonza, Australia) cells/well were seeded with RPMI-1640 medium (Sigma-Aldrich, Australia) containing 10% heat inactivated FCS (Life Technologies, Australia), 120 μg/ml streptomycin (Sigma-Aldrich, Australia), 50 μg/ml Gentamycin (Gibco, Australia), 37 °C, 5% CO2 .

    Techniques: Expressing, Infection, Co-Culture Assay, Isolation

    S. sonnei induces the secretion of IL-1β, IL-18, NLRP3, ASC, and active caspase-1 in macrophages. (A) J774A.1 macrophages, THP-1 macrophages, PBMCs or BMDM were primed with 1 μg/ml LPS for 4 h and then infected with S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. (B–E) J774A.1 macrophages or THP-1 macrophages were primed with 1 μg/ml LPS for 4 h followed and then infected with S. sonnei for an additional 20 h or stimulated with 5 mM ATP for an additional 0.5 h. The levels of IL-1β (B) , IL-18 (C) , caspase-1 (D) , NLRP3, and ASC (E) in the supernatants were measured by Western blotting. The ELISA data are expressed as the mean ± SD of four separate experiments. The Western blotting results are representative of three different experiments. * and *** indicate significant differences at the levels of p

    Journal: Frontiers in Immunology

    Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei-Infected Macrophages

    doi: 10.3389/fimmu.2020.01115

    Figure Lengend Snippet: S. sonnei induces the secretion of IL-1β, IL-18, NLRP3, ASC, and active caspase-1 in macrophages. (A) J774A.1 macrophages, THP-1 macrophages, PBMCs or BMDM were primed with 1 μg/ml LPS for 4 h and then infected with S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. (B–E) J774A.1 macrophages or THP-1 macrophages were primed with 1 μg/ml LPS for 4 h followed and then infected with S. sonnei for an additional 20 h or stimulated with 5 mM ATP for an additional 0.5 h. The levels of IL-1β (B) , IL-18 (C) , caspase-1 (D) , NLRP3, and ASC (E) in the supernatants were measured by Western blotting. The ELISA data are expressed as the mean ± SD of four separate experiments. The Western blotting results are representative of three different experiments. * and *** indicate significant differences at the levels of p

    Article Snippet: THP-1 macrophages were differentiated from THP-1 monocytes by treatment with 50 nM PMA for 48 h. Human peripheral blood mononuclear cells (PBMCs) were separated from whole blood from healthy volunteers by density gradient centrifugation using Histopaque-1077 , and all experimental protocols were performed in accordance with the guidelines and regulations provided and accepted by the Institutional Review Board of the Tri-Service General Hospital, National Defense Medical Center and the volunteers' informed consent (TSGH-IRB-2-106-05-190 and TSGH-IRB-2-106-05-009).

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, Western Blot

    CpG-B DNA, not CpG-A DNA, induces phosphorylation of PKD proteins in murine splenic B cells, cDCs, and pDCs, and human PBMCs

    Journal:

    Article Title: Protein Kinase D1: A New Component in Toll-Like Receptor 9 Signaling

    doi:

    Figure Lengend Snippet: CpG-B DNA, not CpG-A DNA, induces phosphorylation of PKD proteins in murine splenic B cells, cDCs, and pDCs, and human PBMCs

    Article Snippet: Because, in addition to macrophages, other types of cells, including B cells and DCs, also express TLR9 (the CpG motif containing DNA receptor) and respond to CpG motif containing DNA, we further investigated whether CpG motif containing DNA can induce activation of PKD family members in B cells, pDCs, cDCs, and human PBMCs.

    Techniques:

    Gene expression of tumor-suppressor genes. RNA was isolated from peripheral blood mononuclear cells (PBMCs) and K562 cells and quantitative real-time polymerase chain reaction was performed. Relative gene expression was plotted for tumor-suppressor genes. GAPDH was used a reference gene and data were normalized to PBMCs. *p

    Journal: Turkish Journal of Hematology

    Article Title: Tendency of K562 Chronic Myeloid Leukemia Cells Towards Cell Reprogramming

    doi: 10.4274/tjh.2018.0106

    Figure Lengend Snippet: Gene expression of tumor-suppressor genes. RNA was isolated from peripheral blood mononuclear cells (PBMCs) and K562 cells and quantitative real-time polymerase chain reaction was performed. Relative gene expression was plotted for tumor-suppressor genes. GAPDH was used a reference gene and data were normalized to PBMCs. *p

    Article Snippet: Cell Culture Human CML cell line K562 and human peripheral blood mononuclear cells (PBMCs) were obtained from ATCC and Lonza, respectively.

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

    Gene expression of reprogramming factors and pluripotency markers. RNA was isolated from peripheral blood mononuclear cells (PBMCs) and K562 cells and quantitative real-time polymerase chain reaction was performed. Relative gene expression was plotted for A) reprogramming factors and B) pluripotency markers. GAPDH was used as a reference gene and data were normalized to PBMCs. *p

    Journal: Turkish Journal of Hematology

    Article Title: Tendency of K562 Chronic Myeloid Leukemia Cells Towards Cell Reprogramming

    doi: 10.4274/tjh.2018.0106

    Figure Lengend Snippet: Gene expression of reprogramming factors and pluripotency markers. RNA was isolated from peripheral blood mononuclear cells (PBMCs) and K562 cells and quantitative real-time polymerase chain reaction was performed. Relative gene expression was plotted for A) reprogramming factors and B) pluripotency markers. GAPDH was used as a reference gene and data were normalized to PBMCs. *p

    Article Snippet: Cell Culture Human CML cell line K562 and human peripheral blood mononuclear cells (PBMCs) were obtained from ATCC and Lonza, respectively.

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

    Fusion efficiency enhancement . The fusions were performed according to a standard protocol, where the culture media and PEG were reconstituted from powder forms with either NPD or DI water. For each fusion, PBMC from a single batch were divided into two equal fractures and used to prepare two parallel experiments, in NPD or DI based reagents. The figure presents percent of hybridoma-positive wells in each fusion experiment. The percent was calculated as the number of hybridoma-positive wells from 96-well plates where the cells were seeded and grown after the fusion process. The difference between the NPD- and DI-fusion results was found to be statistically significant by Chi-square analysis (p

    Journal: BMC Biotechnology

    Article Title: Enhancement of hybridoma formation, clonability and cell proliferation in a nanoparticle-doped aqueous environment

    doi: 10.1186/1472-6750-8-3

    Figure Lengend Snippet: Fusion efficiency enhancement . The fusions were performed according to a standard protocol, where the culture media and PEG were reconstituted from powder forms with either NPD or DI water. For each fusion, PBMC from a single batch were divided into two equal fractures and used to prepare two parallel experiments, in NPD or DI based reagents. The figure presents percent of hybridoma-positive wells in each fusion experiment. The percent was calculated as the number of hybridoma-positive wells from 96-well plates where the cells were seeded and grown after the fusion process. The difference between the NPD- and DI-fusion results was found to be statistically significant by Chi-square analysis (p

    Article Snippet: For production of hybridoma cells, human peripheral blood mononuclear cells (PBMC) were isolated from 40 mL of freshly drawn whole blood, purified with Histopaque 1077 (Sigma) as previously described, and washed 4 times in DI based culture medium without serum.

    Techniques:

    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

    Human PBMC subsets express receptors for p17. PBMCs were isolated from leukopaks from healthy donors (New York Blood Center) with Lymphoprep. After RBC lysis, cells were stained with p17-Alexa Fluor 430 and surface markers. Live cells were gated as follows:

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Expression of HIV-1 matrix protein p17 and association with B-cell lymphoma in HIV-1 transgenic mice

    doi: 10.1073/pnas.1615258113

    Figure Lengend Snippet: Human PBMC subsets express receptors for p17. PBMCs were isolated from leukopaks from healthy donors (New York Blood Center) with Lymphoprep. After RBC lysis, cells were stained with p17-Alexa Fluor 430 and surface markers. Live cells were gated as follows:

    Article Snippet: Human PBMCs were isolated using Lymphoprep (Axis-Shield) from healthy blood donors' leukopaks obtained from the New York Blood Center, Long Island, NY, in accordance with their guidelines and those of the Institutional Review Board of the University of Maryland School of Medicine.

    Techniques: Isolation, Lysis, Staining

    Infection with Mtb HN878 triggers increased IL-22 production through a TLR2-dependent pathway Lung cells isolated from B6, Il1r-/- and Tlr2-/- mice were infected with either Mtb H37Rv (Rv) or Mtb HN878 (HN) at MOI of 0.1 for 7 days, and the protein levels of ( a ) IL-1β and ( b ) IL-22 were assessed in culture supernatants by ELISA and luminex assay, respectively. Lung cells isolated from B6 mice were treated with cell wall extracted from Mtb H37Rv (Rv) or Mtb HN878 (HN), (10μg/ml each). Protein levels of ( c ) IL-1β and ( d ) IL-22 were assessed by ELISA and luminex assay respectively. PBMCs were isolated from ATB patients and were stimulated with Mtb H37Rv (Rv) or Mtb HN878 (HN) cell wall extracts (20 μg/ml each) and the protein levels of ( e ) IL-22 were measured by luminex assays. Error bars represent means ± s.e.m. n = 3-5 for a-d. n = 17 for human ATB patients for e. ND, not detected; UI, uninfected; UN, untreated. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 by ( a,b ) one-way ANOVA, ( c,d ) Student's t test ( e ) two-way ANOVA

    Journal: Mucosal immunology

    Article Title: Novel role for IL-22 in protection during chronic Mycobacterium tuberculosis HN878 infection

    doi: 10.1038/mi.2017.15

    Figure Lengend Snippet: Infection with Mtb HN878 triggers increased IL-22 production through a TLR2-dependent pathway Lung cells isolated from B6, Il1r-/- and Tlr2-/- mice were infected with either Mtb H37Rv (Rv) or Mtb HN878 (HN) at MOI of 0.1 for 7 days, and the protein levels of ( a ) IL-1β and ( b ) IL-22 were assessed in culture supernatants by ELISA and luminex assay, respectively. Lung cells isolated from B6 mice were treated with cell wall extracted from Mtb H37Rv (Rv) or Mtb HN878 (HN), (10μg/ml each). Protein levels of ( c ) IL-1β and ( d ) IL-22 were assessed by ELISA and luminex assay respectively. PBMCs were isolated from ATB patients and were stimulated with Mtb H37Rv (Rv) or Mtb HN878 (HN) cell wall extracts (20 μg/ml each) and the protein levels of ( e ) IL-22 were measured by luminex assays. Error bars represent means ± s.e.m. n = 3-5 for a-d. n = 17 for human ATB patients for e. ND, not detected; UI, uninfected; UN, untreated. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 by ( a,b ) one-way ANOVA, ( c,d ) Student's t test ( e ) two-way ANOVA

    Article Snippet: Human peripheral blood mononuclear cells (PBMCs) from ATB patients were isolated by Ficoll Hypaque gradient (Lymphoprep; Axis-Shield POC AS, Oslo, Norway ).

    Techniques: Infection, Isolation, Mouse Assay, Enzyme-linked Immunosorbent Assay, Luminex

    Detection of SMN Protein in SMNA Type 1 PBMCs. SMA Type I patient samples (N = 4) were tested in the SMN ELISA at dilutions of 1∶4 with 1.25×10 6 and 2.5×10 6 PBMCs. SMN protein signal was detected in all samples, with an average of 8.32 pg SMN protein per 10 6 cells. Based on the average of 70.2 pg SMN protein per 10 6 cells calculated for adult normal donor PBMCs, the amount of SMN protein in PBMCs of Type I SMA patients in this patient cohort is 88% less than normal. Error bars represent standard deviations.

    Journal: PLoS ONE

    Article Title: Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

    doi: 10.1371/journal.pone.0024269

    Figure Lengend Snippet: Detection of SMN Protein in SMNA Type 1 PBMCs. SMA Type I patient samples (N = 4) were tested in the SMN ELISA at dilutions of 1∶4 with 1.25×10 6 and 2.5×10 6 PBMCs. SMN protein signal was detected in all samples, with an average of 8.32 pg SMN protein per 10 6 cells. Based on the average of 70.2 pg SMN protein per 10 6 cells calculated for adult normal donor PBMCs, the amount of SMN protein in PBMCs of Type I SMA patients in this patient cohort is 88% less than normal. Error bars represent standard deviations.

    Article Snippet: Non-human primate PBMC lysates were run alongside lysates from normal human PBMCs (from AllCells) and mouse PBMCs harvested from 8 week old FVBn animals at PharmOptima.

    Techniques: Enzyme-linked Immunosorbent Assay

    Quantitation of SMN by PBMC subtype. There was no significant difference in SMN protein levels between human lymphocytes and monocytes fractions separated from PBMCs. SMN protein was detectable in the majority of samples for both cell types, ranging from 6.8–25.8 pg/10 6 cells for lymphocytes and 8–17.5 pg/10 6 cells for monocytes. Error bars represent standard deviations.

    Journal: PLoS ONE

    Article Title: Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

    doi: 10.1371/journal.pone.0024269

    Figure Lengend Snippet: Quantitation of SMN by PBMC subtype. There was no significant difference in SMN protein levels between human lymphocytes and monocytes fractions separated from PBMCs. SMN protein was detectable in the majority of samples for both cell types, ranging from 6.8–25.8 pg/10 6 cells for lymphocytes and 8–17.5 pg/10 6 cells for monocytes. Error bars represent standard deviations.

    Article Snippet: Non-human primate PBMC lysates were run alongside lysates from normal human PBMCs (from AllCells) and mouse PBMCs harvested from 8 week old FVBn animals at PharmOptima.

    Techniques: Quantitation Assay

    ELISA Performance, parallelism and detection of SMN in human PBMCs. A : The recombinant hSMN standard curve data developed from N = 6 curves were highly reproducible with standard deviations of about ±0.23 OD unit variations. The dotted lines surrounding the dose-response curve represent 2 standard deviations. B : Comparison of SMN signal detection between recombinant human SMN (dilutions from 1∶4 to 1∶256) and SMN extracted from HeLa cell lysates (1∶4–1∶32) revealed a high degree of parallelism between reagents, allowing for accurate evaluation of native SMN protein using the recombinant SMN ELISA standard. C : The SMN ELISA detected protein in adult donor PBMCs; values ranged from 86 to 229 pg SMN per well at a dilution of 1∶4 and an average of 70.2 pg SMN protein per 10 6 cells. 10 7 PBMCs diluted 1∶4 through 1∶32 produced linear SMN protein levels. Even with only N = 6 normal samples, SMN levels in PBMCs varied by nearly 3-fold. Error bars represent standard deviations.

    Journal: PLoS ONE

    Article Title: Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

    doi: 10.1371/journal.pone.0024269

    Figure Lengend Snippet: ELISA Performance, parallelism and detection of SMN in human PBMCs. A : The recombinant hSMN standard curve data developed from N = 6 curves were highly reproducible with standard deviations of about ±0.23 OD unit variations. The dotted lines surrounding the dose-response curve represent 2 standard deviations. B : Comparison of SMN signal detection between recombinant human SMN (dilutions from 1∶4 to 1∶256) and SMN extracted from HeLa cell lysates (1∶4–1∶32) revealed a high degree of parallelism between reagents, allowing for accurate evaluation of native SMN protein using the recombinant SMN ELISA standard. C : The SMN ELISA detected protein in adult donor PBMCs; values ranged from 86 to 229 pg SMN per well at a dilution of 1∶4 and an average of 70.2 pg SMN protein per 10 6 cells. 10 7 PBMCs diluted 1∶4 through 1∶32 produced linear SMN protein levels. Even with only N = 6 normal samples, SMN levels in PBMCs varied by nearly 3-fold. Error bars represent standard deviations.

    Article Snippet: Non-human primate PBMC lysates were run alongside lysates from normal human PBMCs (from AllCells) and mouse PBMCs harvested from 8 week old FVBn animals at PharmOptima.

    Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Produced

    Smn protein levels by tissue in WT mice. Smn protein levels are distinct across the an array of tissues and regions of the brain. A : Smn levels varied by as much as 10-fold across tissue types in adult FVB mice (14 weeks old). SMN levels by tissue were distributed on a basis loosely ordered by having lesser to greater dividing cell populations. Smn levels for tissues are represented as Smn pg/mg total protein. PBMCs are represented as Smn pg/mg total protein in the lysis buffer extract. On a per cell basis the average Smn level was 67.2 pg/10 6 PBMCs. B : Smn protein in the brain, spinal cord, sciatic nerve and quadriceps muscle of wildtype mice shows marked variations, with spinal cord having 6-fold greater levels than muscle and nerve. In the brain, Smn protein signal in the thalamus was nearly 2.5-fold less than hippocampal levels. Error bars represent standard deviations.

    Journal: PLoS ONE

    Article Title: Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

    doi: 10.1371/journal.pone.0024269

    Figure Lengend Snippet: Smn protein levels by tissue in WT mice. Smn protein levels are distinct across the an array of tissues and regions of the brain. A : Smn levels varied by as much as 10-fold across tissue types in adult FVB mice (14 weeks old). SMN levels by tissue were distributed on a basis loosely ordered by having lesser to greater dividing cell populations. Smn levels for tissues are represented as Smn pg/mg total protein. PBMCs are represented as Smn pg/mg total protein in the lysis buffer extract. On a per cell basis the average Smn level was 67.2 pg/10 6 PBMCs. B : Smn protein in the brain, spinal cord, sciatic nerve and quadriceps muscle of wildtype mice shows marked variations, with spinal cord having 6-fold greater levels than muscle and nerve. In the brain, Smn protein signal in the thalamus was nearly 2.5-fold less than hippocampal levels. Error bars represent standard deviations.

    Article Snippet: Non-human primate PBMC lysates were run alongside lysates from normal human PBMCs (from AllCells) and mouse PBMCs harvested from 8 week old FVBn animals at PharmOptima.

    Techniques: Mouse Assay, Lysis

    Effects of PGRN on the differentiation of human OCs and PGRN as a predictor of osteoporosis. A , HBMCs and PBMCs were differentiated with RANKL in the absence or presence of PGRN. TRAP + MNCs were counted as described above. B , four sh-RNAs targeting human

    Journal: The Journal of Biological Chemistry

    Article Title: Progranulin and a Five Transmembrane Domain-Containing Receptor-like Gene Are the Key Components in Receptor Activator of Nuclear Factor κB (RANK)-dependent Formation of Multinucleated Osteoclasts *

    doi: 10.1074/jbc.M114.608786

    Figure Lengend Snippet: Effects of PGRN on the differentiation of human OCs and PGRN as a predictor of osteoporosis. A , HBMCs and PBMCs were differentiated with RANKL in the absence or presence of PGRN. TRAP + MNCs were counted as described above. B , four sh-RNAs targeting human

    Article Snippet: Human bone marrow cells (HBMCs) and peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers and were separated by density gradient centrifugation using Ficoll-Histopaque (Sigma-Aldrich).

    Techniques:

    Lu AF58786 inhibits phosphorylation of LRRK2, Rab10 and Rab12 in cultured and immune stimulated human PBMCs. ( A ) LRRK2 (pSer910, pSer935, pSer955 and pSer973) phospho-site hits plotted with mean and individual replicate values of the quantified log 10 -transformed CMPD/DMSO ratio as well as the associated q-value of the performed mean rank (n = 5–8). ( B ) Rab10 (pThr73) and Rab12 (pSer106) switch II domain phospho-site hits plotted with mean and individual replicate values of the quantified log 10 -transformed CMPD/DMSO ratio as well as the associated q-value of the performed mean rank (n = 5–8). CMPD, compound (Lu AF58786). DMSO, dimethyl sulfoxide.

    Journal: Scientific Reports

    Article Title: Selective LRRK2 kinase inhibition reduces phosphorylation of endogenous Rab10 and Rab12 in human peripheral mononuclear blood cells

    doi: 10.1038/s41598-017-10501-z

    Figure Lengend Snippet: Lu AF58786 inhibits phosphorylation of LRRK2, Rab10 and Rab12 in cultured and immune stimulated human PBMCs. ( A ) LRRK2 (pSer910, pSer935, pSer955 and pSer973) phospho-site hits plotted with mean and individual replicate values of the quantified log 10 -transformed CMPD/DMSO ratio as well as the associated q-value of the performed mean rank (n = 5–8). ( B ) Rab10 (pThr73) and Rab12 (pSer106) switch II domain phospho-site hits plotted with mean and individual replicate values of the quantified log 10 -transformed CMPD/DMSO ratio as well as the associated q-value of the performed mean rank (n = 5–8). CMPD, compound (Lu AF58786). DMSO, dimethyl sulfoxide.

    Article Snippet: Importantly, we also show that both the abovementioned LRRK2 inhibitor compounds have non-overlapping off-target kinases in human PBMCs at 1 µM concentrations.

    Techniques: Cell Culture, Transformation Assay

    Proteomic and phosphoproteomic study to identify LRRK2 kinase activity-dependent substrates in immune-stimulated human PBMCs. ( A ) % viability after 3 DIV of human immune-stimulated PBMCs treated for 24 hrs with or without 100 nM Lu AF58786 (n = 10 donors; 2 conditions). ( B ) PBMC total protein yield (µg) after 3 DIV of human immune-stimulated PBMCs treated for 24 hrs with or without 100 nM Lu AF58786 (n = 10 donors; 2 conditions). ( C ) Experimental set-up with information on mTRAQ labelling and pairing of samples as well as amount of protein used. ( D ) Experimental flow chart of the proteomic studies. Data was analyzed by either one-way ANOVA with Dunnett’s multiple comparisons test or unpaired t-test. Data is presented as means ± SEM. PMA, phorbol 12-myristate 13-acetate; INF-γ, interferon-γ; DMSO, dimethyl sulfoxide.

    Journal: Scientific Reports

    Article Title: Selective LRRK2 kinase inhibition reduces phosphorylation of endogenous Rab10 and Rab12 in human peripheral mononuclear blood cells

    doi: 10.1038/s41598-017-10501-z

    Figure Lengend Snippet: Proteomic and phosphoproteomic study to identify LRRK2 kinase activity-dependent substrates in immune-stimulated human PBMCs. ( A ) % viability after 3 DIV of human immune-stimulated PBMCs treated for 24 hrs with or without 100 nM Lu AF58786 (n = 10 donors; 2 conditions). ( B ) PBMC total protein yield (µg) after 3 DIV of human immune-stimulated PBMCs treated for 24 hrs with or without 100 nM Lu AF58786 (n = 10 donors; 2 conditions). ( C ) Experimental set-up with information on mTRAQ labelling and pairing of samples as well as amount of protein used. ( D ) Experimental flow chart of the proteomic studies. Data was analyzed by either one-way ANOVA with Dunnett’s multiple comparisons test or unpaired t-test. Data is presented as means ± SEM. PMA, phorbol 12-myristate 13-acetate; INF-γ, interferon-γ; DMSO, dimethyl sulfoxide.

    Article Snippet: Importantly, we also show that both the abovementioned LRRK2 inhibitor compounds have non-overlapping off-target kinases in human PBMCs at 1 µM concentrations.

    Techniques: Activity Assay, Flow Cytometry

    PFE-360 inhibits LRRK2-Ser935 and Rab10-Thr73 phosphorylation in a concentration-dependent manner in non-stimulated PBMCs from human healthy subjects. Determination of LRRK2 inhibitor IC 50 values based on either Rab10-pThr73 or LRRK2-pSer935 levels in human non-stimulated PBMCs. ( A ) Odyssey CLx scan image showing Western Blot analysis of crude lysates from a pool of PBMCs from two donors treated for 1 hour with concentrations of PFE-360 ranging from 4nM-1µM. in duplicate. LRRK2 and Rab10 immunoreactivity ( red panels ), LRRK2-pSer935 and Rab10-pThr73 immunoreactivity ( green panels ) as well as overlay in non-stimulated human PBMCs. Full-length blots are presented in Supplementary Figure 5 . Non-linear regression plot of percentage ( B ) LRRK2-pSer935 inhibition and ( C ) Rab10-pThr73 as a function of log10-transformed PFE-360 concentration. The experiment was repeated three times and the resulting IC50 determination is summarized in Table 2 .

    Journal: Scientific Reports

    Article Title: Selective LRRK2 kinase inhibition reduces phosphorylation of endogenous Rab10 and Rab12 in human peripheral mononuclear blood cells

    doi: 10.1038/s41598-017-10501-z

    Figure Lengend Snippet: PFE-360 inhibits LRRK2-Ser935 and Rab10-Thr73 phosphorylation in a concentration-dependent manner in non-stimulated PBMCs from human healthy subjects. Determination of LRRK2 inhibitor IC 50 values based on either Rab10-pThr73 or LRRK2-pSer935 levels in human non-stimulated PBMCs. ( A ) Odyssey CLx scan image showing Western Blot analysis of crude lysates from a pool of PBMCs from two donors treated for 1 hour with concentrations of PFE-360 ranging from 4nM-1µM. in duplicate. LRRK2 and Rab10 immunoreactivity ( red panels ), LRRK2-pSer935 and Rab10-pThr73 immunoreactivity ( green panels ) as well as overlay in non-stimulated human PBMCs. Full-length blots are presented in Supplementary Figure 5 . Non-linear regression plot of percentage ( B ) LRRK2-pSer935 inhibition and ( C ) Rab10-pThr73 as a function of log10-transformed PFE-360 concentration. The experiment was repeated three times and the resulting IC50 determination is summarized in Table 2 .

    Article Snippet: Importantly, we also show that both the abovementioned LRRK2 inhibitor compounds have non-overlapping off-target kinases in human PBMCs at 1 µM concentrations.

    Techniques: Concentration Assay, Western Blot, Inhibition, Transformation Assay

    LRRK2 kinase-activity dependent LRRK2-Ser935, Rab10-Thr73 and Rab12-Ser106 phosphorylation in cultured and immune stimulated human PBMCs. Quantification of ( A , B ) normalized LRRK2-pSer935/total LRRK2 ( C , D ) Rab10-pThr73/total Rab10 and ( E , F ) Rab12-pSer106/total Rab10 ratios in cultured and immune stimulated human PBMCs treated with either DMSO, 100 nM Lu AF58786 or 100 nM PFE-360 (n = 10 donors; each donor in 3 conditions at left panel , 1hr and right panel , 24hrs). Data was analyzed by one-way ANOVA with Holm-Sidak’s multiple comparisons test. Data is presented as DMSO-normalized means ± SEM; p-values presented are vs. DMSO.

    Journal: Scientific Reports

    Article Title: Selective LRRK2 kinase inhibition reduces phosphorylation of endogenous Rab10 and Rab12 in human peripheral mononuclear blood cells

    doi: 10.1038/s41598-017-10501-z

    Figure Lengend Snippet: LRRK2 kinase-activity dependent LRRK2-Ser935, Rab10-Thr73 and Rab12-Ser106 phosphorylation in cultured and immune stimulated human PBMCs. Quantification of ( A , B ) normalized LRRK2-pSer935/total LRRK2 ( C , D ) Rab10-pThr73/total Rab10 and ( E , F ) Rab12-pSer106/total Rab10 ratios in cultured and immune stimulated human PBMCs treated with either DMSO, 100 nM Lu AF58786 or 100 nM PFE-360 (n = 10 donors; each donor in 3 conditions at left panel , 1hr and right panel , 24hrs). Data was analyzed by one-way ANOVA with Holm-Sidak’s multiple comparisons test. Data is presented as DMSO-normalized means ± SEM; p-values presented are vs. DMSO.

    Article Snippet: Importantly, we also show that both the abovementioned LRRK2 inhibitor compounds have non-overlapping off-target kinases in human PBMCs at 1 µM concentrations.

    Techniques: Activity Assay, Cell Culture

    Acute LRRK2 inhibition with PFE-360 reduces LRRK2-pSer935 and Rab10-Thr73 phosphorylation in non-stimulated PBMCs from human healthy subjects. Odyssey CLx scan Western Blot images showing ( A ) LRRK2 and ( C ) Rab10 immunoreactivity (red panels), LRRK2-pSer935 and Rab10-pThr73 immunoreactivity (green panels) as well as overlay in non-stimulated human PBMCs treated with 2 µM PFE-360. Full-length blots are presented in Supplementary Figure 4 . Quantification of ( B ) relative LRRK2-pSer935/total LRRK2 ratio and ( D ) relative Rab10-pThr73/total Rab10 ratio (n = 6 donors; 2 conditions). Data was analyzed by paired t-test. Data is presented as means ± SEM; ****p

    Journal: Scientific Reports

    Article Title: Selective LRRK2 kinase inhibition reduces phosphorylation of endogenous Rab10 and Rab12 in human peripheral mononuclear blood cells

    doi: 10.1038/s41598-017-10501-z

    Figure Lengend Snippet: Acute LRRK2 inhibition with PFE-360 reduces LRRK2-pSer935 and Rab10-Thr73 phosphorylation in non-stimulated PBMCs from human healthy subjects. Odyssey CLx scan Western Blot images showing ( A ) LRRK2 and ( C ) Rab10 immunoreactivity (red panels), LRRK2-pSer935 and Rab10-pThr73 immunoreactivity (green panels) as well as overlay in non-stimulated human PBMCs treated with 2 µM PFE-360. Full-length blots are presented in Supplementary Figure 4 . Quantification of ( B ) relative LRRK2-pSer935/total LRRK2 ratio and ( D ) relative Rab10-pThr73/total Rab10 ratio (n = 6 donors; 2 conditions). Data was analyzed by paired t-test. Data is presented as means ± SEM; ****p

    Article Snippet: Importantly, we also show that both the abovementioned LRRK2 inhibitor compounds have non-overlapping off-target kinases in human PBMCs at 1 µM concentrations.

    Techniques: Inhibition, Western Blot

    LRRK2 expression and inhibition in cultured human PBMCs immune stimulated with PMA and interferon- γ . ( A ) Schematic representation of the three different treatment conditions. ( B ) Chemical structure of the LRRK2 inhibitor Lu AF58786. ( C ) Determination of LRRK2, G2019S and A2016T IC 50 values using the Odyssey CLx 96-well ICW assay (mean IC 50 , n = 3 experiments). ( D ) Total PBMC yield obtained from human healthy donors after 3 days in vitro (3 DIV) (n = 10 donors; 3 different conditions). ( E ) % viability of human PBMCs (n = 10 donors; 3 different conditions) after 3 DIV. ( F ) Odyssey CLx scan image of Western Blot used for estimation of LRRK2 protein levels (total LRRK2) and LRRK2 phosphorylation (pSer935). The concentration of Lu AF58786 was 100 nM. Full-length blots are presented in Supplementary Figure 1 . ( G ) Quantification of total LRRK2 levels (raw signal). ( H ) Relative LRRK2-pSer935 phosphorylation (pSer935/total LRRK2). Data was analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. Data presented as means ± SEM; ****p

    Journal: Scientific Reports

    Article Title: Selective LRRK2 kinase inhibition reduces phosphorylation of endogenous Rab10 and Rab12 in human peripheral mononuclear blood cells

    doi: 10.1038/s41598-017-10501-z

    Figure Lengend Snippet: LRRK2 expression and inhibition in cultured human PBMCs immune stimulated with PMA and interferon- γ . ( A ) Schematic representation of the three different treatment conditions. ( B ) Chemical structure of the LRRK2 inhibitor Lu AF58786. ( C ) Determination of LRRK2, G2019S and A2016T IC 50 values using the Odyssey CLx 96-well ICW assay (mean IC 50 , n = 3 experiments). ( D ) Total PBMC yield obtained from human healthy donors after 3 days in vitro (3 DIV) (n = 10 donors; 3 different conditions). ( E ) % viability of human PBMCs (n = 10 donors; 3 different conditions) after 3 DIV. ( F ) Odyssey CLx scan image of Western Blot used for estimation of LRRK2 protein levels (total LRRK2) and LRRK2 phosphorylation (pSer935). The concentration of Lu AF58786 was 100 nM. Full-length blots are presented in Supplementary Figure 1 . ( G ) Quantification of total LRRK2 levels (raw signal). ( H ) Relative LRRK2-pSer935 phosphorylation (pSer935/total LRRK2). Data was analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. Data presented as means ± SEM; ****p

    Article Snippet: Importantly, we also show that both the abovementioned LRRK2 inhibitor compounds have non-overlapping off-target kinases in human PBMCs at 1 µM concentrations.

    Techniques: Expressing, Inhibition, Cell Culture, In Vitro, Western Blot, Concentration Assay

    The evaluation of the immunostimulatory effects of NIPAM-hemin. Abbreviations: NIPAM, N -isopropylacrylamide; hemin, ferriprotoporphyrin IX chloride; IFN-γ, interferon-γ; IL, interleukin; NK, natural killer; PBMCs, peripheral blood mononuclear cells; ELISA, enzyme-linked immunosorbent assay; TLRs, toll-like receptors; SEAP, secreted embryonic alkaline phosphatase.

    Journal: International Journal of Nanomedicine

    Article Title: Synthesis of a hemin-containing copolymer as a novel immunostimulator that induces IFN-gamma production

    doi: 10.2147/IJN.S166259

    Figure Lengend Snippet: The evaluation of the immunostimulatory effects of NIPAM-hemin. Abbreviations: NIPAM, N -isopropylacrylamide; hemin, ferriprotoporphyrin IX chloride; IFN-γ, interferon-γ; IL, interleukin; NK, natural killer; PBMCs, peripheral blood mononuclear cells; ELISA, enzyme-linked immunosorbent assay; TLRs, toll-like receptors; SEAP, secreted embryonic alkaline phosphatase.

    Article Snippet: Stimulation of human PBMCs with NIPAM-hemin Commercially available frozen PBMCs were purchased from Cellular Technology Limited (Shaker Heights, OH, USA).

    Techniques: Enzyme-linked Immunosorbent Assay

    NIPAM-hemin induced the production of IFN-γ and IL-6, and had a lesser effect on the induction of IL-1β, in PBMCs. PBMCs were stimulated with NIPAM-hemin, hemin, NIPAM, or poly-NIPAM (MW=66,400). The levels of ( A ) IFN-γ, ( B ) IL-6, and ( C ) IL-1β were determined after 48 h. The concentration of each stimulant was 500 μg/mL. Hemin was dissolved in DMSO, while the other compounds were dissolved in sterilized deionized water. Notes: Data are expressed as the mean±SD (n=3). * P

    Journal: International Journal of Nanomedicine

    Article Title: Synthesis of a hemin-containing copolymer as a novel immunostimulator that induces IFN-gamma production

    doi: 10.2147/IJN.S166259

    Figure Lengend Snippet: NIPAM-hemin induced the production of IFN-γ and IL-6, and had a lesser effect on the induction of IL-1β, in PBMCs. PBMCs were stimulated with NIPAM-hemin, hemin, NIPAM, or poly-NIPAM (MW=66,400). The levels of ( A ) IFN-γ, ( B ) IL-6, and ( C ) IL-1β were determined after 48 h. The concentration of each stimulant was 500 μg/mL. Hemin was dissolved in DMSO, while the other compounds were dissolved in sterilized deionized water. Notes: Data are expressed as the mean±SD (n=3). * P

    Article Snippet: Stimulation of human PBMCs with NIPAM-hemin Commercially available frozen PBMCs were purchased from Cellular Technology Limited (Shaker Heights, OH, USA).

    Techniques: Concentration Assay

    Histone deacetylase (HDAC) inhibitors modulate proinflammatory immune response to Candida albicans. (a–c) Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats provided by Sanquin Blood Bank, Nijmegen, the Netherlands. Cells

    Journal: Clinical and Experimental Immunology

    Article Title: HDAC inhibitors modulate innate immune responses to micro‐organisms relevant to chronic mucocutaneous candidiasis

    doi: 10.1111/cei.13192

    Figure Lengend Snippet: Histone deacetylase (HDAC) inhibitors modulate proinflammatory immune response to Candida albicans. (a–c) Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats provided by Sanquin Blood Bank, Nijmegen, the Netherlands. Cells

    Article Snippet: Human PBMCs were isolated from healthy volunteers (Sanquin Blood Bank, Nijmegen, the Netherlands) by density‐gradient centrifugation over Ficoll‐Paque (GE Healthcare, Chicago, IL, USA), as described previously [ ].

    Techniques: Histone Deacetylase Assay, Isolation

    CD40L expressed by recombinant ALVAC virus enhances specific antiviral CTL activity. CD4 + T cell-containing or -depleted PBMCs from four HIV-1-infected individuals and three HIV-1-uninfected individuals were cocultured with autologous MDDCs that were either pulsed or not with HLA-restricted epitopes of HIV-1 proteins (for HIV-1 infected individuals) or EBV proteins (for HIV-1-uninfected individuals). MDDCs were previously infected or not (medium) with parental ALVAC II or ALVAC recombinant vA3131-2 expressing human CD40L at an MOI of 10 for 48 h. On day 10, specific CTL activity was assessed by intracellular flow cytometric analysis of IFN-γproducing CD8 + T cells and 51 Cr release assay. (A) Representative intracellular IFN-γ flow cytometric data obtained from HIV-1-positive participant #1. (B) Summary data from intracellular IFN-γ flow cytometry of HIV-1-positive participant #2–#4 are graphically depicted. Open bars represent CD4 + T cell-containing condition and dark bar represent CD4 + T cell -depleted conditions. (C) A representative 51 Cr release assay result from HIV-1-positive participant #1 is shown. Similar results were obtained with HIV-1-positive participant #2 (data not shown). (D) Summary data from intracellular IFN-γ flow cytometric analysis of EBV-positive participant #5–#7 are graphically depicted. Open bars represent CD4 + T cell -containing condition and dark bars represent CD4 + T cell -depleted conditions. (E) A representative 51 Cr release assay result from EBV-positive participant #6 is shown. Similar results were obtained with EBV-positive participant #5 (data not shown). DC, MDDCs not pulsed with peptide; DCp, MDDCs pulsed with peptide; vA3131-2/DCp, vA3131-2-infected MDDCs pulsed with peptide; ALVAC/DCp, parental ALVAC II-infected MDDCs pulsed with peptide.

    Journal: Vaccine

    Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8+ T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

    doi: 10.1016/j.vaccine.2008.05.018

    Figure Lengend Snippet: CD40L expressed by recombinant ALVAC virus enhances specific antiviral CTL activity. CD4 + T cell-containing or -depleted PBMCs from four HIV-1-infected individuals and three HIV-1-uninfected individuals were cocultured with autologous MDDCs that were either pulsed or not with HLA-restricted epitopes of HIV-1 proteins (for HIV-1 infected individuals) or EBV proteins (for HIV-1-uninfected individuals). MDDCs were previously infected or not (medium) with parental ALVAC II or ALVAC recombinant vA3131-2 expressing human CD40L at an MOI of 10 for 48 h. On day 10, specific CTL activity was assessed by intracellular flow cytometric analysis of IFN-γproducing CD8 + T cells and 51 Cr release assay. (A) Representative intracellular IFN-γ flow cytometric data obtained from HIV-1-positive participant #1. (B) Summary data from intracellular IFN-γ flow cytometry of HIV-1-positive participant #2–#4 are graphically depicted. Open bars represent CD4 + T cell-containing condition and dark bar represent CD4 + T cell -depleted conditions. (C) A representative 51 Cr release assay result from HIV-1-positive participant #1 is shown. Similar results were obtained with HIV-1-positive participant #2 (data not shown). (D) Summary data from intracellular IFN-γ flow cytometric analysis of EBV-positive participant #5–#7 are graphically depicted. Open bars represent CD4 + T cell -containing condition and dark bars represent CD4 + T cell -depleted conditions. (E) A representative 51 Cr release assay result from EBV-positive participant #6 is shown. Similar results were obtained with EBV-positive participant #5 (data not shown). DC, MDDCs not pulsed with peptide; DCp, MDDCs pulsed with peptide; vA3131-2/DCp, vA3131-2-infected MDDCs pulsed with peptide; ALVAC/DCp, parental ALVAC II-infected MDDCs pulsed with peptide.

    Article Snippet: For flow cytometry, human peripheral blood mononuclear cells (PBMCs) were infected with recombinant viruses or ALVAC II at 5 MOI for 24 h and then stained with PE-conjugated anti-mouse or anti-human CD40L (BD Biosciences, Mississauga, ON, Canada).

    Techniques: Recombinant, CTL Assay, Activity Assay, Infection, Expressing, Flow Cytometry, Release Assay, Cytometry

    Expression of murine CD40L by recombinant canarypox vector. Recombinant canarypox viruses vCPmCD40L and vCPmSP-D-CD40L were generated as described in Materials and Methods. (A) RT-PCR. CEF cells were infected by vCPmCD40L, vCPmSP-D-CD40L or its parental control ALVAC II at 5 MOI for 3 days. Total RNA was isolated from infected CEF cells using Trizol Reagent and subjected to RT-PCR with specific primers amplifying the coding sequence of murine CD40L or SP-D-CD40L. The identity of the amplified product was further verified by DNA sequencing (not shown). (B) Flow cytometry. Human PBMCs were infected by vCPmCD40L, vCPmSP-D-CD40L or ALVAC II at 5 MOI for 24 hrs and stained with PE-labeled anti-mouse CD40L mAb. (C) Western blot. Hela cells were infected by vCPmCD40L, vCPmSP-D-CD40L, or ALVAC II at 10 MOI for 24–48hrs. Cell lysates or magnetic beads incubated with supernatant of infected cells were subjected to Western blot and detected with a goat anti-mouse CD40L or anti-mouse SP-D antibody. As expected, the membrane CD40L was about 35–40kD and soluble multimeric form of CD40L was about 70kD.

    Journal: Vaccine

    Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8+ T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

    doi: 10.1016/j.vaccine.2008.05.018

    Figure Lengend Snippet: Expression of murine CD40L by recombinant canarypox vector. Recombinant canarypox viruses vCPmCD40L and vCPmSP-D-CD40L were generated as described in Materials and Methods. (A) RT-PCR. CEF cells were infected by vCPmCD40L, vCPmSP-D-CD40L or its parental control ALVAC II at 5 MOI for 3 days. Total RNA was isolated from infected CEF cells using Trizol Reagent and subjected to RT-PCR with specific primers amplifying the coding sequence of murine CD40L or SP-D-CD40L. The identity of the amplified product was further verified by DNA sequencing (not shown). (B) Flow cytometry. Human PBMCs were infected by vCPmCD40L, vCPmSP-D-CD40L or ALVAC II at 5 MOI for 24 hrs and stained with PE-labeled anti-mouse CD40L mAb. (C) Western blot. Hela cells were infected by vCPmCD40L, vCPmSP-D-CD40L, or ALVAC II at 10 MOI for 24–48hrs. Cell lysates or magnetic beads incubated with supernatant of infected cells were subjected to Western blot and detected with a goat anti-mouse CD40L or anti-mouse SP-D antibody. As expected, the membrane CD40L was about 35–40kD and soluble multimeric form of CD40L was about 70kD.

    Article Snippet: For flow cytometry, human peripheral blood mononuclear cells (PBMCs) were infected with recombinant viruses or ALVAC II at 5 MOI for 24 h and then stained with PE-conjugated anti-mouse or anti-human CD40L (BD Biosciences, Mississauga, ON, Canada).

    Techniques: Expressing, Recombinant, Plasmid Preparation, Generated, Reverse Transcription Polymerase Chain Reaction, Infection, Isolation, Sequencing, Amplification, DNA Sequencing, Flow Cytometry, Cytometry, Staining, Labeling, Western Blot, Magnetic Beads, Incubation

    The performance of BREM-SC with the public human PBMC CITE-Seq dataset (from 10X Genomics). The UMAP projection of cells are colored by the approximate ground truth ( A ) and BREM-SC clustering results ( B ).

    Journal: Nucleic Acids Research

    Article Title: BREM-SC: a bayesian random effects mixture model for joint clustering single cell multi-omics data

    doi: 10.1093/nar/gkaa314

    Figure Lengend Snippet: The performance of BREM-SC with the public human PBMC CITE-Seq dataset (from 10X Genomics). The UMAP projection of cells are colored by the approximate ground truth ( A ) and BREM-SC clustering results ( B ).

    Article Snippet: Public human peripheral blood mononuclear cells (PBMC) CITE-Seq dataset To assess the performance of BREM-SC, we used a published human PBMC CITE-Seq dataset downloaded from 10X Genomics website ( https://support.10xgenomics.com/single-cell-gene-expression/datasets/3.0.0/pbmc_10k_protein_v3).

    Techniques:

    The performance of BREM-SC for in-house human PBMC CITE-Seq dataset. The UMAP projection of cells are colored by the ground truth ( A ) and BREM-SC clustering results ( B ).

    Journal: Nucleic Acids Research

    Article Title: BREM-SC: a bayesian random effects mixture model for joint clustering single cell multi-omics data

    doi: 10.1093/nar/gkaa314

    Figure Lengend Snippet: The performance of BREM-SC for in-house human PBMC CITE-Seq dataset. The UMAP projection of cells are colored by the ground truth ( A ) and BREM-SC clustering results ( B ).

    Article Snippet: Public human peripheral blood mononuclear cells (PBMC) CITE-Seq dataset To assess the performance of BREM-SC, we used a published human PBMC CITE-Seq dataset downloaded from 10X Genomics website ( https://support.10xgenomics.com/single-cell-gene-expression/datasets/3.0.0/pbmc_10k_protein_v3).

    Techniques:

    (A) Transcriptome analysis of KSHV during de novo infection of human PBMCs. Total RNAs extracted from de novo -infected PBMCs harvested at 4 h, 24 h, 48 h, 72 h, and 120 h postinfection were subjected to cDNA library preparation using a TrueSeq RNA-seq

    Journal: Journal of Virology

    Article Title: Transcriptome Analysis of Kaposi's Sarcoma-Associated Herpesvirus during De Novo Primary Infection of Human B and Endothelial Cells

    doi: 10.1128/JVI.02507-14

    Figure Lengend Snippet: (A) Transcriptome analysis of KSHV during de novo infection of human PBMCs. Total RNAs extracted from de novo -infected PBMCs harvested at 4 h, 24 h, 48 h, 72 h, and 120 h postinfection were subjected to cDNA library preparation using a TrueSeq RNA-seq

    Article Snippet: To determine the profiles of the viral transcripts expressed during the primary infection of B and endothelial cells (natural target cells of KSHV), purified virions were used for the de novo infection of human PBMCs, CD14+ monocytes, and endothelial (TIVE) cells.

    Techniques: Infection, cDNA Library Assay, RNA Sequencing Assay

    Transcriptome analysis of KSHV in de novo -infected PBMCs, TIVE cells, and CD14 + cells. RPKM values, calculated based the number of reads for each gene, were used for analyzing relative expression of KSHV genes as heat maps. Hierarchal clustering of genes

    Journal: Journal of Virology

    Article Title: Transcriptome Analysis of Kaposi's Sarcoma-Associated Herpesvirus during De Novo Primary Infection of Human B and Endothelial Cells

    doi: 10.1128/JVI.02507-14

    Figure Lengend Snippet: Transcriptome analysis of KSHV in de novo -infected PBMCs, TIVE cells, and CD14 + cells. RPKM values, calculated based the number of reads for each gene, were used for analyzing relative expression of KSHV genes as heat maps. Hierarchal clustering of genes

    Article Snippet: To determine the profiles of the viral transcripts expressed during the primary infection of B and endothelial cells (natural target cells of KSHV), purified virions were used for the de novo infection of human PBMCs, CD14+ monocytes, and endothelial (TIVE) cells.

    Techniques: Infection, Expressing

    Viral genes transcribed at 4 hpi and 24 hpi, identified by a nascent RNA capture approach. Human PBMCs infected with KSHV virions were incubated with EdU (alkyne) at 4 hpi and 24 hpi to label the newly transcribing RNA. Total RNA extracted from these

    Journal: Journal of Virology

    Article Title: Transcriptome Analysis of Kaposi's Sarcoma-Associated Herpesvirus during De Novo Primary Infection of Human B and Endothelial Cells

    doi: 10.1128/JVI.02507-14

    Figure Lengend Snippet: Viral genes transcribed at 4 hpi and 24 hpi, identified by a nascent RNA capture approach. Human PBMCs infected with KSHV virions were incubated with EdU (alkyne) at 4 hpi and 24 hpi to label the newly transcribing RNA. Total RNA extracted from these

    Article Snippet: To determine the profiles of the viral transcripts expressed during the primary infection of B and endothelial cells (natural target cells of KSHV), purified virions were used for the de novo infection of human PBMCs, CD14+ monocytes, and endothelial (TIVE) cells.

    Techniques: Infection, Incubation

    KSHV viral transcripts are abundantly present during the primary infection of human PBMCs, CD14+ , and TIVE cells.

    Journal: Journal of Virology

    Article Title: Transcriptome Analysis of Kaposi's Sarcoma-Associated Herpesvirus during De Novo Primary Infection of Human B and Endothelial Cells

    doi: 10.1128/JVI.02507-14

    Figure Lengend Snippet: KSHV viral transcripts are abundantly present during the primary infection of human PBMCs, CD14+ , and TIVE cells.

    Article Snippet: To determine the profiles of the viral transcripts expressed during the primary infection of B and endothelial cells (natural target cells of KSHV), purified virions were used for the de novo infection of human PBMCs, CD14+ monocytes, and endothelial (TIVE) cells.

    Techniques: Infection

    KSHV genome copies exponentially increase after infection. (A) Approximately 8 × 10 7 human PBMCs were infected with KSHV isolated from reactivated TRExBCBL1-RTA, with a multiplicity of infection (MOI) of 10. De novo -infected PBMCs were harvested

    Journal: Journal of Virology

    Article Title: Transcriptome Analysis of Kaposi's Sarcoma-Associated Herpesvirus during De Novo Primary Infection of Human B and Endothelial Cells

    doi: 10.1128/JVI.02507-14

    Figure Lengend Snippet: KSHV genome copies exponentially increase after infection. (A) Approximately 8 × 10 7 human PBMCs were infected with KSHV isolated from reactivated TRExBCBL1-RTA, with a multiplicity of infection (MOI) of 10. De novo -infected PBMCs were harvested

    Article Snippet: To determine the profiles of the viral transcripts expressed during the primary infection of B and endothelial cells (natural target cells of KSHV), purified virions were used for the de novo infection of human PBMCs, CD14+ monocytes, and endothelial (TIVE) cells.

    Techniques: Infection, Isolation