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  • 98
    Millipore pbmc pbmc
    Pbmc Pbmc, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 98 stars, based on 55 article reviews
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    92
    AllCells LLC pbmcs
    Inference and effect of barcode multiplets in single-cell ATAC-seq data. (a) Default t-SNE depiction of public <t>scATAC-seq</t> <t>PBMC</t> 5k dataset. Colors represent cluster annotations from the automated CellRanger output. (b) Quantification of barcodes affected by barcode multiplets for the same dataset (identified by bap). (c) Depiction of two multiplets each composed of 9 oligonucleotide barcodes. Barcodes in each multiplet share a long common subsequence, denoted in black. (d) Visualization of two barcode multiplets from (c) in t-SNE coordinates. (e) Visualization of all implicated barcode multiplets from this dataset. The zoomed panel shows a small group of cells affected by five multiplets, indicated by color. (f) Empirical distribution of the mean restricted longest common subsequence (rLCS) per multiplet. A cutoff of 6 was used to determine either of the two classes of barcode multiplets. (g) Percent difference of the mean log2 fragments between pairs of barcodes within a multiplet. The reported p-value is from a two-sided Kolmogorov–Smirnov test. Boxplots: center line, median; box limits, first and third quartiles; whiskers, 1.5x interquartile range. (h) Overall rates of barcode multiplets from additional scATAC-seq data comparing v1.0 and v1.1 (NextGEM) chip designs.
    Pbmcs, supplied by AllCells LLC, used in various techniques. Bioz Stars score: 92/100, based on 153 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson pbmc
    Proliferation of lymphocytes in <t>LNs</t> and <t>PBMCs</t> as measured by flow cytometry. (A) Ki-67 immunostaining of lymphocytes isolated from LNs of healthy animals on day −18 preexposure ( n = 2) and from LNs of surviving animals ( n = 4) on days 9 and 22 p.i. (B to D) Ki-67 immunostaining of CD4 + T cells (B), CD8 + T cells (C), and CD20 + B cells (D) in PBMCs in treated and untreated survivors versus moribund NHPs. Abbreviations: LN, lymph node; NHP, nonhuman primate; PBMCs, peripheral blood mononuclear cells; p.i., postinfection.
    Pbmc, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 9183 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 9183 article reviews
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    95
    Miltenyi Biotec pbmcs
    NK cells are reduced with shifting distribution of CD3 − <t>CD56</t> dim and CD3 − CD56 bright subsets in patients with chronic HCV infection. <t>PBMCs</t> from 29 HCV patients, 9 SVR individuals, and 8 HS were analyzed by flow cytometry for the frequencies of different subsets of NK cells. The cells were immunostained with PE-CD3 and PerCP-CD56; the gating strategies are described in Materials and Methods. (A) Representative dot plots showing the gating strategy for CD3 − CD56 + NK cells and CD3 − CD56 bright and CD3 − CD56 dim subsets in patients with chronic HCV infection, individuals following treatment with SVR, and HS. Percentage of cell frequency in the gated area is shown in the dot plots. (B) Summary data showing the percentages of CD3 − CD56 + NK cells, CD3 − CD56 bright and CD3 − CD56 dim subsets in the lymphocyte populations (upper panel) and in the NK cell populations (lower panel). Each dot represents one individual, and horizontal bars represent mean values. *, P
    Pbmcs, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 95/100, based on 27366 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    STEMCELL Technologies Inc pbmc
    The allogeneic proliferative response entails a substantial non-T cell bystander component. Panel A. representative proliferative reaction by CFSE analysis. CFSE labeled allogeneic responder <t>PBMC</t> (300,000 cells/well) were cultured with <t>CD3/56</t> depleted PBMC for 6 days. Lymphocyte gate was determined by forward and side scatter. CFSE dye dilution was analyzed on responder alone (left panels) vs. responder and stimulator (right panels) gating on CD4, CD8 and CD4-/CD8- cell fractions. Reactions for 4 separate stimulators (represented on x -axis) and responders (represented in legend) are shown in panels B–E , representing CD4 ( panel B ), CD8 ( panel C ), and CD4-/CD8- ( panel D ) proliferation by CFSE dye dilution analysis, or bulk proliferation by 3 H thymidine incorporation ( panel E ).
    Pbmc, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 94/100, based on 2021 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Sysmex Corporation pbmcs
    The allogeneic proliferative response entails a substantial non-T cell bystander component. Panel A. representative proliferative reaction by CFSE analysis. CFSE labeled allogeneic responder <t>PBMC</t> (300,000 cells/well) were cultured with <t>CD3/56</t> depleted PBMC for 6 days. Lymphocyte gate was determined by forward and side scatter. CFSE dye dilution was analyzed on responder alone (left panels) vs. responder and stimulator (right panels) gating on CD4, CD8 and CD4-/CD8- cell fractions. Reactions for 4 separate stimulators (represented on x -axis) and responders (represented in legend) are shown in panels B–E , representing CD4 ( panel B ), CD8 ( panel C ), and CD4-/CD8- ( panel D ) proliferation by CFSE dye dilution analysis, or bulk proliferation by 3 H thymidine incorporation ( panel E ).
    Pbmcs, supplied by Sysmex Corporation, used in various techniques. Bioz Stars score: 93/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    3H Biomedical pbmc
    Expression of different uPAR splice variant mRNAs in different tissues/cell types normalised to total uPAR . A series of real-time PCR assays was used to measure the expression of different splice variants of uPAR in an extended panel of tissues/cell types (lung, brain, HASM, undifferentiated HBEC, BEAS2B, <t>PMN,</t> <t>PBMC,</t> THP1). Expression of each variant is shown as mean + SEM of three PCR replicates, for two donors or biological replicates as appropriate. Data are shown as 2 -ΔCt normalised to total uPAR and relative to a suitable plasmid control (designated 100%). (A) total classical uPAR (exon 7), (B) classical uPAR exon 6 deletion, (C) classical uPAR exons 5+6 deletion, (D) classical uPAR exon 3 deletion, (E) total alternative uPAR (exon7b), (F) alternative uPAR exon 4+5 deletion. Classical uPAR exon 5 and 4+5 deletions and alternative uPAR exon 5 deletion were not detected.
    Pbmc, supplied by 3H Biomedical, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pfizer Inc pbmc
    Expression of different uPAR splice variant mRNAs in different tissues/cell types normalised to total uPAR . A series of real-time PCR assays was used to measure the expression of different splice variants of uPAR in an extended panel of tissues/cell types (lung, brain, HASM, undifferentiated HBEC, BEAS2B, <t>PMN,</t> <t>PBMC,</t> THP1). Expression of each variant is shown as mean + SEM of three PCR replicates, for two donors or biological replicates as appropriate. Data are shown as 2 -ΔCt normalised to total uPAR and relative to a suitable plasmid control (designated 100%). (A) total classical uPAR (exon 7), (B) classical uPAR exon 6 deletion, (C) classical uPAR exons 5+6 deletion, (D) classical uPAR exon 3 deletion, (E) total alternative uPAR (exon7b), (F) alternative uPAR exon 4+5 deletion. Classical uPAR exon 5 and 4+5 deletions and alternative uPAR exon 5 deletion were not detected.
    Pbmc, supplied by Pfizer Inc, used in various techniques. Bioz Stars score: 92/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Astarte Biologics pbmcs
    Expression of different uPAR splice variant mRNAs in different tissues/cell types normalised to total uPAR . A series of real-time PCR assays was used to measure the expression of different splice variants of uPAR in an extended panel of tissues/cell types (lung, brain, HASM, undifferentiated HBEC, BEAS2B, <t>PMN,</t> <t>PBMC,</t> THP1). Expression of each variant is shown as mean + SEM of three PCR replicates, for two donors or biological replicates as appropriate. Data are shown as 2 -ΔCt normalised to total uPAR and relative to a suitable plasmid control (designated 100%). (A) total classical uPAR (exon 7), (B) classical uPAR exon 6 deletion, (C) classical uPAR exons 5+6 deletion, (D) classical uPAR exon 3 deletion, (E) total alternative uPAR (exon7b), (F) alternative uPAR exon 4+5 deletion. Classical uPAR exon 5 and 4+5 deletions and alternative uPAR exon 5 deletion were not detected.
    Pbmcs, supplied by Astarte Biologics, 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
    Cytoskeleton Inc pbmcs
    Analysis of expression of activation surface markers in <t>PBMCs</t> stimulated with A. hydrophila ATCC ® 7966 TM <t>OMVs.</t> PBMCs from healthy donor were co-cultured with OMVs from A. hydrophila . Activation was measured by flow cytometry using mAbs against surface molecules (CD69, CD86). Monocyte (CD91+) and lymphocytes (CD3+, CD19+) were gated to analyze individually the expression of surface molecules. ∗ P
    Pbmcs, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Focus Biomolecules pbmc
    Analysis of expression of activation surface markers in <t>PBMCs</t> stimulated with A. hydrophila ATCC ® 7966 TM <t>OMVs.</t> PBMCs from healthy donor were co-cultured with OMVs from A. hydrophila . Activation was measured by flow cytometry using mAbs against surface molecules (CD69, CD86). Monocyte (CD91+) and lymphocytes (CD3+, CD19+) were gated to analyze individually the expression of surface molecules. ∗ P
    Pbmc, supplied by Focus Biomolecules, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GraphPad Prism Inc pbmc
    Analysis of expression of activation surface markers in <t>PBMCs</t> stimulated with A. hydrophila ATCC ® 7966 TM <t>OMVs.</t> PBMCs from healthy donor were co-cultured with OMVs from A. hydrophila . Activation was measured by flow cytometry using mAbs against surface molecules (CD69, CD86). Monocyte (CD91+) and lymphocytes (CD3+, CD19+) were gated to analyze individually the expression of surface molecules. ∗ P
    Pbmc, supplied by GraphPad Prism Inc, used in various techniques. Bioz Stars score: 92/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega pbmc
    Analysis of expression of activation surface markers in <t>PBMCs</t> stimulated with A. hydrophila ATCC ® 7966 TM <t>OMVs.</t> PBMCs from healthy donor were co-cultured with OMVs from A. hydrophila . Activation was measured by flow cytometry using mAbs against surface molecules (CD69, CD86). Monocyte (CD91+) and lymphocytes (CD3+, CD19+) were gated to analyze individually the expression of surface molecules. ∗ P
    Pbmc, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 280 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher pbmcs
    Identification of a peptide ligand with the ability to stimulate expression of the latent form of transforming growth factor-β [latency-associated peptide (LAP) (TGF-β 1 )] by primary <t>CD4</t> + T cells. (a) Peripheral blood mononuclear cells
    Pbmcs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 31889 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    10X Genomics pbmcs
    Biologically meaningful DE results for the <t>10x</t> Genomics <t>PBMC</t> dataset. a Scatterplot of the first two t-SNE dimensions obtained from the first ten principal components. Cells are color-coded by clusters found using the SEURAT graph-based clustering method on the first ten principal components. Pseudo-color images on the right display normalized enrichment scores after gene set enrichment analysis for cell types related to CD4+ T cells (see “ Methods ”), for clustering based on b the first ten principal components and c W from ZINB-WaVE with K =20. For dimensionality reduction, ZINB-WaVE was fitted with X = 1 n , V = 1 J , K =20 for W (based on the Akaike information criterion), common dispersion, and ε =10 12 . To compute the weights for differential expression analysis, ZINB-WaVE was fitted with intercept and cell-type covariate in X , V = 1 J , K =0 for W , common dispersion, and ε =10 12 . Normalized enrichment scores for more cell types are shown in Additional file 1 : Figure S17. PCA principal component analysis
    Pbmcs, supplied by 10X Genomics, used in various techniques. Bioz Stars score: 91/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pbmcs  (4Gene)
    92
    4Gene pbmcs
    <t>C(−280)NFAT-binding</t> activity does not depend on AP-1 cooperativity and has a higher affinity than NFAT sites with AP-1 cooperativity. a, NFAT binding to C(−280)NFAT is independent of AP-1. AP-1 sequence failed to compete for binding by C(−280)NFAT in EMSA using human <t>PBMC</t> extracts and vice versa. C(−280)NFAT failed to compete against AP-1 binding. *C-NFAT probe, C(−280)NFAT; *AP-1 probe, AP-1 oligonucleotide. b, NFAT-binding complex does not contain AP-1 components. Abs to Jun-B or c-Fos when incubated with PBMC extracts in EMSAs do not alter binding to the C(−280)NFAT probe. c, NFAT DNA-binding kinetic analysis. NFAT binding to C(−280)NFAT is more stable than to NFAT:AP-1 sites from the human and murine IL-2 promoter in PBMC extracts. Excess competitor for the indicated sites was added and the duration of binding was measured by EMSA. Right panel, The absorbance (OD) of the binding complex after addition of excess competitor. d, NFAT promoter sequence comparison. C(−280)NFAT sequence has four adenosine bases (shadowed) in common when aligned with NFAT IL-2 promoter sequences. Underlined adenosine bases are critical for DNA binding as determined by EMSA ( e ). Sp = species; h = human; m = mouse; D = A,T; N = A,C,T,G; X = A,T,C; a = noncoding sequence; b = EMSA competitor. e, Mutation analysis of C(−280)NFAT to identify nucleotides important for DNA binding. Single-base pair substitutions (C↔A and G↔T) across the C(−280)NFAT sequence were introduced into the indicated nucleotide positions and the corresponding double-stranded oligonucleotides containing mutations were used as competitor sequence in EMSA.
    Pbmcs, supplied by 4Gene, 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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    92
    HiMedia Laboratories pbmcs
    <t>C(−280)NFAT-binding</t> activity does not depend on AP-1 cooperativity and has a higher affinity than NFAT sites with AP-1 cooperativity. a, NFAT binding to C(−280)NFAT is independent of AP-1. AP-1 sequence failed to compete for binding by C(−280)NFAT in EMSA using human <t>PBMC</t> extracts and vice versa. C(−280)NFAT failed to compete against AP-1 binding. *C-NFAT probe, C(−280)NFAT; *AP-1 probe, AP-1 oligonucleotide. b, NFAT-binding complex does not contain AP-1 components. Abs to Jun-B or c-Fos when incubated with PBMC extracts in EMSAs do not alter binding to the C(−280)NFAT probe. c, NFAT DNA-binding kinetic analysis. NFAT binding to C(−280)NFAT is more stable than to NFAT:AP-1 sites from the human and murine IL-2 promoter in PBMC extracts. Excess competitor for the indicated sites was added and the duration of binding was measured by EMSA. Right panel, The absorbance (OD) of the binding complex after addition of excess competitor. d, NFAT promoter sequence comparison. C(−280)NFAT sequence has four adenosine bases (shadowed) in common when aligned with NFAT IL-2 promoter sequences. Underlined adenosine bases are critical for DNA binding as determined by EMSA ( e ). Sp = species; h = human; m = mouse; D = A,T; N = A,C,T,G; X = A,T,C; a = noncoding sequence; b = EMSA competitor. e, Mutation analysis of C(−280)NFAT to identify nucleotides important for DNA binding. Single-base pair substitutions (C↔A and G↔T) across the C(−280)NFAT sequence were introduced into the indicated nucleotide positions and the corresponding double-stranded oligonucleotides containing mutations were used as competitor sequence in EMSA.
    Pbmcs, supplied by HiMedia Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 101 article reviews
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    92
    Sanquin pbmc
    Spontaneously arising survivin specific T cells in patients. A ) Direct ex vivo tetramer staining of T cells derived from HNSCC patients. The patients (1 thru 5) were all <t>HLA-A2</t> positive and the CD8 positive T cells were stained with HLA-A2 tetramers containing either the survivin 5 (light grey bars), survivin 96 (dark grey bars) or the modified survivin 96M (black bars) epitope. The percentage tetramer positive T cells in incidated on the Y-axis. B ) <t>PBMC</t> from HNSCC patients were stained with HLA-A2 tetramers containing the survivin 5 epitope and subsequently sorted by flow cytometry. Cells were plated out at a concentration of 0.2 cells per well and stimulated with feeder mix weekly. The left hand panel shows the forward scatter (X-axis; FSC) and side scatter (Y-axis; SSC) of the cells after four weeks of stimulation with feeder mix. A boxed live gate is indicated. The right hand panel shows tetramer staining of the cells in the live gates. Survivin 5 tetramer labeled with either APC (X-axis) or PE (Y-axis) were used to stain the T cells. The percentage of double tetramer positive cells is shown in the upper right quadrant. C ) Draining lymph nodes were obtained from a patient with locally advanced breast cancer, who had received neo-adjuvant chemotherapy before surgery. The spontaneously arising T cells were tested in an ELIspot assay against a number of different HLA-A2 binding peptides representing CTL epitopes; Survivin(95–104), CEA(571–579), Her-2/Neu(369–377) and Her-2/Neu(654–662) respectively and HPV16E7-(11–20) peptide as a negative control. Indicated are the number of spots per 100.000 T cells.
    Pbmc, supplied by Sanquin, used in various techniques. Bioz Stars score: 92/100, based on 222 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pbmc  (ZenBio)
    90
    ZenBio pbmc
    EphA3 co-stains with macrophage/leukocyte markers A. Immunofluorescent staining of CD68, GFAP and Von Willebrand factor in a GBM specimen. B. Immunofluorescent staining of EphA3 (red) and CD31, GFAP, CD68, CD163, and CD206 on consecutive frozen sections of BTCOE4443 human GBM specimen. Nuclei are stained with DAPI (blue). Selected areas were magnified (last column on the right). C. Confocal immunofluorescent staining of EphA3 and CD68 in a GBM specimen using stacked 2D images. D, E. -stacked 2D images, and F. Confocal microscopy of co-staining of EphA3 and CD45 in GBM specimens. G. <t>CD4+</t> T cells do not express the EphA3 receptor. Western blot analysis of peripheral blood mononuclear cells <t>(PBMC),</t> CD4+ and CD4+ activated T cells were probed for EphA3 immunoreactivity.
    Pbmc, supplied by ZenBio, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    BioreclamationIVT pbmcs
    EphA3 co-stains with macrophage/leukocyte markers A. Immunofluorescent staining of CD68, GFAP and Von Willebrand factor in a GBM specimen. B. Immunofluorescent staining of EphA3 (red) and CD31, GFAP, CD68, CD163, and CD206 on consecutive frozen sections of BTCOE4443 human GBM specimen. Nuclei are stained with DAPI (blue). Selected areas were magnified (last column on the right). C. Confocal immunofluorescent staining of EphA3 and CD68 in a GBM specimen using stacked 2D images. D, E. -stacked 2D images, and F. Confocal microscopy of co-staining of EphA3 and CD45 in GBM specimens. G. <t>CD4+</t> T cells do not express the EphA3 receptor. Western blot analysis of peripheral blood mononuclear cells <t>(PBMC),</t> CD4+ and CD4+ activated T cells were probed for EphA3 immunoreactivity.
    Pbmcs, supplied by BioreclamationIVT, used in various techniques. Bioz Stars score: 92/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cellular Technology Ltd pbmc
    CD8 cells respond to CEF peptides ( A ), and CD4 cells to mumps antigen ( B ): these responses seen ex vivo are preserved after freeze thawing ( C D ). To determine T cell subsets responding to the CEF and mumps antigens, magnetic affinity bead-based separation was performed depleting more than 95% of CD4 or CD8 T cells, as specified. The <t>PBMC</t> and cell fractions were stimulated with CEF peptides ( A ) or mumps antigen ( B ). For each condition, triplicate wells were tested with the mean and standard deviation shown. The data shown are from one of two experiments performed with similar results. Freshly isolated, <t>non-cryopreserved</t> PBMC ( ex vivo ) and cryopreserved PBMC that were tested directly without resting following thawing (fresh) were tested in an IFN-γ ELISPOT assay against CEF peptides ( C ) and mumps antigen ( D ). Data points obtained for individual donors are connected with a line. Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or freeze-thawed, the medium background was less than 10 spots per well. Nonparametric Wilcoxon signed-rank test was used to compare matched ex vivo vs . fresh responses with a p -value ≤ 0.05 being considered significant.
    Pbmc, supplied by Cellular Technology Ltd, used in various techniques. Bioz Stars score: 93/100, based on 177 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbmc/product/Cellular Technology Ltd
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    92
    Ficoll-Paque Pharmacia pbmcs
    Semiquantitative <t>PCR</t> analysis to evaluate differences in proviral load. (A) PCR to detect BLV pol in <t>PBMC</t> from treated rabbits. Rabbit PBMC were harvested at 1, 4, and 10 months postinoculation and subjected to PCR with BLV pol primers KB560 and KB561 (10 × 10 4 PBMC) or β- actin to control for sample variation (2 × 10 4 PBMC). Each panel is labeled with month of sample harvest. Lanes are labeled with the source of PBMC DNA by rabbit number and treatment, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). In lower panels, the corresponding rabbit samples are designated by the matching parallel black lines. Positions of BLV pol amplicon (591 bp) and β- actin amplicon (594 bp) are designated. *, not determined (ND) because sample was not available; †, not determined because the animal died before harvest. (B) PCR standard curve. PBMC lysate harvested at 1 month postinoculation from BLV rabbit 44-10 was serially diluted in a range of 5 × 10 2 to 5 × 10 4 PBMC and subjected to PCR with BLV pol primers KB560 and KB561. Each lane is labeled with the number of cells used for PCR amplification, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). The arrow on the left indicates the position of BLV pol amplicon (591 bp), and the lines on the right indicate positions of 676- and 571-bp DNA size markers.
    Pbmcs, supplied by Ficoll-Paque Pharmacia, used in various techniques. Bioz Stars score: 92/100, based on 338 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Hemacare pbmcs
    Semiquantitative <t>PCR</t> analysis to evaluate differences in proviral load. (A) PCR to detect BLV pol in <t>PBMC</t> from treated rabbits. Rabbit PBMC were harvested at 1, 4, and 10 months postinoculation and subjected to PCR with BLV pol primers KB560 and KB561 (10 × 10 4 PBMC) or β- actin to control for sample variation (2 × 10 4 PBMC). Each panel is labeled with month of sample harvest. Lanes are labeled with the source of PBMC DNA by rabbit number and treatment, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). In lower panels, the corresponding rabbit samples are designated by the matching parallel black lines. Positions of BLV pol amplicon (591 bp) and β- actin amplicon (594 bp) are designated. *, not determined (ND) because sample was not available; †, not determined because the animal died before harvest. (B) PCR standard curve. PBMC lysate harvested at 1 month postinoculation from BLV rabbit 44-10 was serially diluted in a range of 5 × 10 2 to 5 × 10 4 PBMC and subjected to PCR with BLV pol primers KB560 and KB561. Each lane is labeled with the number of cells used for PCR amplification, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). The arrow on the left indicates the position of BLV pol amplicon (591 bp), and the lines on the right indicate positions of 676- and 571-bp DNA size markers.
    Pbmcs, supplied by Hemacare, used in various techniques. Bioz Stars score: 90/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pbmcs  (Roche)
    94
    Roche pbmcs
    Semiquantitative <t>PCR</t> analysis to evaluate differences in proviral load. (A) PCR to detect BLV pol in <t>PBMC</t> from treated rabbits. Rabbit PBMC were harvested at 1, 4, and 10 months postinoculation and subjected to PCR with BLV pol primers KB560 and KB561 (10 × 10 4 PBMC) or β- actin to control for sample variation (2 × 10 4 PBMC). Each panel is labeled with month of sample harvest. Lanes are labeled with the source of PBMC DNA by rabbit number and treatment, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). In lower panels, the corresponding rabbit samples are designated by the matching parallel black lines. Positions of BLV pol amplicon (591 bp) and β- actin amplicon (594 bp) are designated. *, not determined (ND) because sample was not available; †, not determined because the animal died before harvest. (B) PCR standard curve. PBMC lysate harvested at 1 month postinoculation from BLV rabbit 44-10 was serially diluted in a range of 5 × 10 2 to 5 × 10 4 PBMC and subjected to PCR with BLV pol primers KB560 and KB561. Each lane is labeled with the number of cells used for PCR amplification, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). The arrow on the left indicates the position of BLV pol amplicon (591 bp), and the lines on the right indicate positions of 676- and 571-bp DNA size markers.
    Pbmcs, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 1790 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SLIT2 LTD pbmcs
    Fractionation of FCM and identification of <t>Slit2.</t> ( A ) FCM was fractionated by centrifugation using 100-kDa filters. <t>PBMCs</t> were cultured for 5 d in the presence or absence of 10% (vol/vol) FCM, 10-fold concentrated 100-kDa retentate (Ret), or 100-kDa flow through (FT). After a 5-d incubation, cells were air-dried, fixed, and stained, and the number of fibrocytes was counted. Compared with PBMCs cultured in the absence of conditioned medium (SFM), FCM and the 100-kDa retentate significantly decreased the number of fibrocytes. Values are mean ± SEM ( n = 4). * P
    Pbmcs, supplied by SLIT2 LTD, used in various techniques. Bioz Stars score: 92/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Inference and effect of barcode multiplets in single-cell ATAC-seq data. (a) Default t-SNE depiction of public scATAC-seq PBMC 5k dataset. Colors represent cluster annotations from the automated CellRanger output. (b) Quantification of barcodes affected by barcode multiplets for the same dataset (identified by bap). (c) Depiction of two multiplets each composed of 9 oligonucleotide barcodes. Barcodes in each multiplet share a long common subsequence, denoted in black. (d) Visualization of two barcode multiplets from (c) in t-SNE coordinates. (e) Visualization of all implicated barcode multiplets from this dataset. The zoomed panel shows a small group of cells affected by five multiplets, indicated by color. (f) Empirical distribution of the mean restricted longest common subsequence (rLCS) per multiplet. A cutoff of 6 was used to determine either of the two classes of barcode multiplets. (g) Percent difference of the mean log2 fragments between pairs of barcodes within a multiplet. The reported p-value is from a two-sided Kolmogorov–Smirnov test. Boxplots: center line, median; box limits, first and third quartiles; whiskers, 1.5x interquartile range. (h) Overall rates of barcode multiplets from additional scATAC-seq data comparing v1.0 and v1.1 (NextGEM) chip designs.

    Journal: bioRxiv

    Article Title: Inference and effects of barcode multiplets in droplet-based single-cell assays

    doi: 10.1101/824003

    Figure Lengend Snippet: Inference and effect of barcode multiplets in single-cell ATAC-seq data. (a) Default t-SNE depiction of public scATAC-seq PBMC 5k dataset. Colors represent cluster annotations from the automated CellRanger output. (b) Quantification of barcodes affected by barcode multiplets for the same dataset (identified by bap). (c) Depiction of two multiplets each composed of 9 oligonucleotide barcodes. Barcodes in each multiplet share a long common subsequence, denoted in black. (d) Visualization of two barcode multiplets from (c) in t-SNE coordinates. (e) Visualization of all implicated barcode multiplets from this dataset. The zoomed panel shows a small group of cells affected by five multiplets, indicated by color. (f) Empirical distribution of the mean restricted longest common subsequence (rLCS) per multiplet. A cutoff of 6 was used to determine either of the two classes of barcode multiplets. (g) Percent difference of the mean log2 fragments between pairs of barcodes within a multiplet. The reported p-value is from a two-sided Kolmogorov–Smirnov test. Boxplots: center line, median; box limits, first and third quartiles; whiskers, 1.5x interquartile range. (h) Overall rates of barcode multiplets from additional scATAC-seq data comparing v1.0 and v1.1 (NextGEM) chip designs.

    Article Snippet: Profiling PBMCs using 10x scATAC-seq For 10x scATAC-seq experiments with PBMCs (PB003F, Allcells), frozen cells were quickly thawed in a 37°C water bath for about 30s and transferred to a 15 mL tube.

    Techniques: Chromatin Immunoprecipitation

    Supporting information for Figure 3 . (a) Quantification of barcodes affected by barcode multiplets for the PBMC dataset generated with this work (“This Study”). (b) Percentage of barcode multiplets identified for different numbers of input barcodes (see Methods ). (c) Visualization of seven additional barcode multiplets from the Public dataset. (d) Proportion of bead pairs occurring in the same chromatin accessibility-defined Louvain cluster compared to a permuted background. Error bars represent standard error of mean over 100 permutations per dataset. (e) Downsampling analysis of the dataset generated in this work (“This Study”). Barcode multiplets were examined at downsampled intervals from 10%-90% by units of 10%. The highlighted sample represents 40% downsampling and corresponds to a median 10,000 fragments detected per barcode. At all downsampled thresholds, we detected 0 pairs that were not present in the 100% sample. (f) Distribution of the restricted longest common subsequence (rLCS) for 1,000,000 randomly-sampled barcode pairs in the 10x barcode universe. A threshold at 6 is drawn for use in other analyses.

    Journal: bioRxiv

    Article Title: Inference and effects of barcode multiplets in droplet-based single-cell assays

    doi: 10.1101/824003

    Figure Lengend Snippet: Supporting information for Figure 3 . (a) Quantification of barcodes affected by barcode multiplets for the PBMC dataset generated with this work (“This Study”). (b) Percentage of barcode multiplets identified for different numbers of input barcodes (see Methods ). (c) Visualization of seven additional barcode multiplets from the Public dataset. (d) Proportion of bead pairs occurring in the same chromatin accessibility-defined Louvain cluster compared to a permuted background. Error bars represent standard error of mean over 100 permutations per dataset. (e) Downsampling analysis of the dataset generated in this work (“This Study”). Barcode multiplets were examined at downsampled intervals from 10%-90% by units of 10%. The highlighted sample represents 40% downsampling and corresponds to a median 10,000 fragments detected per barcode. At all downsampled thresholds, we detected 0 pairs that were not present in the 100% sample. (f) Distribution of the restricted longest common subsequence (rLCS) for 1,000,000 randomly-sampled barcode pairs in the 10x barcode universe. A threshold at 6 is drawn for use in other analyses.

    Article Snippet: Profiling PBMCs using 10x scATAC-seq For 10x scATAC-seq experiments with PBMCs (PB003F, Allcells), frozen cells were quickly thawed in a 37°C water bath for about 30s and transferred to a 15 mL tube.

    Techniques: Generated

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

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.00897-17

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

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

    Techniques: Flow Cytometry, Cytometry, Immunostaining, Isolation

    NK cells are reduced with shifting distribution of CD3 − CD56 dim and CD3 − CD56 bright subsets in patients with chronic HCV infection. PBMCs from 29 HCV patients, 9 SVR individuals, and 8 HS were analyzed by flow cytometry for the frequencies of different subsets of NK cells. The cells were immunostained with PE-CD3 and PerCP-CD56; the gating strategies are described in Materials and Methods. (A) Representative dot plots showing the gating strategy for CD3 − CD56 + NK cells and CD3 − CD56 bright and CD3 − CD56 dim subsets in patients with chronic HCV infection, individuals following treatment with SVR, and HS. Percentage of cell frequency in the gated area is shown in the dot plots. (B) Summary data showing the percentages of CD3 − CD56 + NK cells, CD3 − CD56 bright and CD3 − CD56 dim subsets in the lymphocyte populations (upper panel) and in the NK cell populations (lower panel). Each dot represents one individual, and horizontal bars represent mean values. *, P

    Journal: Journal of Virology

    Article Title: KLRG1 Negatively Regulates Natural Killer Cell Functions through the Akt Pathway in Individuals with Chronic Hepatitis C Virus Infection

    doi: 10.1128/JVI.01515-13

    Figure Lengend Snippet: NK cells are reduced with shifting distribution of CD3 − CD56 dim and CD3 − CD56 bright subsets in patients with chronic HCV infection. PBMCs from 29 HCV patients, 9 SVR individuals, and 8 HS were analyzed by flow cytometry for the frequencies of different subsets of NK cells. The cells were immunostained with PE-CD3 and PerCP-CD56; the gating strategies are described in Materials and Methods. (A) Representative dot plots showing the gating strategy for CD3 − CD56 + NK cells and CD3 − CD56 bright and CD3 − CD56 dim subsets in patients with chronic HCV infection, individuals following treatment with SVR, and HS. Percentage of cell frequency in the gated area is shown in the dot plots. (B) Summary data showing the percentages of CD3 − CD56 + NK cells, CD3 − CD56 bright and CD3 − CD56 dim subsets in the lymphocyte populations (upper panel) and in the NK cell populations (lower panel). Each dot represents one individual, and horizontal bars represent mean values. *, P

    Article Snippet: Where indicated, CD56+ NK cells were further purified from PBMCs by magnetic beads conjugated to anti-CD56 antibody; CD3− CD56+ NKs were also purified from PBMCs by negative selection according to the manufacturer's instructions (purity, > 95%; Miltenyi Biotec Inc., Auburn, CA).

    Techniques: Infection, Flow Cytometry, Cytometry

    KLRG1 is upregulated on NK cells by HCV infection. (A) PBMCs from 24 HCV patients, 9 SVR individuals, and 8 HS were analyzed by flow cytometry for expression of KLRG1 on different subsets of NK cells. Representative dot plots showing the gating strategy for KLRG1 expression on total CD3 − CD56 + NK cells and CD3 − CD56 bright and CD3 − CD56 dim subsets in patients with chronic HCV infection, individuals after treatment with SVR, and HS. Percent cell frequency in the gated area is shown in the dot plots. (B) Summary data showing the percentages of KLRG1 expression in total CD3 − CD56 + NK cells and CD3 − CD56 bright and CD3 − CD56 dim subsets. Each dot represents one individual, and the horizontal bars represent mean values. *, P

    Journal: Journal of Virology

    Article Title: KLRG1 Negatively Regulates Natural Killer Cell Functions through the Akt Pathway in Individuals with Chronic Hepatitis C Virus Infection

    doi: 10.1128/JVI.01515-13

    Figure Lengend Snippet: KLRG1 is upregulated on NK cells by HCV infection. (A) PBMCs from 24 HCV patients, 9 SVR individuals, and 8 HS were analyzed by flow cytometry for expression of KLRG1 on different subsets of NK cells. Representative dot plots showing the gating strategy for KLRG1 expression on total CD3 − CD56 + NK cells and CD3 − CD56 bright and CD3 − CD56 dim subsets in patients with chronic HCV infection, individuals after treatment with SVR, and HS. Percent cell frequency in the gated area is shown in the dot plots. (B) Summary data showing the percentages of KLRG1 expression in total CD3 − CD56 + NK cells and CD3 − CD56 bright and CD3 − CD56 dim subsets. Each dot represents one individual, and the horizontal bars represent mean values. *, P

    Article Snippet: Where indicated, CD56+ NK cells were further purified from PBMCs by magnetic beads conjugated to anti-CD56 antibody; CD3− CD56+ NKs were also purified from PBMCs by negative selection according to the manufacturer's instructions (purity, > 95%; Miltenyi Biotec Inc., Auburn, CA).

    Techniques: Infection, Flow Cytometry, Cytometry, Expressing

    KLRG1 expression is inversely associated with the low levels of IFN-γ production by NK cells in HCV infection. (A) PBMCs from chronically HCV-infected patients and HS were stimulated with rhIL-12 (10 ng/ml) for 18 h and incubated for the last 4 h with brefeldin A (10 μg/ml) to halt cytokine secretion. The cells were immunostained with PE-CD3, PerCP-CD56, APC–IFN-γ, and Alexa Fluor 488-KLRG1, and the IFN-γ production by KLRG1 + and KLRG1 − NK cells was analyzed by flow cytometry. Representative dot plots for IFN-γ production by KLRG1 + and KLRG1 − NK cells, including total CD3 − CD56 + , CD3 − CD56 bright , and CD3 − CD56 dim subsets from an HCV-infected subject and HS. (B) The average percentage of IFN-γ production by the KLRG1 + versus KLRG1 − fraction of total CD3 − CD56 + NK cells, or in the CD3 − CD56 bright and CD3 − CD56 dim subsets. Results are expressed as means ± SD of the percentages of IFN-γ production by NK cells from 24 HCV-infected patients versus 8 HS. *, P

    Journal: Journal of Virology

    Article Title: KLRG1 Negatively Regulates Natural Killer Cell Functions through the Akt Pathway in Individuals with Chronic Hepatitis C Virus Infection

    doi: 10.1128/JVI.01515-13

    Figure Lengend Snippet: KLRG1 expression is inversely associated with the low levels of IFN-γ production by NK cells in HCV infection. (A) PBMCs from chronically HCV-infected patients and HS were stimulated with rhIL-12 (10 ng/ml) for 18 h and incubated for the last 4 h with brefeldin A (10 μg/ml) to halt cytokine secretion. The cells were immunostained with PE-CD3, PerCP-CD56, APC–IFN-γ, and Alexa Fluor 488-KLRG1, and the IFN-γ production by KLRG1 + and KLRG1 − NK cells was analyzed by flow cytometry. Representative dot plots for IFN-γ production by KLRG1 + and KLRG1 − NK cells, including total CD3 − CD56 + , CD3 − CD56 bright , and CD3 − CD56 dim subsets from an HCV-infected subject and HS. (B) The average percentage of IFN-γ production by the KLRG1 + versus KLRG1 − fraction of total CD3 − CD56 + NK cells, or in the CD3 − CD56 bright and CD3 − CD56 dim subsets. Results are expressed as means ± SD of the percentages of IFN-γ production by NK cells from 24 HCV-infected patients versus 8 HS. *, P

    Article Snippet: Where indicated, CD56+ NK cells were further purified from PBMCs by magnetic beads conjugated to anti-CD56 antibody; CD3− CD56+ NKs were also purified from PBMCs by negative selection according to the manufacturer's instructions (purity, > 95%; Miltenyi Biotec Inc., Auburn, CA).

    Techniques: Expressing, Infection, Incubation, Flow Cytometry, Cytometry

    KLRG1 expression is positively associated with apoptosis of NK cells following HCV infection. (A) PBMCs from HCV-infected patients and HS were stimulated with 10 ng/ml rhIL-12 for 18 h, followed by 1 μg/ml brefeldin A 4 h prior to harvest of the cells (to block cytokine secretion). PBMCs were immunostained and then analyzed for Annexin V expression on total CD3 − CD56 + and CD3 − CD56 bright and CD3 − CD56 dim NK cells by flow cytometry. Representative dot plots for Annexin V expression on KLRG1 + and KLRG1 − NK cells, including CD3 − CD56 + and the CD3 − CD56 bright and CD3 − CD56 dim subsets from an HCV-infected patient and HS are shown. (B) Average percentage of Annexin V expression from the KLRG1 + versus KLRG1 − cell fraction of total CD3 − CD56 + NK cells and the CD3 − CD56 bright and CD3 − CD56 dim subsets. Results are expressed as means ± SD of the percentages of Annexin V expression by NK cells from 24 HCV-infected patients versus 8 HS. *, P

    Journal: Journal of Virology

    Article Title: KLRG1 Negatively Regulates Natural Killer Cell Functions through the Akt Pathway in Individuals with Chronic Hepatitis C Virus Infection

    doi: 10.1128/JVI.01515-13

    Figure Lengend Snippet: KLRG1 expression is positively associated with apoptosis of NK cells following HCV infection. (A) PBMCs from HCV-infected patients and HS were stimulated with 10 ng/ml rhIL-12 for 18 h, followed by 1 μg/ml brefeldin A 4 h prior to harvest of the cells (to block cytokine secretion). PBMCs were immunostained and then analyzed for Annexin V expression on total CD3 − CD56 + and CD3 − CD56 bright and CD3 − CD56 dim NK cells by flow cytometry. Representative dot plots for Annexin V expression on KLRG1 + and KLRG1 − NK cells, including CD3 − CD56 + and the CD3 − CD56 bright and CD3 − CD56 dim subsets from an HCV-infected patient and HS are shown. (B) Average percentage of Annexin V expression from the KLRG1 + versus KLRG1 − cell fraction of total CD3 − CD56 + NK cells and the CD3 − CD56 bright and CD3 − CD56 dim subsets. Results are expressed as means ± SD of the percentages of Annexin V expression by NK cells from 24 HCV-infected patients versus 8 HS. *, P

    Article Snippet: Where indicated, CD56+ NK cells were further purified from PBMCs by magnetic beads conjugated to anti-CD56 antibody; CD3− CD56+ NKs were also purified from PBMCs by negative selection according to the manufacturer's instructions (purity, > 95%; Miltenyi Biotec Inc., Auburn, CA).

    Techniques: Expressing, Infection, Blocking Assay, Flow Cytometry, Cytometry

    The allogeneic proliferative response entails a substantial non-T cell bystander component. Panel A. representative proliferative reaction by CFSE analysis. CFSE labeled allogeneic responder PBMC (300,000 cells/well) were cultured with CD3/56 depleted PBMC for 6 days. Lymphocyte gate was determined by forward and side scatter. CFSE dye dilution was analyzed on responder alone (left panels) vs. responder and stimulator (right panels) gating on CD4, CD8 and CD4-/CD8- cell fractions. Reactions for 4 separate stimulators (represented on x -axis) and responders (represented in legend) are shown in panels B–E , representing CD4 ( panel B ), CD8 ( panel C ), and CD4-/CD8- ( panel D ) proliferation by CFSE dye dilution analysis, or bulk proliferation by 3 H thymidine incorporation ( panel E ).

    Journal: Cells

    Article Title: Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

    doi: 10.3390/cells1020127

    Figure Lengend Snippet: The allogeneic proliferative response entails a substantial non-T cell bystander component. Panel A. representative proliferative reaction by CFSE analysis. CFSE labeled allogeneic responder PBMC (300,000 cells/well) were cultured with CD3/56 depleted PBMC for 6 days. Lymphocyte gate was determined by forward and side scatter. CFSE dye dilution was analyzed on responder alone (left panels) vs. responder and stimulator (right panels) gating on CD4, CD8 and CD4-/CD8- cell fractions. Reactions for 4 separate stimulators (represented on x -axis) and responders (represented in legend) are shown in panels B–E , representing CD4 ( panel B ), CD8 ( panel C ), and CD4-/CD8- ( panel D ) proliferation by CFSE dye dilution analysis, or bulk proliferation by 3 H thymidine incorporation ( panel E ).

    Article Snippet: PBMC, CD3- depleted PBMC ( > 97% CD3- cells; RosetteSep CD3 depletion reagent; StemCell Technologies, Vancouver BC, Canada), CD3/56 depleted PBMC ( > 95% CD3/56- cells; RosetteSep reagent), CD4 T cells (negative selection method, RosetteSep reagent), and CD8 T cells (negative selection method using R & D systems, Inc., Minneapolis MN, USA) were freshly prepared from peripheral blood specimens.

    Techniques: Labeling, Cell Culture

    Allo-antigen-specific T cells show dissociated IL-2-IFN-γ production. Allogeneic 20 h cultures were performed as 2-way MLR with 3 × 10 5 PBMC ( panels A–C ), or as a 1-way allogeneic response using CD3/CD56 depleted PBMC stimulators (300,000 cells/well) and CD4 ( panels D–F ), or CD8 ( panels G–I ) cell responders (300,000 cells/well). IL-2 sfu ( panels A, D, G ), IFN-γ sfu ( panels B, E, H ) and proliferation cpm ( panels C, F, I ) are shown. Stimulator identification is shown on the x-axis, while responder identification is shown in the legend. Shaded regions highlight discordance between IL-2 and IFN-γ producing effector function. Stimulator or responder cell alone control cultures resulted in

    Journal: Cells

    Article Title: Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

    doi: 10.3390/cells1020127

    Figure Lengend Snippet: Allo-antigen-specific T cells show dissociated IL-2-IFN-γ production. Allogeneic 20 h cultures were performed as 2-way MLR with 3 × 10 5 PBMC ( panels A–C ), or as a 1-way allogeneic response using CD3/CD56 depleted PBMC stimulators (300,000 cells/well) and CD4 ( panels D–F ), or CD8 ( panels G–I ) cell responders (300,000 cells/well). IL-2 sfu ( panels A, D, G ), IFN-γ sfu ( panels B, E, H ) and proliferation cpm ( panels C, F, I ) are shown. Stimulator identification is shown on the x-axis, while responder identification is shown in the legend. Shaded regions highlight discordance between IL-2 and IFN-γ producing effector function. Stimulator or responder cell alone control cultures resulted in

    Article Snippet: PBMC, CD3- depleted PBMC ( > 97% CD3- cells; RosetteSep CD3 depletion reagent; StemCell Technologies, Vancouver BC, Canada), CD3/56 depleted PBMC ( > 95% CD3/56- cells; RosetteSep reagent), CD4 T cells (negative selection method, RosetteSep reagent), and CD8 T cells (negative selection method using R & D systems, Inc., Minneapolis MN, USA) were freshly prepared from peripheral blood specimens.

    Techniques:

    Expression of different uPAR splice variant mRNAs in different tissues/cell types normalised to total uPAR . A series of real-time PCR assays was used to measure the expression of different splice variants of uPAR in an extended panel of tissues/cell types (lung, brain, HASM, undifferentiated HBEC, BEAS2B, PMN, PBMC, THP1). Expression of each variant is shown as mean + SEM of three PCR replicates, for two donors or biological replicates as appropriate. Data are shown as 2 -ΔCt normalised to total uPAR and relative to a suitable plasmid control (designated 100%). (A) total classical uPAR (exon 7), (B) classical uPAR exon 6 deletion, (C) classical uPAR exons 5+6 deletion, (D) classical uPAR exon 3 deletion, (E) total alternative uPAR (exon7b), (F) alternative uPAR exon 4+5 deletion. Classical uPAR exon 5 and 4+5 deletions and alternative uPAR exon 5 deletion were not detected.

    Journal: BMC Molecular Biology

    Article Title: Characterisation of urokinase plasminogen activator receptor variants in human airway and peripheral cells

    doi: 10.1186/1471-2199-10-75

    Figure Lengend Snippet: Expression of different uPAR splice variant mRNAs in different tissues/cell types normalised to total uPAR . A series of real-time PCR assays was used to measure the expression of different splice variants of uPAR in an extended panel of tissues/cell types (lung, brain, HASM, undifferentiated HBEC, BEAS2B, PMN, PBMC, THP1). Expression of each variant is shown as mean + SEM of three PCR replicates, for two donors or biological replicates as appropriate. Data are shown as 2 -ΔCt normalised to total uPAR and relative to a suitable plasmid control (designated 100%). (A) total classical uPAR (exon 7), (B) classical uPAR exon 6 deletion, (C) classical uPAR exons 5+6 deletion, (D) classical uPAR exon 3 deletion, (E) total alternative uPAR (exon7b), (F) alternative uPAR exon 4+5 deletion. Classical uPAR exon 5 and 4+5 deletions and alternative uPAR exon 5 deletion were not detected.

    Article Snippet: RNA and lysates from PMN and PBMC were also obtained commercially (3 H Biomedical, Uppsala, Sweden).

    Techniques: Expressing, Variant Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Plasmid Preparation

    Expression of uPAR splice variant mRNA in different tissues/cell types . A series of real-time PCR (TaqMan) assays was used to measure the expression of different splice variants of uPAR in an extended panel of tissues/cell types (lung, brain, HASM, undifferentiated HBEC, BEAS2B, PMN, PBMC, THP1). Expression of each variant is shown as mean + SEM of three PCR replicates, for two donors or biological replicates as appropriate. Data are shown as 2 -ΔCt normalised to HPRT1 and relative to a suitable plasmid positive control containing the specific splice variant cDNA (designated 100%). (A) total uPAR, (B) total classical uPAR (exon 7), (C) classical uPAR exon 6 deletion, (D) classical uPAR exons 5+6 deletion, (E) classical uPAR exon 3 deletion, (F) total alternative uPAR (exon7b), (G) alternative uPAR exon 4+5 deletion. Classical uPAR exon 5 and 4+5 deletions and alternative uPAR exon 5 deletion were not detected.

    Journal: BMC Molecular Biology

    Article Title: Characterisation of urokinase plasminogen activator receptor variants in human airway and peripheral cells

    doi: 10.1186/1471-2199-10-75

    Figure Lengend Snippet: Expression of uPAR splice variant mRNA in different tissues/cell types . A series of real-time PCR (TaqMan) assays was used to measure the expression of different splice variants of uPAR in an extended panel of tissues/cell types (lung, brain, HASM, undifferentiated HBEC, BEAS2B, PMN, PBMC, THP1). Expression of each variant is shown as mean + SEM of three PCR replicates, for two donors or biological replicates as appropriate. Data are shown as 2 -ΔCt normalised to HPRT1 and relative to a suitable plasmid positive control containing the specific splice variant cDNA (designated 100%). (A) total uPAR, (B) total classical uPAR (exon 7), (C) classical uPAR exon 6 deletion, (D) classical uPAR exons 5+6 deletion, (E) classical uPAR exon 3 deletion, (F) total alternative uPAR (exon7b), (G) alternative uPAR exon 4+5 deletion. Classical uPAR exon 5 and 4+5 deletions and alternative uPAR exon 5 deletion were not detected.

    Article Snippet: RNA and lysates from PMN and PBMC were also obtained commercially (3 H Biomedical, Uppsala, Sweden).

    Techniques: Expressing, Variant Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Plasmid Preparation, Positive Control

    Expression of uPAR protein in different cell types . Western blotting of cell lysates was performed for an extended panel of cell types (HASM, undifferentiated HBEC, BEAS2B, THP1, PMN, PBMC) plus recombinant uPAR (ruPAR) using domain I specific (A) and domain II specific (B) antibodies to identify different variants. A β-actin antibody was used as a loading control (C). An ELISA assay, with a sensitivity of 30 pg/mL, was used to detect soluble uPAR in the culture supernatant where appropriate (D).

    Journal: BMC Molecular Biology

    Article Title: Characterisation of urokinase plasminogen activator receptor variants in human airway and peripheral cells

    doi: 10.1186/1471-2199-10-75

    Figure Lengend Snippet: Expression of uPAR protein in different cell types . Western blotting of cell lysates was performed for an extended panel of cell types (HASM, undifferentiated HBEC, BEAS2B, THP1, PMN, PBMC) plus recombinant uPAR (ruPAR) using domain I specific (A) and domain II specific (B) antibodies to identify different variants. A β-actin antibody was used as a loading control (C). An ELISA assay, with a sensitivity of 30 pg/mL, was used to detect soluble uPAR in the culture supernatant where appropriate (D).

    Article Snippet: RNA and lysates from PMN and PBMC were also obtained commercially (3 H Biomedical, Uppsala, Sweden).

    Techniques: Expressing, Western Blot, Recombinant, Enzyme-linked Immunosorbent Assay

    Analysis of expression of activation surface markers in PBMCs stimulated with A. hydrophila ATCC ® 7966 TM OMVs. PBMCs from healthy donor were co-cultured with OMVs from A. hydrophila . Activation was measured by flow cytometry using mAbs against surface molecules (CD69, CD86). Monocyte (CD91+) and lymphocytes (CD3+, CD19+) were gated to analyze individually the expression of surface molecules. ∗ P

    Journal: Frontiers in Microbiology

    Article Title: The Outer Membrane Vesicles of Aeromonas hydrophila ATCC® 7966TM: A Proteomic Analysis and Effect on Host Cells

    doi: 10.3389/fmicb.2018.02765

    Figure Lengend Snippet: Analysis of expression of activation surface markers in PBMCs stimulated with A. hydrophila ATCC ® 7966 TM OMVs. PBMCs from healthy donor were co-cultured with OMVs from A. hydrophila . Activation was measured by flow cytometry using mAbs against surface molecules (CD69, CD86). Monocyte (CD91+) and lymphocytes (CD3+, CD19+) were gated to analyze individually the expression of surface molecules. ∗ P

    Article Snippet: Effect of OMVs on PBMCs Cytoskeleton PBMCs were fixed onto cover slips by duplicated and permeabilized with Triton X-100 0.5% in PBS.

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

    Cytokine quantification from PBMCs stimulated with A. hydrophila ATCC ® 7966 TM OMVs. Inflammatory cytokines were determined by CBA cytometric assay at different time points. A. hydrophila OMVs induced high levels of inflammatory cytokines such as IL-8, IL-1β, IL-6, and TNFα. ∗ P

    Journal: Frontiers in Microbiology

    Article Title: The Outer Membrane Vesicles of Aeromonas hydrophila ATCC® 7966TM: A Proteomic Analysis and Effect on Host Cells

    doi: 10.3389/fmicb.2018.02765

    Figure Lengend Snippet: Cytokine quantification from PBMCs stimulated with A. hydrophila ATCC ® 7966 TM OMVs. Inflammatory cytokines were determined by CBA cytometric assay at different time points. A. hydrophila OMVs induced high levels of inflammatory cytokines such as IL-8, IL-1β, IL-6, and TNFα. ∗ P

    Article Snippet: Effect of OMVs on PBMCs Cytoskeleton PBMCs were fixed onto cover slips by duplicated and permeabilized with Triton X-100 0.5% in PBS.

    Techniques: Crocin Bleaching Assay

    Analysis of expression of inhibition surface markers in PBMCs stimulated with A. hydrophila ATCC ® 7966 TM OMVs. PBMCs from healthy donor were co-cultured with OMVs from A. hydrophila . Inhibition was measured by flow cytometry using mAbs against surface molecules (PD-1, PD-L1). Monocyte (CD91+) and lymphocytes (CD3+, CD19+) were gated to analyze individually the expression of surface molecules. FKB were used as control for the expression of surface markers. ∗ P

    Journal: Frontiers in Microbiology

    Article Title: The Outer Membrane Vesicles of Aeromonas hydrophila ATCC® 7966TM: A Proteomic Analysis and Effect on Host Cells

    doi: 10.3389/fmicb.2018.02765

    Figure Lengend Snippet: Analysis of expression of inhibition surface markers in PBMCs stimulated with A. hydrophila ATCC ® 7966 TM OMVs. PBMCs from healthy donor were co-cultured with OMVs from A. hydrophila . Inhibition was measured by flow cytometry using mAbs against surface molecules (PD-1, PD-L1). Monocyte (CD91+) and lymphocytes (CD3+, CD19+) were gated to analyze individually the expression of surface molecules. FKB were used as control for the expression of surface markers. ∗ P

    Article Snippet: Effect of OMVs on PBMCs Cytoskeleton PBMCs were fixed onto cover slips by duplicated and permeabilized with Triton X-100 0.5% in PBS.

    Techniques: Expressing, Inhibition, Cell Culture, Flow Cytometry, Cytometry

    Apoptosis and DNA damage of PBMCs induced by A. hydrophila ATCC ® 7966 TM OMVs. PBMCs from healthy donor were co-cultured with different concentrations of A. hydrophila OMVs during 24 h. Apoptosis and DNA damaged was evaluated by flow cytometry with mAbs anti-PARP and anti-H2AX. CCCP was used as control for apoptosis induction. ∗ P

    Journal: Frontiers in Microbiology

    Article Title: The Outer Membrane Vesicles of Aeromonas hydrophila ATCC® 7966TM: A Proteomic Analysis and Effect on Host Cells

    doi: 10.3389/fmicb.2018.02765

    Figure Lengend Snippet: Apoptosis and DNA damage of PBMCs induced by A. hydrophila ATCC ® 7966 TM OMVs. PBMCs from healthy donor were co-cultured with different concentrations of A. hydrophila OMVs during 24 h. Apoptosis and DNA damaged was evaluated by flow cytometry with mAbs anti-PARP and anti-H2AX. CCCP was used as control for apoptosis induction. ∗ P

    Article Snippet: Effect of OMVs on PBMCs Cytoskeleton PBMCs were fixed onto cover slips by duplicated and permeabilized with Triton X-100 0.5% in PBS.

    Techniques: Cell Culture, Flow Cytometry, Cytometry

    Morphological alterations induced by A. hydrophila ATCC ® 7966 TM OMVs in PBMCs. (A) After 2 h post-stimulation with A. hydrophila OMVs, no morphology changes of the PBMCs were observed compared with PBMCs after 24 h post-stimulation with vesicles, after 24 h post-stimulation conglomerated cells were observed (arrowheads) (B) . Giemsa staining revealed differences in the affinity of the dye to the nucleus and cytoplasm, bigger cells, and vacuolization of the stimulated PBMCs with A. hydrophila OMVs (arrowheads) (C) . Confocal microscopy, showed defective tubulin polymerization in PBMCs stimulated with OMVs (D) . bars = 50 μm.

    Journal: Frontiers in Microbiology

    Article Title: The Outer Membrane Vesicles of Aeromonas hydrophila ATCC® 7966TM: A Proteomic Analysis and Effect on Host Cells

    doi: 10.3389/fmicb.2018.02765

    Figure Lengend Snippet: Morphological alterations induced by A. hydrophila ATCC ® 7966 TM OMVs in PBMCs. (A) After 2 h post-stimulation with A. hydrophila OMVs, no morphology changes of the PBMCs were observed compared with PBMCs after 24 h post-stimulation with vesicles, after 24 h post-stimulation conglomerated cells were observed (arrowheads) (B) . Giemsa staining revealed differences in the affinity of the dye to the nucleus and cytoplasm, bigger cells, and vacuolization of the stimulated PBMCs with A. hydrophila OMVs (arrowheads) (C) . Confocal microscopy, showed defective tubulin polymerization in PBMCs stimulated with OMVs (D) . bars = 50 μm.

    Article Snippet: Effect of OMVs on PBMCs Cytoskeleton PBMCs were fixed onto cover slips by duplicated and permeabilized with Triton X-100 0.5% in PBS.

    Techniques: Staining, Confocal Microscopy

    Identification of a peptide ligand with the ability to stimulate expression of the latent form of transforming growth factor-β [latency-associated peptide (LAP) (TGF-β 1 )] by primary CD4 + T cells. (a) Peripheral blood mononuclear cells

    Journal: Immunology

    Article Title: Induction of latency-associated peptide (transforming growth factor-?1) expression on CD4+ T cells reduces Toll-like receptor 4 ligand-induced tumour necrosis factor-? production in a transforming growth factor-?-dependent manner

    doi: 10.1111/j.1365-2567.2011.03425.x

    Figure Lengend Snippet: Identification of a peptide ligand with the ability to stimulate expression of the latent form of transforming growth factor-β [latency-associated peptide (LAP) (TGF-β 1 )] by primary CD4 + T cells. (a) Peripheral blood mononuclear cells

    Article Snippet: CD4 and CD8 T cells were depleted from PBMCs as described by the manufacturer (Dynal, Oslo, Norway).

    Techniques: Expressing

    The anti-inflammatory property of GPC 81–95 is mediated by CD4 T cells and is transforming growth factor-β 1 (TGF-β 1 )-dependent. (a) Peripheral blood mononuclear cells (PBMCs) were incubated with GPC 81–95 or irrelevant peptide

    Journal: Immunology

    Article Title: Induction of latency-associated peptide (transforming growth factor-?1) expression on CD4+ T cells reduces Toll-like receptor 4 ligand-induced tumour necrosis factor-? production in a transforming growth factor-?-dependent manner

    doi: 10.1111/j.1365-2567.2011.03425.x

    Figure Lengend Snippet: The anti-inflammatory property of GPC 81–95 is mediated by CD4 T cells and is transforming growth factor-β 1 (TGF-β 1 )-dependent. (a) Peripheral blood mononuclear cells (PBMCs) were incubated with GPC 81–95 or irrelevant peptide

    Article Snippet: CD4 and CD8 T cells were depleted from PBMCs as described by the manufacturer (Dynal, Oslo, Norway).

    Techniques: Gel Permeation Chromatography, Incubation

    Biologically meaningful DE results for the 10x Genomics PBMC dataset. a Scatterplot of the first two t-SNE dimensions obtained from the first ten principal components. Cells are color-coded by clusters found using the SEURAT graph-based clustering method on the first ten principal components. Pseudo-color images on the right display normalized enrichment scores after gene set enrichment analysis for cell types related to CD4+ T cells (see “ Methods ”), for clustering based on b the first ten principal components and c W from ZINB-WaVE with K =20. For dimensionality reduction, ZINB-WaVE was fitted with X = 1 n , V = 1 J , K =20 for W (based on the Akaike information criterion), common dispersion, and ε =10 12 . To compute the weights for differential expression analysis, ZINB-WaVE was fitted with intercept and cell-type covariate in X , V = 1 J , K =0 for W , common dispersion, and ε =10 12 . Normalized enrichment scores for more cell types are shown in Additional file 1 : Figure S17. PCA principal component analysis

    Journal: Genome Biology

    Article Title: Observation weights unlock bulk RNA-seq tools for zero inflation and single-cell applications

    doi: 10.1186/s13059-018-1406-4

    Figure Lengend Snippet: Biologically meaningful DE results for the 10x Genomics PBMC dataset. a Scatterplot of the first two t-SNE dimensions obtained from the first ten principal components. Cells are color-coded by clusters found using the SEURAT graph-based clustering method on the first ten principal components. Pseudo-color images on the right display normalized enrichment scores after gene set enrichment analysis for cell types related to CD4+ T cells (see “ Methods ”), for clustering based on b the first ten principal components and c W from ZINB-WaVE with K =20. For dimensionality reduction, ZINB-WaVE was fitted with X = 1 n , V = 1 J , K =20 for W (based on the Akaike information criterion), common dispersion, and ε =10 12 . To compute the weights for differential expression analysis, ZINB-WaVE was fitted with intercept and cell-type covariate in X , V = 1 J , K =0 for W , common dispersion, and ε =10 12 . Normalized enrichment scores for more cell types are shown in Additional file 1 : Figure S17. PCA principal component analysis

    Article Snippet: 10x Genomics PBMC dataset We analyzed a dataset of PBMCs that is freely available from 10x Genomics ( https://support.10xgenomics.com/single-cell-gene-expression/datasets/1.1.0/pbmc3k ) [ ].

    Techniques: Expressing

    Comparison of differential expression methods on simulated scRNA-seq datasets. Differential expression methods are compared based on FDP-TPR curves for data simulated from a 10x Genomics PBMC single-cell RNA-seq dataset ( n =1200). Zoomed versions of the FDP-TPR curves are shown here and full curves are in Additional file 1 : Figure S12. Circles represent working points on a nominal 5% FDR level and are filled if the empirical FDR (i.e., FDP) is below the nominal FDR. 10x Genomics sequencing typically involves high-throughput and massive multiplexing, resulting in very shallow sequencing depths and thus, low counts, making it extremely difficult to identify excess zeros. Unweighted and ZINB-WaVE-weighted EDGER are tied for best performance, followed by ZINB-WaVE-weighted DESEQ2 . In general, bulk RNA-seq methods perform well in this simulation, probably because the extremely high zero abundance in combination with low counts can be reasonably accommodated by the negative binomial distribution. The behavior in the lower half of the curve for NODES is due to a smooth increase in true positives with an identical number of false positives over a range of low FDR cut-offs. FDP false discovery proportion, FDR false discovery rate, PBMC peripheral blood mononuclear cell, TPR true positive rate

    Journal: Genome Biology

    Article Title: Observation weights unlock bulk RNA-seq tools for zero inflation and single-cell applications

    doi: 10.1186/s13059-018-1406-4

    Figure Lengend Snippet: Comparison of differential expression methods on simulated scRNA-seq datasets. Differential expression methods are compared based on FDP-TPR curves for data simulated from a 10x Genomics PBMC single-cell RNA-seq dataset ( n =1200). Zoomed versions of the FDP-TPR curves are shown here and full curves are in Additional file 1 : Figure S12. Circles represent working points on a nominal 5% FDR level and are filled if the empirical FDR (i.e., FDP) is below the nominal FDR. 10x Genomics sequencing typically involves high-throughput and massive multiplexing, resulting in very shallow sequencing depths and thus, low counts, making it extremely difficult to identify excess zeros. Unweighted and ZINB-WaVE-weighted EDGER are tied for best performance, followed by ZINB-WaVE-weighted DESEQ2 . In general, bulk RNA-seq methods perform well in this simulation, probably because the extremely high zero abundance in combination with low counts can be reasonably accommodated by the negative binomial distribution. The behavior in the lower half of the curve for NODES is due to a smooth increase in true positives with an identical number of false positives over a range of low FDR cut-offs. FDP false discovery proportion, FDR false discovery rate, PBMC peripheral blood mononuclear cell, TPR true positive rate

    Article Snippet: 10x Genomics PBMC dataset We analyzed a dataset of PBMCs that is freely available from 10x Genomics ( https://support.10xgenomics.com/single-cell-gene-expression/datasets/1.1.0/pbmc3k ) [ ].

    Techniques: Expressing, RNA Sequencing Assay, Sequencing, High Throughput Screening Assay, Multiplexing

    C(−280)NFAT-binding activity does not depend on AP-1 cooperativity and has a higher affinity than NFAT sites with AP-1 cooperativity. a, NFAT binding to C(−280)NFAT is independent of AP-1. AP-1 sequence failed to compete for binding by C(−280)NFAT in EMSA using human PBMC extracts and vice versa. C(−280)NFAT failed to compete against AP-1 binding. *C-NFAT probe, C(−280)NFAT; *AP-1 probe, AP-1 oligonucleotide. b, NFAT-binding complex does not contain AP-1 components. Abs to Jun-B or c-Fos when incubated with PBMC extracts in EMSAs do not alter binding to the C(−280)NFAT probe. c, NFAT DNA-binding kinetic analysis. NFAT binding to C(−280)NFAT is more stable than to NFAT:AP-1 sites from the human and murine IL-2 promoter in PBMC extracts. Excess competitor for the indicated sites was added and the duration of binding was measured by EMSA. Right panel, The absorbance (OD) of the binding complex after addition of excess competitor. d, NFAT promoter sequence comparison. C(−280)NFAT sequence has four adenosine bases (shadowed) in common when aligned with NFAT IL-2 promoter sequences. Underlined adenosine bases are critical for DNA binding as determined by EMSA ( e ). Sp = species; h = human; m = mouse; D = A,T; N = A,C,T,G; X = A,T,C; a = noncoding sequence; b = EMSA competitor. e, Mutation analysis of C(−280)NFAT to identify nucleotides important for DNA binding. Single-base pair substitutions (C↔A and G↔T) across the C(−280)NFAT sequence were introduced into the indicated nucleotide positions and the corresponding double-stranded oligonucleotides containing mutations were used as competitor sequence in EMSA.

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

    Article Title: Induction of the CTLA-4 Gene in Human Lymphocytes Is Dependent on NFAT Binding the Proximal Promoter

    doi:

    Figure Lengend Snippet: C(−280)NFAT-binding activity does not depend on AP-1 cooperativity and has a higher affinity than NFAT sites with AP-1 cooperativity. a, NFAT binding to C(−280)NFAT is independent of AP-1. AP-1 sequence failed to compete for binding by C(−280)NFAT in EMSA using human PBMC extracts and vice versa. C(−280)NFAT failed to compete against AP-1 binding. *C-NFAT probe, C(−280)NFAT; *AP-1 probe, AP-1 oligonucleotide. b, NFAT-binding complex does not contain AP-1 components. Abs to Jun-B or c-Fos when incubated with PBMC extracts in EMSAs do not alter binding to the C(−280)NFAT probe. c, NFAT DNA-binding kinetic analysis. NFAT binding to C(−280)NFAT is more stable than to NFAT:AP-1 sites from the human and murine IL-2 promoter in PBMC extracts. Excess competitor for the indicated sites was added and the duration of binding was measured by EMSA. Right panel, The absorbance (OD) of the binding complex after addition of excess competitor. d, NFAT promoter sequence comparison. C(−280)NFAT sequence has four adenosine bases (shadowed) in common when aligned with NFAT IL-2 promoter sequences. Underlined adenosine bases are critical for DNA binding as determined by EMSA ( e ). Sp = species; h = human; m = mouse; D = A,T; N = A,C,T,G; X = A,T,C; a = noncoding sequence; b = EMSA competitor. e, Mutation analysis of C(−280)NFAT to identify nucleotides important for DNA binding. Single-base pair substitutions (C↔A and G↔T) across the C(−280)NFAT sequence were introduced into the indicated nucleotide positions and the corresponding double-stranded oligonucleotides containing mutations were used as competitor sequence in EMSA.

    Article Snippet: To study the dependence of CTLA-4 expression on NFAT activity in PBMCs, cell-permeable inhibitors that affect NFAT specifically were added to PBMCs and CTLA-4 gene expression was measured.

    Techniques: Binding Assay, Activity Assay, Sequencing, Incubation, Mutagenesis

    Identifying NFAT as the factor that binds to the proximal CTLA-4 promoter. a, Sequence of the proximal CTLA-4 promoter important for transcription and potential transcription factor recognition motifs predicted by Transcription ESS is shown from nucleotides −264 to −297 from the translational start site. This sequence has consensus sites for NFAT, c-Myb, and SRE and is defined as C(−280)NFAT. b, NFAT sequences compete away DNA binding to C(−280)NFAT probe. EMSA results with γ - 32 P-labeled dsC(−280)NFAT probe, with extracts prepared from stimulated human PBMCs. Competition assays were performed using excess double-stranded unlabeled oligonucleotides for C(−280)NFAT = C-NFAT, human IL-2 NFAT = hNFAT, Sp-1, or GAS sites. c, C(−280)NFAT oligonucleotide interacts with DNA-binding activity for NFAT and is competed by other NFAT sequences (hIL-2 NFAT and mIL-2 NFAT). Competition with excess c-Myb or SRE oligonucleotide does not alter DNA binding to the C(−280)NFAT probe. d, NFAT1 binds to C(−280)NFAT. EMSA performed in the presence of Abs specific to NFAT1 yields a supershifted band (arrow) whereas nonspecific Ab (Ns Ab) or anti-NFAT2 does not.

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

    Article Title: Induction of the CTLA-4 Gene in Human Lymphocytes Is Dependent on NFAT Binding the Proximal Promoter

    doi:

    Figure Lengend Snippet: Identifying NFAT as the factor that binds to the proximal CTLA-4 promoter. a, Sequence of the proximal CTLA-4 promoter important for transcription and potential transcription factor recognition motifs predicted by Transcription ESS is shown from nucleotides −264 to −297 from the translational start site. This sequence has consensus sites for NFAT, c-Myb, and SRE and is defined as C(−280)NFAT. b, NFAT sequences compete away DNA binding to C(−280)NFAT probe. EMSA results with γ - 32 P-labeled dsC(−280)NFAT probe, with extracts prepared from stimulated human PBMCs. Competition assays were performed using excess double-stranded unlabeled oligonucleotides for C(−280)NFAT = C-NFAT, human IL-2 NFAT = hNFAT, Sp-1, or GAS sites. c, C(−280)NFAT oligonucleotide interacts with DNA-binding activity for NFAT and is competed by other NFAT sequences (hIL-2 NFAT and mIL-2 NFAT). Competition with excess c-Myb or SRE oligonucleotide does not alter DNA binding to the C(−280)NFAT probe. d, NFAT1 binds to C(−280)NFAT. EMSA performed in the presence of Abs specific to NFAT1 yields a supershifted band (arrow) whereas nonspecific Ab (Ns Ab) or anti-NFAT2 does not.

    Article Snippet: To study the dependence of CTLA-4 expression on NFAT activity in PBMCs, cell-permeable inhibitors that affect NFAT specifically were added to PBMCs and CTLA-4 gene expression was measured.

    Techniques: Sequencing, Binding Assay, Labeling, Activity Assay

    Spontaneously arising survivin specific T cells in patients. A ) Direct ex vivo tetramer staining of T cells derived from HNSCC patients. The patients (1 thru 5) were all HLA-A2 positive and the CD8 positive T cells were stained with HLA-A2 tetramers containing either the survivin 5 (light grey bars), survivin 96 (dark grey bars) or the modified survivin 96M (black bars) epitope. The percentage tetramer positive T cells in incidated on the Y-axis. B ) PBMC from HNSCC patients were stained with HLA-A2 tetramers containing the survivin 5 epitope and subsequently sorted by flow cytometry. Cells were plated out at a concentration of 0.2 cells per well and stimulated with feeder mix weekly. The left hand panel shows the forward scatter (X-axis; FSC) and side scatter (Y-axis; SSC) of the cells after four weeks of stimulation with feeder mix. A boxed live gate is indicated. The right hand panel shows tetramer staining of the cells in the live gates. Survivin 5 tetramer labeled with either APC (X-axis) or PE (Y-axis) were used to stain the T cells. The percentage of double tetramer positive cells is shown in the upper right quadrant. C ) Draining lymph nodes were obtained from a patient with locally advanced breast cancer, who had received neo-adjuvant chemotherapy before surgery. The spontaneously arising T cells were tested in an ELIspot assay against a number of different HLA-A2 binding peptides representing CTL epitopes; Survivin(95–104), CEA(571–579), Her-2/Neu(369–377) and Her-2/Neu(654–662) respectively and HPV16E7-(11–20) peptide as a negative control. Indicated are the number of spots per 100.000 T cells.

    Journal: Journal of Translational Medicine

    Article Title: Exploring dendritic cell based vaccines targeting survivin for the treatment of head and neck cancer patients

    doi: 10.1186/1479-5876-11-152

    Figure Lengend Snippet: Spontaneously arising survivin specific T cells in patients. A ) Direct ex vivo tetramer staining of T cells derived from HNSCC patients. The patients (1 thru 5) were all HLA-A2 positive and the CD8 positive T cells were stained with HLA-A2 tetramers containing either the survivin 5 (light grey bars), survivin 96 (dark grey bars) or the modified survivin 96M (black bars) epitope. The percentage tetramer positive T cells in incidated on the Y-axis. B ) PBMC from HNSCC patients were stained with HLA-A2 tetramers containing the survivin 5 epitope and subsequently sorted by flow cytometry. Cells were plated out at a concentration of 0.2 cells per well and stimulated with feeder mix weekly. The left hand panel shows the forward scatter (X-axis; FSC) and side scatter (Y-axis; SSC) of the cells after four weeks of stimulation with feeder mix. A boxed live gate is indicated. The right hand panel shows tetramer staining of the cells in the live gates. Survivin 5 tetramer labeled with either APC (X-axis) or PE (Y-axis) were used to stain the T cells. The percentage of double tetramer positive cells is shown in the upper right quadrant. C ) Draining lymph nodes were obtained from a patient with locally advanced breast cancer, who had received neo-adjuvant chemotherapy before surgery. The spontaneously arising T cells were tested in an ELIspot assay against a number of different HLA-A2 binding peptides representing CTL epitopes; Survivin(95–104), CEA(571–579), Her-2/Neu(369–377) and Her-2/Neu(654–662) respectively and HPV16E7-(11–20) peptide as a negative control. Indicated are the number of spots per 100.000 T cells.

    Article Snippet: T cell activation and survivin protein detection Isolation of resting CD8β positive T cells from HLA-A2 positive, healthy donor PBMC (Sanquin) was performed by positive selection using a MACS column (Miltenyi Biotec).

    Techniques: Ex Vivo, Staining, Derivative Assay, Modification, Flow Cytometry, Cytometry, Concentration Assay, Labeling, Enzyme-linked Immunospot, Binding Assay, CTL Assay, Negative Control

    EphA3 co-stains with macrophage/leukocyte markers A. Immunofluorescent staining of CD68, GFAP and Von Willebrand factor in a GBM specimen. B. Immunofluorescent staining of EphA3 (red) and CD31, GFAP, CD68, CD163, and CD206 on consecutive frozen sections of BTCOE4443 human GBM specimen. Nuclei are stained with DAPI (blue). Selected areas were magnified (last column on the right). C. Confocal immunofluorescent staining of EphA3 and CD68 in a GBM specimen using stacked 2D images. D, E. -stacked 2D images, and F. Confocal microscopy of co-staining of EphA3 and CD45 in GBM specimens. G. CD4+ T cells do not express the EphA3 receptor. Western blot analysis of peripheral blood mononuclear cells (PBMC), CD4+ and CD4+ activated T cells were probed for EphA3 immunoreactivity.

    Journal: Oncotarget

    Article Title: Simultaneous targeting of Eph receptors in glioblastoma

    doi: 10.18632/oncotarget.10978

    Figure Lengend Snippet: EphA3 co-stains with macrophage/leukocyte markers A. Immunofluorescent staining of CD68, GFAP and Von Willebrand factor in a GBM specimen. B. Immunofluorescent staining of EphA3 (red) and CD31, GFAP, CD68, CD163, and CD206 on consecutive frozen sections of BTCOE4443 human GBM specimen. Nuclei are stained with DAPI (blue). Selected areas were magnified (last column on the right). C. Confocal immunofluorescent staining of EphA3 and CD68 in a GBM specimen using stacked 2D images. D, E. -stacked 2D images, and F. Confocal microscopy of co-staining of EphA3 and CD45 in GBM specimens. G. CD4+ T cells do not express the EphA3 receptor. Western blot analysis of peripheral blood mononuclear cells (PBMC), CD4+ and CD4+ activated T cells were probed for EphA3 immunoreactivity.

    Article Snippet: Isolation and activation of CD4+ T cells Total CD4+ T Cells were isolated from PBMC (ZenBio, Durham, NC) using the Dynabeads Untouched human CD4+ T-Cell Kit (Invitrogen) according to the manufacturer's instructions.

    Techniques: Staining, Confocal Microscopy, Western Blot

    CD8 cells respond to CEF peptides ( A ), and CD4 cells to mumps antigen ( B ): these responses seen ex vivo are preserved after freeze thawing ( C D ). To determine T cell subsets responding to the CEF and mumps antigens, magnetic affinity bead-based separation was performed depleting more than 95% of CD4 or CD8 T cells, as specified. The PBMC and cell fractions were stimulated with CEF peptides ( A ) or mumps antigen ( B ). For each condition, triplicate wells were tested with the mean and standard deviation shown. The data shown are from one of two experiments performed with similar results. Freshly isolated, non-cryopreserved PBMC ( ex vivo ) and cryopreserved PBMC that were tested directly without resting following thawing (fresh) were tested in an IFN-γ ELISPOT assay against CEF peptides ( C ) and mumps antigen ( D ). Data points obtained for individual donors are connected with a line. Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or freeze-thawed, the medium background was less than 10 spots per well. Nonparametric Wilcoxon signed-rank test was used to compare matched ex vivo vs . fresh responses with a p -value ≤ 0.05 being considered significant.

    Journal: Cells

    Article Title: Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays

    doi: 10.3390/cells1030409

    Figure Lengend Snippet: CD8 cells respond to CEF peptides ( A ), and CD4 cells to mumps antigen ( B ): these responses seen ex vivo are preserved after freeze thawing ( C D ). To determine T cell subsets responding to the CEF and mumps antigens, magnetic affinity bead-based separation was performed depleting more than 95% of CD4 or CD8 T cells, as specified. The PBMC and cell fractions were stimulated with CEF peptides ( A ) or mumps antigen ( B ). For each condition, triplicate wells were tested with the mean and standard deviation shown. The data shown are from one of two experiments performed with similar results. Freshly isolated, non-cryopreserved PBMC ( ex vivo ) and cryopreserved PBMC that were tested directly without resting following thawing (fresh) were tested in an IFN-γ ELISPOT assay against CEF peptides ( C ) and mumps antigen ( D ). Data points obtained for individual donors are connected with a line. Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or freeze-thawed, the medium background was less than 10 spots per well. Nonparametric Wilcoxon signed-rank test was used to compare matched ex vivo vs . fresh responses with a p -value ≤ 0.05 being considered significant.

    Article Snippet: Thawing and Resting of Cryopreserved PBMC Cryopreserved human PBMC of 25 donors were acquired from a library of PBMC (ePBMC, CTL‑CP1) offered by Cellular Technologies Ltd. (CTL, Shaker Heights, OH, USA).

    Techniques: Ex Vivo, Standard Deviation, Isolation, Enzyme-linked Immunospot

    Comparison of mumps antigen elicited IFN-γ in freshly thawed and rested cryopreserved PBMC for low ( A ) and high responder subjects ( B ). Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or rested, the medium background was less than 10 spots per well. Non-parametric Wilcoxon signed-rank test was used to compare matched fresh vs . rested responses with a p -value ≤ 0.05 being considered significant.

    Journal: Cells

    Article Title: Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays

    doi: 10.3390/cells1030409

    Figure Lengend Snippet: Comparison of mumps antigen elicited IFN-γ in freshly thawed and rested cryopreserved PBMC for low ( A ) and high responder subjects ( B ). Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or rested, the medium background was less than 10 spots per well. Non-parametric Wilcoxon signed-rank test was used to compare matched fresh vs . rested responses with a p -value ≤ 0.05 being considered significant.

    Article Snippet: Thawing and Resting of Cryopreserved PBMC Cryopreserved human PBMC of 25 donors were acquired from a library of PBMC (ePBMC, CTL‑CP1) offered by Cellular Technologies Ltd. (CTL, Shaker Heights, OH, USA).

    Techniques:

    Comparison of CEF responses elicited by freshly thawed (“fresh”) and rested cryopreserved PBMC in ELISPOT assays. ( A ) CEF-specific responses among “low responders” who were defined as subjects who had fewer than 50 SFU in freshly thawed PMBC. ( B ) CEF-specific responses among “high responders” who were defined as subjects who had greater than 50 spots forming units (SFU) in freshly thawed PMBC. Data points obtained from the same donor before and after resting are connected by a line. Each data point represents the mean of triplicate antigen-stimulated wells. For each donor and antigen tested, the standard deviation was

    Journal: Cells

    Article Title: Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays

    doi: 10.3390/cells1030409

    Figure Lengend Snippet: Comparison of CEF responses elicited by freshly thawed (“fresh”) and rested cryopreserved PBMC in ELISPOT assays. ( A ) CEF-specific responses among “low responders” who were defined as subjects who had fewer than 50 SFU in freshly thawed PMBC. ( B ) CEF-specific responses among “high responders” who were defined as subjects who had greater than 50 spots forming units (SFU) in freshly thawed PMBC. Data points obtained from the same donor before and after resting are connected by a line. Each data point represents the mean of triplicate antigen-stimulated wells. For each donor and antigen tested, the standard deviation was

    Article Snippet: Thawing and Resting of Cryopreserved PBMC Cryopreserved human PBMC of 25 donors were acquired from a library of PBMC (ePBMC, CTL‑CP1) offered by Cellular Technologies Ltd. (CTL, Shaker Heights, OH, USA).

    Techniques: Enzyme-linked Immunospot, Standard Deviation

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

    Journal: Journal of Virology

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

    doi:

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

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

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

    Fractionation of FCM and identification of Slit2. ( A ) FCM was fractionated by centrifugation using 100-kDa filters. PBMCs were cultured for 5 d in the presence or absence of 10% (vol/vol) FCM, 10-fold concentrated 100-kDa retentate (Ret), or 100-kDa flow through (FT). After a 5-d incubation, cells were air-dried, fixed, and stained, and the number of fibrocytes was counted. Compared with PBMCs cultured in the absence of conditioned medium (SFM), FCM and the 100-kDa retentate significantly decreased the number of fibrocytes. Values are mean ± SEM ( n = 4). * P

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

    Article Title: Fibroblasts secrete Slit2 to inhibit fibrocyte differentiation and fibrosis

    doi: 10.1073/pnas.1417426112

    Figure Lengend Snippet: Fractionation of FCM and identification of Slit2. ( A ) FCM was fractionated by centrifugation using 100-kDa filters. PBMCs were cultured for 5 d in the presence or absence of 10% (vol/vol) FCM, 10-fold concentrated 100-kDa retentate (Ret), or 100-kDa flow through (FT). After a 5-d incubation, cells were air-dried, fixed, and stained, and the number of fibrocytes was counted. Compared with PBMCs cultured in the absence of conditioned medium (SFM), FCM and the 100-kDa retentate significantly decreased the number of fibrocytes. Values are mean ± SEM ( n = 4). * P

    Article Snippet: We observed that Robo1 is expressed on fibrocytes and macrophages and that incubating PBMCs with Slit2 led to reduced fibrocytes, but no obvious changes in Robo1 levels on macrophages ( ).

    Techniques: Fractionation, Centrifugation, Cell Culture, Flow Cytometry, Incubation, Staining

    The effect of recombinant Slit2 (rSlit2) on human fibrocyte differentiation. ( A ) Human PBMCs and isolated monocytes were cultured in the presence or absence of rSlit2 for 5 d. Cells were then air-dried, fixed, and stained, and fibrocytes were counted. Values are mean ± SEM ( n = 3). The absence of error bars indicates that the error was smaller than the plot symbol. rSlit2 concentrations at 0.1 ng/mL and above significantly inhibited fibrocyte differentiation ( t test). Lines are fits to sigmoidal dose–response curves with variable Hill coefficients. ( B ) MRC-5 FCM was incubated with beads labeled with anti-Slit2 antibodies, anti-HSP-47 (a control protein depletion), rabbit IgG (a control IgG), or unlabeled beads. Human PBMCs were cultured in the presence or absence of 10% (vol/vol) antibody-depleted FCM for 5 d. Values are mean ± SEM ( n = 4). * P

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

    Article Title: Fibroblasts secrete Slit2 to inhibit fibrocyte differentiation and fibrosis

    doi: 10.1073/pnas.1417426112

    Figure Lengend Snippet: The effect of recombinant Slit2 (rSlit2) on human fibrocyte differentiation. ( A ) Human PBMCs and isolated monocytes were cultured in the presence or absence of rSlit2 for 5 d. Cells were then air-dried, fixed, and stained, and fibrocytes were counted. Values are mean ± SEM ( n = 3). The absence of error bars indicates that the error was smaller than the plot symbol. rSlit2 concentrations at 0.1 ng/mL and above significantly inhibited fibrocyte differentiation ( t test). Lines are fits to sigmoidal dose–response curves with variable Hill coefficients. ( B ) MRC-5 FCM was incubated with beads labeled with anti-Slit2 antibodies, anti-HSP-47 (a control protein depletion), rabbit IgG (a control IgG), or unlabeled beads. Human PBMCs were cultured in the presence or absence of 10% (vol/vol) antibody-depleted FCM for 5 d. Values are mean ± SEM ( n = 4). * P

    Article Snippet: We observed that Robo1 is expressed on fibrocytes and macrophages and that incubating PBMCs with Slit2 led to reduced fibrocytes, but no obvious changes in Robo1 levels on macrophages ( ).

    Techniques: Recombinant, Isolation, Cell Culture, Staining, Incubation, Labeling