15-aa-long peptides Search Results


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  • 99
    Thermo Fisher phusion dna polymerase
    Phusion Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11374 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore l ascorbic acid 2 phosphate
    L Ascorbic Acid 2 Phosphate, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2967 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher platinum taq dna polymerase high fidelity
    Platinum Taq Dna Polymerase High Fidelity, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6479 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 7aad viability staining solution
    7aad Viability Staining Solution, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    INTAVIS automatic multiprep robot
    Automatic Multiprep Robot, supplied by INTAVIS, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cfse
    BDCA3 + DCs and BDCA1 + DCs have a similar ability to present endogenous antigen on MHCI. (A) Presentation of preprocessed peptide by DCs. Day 1 DCs from HLA-A*0201 donors were incubated with Flu-M1 (aa 58–66) or HIV-p17 (aa 77–85, negative control) peptide at 25 ng/ml for 3 h at 37°C. The cells were then washed and cultured with autologous <t>CFSE-labeled</t> <t>CD8</t> + T cells at indicated DC/T cell ratio in the presence of TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown data are normalized to BDCA1 + DC/T cells at a 1:30 ratio and the mean ± SD ( n = 4 independent experiments) is depicted. (B) EGFP fluorescence intensity in DC subsets. DCs from HLA-A*0201 donors were transfected directly after isolation with a plasmid encoding for Flu-M1 (aa 55–72)–EGFP fusion protein. DCs were cultured overnight in the presence or absence of TLR7/8 L. 16 h after transfection, DCs were analyzed by flow cytometry for the expression of EGFP. Shown histograms are DCs gated on live EGFP + cells. Shown is one representative experiment of three. (C) Presentation of endogenous antigen by DCs. Flu-M1 (aa 55–72)-EGFP–transfected DCs were cultured overnight in the presence or absence of TLR7/8 L and then cultured with autologous CFSE-labeled CD8 + T cells at indicated DC/T cell ratios, normalized to percentage of EGFP + DCs. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated as in A. Shown is one representative experiment of three.
    Cfse, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 30979 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    INTAVIS fmoc chemistry
    BDCA3 + DCs and BDCA1 + DCs have a similar ability to present endogenous antigen on MHCI. (A) Presentation of preprocessed peptide by DCs. Day 1 DCs from HLA-A*0201 donors were incubated with Flu-M1 (aa 58–66) or HIV-p17 (aa 77–85, negative control) peptide at 25 ng/ml for 3 h at 37°C. The cells were then washed and cultured with autologous <t>CFSE-labeled</t> <t>CD8</t> + T cells at indicated DC/T cell ratio in the presence of TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown data are normalized to BDCA1 + DC/T cells at a 1:30 ratio and the mean ± SD ( n = 4 independent experiments) is depicted. (B) EGFP fluorescence intensity in DC subsets. DCs from HLA-A*0201 donors were transfected directly after isolation with a plasmid encoding for Flu-M1 (aa 55–72)–EGFP fusion protein. DCs were cultured overnight in the presence or absence of TLR7/8 L. 16 h after transfection, DCs were analyzed by flow cytometry for the expression of EGFP. Shown histograms are DCs gated on live EGFP + cells. Shown is one representative experiment of three. (C) Presentation of endogenous antigen by DCs. Flu-M1 (aa 55–72)-EGFP–transfected DCs were cultured overnight in the presence or absence of TLR7/8 L and then cultured with autologous CFSE-labeled CD8 + T cells at indicated DC/T cell ratios, normalized to percentage of EGFP + DCs. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated as in A. Shown is one representative experiment of three.
    Fmoc Chemistry, supplied by INTAVIS, used in various techniques. Bioz Stars score: 93/100, based on 140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phusion high fidelity pcr master mix
    BDCA3 + DCs and BDCA1 + DCs have a similar ability to present endogenous antigen on MHCI. (A) Presentation of preprocessed peptide by DCs. Day 1 DCs from HLA-A*0201 donors were incubated with Flu-M1 (aa 58–66) or HIV-p17 (aa 77–85, negative control) peptide at 25 ng/ml for 3 h at 37°C. The cells were then washed and cultured with autologous <t>CFSE-labeled</t> <t>CD8</t> + T cells at indicated DC/T cell ratio in the presence of TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown data are normalized to BDCA1 + DC/T cells at a 1:30 ratio and the mean ± SD ( n = 4 independent experiments) is depicted. (B) EGFP fluorescence intensity in DC subsets. DCs from HLA-A*0201 donors were transfected directly after isolation with a plasmid encoding for Flu-M1 (aa 55–72)–EGFP fusion protein. DCs were cultured overnight in the presence or absence of TLR7/8 L. 16 h after transfection, DCs were analyzed by flow cytometry for the expression of EGFP. Shown histograms are DCs gated on live EGFP + cells. Shown is one representative experiment of three. (C) Presentation of endogenous antigen by DCs. Flu-M1 (aa 55–72)-EGFP–transfected DCs were cultured overnight in the presence or absence of TLR7/8 L and then cultured with autologous CFSE-labeled CD8 + T cells at indicated DC/T cell ratios, normalized to percentage of EGFP + DCs. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated as in A. Shown is one representative experiment of three.
    Phusion High Fidelity Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1884 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher viia 7 real time pcr system
    BDCA3 + DCs and BDCA1 + DCs have a similar ability to present endogenous antigen on MHCI. (A) Presentation of preprocessed peptide by DCs. Day 1 DCs from HLA-A*0201 donors were incubated with Flu-M1 (aa 58–66) or HIV-p17 (aa 77–85, negative control) peptide at 25 ng/ml for 3 h at 37°C. The cells were then washed and cultured with autologous <t>CFSE-labeled</t> <t>CD8</t> + T cells at indicated DC/T cell ratio in the presence of TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown data are normalized to BDCA1 + DC/T cells at a 1:30 ratio and the mean ± SD ( n = 4 independent experiments) is depicted. (B) EGFP fluorescence intensity in DC subsets. DCs from HLA-A*0201 donors were transfected directly after isolation with a plasmid encoding for Flu-M1 (aa 55–72)–EGFP fusion protein. DCs were cultured overnight in the presence or absence of TLR7/8 L. 16 h after transfection, DCs were analyzed by flow cytometry for the expression of EGFP. Shown histograms are DCs gated on live EGFP + cells. Shown is one representative experiment of three. (C) Presentation of endogenous antigen by DCs. Flu-M1 (aa 55–72)-EGFP–transfected DCs were cultured overnight in the presence or absence of TLR7/8 L and then cultured with autologous CFSE-labeled CD8 + T cells at indicated DC/T cell ratios, normalized to percentage of EGFP + DCs. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated as in A. Shown is one representative experiment of three.
    Viia 7 Real Time Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14540 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Synthesis Inc aa 15 mer long hiv gagp24 peptides
    <t>Anti-HIV</t> <t>Gagp24</t> antibody avidity following vaccination. Animals were immunized i.d. three times at week 0, 6 and 15 with αDCIR.Gag24 or the molar equivalent of the Gagp24 protein with or without poly(I:C). Serum HIV Gagp24-specific IgG antibody avidity was measured by ELISA 2 weeks after the third injection (week 17) with αDCIR.Gagp24 or Gagp24 without adjuvant (A) or with poly(I:C) (B) . Relative avidity index (RAI) (%) of Gagp24-specific IgG for the individual monkeys is presented.
    Aa 15 Mer Long Hiv Gagp24 Peptides, supplied by Bio-Synthesis Inc, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher amplitaq gold 360 dna polymerase
    Inhibition and the effect of hot-start. ( a ) Inhibition profiles of 2D9 versus <t>AmpliTaq</t> <t>Gold</t> 360 (ATaqG) in PCR. The effect of various inhibitors (SP61: bone dust, Tar, Soil, 1736; cave sediment) on polymerase activity of 2D9 and ATaqG as determined by a PCR assay in the presence of decreasing amounts of inhibitor and plotted as the average relative resistance ( n = 3, standard error) (as in Figure 4 ) of 2D9 and ATaqG with respect to ATaqG as a reference (resistance = 1). ( b ) The effect of manual hot-start on resistance of 2D9 polymerase to inhibitors tar and soil at minimal limiting amounts of inhibitor. Hot-start increases inhibitor resistance by at least a factor of two.
    Amplitaq Gold 360 Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 455 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti cd49d antibodies
    Inhibition and the effect of hot-start. ( a ) Inhibition profiles of 2D9 versus <t>AmpliTaq</t> <t>Gold</t> 360 (ATaqG) in PCR. The effect of various inhibitors (SP61: bone dust, Tar, Soil, 1736; cave sediment) on polymerase activity of 2D9 and ATaqG as determined by a PCR assay in the presence of decreasing amounts of inhibitor and plotted as the average relative resistance ( n = 3, standard error) (as in Figure 4 ) of 2D9 and ATaqG with respect to ATaqG as a reference (resistance = 1). ( b ) The effect of manual hot-start on resistance of 2D9 polymerase to inhibitors tar and soil at minimal limiting amounts of inhibitor. Hot-start increases inhibitor resistance by at least a factor of two.
    Anti Cd49d Antibodies, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 361 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fgf basic aa 1 155 recombinant human protein
    Inhibition and the effect of hot-start. ( a ) Inhibition profiles of 2D9 versus <t>AmpliTaq</t> <t>Gold</t> 360 (ATaqG) in PCR. The effect of various inhibitors (SP61: bone dust, Tar, Soil, 1736; cave sediment) on polymerase activity of 2D9 and ATaqG as determined by a PCR assay in the presence of decreasing amounts of inhibitor and plotted as the average relative resistance ( n = 3, standard error) (as in Figure 4 ) of 2D9 and ATaqG with respect to ATaqG as a reference (resistance = 1). ( b ) The effect of manual hot-start on resistance of 2D9 polymerase to inhibitors tar and soil at minimal limiting amounts of inhibitor. Hot-start increases inhibitor resistance by at least a factor of two.
    Fgf Basic Aa 1 155 Recombinant Human Protein, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 237 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cd28
    Protection against heterologous challenge with a Puerto Rican ZIKV isolate. ( a ) Six animals (three females and three males) were re-challenged subcutaneously with 1 × 10 5 TCID 50 of a PR ZIKV isolate 45 d after primary infection. Viral RNA levels in blood plasma were monitored between days 1 and 21 after the secondary challenge. ZIKV RNA copies in blood plasma are indicated. A single positive sample, detected in one male monkey 1 d after infection, is indicated (blue solid line). Lymphocyte activation and frequencies were measured during re-challenge. Activation and numbers of T cells, B cells, NK cells and monocytes were measured by flow cytometry on days 0, 1, 2, 3, 4, 5 and 7 after ZIKV infection of three male and three female monkeys. ( b , c ) Percentage of activated (CD69 + ) and total numbers of naive <t>(CD28</t> + CD95 − ) (left), central memory (CD28 + CD95 + ) (middle), and effector and effector memory (CD28 − CD95 + ) (right) CD4 + ( b ) and CD8 + ( c ) T cell subsets in whole blood. ( d ) Percentage of activated (CD69 + ) and total numbers of CD16 + (left), CD16 − CD56 + (middle) and CD16 − CD56 − (right) NK cells in whole blood. ( e ) CD38 expression (geometric mean fluorescence of CD38) and total numbers of naive (CD27 − ) (left) and memory (CD27 + ) (right) B cells in whole blood. ( f ) Percentage of activated and total numbers of monocytes in whole blood. Black line indicates median. Individual values are shown for males (blue) and females (red). ( g . Values below this threshold were set to a value of 0. Values greater than 1.5-fold above that of the day of re-challenge were considered to be anamnestic. Each monkey is indicated by a different color symbol. Triangles indicate males, and circles indicate females.
    Cd28, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1294 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti gm130
    Protection against heterologous challenge with a Puerto Rican ZIKV isolate. ( a ) Six animals (three females and three males) were re-challenged subcutaneously with 1 × 10 5 TCID 50 of a PR ZIKV isolate 45 d after primary infection. Viral RNA levels in blood plasma were monitored between days 1 and 21 after the secondary challenge. ZIKV RNA copies in blood plasma are indicated. A single positive sample, detected in one male monkey 1 d after infection, is indicated (blue solid line). Lymphocyte activation and frequencies were measured during re-challenge. Activation and numbers of T cells, B cells, NK cells and monocytes were measured by flow cytometry on days 0, 1, 2, 3, 4, 5 and 7 after ZIKV infection of three male and three female monkeys. ( b , c ) Percentage of activated (CD69 + ) and total numbers of naive <t>(CD28</t> + CD95 − ) (left), central memory (CD28 + CD95 + ) (middle), and effector and effector memory (CD28 − CD95 + ) (right) CD4 + ( b ) and CD8 + ( c ) T cell subsets in whole blood. ( d ) Percentage of activated (CD69 + ) and total numbers of CD16 + (left), CD16 − CD56 + (middle) and CD16 − CD56 − (right) NK cells in whole blood. ( e ) CD38 expression (geometric mean fluorescence of CD38) and total numbers of naive (CD27 − ) (left) and memory (CD27 + ) (right) B cells in whole blood. ( f ) Percentage of activated and total numbers of monocytes in whole blood. Black line indicates median. Individual values are shown for males (blue) and females (red). ( g . Values below this threshold were set to a value of 0. Values greater than 1.5-fold above that of the day of re-challenge were considered to be anamnestic. Each monkey is indicated by a different color symbol. Triangles indicate males, and circles indicate females.
    Anti Gm130, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 162 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Kinexus Bioinformatics Corporation peptide array
    Protection against heterologous challenge with a Puerto Rican ZIKV isolate. ( a ) Six animals (three females and three males) were re-challenged subcutaneously with 1 × 10 5 TCID 50 of a PR ZIKV isolate 45 d after primary infection. Viral RNA levels in blood plasma were monitored between days 1 and 21 after the secondary challenge. ZIKV RNA copies in blood plasma are indicated. A single positive sample, detected in one male monkey 1 d after infection, is indicated (blue solid line). Lymphocyte activation and frequencies were measured during re-challenge. Activation and numbers of T cells, B cells, NK cells and monocytes were measured by flow cytometry on days 0, 1, 2, 3, 4, 5 and 7 after ZIKV infection of three male and three female monkeys. ( b , c ) Percentage of activated (CD69 + ) and total numbers of naive <t>(CD28</t> + CD95 − ) (left), central memory (CD28 + CD95 + ) (middle), and effector and effector memory (CD28 − CD95 + ) (right) CD4 + ( b ) and CD8 + ( c ) T cell subsets in whole blood. ( d ) Percentage of activated (CD69 + ) and total numbers of CD16 + (left), CD16 − CD56 + (middle) and CD16 − CD56 − (right) NK cells in whole blood. ( e ) CD38 expression (geometric mean fluorescence of CD38) and total numbers of naive (CD27 − ) (left) and memory (CD27 + ) (right) B cells in whole blood. ( f ) Percentage of activated and total numbers of monocytes in whole blood. Black line indicates median. Individual values are shown for males (blue) and females (red). ( g . Values below this threshold were set to a value of 0. Values greater than 1.5-fold above that of the day of re-challenge were considered to be anamnestic. Each monkey is indicated by a different color symbol. Triangles indicate males, and circles indicate females.
    Peptide Array, supplied by Kinexus Bioinformatics Corporation, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher bis tris gels
    Protection against heterologous challenge with a Puerto Rican ZIKV isolate. ( a ) Six animals (three females and three males) were re-challenged subcutaneously with 1 × 10 5 TCID 50 of a PR ZIKV isolate 45 d after primary infection. Viral RNA levels in blood plasma were monitored between days 1 and 21 after the secondary challenge. ZIKV RNA copies in blood plasma are indicated. A single positive sample, detected in one male monkey 1 d after infection, is indicated (blue solid line). Lymphocyte activation and frequencies were measured during re-challenge. Activation and numbers of T cells, B cells, NK cells and monocytes were measured by flow cytometry on days 0, 1, 2, 3, 4, 5 and 7 after ZIKV infection of three male and three female monkeys. ( b , c ) Percentage of activated (CD69 + ) and total numbers of naive <t>(CD28</t> + CD95 − ) (left), central memory (CD28 + CD95 + ) (middle), and effector and effector memory (CD28 − CD95 + ) (right) CD4 + ( b ) and CD8 + ( c ) T cell subsets in whole blood. ( d ) Percentage of activated (CD69 + ) and total numbers of CD16 + (left), CD16 − CD56 + (middle) and CD16 − CD56 − (right) NK cells in whole blood. ( e ) CD38 expression (geometric mean fluorescence of CD38) and total numbers of naive (CD27 − ) (left) and memory (CD27 + ) (right) B cells in whole blood. ( f ) Percentage of activated and total numbers of monocytes in whole blood. Black line indicates median. Individual values are shown for males (blue) and females (red). ( g . Values below this threshold were set to a value of 0. Values greater than 1.5-fold above that of the day of re-challenge were considered to be anamnestic. Each monkey is indicated by a different color symbol. Triangles indicate males, and circles indicate females.
    Bis Tris Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 23849 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cd4
    Protection against heterologous challenge with a Puerto Rican ZIKV isolate. ( a ) Six animals (three females and three males) were re-challenged subcutaneously with 1 × 10 5 TCID 50 of a PR ZIKV isolate 45 d after primary infection. Viral RNA levels in blood plasma were monitored between days 1 and 21 after the secondary challenge. ZIKV RNA copies in blood plasma are indicated. A single positive sample, detected in one male monkey 1 d after infection, is indicated (blue solid line). Lymphocyte activation and frequencies were measured during re-challenge. Activation and numbers of T cells, B cells, NK cells and monocytes were measured by flow cytometry on days 0, 1, 2, 3, 4, 5 and 7 after ZIKV infection of three male and three female monkeys. ( b , c ) Percentage of activated (CD69 + ) and total numbers of naive (CD28 + CD95 − ) (left), central memory (CD28 + CD95 + ) (middle), and effector and effector memory (CD28 − CD95 + ) (right) <t>CD4</t> + ( b ) and CD8 + ( c ) T cell subsets in whole blood. ( d ) Percentage of activated (CD69 + ) and total numbers of CD16 + (left), CD16 − CD56 + (middle) and CD16 − CD56 − (right) NK cells in whole blood. ( e ) CD38 expression (geometric mean fluorescence of CD38) and total numbers of naive (CD27 − ) (left) and memory (CD27 + ) (right) B cells in whole blood. ( f ) Percentage of activated and total numbers of monocytes in whole blood. Black line indicates median. Individual values are shown for males (blue) and females (red). ( g . Values below this threshold were set to a value of 0. Values greater than 1.5-fold above that of the day of re-challenge were considered to be anamnestic. Each monkey is indicated by a different color symbol. Triangles indicate males, and circles indicate females.
    Cd4, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14912 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher celltrace violet cell proliferation kit
    Proliferative capacity of CD4 T cells is not reversible by PD-1 blockade in human immunodeficiency virus (HIV)–infected children. <t>CellTrace</t> Violet (CTV)–labeled peripheral blood mononuclear cells from HIV-infected children were stimulated with anti-CD3 or HIV Gag peptide pool in the presence of PD-1 blocking antibody or isotype control antibody. Flow cytometry from a representative donor indicating the percentage of CTV dim CD4 + T cells after 7 days of anti-CD3( A ) or HIV Gag ( C ) peptide stimulation is shown. Graphs depict summary of proliferative responses to anti-CD3 ( B ) and HIV Gag ( D ) peptide pool in the presence of PD-1 blocking antibody in HIV-infected children. Statistical analysis was performed using the ratio paired t test. Triangles indicate viremic subjects.
    Celltrace Violet Cell Proliferation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1904 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    INTAVIS celluspots peptide arrays
    Proliferative capacity of CD4 T cells is not reversible by PD-1 blockade in human immunodeficiency virus (HIV)–infected children. <t>CellTrace</t> Violet (CTV)–labeled peripheral blood mononuclear cells from HIV-infected children were stimulated with anti-CD3 or HIV Gag peptide pool in the presence of PD-1 blocking antibody or isotype control antibody. Flow cytometry from a representative donor indicating the percentage of CTV dim CD4 + T cells after 7 days of anti-CD3( A ) or HIV Gag ( C ) peptide stimulation is shown. Graphs depict summary of proliferative responses to anti-CD3 ( B ) and HIV Gag ( D ) peptide pool in the presence of PD-1 blocking antibody in HIV-infected children. Statistical analysis was performed using the ratio paired t test. Triangles indicate viremic subjects.
    Celluspots Peptide Arrays, supplied by INTAVIS, used in various techniques. Bioz Stars score: 88/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pepscan coating antigen
    Proliferative capacity of CD4 T cells is not reversible by PD-1 blockade in human immunodeficiency virus (HIV)–infected children. <t>CellTrace</t> Violet (CTV)–labeled peripheral blood mononuclear cells from HIV-infected children were stimulated with anti-CD3 or HIV Gag peptide pool in the presence of PD-1 blocking antibody or isotype control antibody. Flow cytometry from a representative donor indicating the percentage of CTV dim CD4 + T cells after 7 days of anti-CD3( A ) or HIV Gag ( C ) peptide stimulation is shown. Graphs depict summary of proliferative responses to anti-CD3 ( B ) and HIV Gag ( D ) peptide pool in the presence of PD-1 blocking antibody in HIV-infected children. Statistical analysis was performed using the ratio paired t test. Triangles indicate viremic subjects.
    Coating Antigen, supplied by Pepscan, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher adhesive multiwell gene frames
    Proliferative capacity of CD4 T cells is not reversible by PD-1 blockade in human immunodeficiency virus (HIV)–infected children. <t>CellTrace</t> Violet (CTV)–labeled peripheral blood mononuclear cells from HIV-infected children were stimulated with anti-CD3 or HIV Gag peptide pool in the presence of PD-1 blocking antibody or isotype control antibody. Flow cytometry from a representative donor indicating the percentage of CTV dim CD4 + T cells after 7 days of anti-CD3( A ) or HIV Gag ( C ) peptide stimulation is shown. Graphs depict summary of proliferative responses to anti-CD3 ( B ) and HIV Gag ( D ) peptide pool in the presence of PD-1 blocking antibody in HIV-infected children. Statistical analysis was performed using the ratio paired t test. Triangles indicate viremic subjects.
    Adhesive Multiwell Gene Frames, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    BDCA3 + DCs and BDCA1 + DCs have a similar ability to present endogenous antigen on MHCI. (A) Presentation of preprocessed peptide by DCs. Day 1 DCs from HLA-A*0201 donors were incubated with Flu-M1 (aa 58–66) or HIV-p17 (aa 77–85, negative control) peptide at 25 ng/ml for 3 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells at indicated DC/T cell ratio in the presence of TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown data are normalized to BDCA1 + DC/T cells at a 1:30 ratio and the mean ± SD ( n = 4 independent experiments) is depicted. (B) EGFP fluorescence intensity in DC subsets. DCs from HLA-A*0201 donors were transfected directly after isolation with a plasmid encoding for Flu-M1 (aa 55–72)–EGFP fusion protein. DCs were cultured overnight in the presence or absence of TLR7/8 L. 16 h after transfection, DCs were analyzed by flow cytometry for the expression of EGFP. Shown histograms are DCs gated on live EGFP + cells. Shown is one representative experiment of three. (C) Presentation of endogenous antigen by DCs. Flu-M1 (aa 55–72)-EGFP–transfected DCs were cultured overnight in the presence or absence of TLR7/8 L and then cultured with autologous CFSE-labeled CD8 + T cells at indicated DC/T cell ratios, normalized to percentage of EGFP + DCs. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated as in A. Shown is one representative experiment of three.

    Journal: The Journal of Experimental Medicine

    Article Title: Antigen delivery to early endosomes eliminates the superiority of human blood BDCA3+ dendritic cells at cross presentation

    doi: 10.1084/jem.20121251

    Figure Lengend Snippet: BDCA3 + DCs and BDCA1 + DCs have a similar ability to present endogenous antigen on MHCI. (A) Presentation of preprocessed peptide by DCs. Day 1 DCs from HLA-A*0201 donors were incubated with Flu-M1 (aa 58–66) or HIV-p17 (aa 77–85, negative control) peptide at 25 ng/ml for 3 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells at indicated DC/T cell ratio in the presence of TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown data are normalized to BDCA1 + DC/T cells at a 1:30 ratio and the mean ± SD ( n = 4 independent experiments) is depicted. (B) EGFP fluorescence intensity in DC subsets. DCs from HLA-A*0201 donors were transfected directly after isolation with a plasmid encoding for Flu-M1 (aa 55–72)–EGFP fusion protein. DCs were cultured overnight in the presence or absence of TLR7/8 L. 16 h after transfection, DCs were analyzed by flow cytometry for the expression of EGFP. Shown histograms are DCs gated on live EGFP + cells. Shown is one representative experiment of three. (C) Presentation of endogenous antigen by DCs. Flu-M1 (aa 55–72)-EGFP–transfected DCs were cultured overnight in the presence or absence of TLR7/8 L and then cultured with autologous CFSE-labeled CD8 + T cells at indicated DC/T cell ratios, normalized to percentage of EGFP + DCs. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated as in A. Shown is one representative experiment of three.

    Article Snippet: After antigen uptake, DCs were washed extensively to remove free antibody or peptide and cultured with 1.5 × 106 CFSE (Invitrogen)-labeled CD8+ T cells at a DC/T cell ratio of 1:30 (unless otherwise indicated), in the presence of 20 U/ml IL-2 (Roche) and 1 µg/ml TLR7/8 L. After 8–10 d, cells were harvested and stained with Flu-M1 (aa 58–66) or CMV-pp65 (aa 495–503) pentamer for 15 min at room temperature, followed by labeling with antibodies against CD3, CD8, CD4, and CD19, fixation, and analysis by flow cytometry.

    Techniques: Incubation, Negative Control, Cell Culture, Labeling, Fluorescence, Transfection, Isolation, Plasmid Preparation, Flow Cytometry, Cytometry, Expressing

    Antigen targeted to early endosomes via CD40 is cross presented by both DC subsets with similar efficacy. (A) Anti-CD40 antibody intracellular trafficking. Day 1 BDCA1 + DCs (top) or BDCA3 + DCs (bottom) were fed with Alexa Fluor 488–labeled anti-CD40 antibody (green) continuously for 3 h at 37°C, washed, and allowed to adhere to coverslips. After fixation and permeabilization, the lysosomes or early endosomes were stained using anti-Lamp1 or anti-EEA1 (red), respectively. Plasma membrane was stained using anti–HLA-DR (blue) antibodies. Cells were then analyzed using confocal microscopy. Bars, 5 µm. Shown is one representative experiment of three. (B) Accumulation of anti-CD40 antibody. Day 1 DCs were fed with Alexa Fluor 488–labeled anti-CD40 antibody continuously for 4–6 h at 4°C or 37°C. Results were analyzed by flow cytometry. The 4°C MFI was subtracted from the 37°C MFI, and the resulting MFI was normalized to BDCA1 + DCs. Data shown are the mean MFI ± SD ( n = 3 independent experiments). (C) Antigen cross presentation via CD40 in DC subsets. Day 1 isolated DCs from HLA-A*0201 donors were fed with anti-CD40, or control isotype antibody conjugated to Flu-M1 (aa 55–72) at 1 µg/ml for 4 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells in the presence of IL-2 and TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown is one representative experiment of more than five. (D) As in C; anti-CD40 and control isotype antibodies were conjugated to CMV-pp65 (aa 488–508) at the indicated doses. Shown is one representative experiment of two.

    Journal: The Journal of Experimental Medicine

    Article Title: Antigen delivery to early endosomes eliminates the superiority of human blood BDCA3+ dendritic cells at cross presentation

    doi: 10.1084/jem.20121251

    Figure Lengend Snippet: Antigen targeted to early endosomes via CD40 is cross presented by both DC subsets with similar efficacy. (A) Anti-CD40 antibody intracellular trafficking. Day 1 BDCA1 + DCs (top) or BDCA3 + DCs (bottom) were fed with Alexa Fluor 488–labeled anti-CD40 antibody (green) continuously for 3 h at 37°C, washed, and allowed to adhere to coverslips. After fixation and permeabilization, the lysosomes or early endosomes were stained using anti-Lamp1 or anti-EEA1 (red), respectively. Plasma membrane was stained using anti–HLA-DR (blue) antibodies. Cells were then analyzed using confocal microscopy. Bars, 5 µm. Shown is one representative experiment of three. (B) Accumulation of anti-CD40 antibody. Day 1 DCs were fed with Alexa Fluor 488–labeled anti-CD40 antibody continuously for 4–6 h at 4°C or 37°C. Results were analyzed by flow cytometry. The 4°C MFI was subtracted from the 37°C MFI, and the resulting MFI was normalized to BDCA1 + DCs. Data shown are the mean MFI ± SD ( n = 3 independent experiments). (C) Antigen cross presentation via CD40 in DC subsets. Day 1 isolated DCs from HLA-A*0201 donors were fed with anti-CD40, or control isotype antibody conjugated to Flu-M1 (aa 55–72) at 1 µg/ml for 4 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells in the presence of IL-2 and TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown is one representative experiment of more than five. (D) As in C; anti-CD40 and control isotype antibodies were conjugated to CMV-pp65 (aa 488–508) at the indicated doses. Shown is one representative experiment of two.

    Article Snippet: After antigen uptake, DCs were washed extensively to remove free antibody or peptide and cultured with 1.5 × 106 CFSE (Invitrogen)-labeled CD8+ T cells at a DC/T cell ratio of 1:30 (unless otherwise indicated), in the presence of 20 U/ml IL-2 (Roche) and 1 µg/ml TLR7/8 L. After 8–10 d, cells were harvested and stained with Flu-M1 (aa 58–66) or CMV-pp65 (aa 495–503) pentamer for 15 min at room temperature, followed by labeling with antibodies against CD3, CD8, CD4, and CD19, fixation, and analysis by flow cytometry.

    Techniques: Labeling, Staining, Confocal Microscopy, Flow Cytometry, Cytometry, Isolation, Cell Culture

    BDCA3 + DC exhibit an enhanced ability to cross present antigen delivered to lysosomes. (A) Antigen cross presentation via DEC205 by BDCA3 + DCs and BDCA1 + DCs. Day 1 DCs from HLA-A*0201 donors were fed with anti-DEC205 or control isotype antibody conjugated to Flu-M1 (aa 55–72) at the indicated doses for 4 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells in the presence of IL-2 and TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown is one representative experiment of n > 6. (B) As in A; anti-DEC205 and the control isotype antibodies were conjugated to CMV-pp65 (aa 488–508). Shown is one representative experiment of two. (C) Accumulation of anti-DEC205 antibody. Day 1 DCs were fed with Alexa Fluor 488–labeled anti-DEC205 antibody continuously for 4–6 h at 4 or 37°C. Results were analyzed by flow cytometry. 4°C MFI was subtracted from the 37°C MFI, and the resulting MFI was normalized to BDCA1 + DCs. Data shown are the mean MFI ± SD ( n = 7 independent experiments). (D) Internalization of anti-DEC205 antibody. Day 1 DCs were incubated with Alexa Fluor 488–labeled anti-DEC205 antibody for 30 min at 4°C. The cells were then washed and cultured at 37°C for the indicated times. At each time point, cells were labeled with an Alexa Fluor 647–labeled anti–human IgG antibody to label remaining surface bound antibody. Cells were then analyzed by flow cytometry. The total DEC205 is Alexa Fluor 488–labeled antibody, whereas the surface DEC205 is the Alexa Fluor 647–labeled anti–human antibody. Shown is one representative experiment of four. (E) Anti-DEC205 antibody trafficking. Day 1 DCs were fed Alexa Fluor 488–labeled anti-DEC205 antibody (green) continuously for 3 h at 37°C, washed, and allowed to adhere to coverslips. After fixation and permeabilization, the lysosomes and cell membrane were stained using anti-Lamp1 (red) and anti–HLA-DR (blue) antibodies, respectively. Cells were then analyzed using confocal microscopy. Bars, 7.5 µm. Shown is one representative experiment of five. (F) Detection of the indicated lysosomal proteases by Western blot of day 1 BDCA1 + DCs, BDCA3 + DCs, and mo-DCs. DC subsets were lysed and analyzed by Western blot for lysosomal protease expression. Shown is one representative experiment of three.

    Journal: The Journal of Experimental Medicine

    Article Title: Antigen delivery to early endosomes eliminates the superiority of human blood BDCA3+ dendritic cells at cross presentation

    doi: 10.1084/jem.20121251

    Figure Lengend Snippet: BDCA3 + DC exhibit an enhanced ability to cross present antigen delivered to lysosomes. (A) Antigen cross presentation via DEC205 by BDCA3 + DCs and BDCA1 + DCs. Day 1 DCs from HLA-A*0201 donors were fed with anti-DEC205 or control isotype antibody conjugated to Flu-M1 (aa 55–72) at the indicated doses for 4 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells in the presence of IL-2 and TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown is one representative experiment of n > 6. (B) As in A; anti-DEC205 and the control isotype antibodies were conjugated to CMV-pp65 (aa 488–508). Shown is one representative experiment of two. (C) Accumulation of anti-DEC205 antibody. Day 1 DCs were fed with Alexa Fluor 488–labeled anti-DEC205 antibody continuously for 4–6 h at 4 or 37°C. Results were analyzed by flow cytometry. 4°C MFI was subtracted from the 37°C MFI, and the resulting MFI was normalized to BDCA1 + DCs. Data shown are the mean MFI ± SD ( n = 7 independent experiments). (D) Internalization of anti-DEC205 antibody. Day 1 DCs were incubated with Alexa Fluor 488–labeled anti-DEC205 antibody for 30 min at 4°C. The cells were then washed and cultured at 37°C for the indicated times. At each time point, cells were labeled with an Alexa Fluor 647–labeled anti–human IgG antibody to label remaining surface bound antibody. Cells were then analyzed by flow cytometry. The total DEC205 is Alexa Fluor 488–labeled antibody, whereas the surface DEC205 is the Alexa Fluor 647–labeled anti–human antibody. Shown is one representative experiment of four. (E) Anti-DEC205 antibody trafficking. Day 1 DCs were fed Alexa Fluor 488–labeled anti-DEC205 antibody (green) continuously for 3 h at 37°C, washed, and allowed to adhere to coverslips. After fixation and permeabilization, the lysosomes and cell membrane were stained using anti-Lamp1 (red) and anti–HLA-DR (blue) antibodies, respectively. Cells were then analyzed using confocal microscopy. Bars, 7.5 µm. Shown is one representative experiment of five. (F) Detection of the indicated lysosomal proteases by Western blot of day 1 BDCA1 + DCs, BDCA3 + DCs, and mo-DCs. DC subsets were lysed and analyzed by Western blot for lysosomal protease expression. Shown is one representative experiment of three.

    Article Snippet: After antigen uptake, DCs were washed extensively to remove free antibody or peptide and cultured with 1.5 × 106 CFSE (Invitrogen)-labeled CD8+ T cells at a DC/T cell ratio of 1:30 (unless otherwise indicated), in the presence of 20 U/ml IL-2 (Roche) and 1 µg/ml TLR7/8 L. After 8–10 d, cells were harvested and stained with Flu-M1 (aa 58–66) or CMV-pp65 (aa 495–503) pentamer for 15 min at room temperature, followed by labeling with antibodies against CD3, CD8, CD4, and CD19, fixation, and analysis by flow cytometry.

    Techniques: Cell Culture, Labeling, Flow Cytometry, Cytometry, Incubation, Staining, Confocal Microscopy, Western Blot, Expressing

    Antigens escaping from lysosomes are cross presented equally by both DC subsets. (A) Number of IAV particles associated with DCs. Day 1 DCs were fed either with fusion-competent or -incompetent replication-defective (heat inactivated, HI) IAV for 6 h at 37°C. The cells were then washed and allowed to adhere to coverslips. After fixation and permeabilization, the cells were labeled with an anti-NP antibody. Cells were analyzed by confocal microscopy. Numbers of cells associated with IAV particles were counted and quantified. Shown is one representative experiment of three. (B) Cross presentation of fusion-competent and fusion-incompetent HI IAV. Day 1 DCs from HLA-A*0201 donors were fed with fusion-competent pH 7.4–treated HI IAV or fusion-incompetent pH 4.5–treated HI IAV for 6 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells. 8–10 d later, CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown is one representative experiment of three. (C) Cross presentation of escape-competent and escape-incompetent KBMA L. monocytogenes . Day 1 DCs from HLA-A*0201 donors were fed for 1 h with escape-incompetent (LLO − ) and escape-competent (LLO + ) KBMA L. monocytogenes strains engineered to secrete ActAN100-Flu-M1 (aa 58–66) fusion protein. The cells were then washed and, as in B, cultured with autologous T cells to measure antigen cross presentation. Shown is one representative experiment of three.

    Journal: The Journal of Experimental Medicine

    Article Title: Antigen delivery to early endosomes eliminates the superiority of human blood BDCA3+ dendritic cells at cross presentation

    doi: 10.1084/jem.20121251

    Figure Lengend Snippet: Antigens escaping from lysosomes are cross presented equally by both DC subsets. (A) Number of IAV particles associated with DCs. Day 1 DCs were fed either with fusion-competent or -incompetent replication-defective (heat inactivated, HI) IAV for 6 h at 37°C. The cells were then washed and allowed to adhere to coverslips. After fixation and permeabilization, the cells were labeled with an anti-NP antibody. Cells were analyzed by confocal microscopy. Numbers of cells associated with IAV particles were counted and quantified. Shown is one representative experiment of three. (B) Cross presentation of fusion-competent and fusion-incompetent HI IAV. Day 1 DCs from HLA-A*0201 donors were fed with fusion-competent pH 7.4–treated HI IAV or fusion-incompetent pH 4.5–treated HI IAV for 6 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells. 8–10 d later, CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown is one representative experiment of three. (C) Cross presentation of escape-competent and escape-incompetent KBMA L. monocytogenes . Day 1 DCs from HLA-A*0201 donors were fed for 1 h with escape-incompetent (LLO − ) and escape-competent (LLO + ) KBMA L. monocytogenes strains engineered to secrete ActAN100-Flu-M1 (aa 58–66) fusion protein. The cells were then washed and, as in B, cultured with autologous T cells to measure antigen cross presentation. Shown is one representative experiment of three.

    Article Snippet: After antigen uptake, DCs were washed extensively to remove free antibody or peptide and cultured with 1.5 × 106 CFSE (Invitrogen)-labeled CD8+ T cells at a DC/T cell ratio of 1:30 (unless otherwise indicated), in the presence of 20 U/ml IL-2 (Roche) and 1 µg/ml TLR7/8 L. After 8–10 d, cells were harvested and stained with Flu-M1 (aa 58–66) or CMV-pp65 (aa 495–503) pentamer for 15 min at room temperature, followed by labeling with antibodies against CD3, CD8, CD4, and CD19, fixation, and analysis by flow cytometry.

    Techniques: Labeling, Confocal Microscopy, Cell Culture

    Antigen delivered to early endosomes via CD40 is more efficiently cross presented by all DC subsets than antigen delivered to lysosomes via DEC205. (A) Antibody accumulation. Day 1 DCs were fed with Alexa Fluor 488–labeled anti-DEC205 or anti-CD40 antibody continuously for 3–4 h at 4°C or 37°C. Results were analyzed by flow cytometry. The 4°C MFI was subtracted from the 37°C MFI, and resultant MFI was normalized to BDCA1 + DCs fed with anti-DEC205. MFI was also normalized for the number of fluorophores per antibody. Data shown are the mean MFI ± SD ( n = 5 independent experiments). (B and C) Antigen cross presentation via CD40 and DEC205 in DC subsets. Day 1 isolated DCs from HLA-A*0201 donors were fed with anti-DEC205, anti-CD40, or control isotype antibodies conjugated to Flu-M1 (aa 55–72) at indicated doses for 4 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells in the presence of IL-2 and TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cells were detected by staining with Flu-M1 (aa 58–66) pentamer. T cell proliferation was measured by CFSE dilution. The graphs shown in B show frequency of CFSE low , Flu-M1 (aa 58–66) pentamer-positive CD8 + T cells. In C, FACS plots of CD8 + T cells showing CFSE dilution in response to antigen presentation and numbers indicate frequency of CD8 + T cells in each quadrant. Shown is one representative experiment of three.

    Journal: The Journal of Experimental Medicine

    Article Title: Antigen delivery to early endosomes eliminates the superiority of human blood BDCA3+ dendritic cells at cross presentation

    doi: 10.1084/jem.20121251

    Figure Lengend Snippet: Antigen delivered to early endosomes via CD40 is more efficiently cross presented by all DC subsets than antigen delivered to lysosomes via DEC205. (A) Antibody accumulation. Day 1 DCs were fed with Alexa Fluor 488–labeled anti-DEC205 or anti-CD40 antibody continuously for 3–4 h at 4°C or 37°C. Results were analyzed by flow cytometry. The 4°C MFI was subtracted from the 37°C MFI, and resultant MFI was normalized to BDCA1 + DCs fed with anti-DEC205. MFI was also normalized for the number of fluorophores per antibody. Data shown are the mean MFI ± SD ( n = 5 independent experiments). (B and C) Antigen cross presentation via CD40 and DEC205 in DC subsets. Day 1 isolated DCs from HLA-A*0201 donors were fed with anti-DEC205, anti-CD40, or control isotype antibodies conjugated to Flu-M1 (aa 55–72) at indicated doses for 4 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells in the presence of IL-2 and TLR7/8 L. 8–10 d later, Flu-M1–specific CD8 + T cells were detected by staining with Flu-M1 (aa 58–66) pentamer. T cell proliferation was measured by CFSE dilution. The graphs shown in B show frequency of CFSE low , Flu-M1 (aa 58–66) pentamer-positive CD8 + T cells. In C, FACS plots of CD8 + T cells showing CFSE dilution in response to antigen presentation and numbers indicate frequency of CD8 + T cells in each quadrant. Shown is one representative experiment of three.

    Article Snippet: After antigen uptake, DCs were washed extensively to remove free antibody or peptide and cultured with 1.5 × 106 CFSE (Invitrogen)-labeled CD8+ T cells at a DC/T cell ratio of 1:30 (unless otherwise indicated), in the presence of 20 U/ml IL-2 (Roche) and 1 µg/ml TLR7/8 L. After 8–10 d, cells were harvested and stained with Flu-M1 (aa 58–66) or CMV-pp65 (aa 495–503) pentamer for 15 min at room temperature, followed by labeling with antibodies against CD3, CD8, CD4, and CD19, fixation, and analysis by flow cytometry.

    Techniques: Labeling, Flow Cytometry, Cytometry, Isolation, Cell Culture, Staining, FACS

    Antigen targeted to early endosomes via CD11c is cross presented by both DC subsets with similar efficacy. (A) Anti-CD11c antibody intracellular trafficking. Day 1 BDCA1 + DCs were fed with Alexa Fluor 488–labeled anti-CD11c antibody (green) continuously for 3 h, washed, and allowed to adhere to coverslips. After fixation and permeabilization, the early endosomes and plasma membrane were stained using EEA1 (red) and anti–HLA-DR (blue) antibodies, respectively. Cells were then analyzed using confocal microscopy. Bar, 5 µm. Shown is one representative experiment of three. (B) Antigen cross presentation via CD11c in DC subsets. Day 1 isolated DCs from HLA-A*0201 donors were fed with anti-CD11c or control isotype antibody conjugated to Flu-M1 (aa 55–72) at indicated doses for 4 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells in the presence of IL-2 and TLR7/8 L. 8–10 d later, CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown is one representative experiment of three.

    Journal: The Journal of Experimental Medicine

    Article Title: Antigen delivery to early endosomes eliminates the superiority of human blood BDCA3+ dendritic cells at cross presentation

    doi: 10.1084/jem.20121251

    Figure Lengend Snippet: Antigen targeted to early endosomes via CD11c is cross presented by both DC subsets with similar efficacy. (A) Anti-CD11c antibody intracellular trafficking. Day 1 BDCA1 + DCs were fed with Alexa Fluor 488–labeled anti-CD11c antibody (green) continuously for 3 h, washed, and allowed to adhere to coverslips. After fixation and permeabilization, the early endosomes and plasma membrane were stained using EEA1 (red) and anti–HLA-DR (blue) antibodies, respectively. Cells were then analyzed using confocal microscopy. Bar, 5 µm. Shown is one representative experiment of three. (B) Antigen cross presentation via CD11c in DC subsets. Day 1 isolated DCs from HLA-A*0201 donors were fed with anti-CD11c or control isotype antibody conjugated to Flu-M1 (aa 55–72) at indicated doses for 4 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8 + T cells in the presence of IL-2 and TLR7/8 L. 8–10 d later, CD8 + T cell expansion was evaluated by gating on CFSE low cells positive for Flu-M1 (aa 58–66) pentamer. Shown is one representative experiment of three.

    Article Snippet: After antigen uptake, DCs were washed extensively to remove free antibody or peptide and cultured with 1.5 × 106 CFSE (Invitrogen)-labeled CD8+ T cells at a DC/T cell ratio of 1:30 (unless otherwise indicated), in the presence of 20 U/ml IL-2 (Roche) and 1 µg/ml TLR7/8 L. After 8–10 d, cells were harvested and stained with Flu-M1 (aa 58–66) or CMV-pp65 (aa 495–503) pentamer for 15 min at room temperature, followed by labeling with antibodies against CD3, CD8, CD4, and CD19, fixation, and analysis by flow cytometry.

    Techniques: Labeling, Staining, Confocal Microscopy, Isolation, Cell Culture

    Anti-HIV Gagp24 antibody avidity following vaccination. Animals were immunized i.d. three times at week 0, 6 and 15 with αDCIR.Gag24 or the molar equivalent of the Gagp24 protein with or without poly(I:C). Serum HIV Gagp24-specific IgG antibody avidity was measured by ELISA 2 weeks after the third injection (week 17) with αDCIR.Gagp24 or Gagp24 without adjuvant (A) or with poly(I:C) (B) . Relative avidity index (RAI) (%) of Gagp24-specific IgG for the individual monkeys is presented.

    Journal: PLoS ONE

    Article Title: Delivering HIV Gagp24 to DCIR Induces Strong Antibody Responses In Vivo

    doi: 10.1371/journal.pone.0135513

    Figure Lengend Snippet: Anti-HIV Gagp24 antibody avidity following vaccination. Animals were immunized i.d. three times at week 0, 6 and 15 with αDCIR.Gag24 or the molar equivalent of the Gagp24 protein with or without poly(I:C). Serum HIV Gagp24-specific IgG antibody avidity was measured by ELISA 2 weeks after the third injection (week 17) with αDCIR.Gagp24 or Gagp24 without adjuvant (A) or with poly(I:C) (B) . Relative avidity index (RAI) (%) of Gagp24-specific IgG for the individual monkeys is presented.

    Article Snippet: HIV Gagp24 peptides Pools of 11 overlapping (staggered by 4 aa) 15-mer long HIV Gagp24 peptides (Bio-Synthesis, Lewisville, TX) were resuspended in DMSO (Sigma-Aldrich, St. Louis, MO).

    Techniques: Enzyme-linked Immunosorbent Assay, Injection

    Immunization with αDCIR.Gagp24 without adjuvant generates high titers of HIV Gagp24 antibody responses in NHPs. Animals were immunized i.d. three times at week 0, 6 and 15 with αDCIR.Gag24 or control hIgG4.Gagp24 or the molar equivalent of the HIV Gagp24 protein with or without poly(I:C). HIV Gagp24-specific IgG antibodies in serum were assessed at indicated time points post-immunization with αDCIR.Gagp24 or hIgG4.Gagp24 or Gagp24 without adjuvant (A) or with poly(I:C) (B) . Dashed lines indicates times of immunization. The serial measurements of Gagp24-specific antibody responses collected on individual monkeys over time among the vaccine groups were summarized by calculating the area under the titration curves for each monkey at each time point, and longitudinal differences between the vaccine groups were assessed by linear mixed model analysis. Data are presented as mean ± SEM. *, p

    Journal: PLoS ONE

    Article Title: Delivering HIV Gagp24 to DCIR Induces Strong Antibody Responses In Vivo

    doi: 10.1371/journal.pone.0135513

    Figure Lengend Snippet: Immunization with αDCIR.Gagp24 without adjuvant generates high titers of HIV Gagp24 antibody responses in NHPs. Animals were immunized i.d. three times at week 0, 6 and 15 with αDCIR.Gag24 or control hIgG4.Gagp24 or the molar equivalent of the HIV Gagp24 protein with or without poly(I:C). HIV Gagp24-specific IgG antibodies in serum were assessed at indicated time points post-immunization with αDCIR.Gagp24 or hIgG4.Gagp24 or Gagp24 without adjuvant (A) or with poly(I:C) (B) . Dashed lines indicates times of immunization. The serial measurements of Gagp24-specific antibody responses collected on individual monkeys over time among the vaccine groups were summarized by calculating the area under the titration curves for each monkey at each time point, and longitudinal differences between the vaccine groups were assessed by linear mixed model analysis. Data are presented as mean ± SEM. *, p

    Article Snippet: HIV Gagp24 peptides Pools of 11 overlapping (staggered by 4 aa) 15-mer long HIV Gagp24 peptides (Bio-Synthesis, Lewisville, TX) were resuspended in DMSO (Sigma-Aldrich, St. Louis, MO).

    Techniques: Titration

    Low doses of αDCIR.Gagp24 expand a broad repertoire of HIV Gagp24-specific T cells. A. Experimental procedure. ICS, intracellular staining. B. PBMCs from an HIV-infected patient (patient A15) were cultured for 10 days in medium alone (top panel), or with 0.3 nM hIgG4.Gagp24 (middle panel), or 0.3 nM αDCIR.Gagp24 (lower panel) and then rechallenged for 6 h with or without (-C) 5 clusters (CL) of 15-mer overlapping peptides covering HIV Gagp24 in the presence of brefeldin A prior to intracellular IFNγ staining. The flow cytometry profiles are gated on the overall CD3 + T cell population. Data are representative of 4 different patients. C. PBMCs from 4 HIV-infected patients (patient A1, A2. A12 and A15) were cultured for 10 days with 0.3 nM (filed symbols) or 30 pM (open symbols) of αDCIR.Gagp24 or hIgG4.Gagp24 and then restimulated for 48 hrs with 5 clusters (CL) of 15-mer overlapping peptides covering HIV Gagp24. The culture supernatants were then harvested and IFNγ produced by total T cells was analyzed by multiplex bead-based assay. Each patient is represented by a different symbol. Data are presented as mean of sum of HIV Gagp24 clusters. Differences between αDCIR.Gagp24 and hIgG4.Gagp24 were assessed by a matched paired t-test.

    Journal: PLoS ONE

    Article Title: Delivering HIV Gagp24 to DCIR Induces Strong Antibody Responses In Vivo

    doi: 10.1371/journal.pone.0135513

    Figure Lengend Snippet: Low doses of αDCIR.Gagp24 expand a broad repertoire of HIV Gagp24-specific T cells. A. Experimental procedure. ICS, intracellular staining. B. PBMCs from an HIV-infected patient (patient A15) were cultured for 10 days in medium alone (top panel), or with 0.3 nM hIgG4.Gagp24 (middle panel), or 0.3 nM αDCIR.Gagp24 (lower panel) and then rechallenged for 6 h with or without (-C) 5 clusters (CL) of 15-mer overlapping peptides covering HIV Gagp24 in the presence of brefeldin A prior to intracellular IFNγ staining. The flow cytometry profiles are gated on the overall CD3 + T cell population. Data are representative of 4 different patients. C. PBMCs from 4 HIV-infected patients (patient A1, A2. A12 and A15) were cultured for 10 days with 0.3 nM (filed symbols) or 30 pM (open symbols) of αDCIR.Gagp24 or hIgG4.Gagp24 and then restimulated for 48 hrs with 5 clusters (CL) of 15-mer overlapping peptides covering HIV Gagp24. The culture supernatants were then harvested and IFNγ produced by total T cells was analyzed by multiplex bead-based assay. Each patient is represented by a different symbol. Data are presented as mean of sum of HIV Gagp24 clusters. Differences between αDCIR.Gagp24 and hIgG4.Gagp24 were assessed by a matched paired t-test.

    Article Snippet: HIV Gagp24 peptides Pools of 11 overlapping (staggered by 4 aa) 15-mer long HIV Gagp24 peptides (Bio-Synthesis, Lewisville, TX) were resuspended in DMSO (Sigma-Aldrich, St. Louis, MO).

    Techniques: Staining, Infection, Cell Culture, Flow Cytometry, Cytometry, Produced, Multiplex Assay, Bead-based Assay

    Characterization of the αDCIR.Gagp24 fusion rAbs. A. Schematic representation and SDS-PAGE analysis under reducing conditions of the αDCIR.Gagp24 fusion protein. Expression constructs for chimeric mouse variable (V) region-human IgG4 constant (C) region αDCIR and isotype control recombinant antibodies (rAbs) were engineered with the HIV Gagp24 coding region fused in frame to the heavy (H) chain C-terminus (left panel). These constructs were co-transfected with a matching light (k) chain expression vector into stable CHO-S cell lines and the secreted fusion rAbs were purified from the culture supernatants by protein A-affinity chromatography and analyzed on reducing SDS-PAGE. Proteins were stained with Coomassie Blue (right panel). B. Specific binding of αDCIR.Gagp24 rAb. Monocytes (left panel), B cells (middle panel) and T cells (right panel) from an HIV-infected patient PBMCs were treated with 3 nM, 0.3 nM and 30 pM of αDCIR.Gagp24 and 3 nM hIgG4.Gagp24 fusion proteins, followed by incubation with an anti-HIV Gagp24 PE-conjugated antibody and then analyzed by flow cytometry. Blue traces are staining from αDCIR.Gagp24. The blue color gradient represents the different concentrations from light blue (3 nM) to dark blue (30 pM). Grey traces (staining by 3 nM hIgG4.Gagp24) and grey solid traces (untreated cells) are the background staining. Data are representative of 2 different patients.

    Journal: PLoS ONE

    Article Title: Delivering HIV Gagp24 to DCIR Induces Strong Antibody Responses In Vivo

    doi: 10.1371/journal.pone.0135513

    Figure Lengend Snippet: Characterization of the αDCIR.Gagp24 fusion rAbs. A. Schematic representation and SDS-PAGE analysis under reducing conditions of the αDCIR.Gagp24 fusion protein. Expression constructs for chimeric mouse variable (V) region-human IgG4 constant (C) region αDCIR and isotype control recombinant antibodies (rAbs) were engineered with the HIV Gagp24 coding region fused in frame to the heavy (H) chain C-terminus (left panel). These constructs were co-transfected with a matching light (k) chain expression vector into stable CHO-S cell lines and the secreted fusion rAbs were purified from the culture supernatants by protein A-affinity chromatography and analyzed on reducing SDS-PAGE. Proteins were stained with Coomassie Blue (right panel). B. Specific binding of αDCIR.Gagp24 rAb. Monocytes (left panel), B cells (middle panel) and T cells (right panel) from an HIV-infected patient PBMCs were treated with 3 nM, 0.3 nM and 30 pM of αDCIR.Gagp24 and 3 nM hIgG4.Gagp24 fusion proteins, followed by incubation with an anti-HIV Gagp24 PE-conjugated antibody and then analyzed by flow cytometry. Blue traces are staining from αDCIR.Gagp24. The blue color gradient represents the different concentrations from light blue (3 nM) to dark blue (30 pM). Grey traces (staining by 3 nM hIgG4.Gagp24) and grey solid traces (untreated cells) are the background staining. Data are representative of 2 different patients.

    Article Snippet: HIV Gagp24 peptides Pools of 11 overlapping (staggered by 4 aa) 15-mer long HIV Gagp24 peptides (Bio-Synthesis, Lewisville, TX) were resuspended in DMSO (Sigma-Aldrich, St. Louis, MO).

    Techniques: SDS Page, Expressing, Construct, Recombinant, Transfection, Plasmid Preparation, Purification, Affinity Chromatography, Staining, Binding Assay, Infection, Incubation, Flow Cytometry, Cytometry

    αDCIR.Gagp24 expands multifunctional HIV Gagp24-specific CD4 + T cells in vitro . PBMCs from HIV-infected patients (panel A , patient A15; panel B , patients A2, A1, A15) were cultured for 10 days with 0.3 nM of αDCIR.Gagp24 or hIgG4.Gag p24 or left unstimulated and then restimulated for 6 hrs with or without clusters of 15-mer overlapping peptides covering HIV Gagp24 in the presence of brefeldin A prior to intracellular cytokine staining. The profiles are gated on the overall CD4 + T cell populations, with the percentages of populations responding to the designated peptides shown in each plot. A . Coordinate analysis of TNFα versus MIP-1β, IFNγ or CD154 expression (patient A15). Data are representative of 4 different patients. B . Multifunctionality of the cytokine responses. Combined percentages of 5 CD4 + T cell populations producing different combinations of at least 3 cytokines (IFNγ, TNFα, MIP-1β or CD154). Data are presented as mean of sum of HIV Gagp24 clusters ± SEM for 3 patients. Differences between αDCIR.Gagp24 and hIgG4.Gagp24 were assessed by a matched paired t-test.

    Journal: PLoS ONE

    Article Title: Delivering HIV Gagp24 to DCIR Induces Strong Antibody Responses In Vivo

    doi: 10.1371/journal.pone.0135513

    Figure Lengend Snippet: αDCIR.Gagp24 expands multifunctional HIV Gagp24-specific CD4 + T cells in vitro . PBMCs from HIV-infected patients (panel A , patient A15; panel B , patients A2, A1, A15) were cultured for 10 days with 0.3 nM of αDCIR.Gagp24 or hIgG4.Gag p24 or left unstimulated and then restimulated for 6 hrs with or without clusters of 15-mer overlapping peptides covering HIV Gagp24 in the presence of brefeldin A prior to intracellular cytokine staining. The profiles are gated on the overall CD4 + T cell populations, with the percentages of populations responding to the designated peptides shown in each plot. A . Coordinate analysis of TNFα versus MIP-1β, IFNγ or CD154 expression (patient A15). Data are representative of 4 different patients. B . Multifunctionality of the cytokine responses. Combined percentages of 5 CD4 + T cell populations producing different combinations of at least 3 cytokines (IFNγ, TNFα, MIP-1β or CD154). Data are presented as mean of sum of HIV Gagp24 clusters ± SEM for 3 patients. Differences between αDCIR.Gagp24 and hIgG4.Gagp24 were assessed by a matched paired t-test.

    Article Snippet: HIV Gagp24 peptides Pools of 11 overlapping (staggered by 4 aa) 15-mer long HIV Gagp24 peptides (Bio-Synthesis, Lewisville, TX) were resuspended in DMSO (Sigma-Aldrich, St. Louis, MO).

    Techniques: In Vitro, Infection, Cell Culture, Staining, Expressing

    Inhibition and the effect of hot-start. ( a ) Inhibition profiles of 2D9 versus AmpliTaq Gold 360 (ATaqG) in PCR. The effect of various inhibitors (SP61: bone dust, Tar, Soil, 1736; cave sediment) on polymerase activity of 2D9 and ATaqG as determined by a PCR assay in the presence of decreasing amounts of inhibitor and plotted as the average relative resistance ( n = 3, standard error) (as in Figure 4 ) of 2D9 and ATaqG with respect to ATaqG as a reference (resistance = 1). ( b ) The effect of manual hot-start on resistance of 2D9 polymerase to inhibitors tar and soil at minimal limiting amounts of inhibitor. Hot-start increases inhibitor resistance by at least a factor of two.

    Journal: Nucleic Acids Research

    Article Title: Molecular breeding of polymerases for resistance to environmental inhibitors

    doi: 10.1093/nar/gkq1360

    Figure Lengend Snippet: Inhibition and the effect of hot-start. ( a ) Inhibition profiles of 2D9 versus AmpliTaq Gold 360 (ATaqG) in PCR. The effect of various inhibitors (SP61: bone dust, Tar, Soil, 1736; cave sediment) on polymerase activity of 2D9 and ATaqG as determined by a PCR assay in the presence of decreasing amounts of inhibitor and plotted as the average relative resistance ( n = 3, standard error) (as in Figure 4 ) of 2D9 and ATaqG with respect to ATaqG as a reference (resistance = 1). ( b ) The effect of manual hot-start on resistance of 2D9 polymerase to inhibitors tar and soil at minimal limiting amounts of inhibitor. Hot-start increases inhibitor resistance by at least a factor of two.

    Article Snippet: Inhibition profiles for AmpliTaq Gold 360 DNA polymerase (Applied Biosystems) were determined using an identical PCR set-up but using AmpliTaq Gold buffer and thermocycling conditions according to manufacturer’s recommendations.

    Techniques: Inhibition, Polymerase Chain Reaction, Activity Assay

    Protection against heterologous challenge with a Puerto Rican ZIKV isolate. ( a ) Six animals (three females and three males) were re-challenged subcutaneously with 1 × 10 5 TCID 50 of a PR ZIKV isolate 45 d after primary infection. Viral RNA levels in blood plasma were monitored between days 1 and 21 after the secondary challenge. ZIKV RNA copies in blood plasma are indicated. A single positive sample, detected in one male monkey 1 d after infection, is indicated (blue solid line). Lymphocyte activation and frequencies were measured during re-challenge. Activation and numbers of T cells, B cells, NK cells and monocytes were measured by flow cytometry on days 0, 1, 2, 3, 4, 5 and 7 after ZIKV infection of three male and three female monkeys. ( b , c ) Percentage of activated (CD69 + ) and total numbers of naive (CD28 + CD95 − ) (left), central memory (CD28 + CD95 + ) (middle), and effector and effector memory (CD28 − CD95 + ) (right) CD4 + ( b ) and CD8 + ( c ) T cell subsets in whole blood. ( d ) Percentage of activated (CD69 + ) and total numbers of CD16 + (left), CD16 − CD56 + (middle) and CD16 − CD56 − (right) NK cells in whole blood. ( e ) CD38 expression (geometric mean fluorescence of CD38) and total numbers of naive (CD27 − ) (left) and memory (CD27 + ) (right) B cells in whole blood. ( f ) Percentage of activated and total numbers of monocytes in whole blood. Black line indicates median. Individual values are shown for males (blue) and females (red). ( g . Values below this threshold were set to a value of 0. Values greater than 1.5-fold above that of the day of re-challenge were considered to be anamnestic. Each monkey is indicated by a different color symbol. Triangles indicate males, and circles indicate females.

    Journal: Nature medicine

    Article Title: Zika viral dynamics and shedding in rhesus and cynomolgus macaques

    doi: 10.1038/nm.4206

    Figure Lengend Snippet: Protection against heterologous challenge with a Puerto Rican ZIKV isolate. ( a ) Six animals (three females and three males) were re-challenged subcutaneously with 1 × 10 5 TCID 50 of a PR ZIKV isolate 45 d after primary infection. Viral RNA levels in blood plasma were monitored between days 1 and 21 after the secondary challenge. ZIKV RNA copies in blood plasma are indicated. A single positive sample, detected in one male monkey 1 d after infection, is indicated (blue solid line). Lymphocyte activation and frequencies were measured during re-challenge. Activation and numbers of T cells, B cells, NK cells and monocytes were measured by flow cytometry on days 0, 1, 2, 3, 4, 5 and 7 after ZIKV infection of three male and three female monkeys. ( b , c ) Percentage of activated (CD69 + ) and total numbers of naive (CD28 + CD95 − ) (left), central memory (CD28 + CD95 + ) (middle), and effector and effector memory (CD28 − CD95 + ) (right) CD4 + ( b ) and CD8 + ( c ) T cell subsets in whole blood. ( d ) Percentage of activated (CD69 + ) and total numbers of CD16 + (left), CD16 − CD56 + (middle) and CD16 − CD56 − (right) NK cells in whole blood. ( e ) CD38 expression (geometric mean fluorescence of CD38) and total numbers of naive (CD27 − ) (left) and memory (CD27 + ) (right) B cells in whole blood. ( f ) Percentage of activated and total numbers of monocytes in whole blood. Black line indicates median. Individual values are shown for males (blue) and females (red). ( g . Values below this threshold were set to a value of 0. Values greater than 1.5-fold above that of the day of re-challenge were considered to be anamnestic. Each monkey is indicated by a different color symbol. Triangles indicate males, and circles indicate females.

    Article Snippet: The following conjugated antibodies and staining reagents were used for surface staining: CD4 (L200), CD95 (DX2), CD28 (L293), CD8 (SK1) and Yellow LIVE/DEAD Fixable Dead Cell Stain (Invitrogen).

    Techniques: Infection, Activation Assay, Flow Cytometry, Cytometry, Expressing, Fluorescence

    Protection against heterologous challenge with a Puerto Rican ZIKV isolate. ( a ) Six animals (three females and three males) were re-challenged subcutaneously with 1 × 10 5 TCID 50 of a PR ZIKV isolate 45 d after primary infection. Viral RNA levels in blood plasma were monitored between days 1 and 21 after the secondary challenge. ZIKV RNA copies in blood plasma are indicated. A single positive sample, detected in one male monkey 1 d after infection, is indicated (blue solid line). Lymphocyte activation and frequencies were measured during re-challenge. Activation and numbers of T cells, B cells, NK cells and monocytes were measured by flow cytometry on days 0, 1, 2, 3, 4, 5 and 7 after ZIKV infection of three male and three female monkeys. ( b , c ) Percentage of activated (CD69 + ) and total numbers of naive (CD28 + CD95 − ) (left), central memory (CD28 + CD95 + ) (middle), and effector and effector memory (CD28 − CD95 + ) (right) CD4 + ( b ) and CD8 + ( c ) T cell subsets in whole blood. ( d ) Percentage of activated (CD69 + ) and total numbers of CD16 + (left), CD16 − CD56 + (middle) and CD16 − CD56 − (right) NK cells in whole blood. ( e ) CD38 expression (geometric mean fluorescence of CD38) and total numbers of naive (CD27 − ) (left) and memory (CD27 + ) (right) B cells in whole blood. ( f ) Percentage of activated and total numbers of monocytes in whole blood. Black line indicates median. Individual values are shown for males (blue) and females (red). ( g . Values below this threshold were set to a value of 0. Values greater than 1.5-fold above that of the day of re-challenge were considered to be anamnestic. Each monkey is indicated by a different color symbol. Triangles indicate males, and circles indicate females.

    Journal: Nature medicine

    Article Title: Zika viral dynamics and shedding in rhesus and cynomolgus macaques

    doi: 10.1038/nm.4206

    Figure Lengend Snippet: Protection against heterologous challenge with a Puerto Rican ZIKV isolate. ( a ) Six animals (three females and three males) were re-challenged subcutaneously with 1 × 10 5 TCID 50 of a PR ZIKV isolate 45 d after primary infection. Viral RNA levels in blood plasma were monitored between days 1 and 21 after the secondary challenge. ZIKV RNA copies in blood plasma are indicated. A single positive sample, detected in one male monkey 1 d after infection, is indicated (blue solid line). Lymphocyte activation and frequencies were measured during re-challenge. Activation and numbers of T cells, B cells, NK cells and monocytes were measured by flow cytometry on days 0, 1, 2, 3, 4, 5 and 7 after ZIKV infection of three male and three female monkeys. ( b , c ) Percentage of activated (CD69 + ) and total numbers of naive (CD28 + CD95 − ) (left), central memory (CD28 + CD95 + ) (middle), and effector and effector memory (CD28 − CD95 + ) (right) CD4 + ( b ) and CD8 + ( c ) T cell subsets in whole blood. ( d ) Percentage of activated (CD69 + ) and total numbers of CD16 + (left), CD16 − CD56 + (middle) and CD16 − CD56 − (right) NK cells in whole blood. ( e ) CD38 expression (geometric mean fluorescence of CD38) and total numbers of naive (CD27 − ) (left) and memory (CD27 + ) (right) B cells in whole blood. ( f ) Percentage of activated and total numbers of monocytes in whole blood. Black line indicates median. Individual values are shown for males (blue) and females (red). ( g . Values below this threshold were set to a value of 0. Values greater than 1.5-fold above that of the day of re-challenge were considered to be anamnestic. Each monkey is indicated by a different color symbol. Triangles indicate males, and circles indicate females.

    Article Snippet: The following conjugated antibodies and staining reagents were used for surface staining: CD4 (L200), CD95 (DX2), CD28 (L293), CD8 (SK1) and Yellow LIVE/DEAD Fixable Dead Cell Stain (Invitrogen).

    Techniques: Infection, Activation Assay, Flow Cytometry, Cytometry, Expressing, Fluorescence

    Proliferative capacity of CD4 T cells is not reversible by PD-1 blockade in human immunodeficiency virus (HIV)–infected children. CellTrace Violet (CTV)–labeled peripheral blood mononuclear cells from HIV-infected children were stimulated with anti-CD3 or HIV Gag peptide pool in the presence of PD-1 blocking antibody or isotype control antibody. Flow cytometry from a representative donor indicating the percentage of CTV dim CD4 + T cells after 7 days of anti-CD3( A ) or HIV Gag ( C ) peptide stimulation is shown. Graphs depict summary of proliferative responses to anti-CD3 ( B ) and HIV Gag ( D ) peptide pool in the presence of PD-1 blocking antibody in HIV-infected children. Statistical analysis was performed using the ratio paired t test. Triangles indicate viremic subjects.

    Journal: The Journal of Infectious Diseases

    Article Title: HIV-Infected Children Have Elevated Levels of PD-1+ Memory CD4 T Cells With Low Proliferative Capacity and High Inflammatory Cytokine Effector Functions

    doi: 10.1093/infdis/jix341

    Figure Lengend Snippet: Proliferative capacity of CD4 T cells is not reversible by PD-1 blockade in human immunodeficiency virus (HIV)–infected children. CellTrace Violet (CTV)–labeled peripheral blood mononuclear cells from HIV-infected children were stimulated with anti-CD3 or HIV Gag peptide pool in the presence of PD-1 blocking antibody or isotype control antibody. Flow cytometry from a representative donor indicating the percentage of CTV dim CD4 + T cells after 7 days of anti-CD3( A ) or HIV Gag ( C ) peptide stimulation is shown. Graphs depict summary of proliferative responses to anti-CD3 ( B ) and HIV Gag ( D ) peptide pool in the presence of PD-1 blocking antibody in HIV-infected children. Statistical analysis was performed using the ratio paired t test. Triangles indicate viremic subjects.

    Article Snippet: PBMCs were labeled with CellTrace Violet Cell Proliferation kit (Invitrogen), then stimulated with soluble anti-CD3 or HIV-specific pooled Gag peptide (2 μg/mL) and CD28/CD49 (1 μg/mL).

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

    PD-1 + CD4 memory T cells (T M ) have decreased proliferative capacity in healthy adults and predict proliferative capacity in human immunodeficiency virus (HIV)–infected children. Peripheral blood mononuclear cells (PBMCs) from healthy adult donors were sorted into PD-1 – and PD-1 + memory CD4 T-cell subsets by live cell sorting. A and B , Carboxyfluorescein succinimidyl ester (CFSE)-labeled PD-1 – and PD-1 + CD4 memory T cells were stimulated with anti-CD3 (Clone OKT3) and monocyte-derived dendritic cells; cell proliferation was assessed on day 4. A , Representative flow plots show percent of proliferating cells measured by dilution of CFSE in PD-1 – and PD-1 + CD4 memory T cells sorted from the same donor. B , Paired comparison of proliferation between PD-1 – and PD-1 + sorted memory CD4 T cells from 3 adult donors. P value was calculated with a ratio paired t test. C , PBMCs from HIV-infected children were labeled with CellTrace Violet then either left unstimulated (no antigen), or stimulated with anti-CD3 or HIV Gag peptide pool. Representative flow plots of proliferation of CD4 + T cells on day 7 are shown from 1 donor. D , Linear regression analysis of baseline percentage of PD-1 + CD4 memory T cells vs the percentage of proliferating CD4 T cells after anti-CD3 and HIV Gag peptide stimulation. Triangles indicate viremic subjects.

    Journal: The Journal of Infectious Diseases

    Article Title: HIV-Infected Children Have Elevated Levels of PD-1+ Memory CD4 T Cells With Low Proliferative Capacity and High Inflammatory Cytokine Effector Functions

    doi: 10.1093/infdis/jix341

    Figure Lengend Snippet: PD-1 + CD4 memory T cells (T M ) have decreased proliferative capacity in healthy adults and predict proliferative capacity in human immunodeficiency virus (HIV)–infected children. Peripheral blood mononuclear cells (PBMCs) from healthy adult donors were sorted into PD-1 – and PD-1 + memory CD4 T-cell subsets by live cell sorting. A and B , Carboxyfluorescein succinimidyl ester (CFSE)-labeled PD-1 – and PD-1 + CD4 memory T cells were stimulated with anti-CD3 (Clone OKT3) and monocyte-derived dendritic cells; cell proliferation was assessed on day 4. A , Representative flow plots show percent of proliferating cells measured by dilution of CFSE in PD-1 – and PD-1 + CD4 memory T cells sorted from the same donor. B , Paired comparison of proliferation between PD-1 – and PD-1 + sorted memory CD4 T cells from 3 adult donors. P value was calculated with a ratio paired t test. C , PBMCs from HIV-infected children were labeled with CellTrace Violet then either left unstimulated (no antigen), or stimulated with anti-CD3 or HIV Gag peptide pool. Representative flow plots of proliferation of CD4 + T cells on day 7 are shown from 1 donor. D , Linear regression analysis of baseline percentage of PD-1 + CD4 memory T cells vs the percentage of proliferating CD4 T cells after anti-CD3 and HIV Gag peptide stimulation. Triangles indicate viremic subjects.

    Article Snippet: PBMCs were labeled with CellTrace Violet Cell Proliferation kit (Invitrogen), then stimulated with soluble anti-CD3 or HIV-specific pooled Gag peptide (2 μg/mL) and CD28/CD49 (1 μg/mL).

    Techniques: Infection, FACS, Labeling, Derivative Assay, Flow Cytometry