cy5 Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher cy5
    Cy5, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3652 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cy5/product/Thermo Fisher
    Average 99 stars, based on 3652 article reviews
    Price from $9.99 to $1999.99
    cy5 - by Bioz Stars, 2020-12
    99/100 stars
      Buy from Supplier

    99
    GE Healthcare cy5
    Comparative analyses of biomolecular activities on the DT20 and PEG surfaces. (a) Detection of the dynamics of Holliday junction through FRET. (b) The FRET histograms of single Holliday junctions obtained at 80 mM Mg 2+ . (c) The transition rates of Holliday junction decreases as the Mg 2+ concentration increases. Error bars indicate s.d. (d) PcrA reeling causes the looping of 5’ ssDNA tail. (e) Representative asymmetric sawtooth-shaped intensity and FRET-time traces of 5’ ssDNA tail looping (green and magenta for Cy3 and <t>Cy5</t> signals, respectively). (f) ∆t histograms obtained at different ATP concentrations, 10 µM ATP (top) and 500 µM ATP (bottom). (g) The FRET signal distinguishes PCNA bound DinB from non-specifically bound DinB. A Holliday junction structure is used to prevent PCNA from sliding off the ssDNA region. 14 (h) Fluorescence images of DinB-PCNA interaction showing the overlay of the Cy3 (green) and Cy5 (magenta) channels for both surfaces. The scale bar indicates 4.5 µm.
    Cy5, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 7791 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cy5/product/GE Healthcare
    Average 99 stars, based on 7791 article reviews
    Price from $9.99 to $1999.99
    cy5 - by Bioz Stars, 2020-12
    99/100 stars
      Buy from Supplier

    94
    Agilent technologies cy5
    Validation of Whole Genome Transposon Analysis Using Two Sequenced Strains of S. cerevisiae (A) Whole genome comparison of full-length Ty1 and Ty2 elements from yeast strains RM11 and S288c after hybridization to the same Agilent yeast whole genome microarray. Black circles indicate the position of Ty1 or Ty2 full-length elements annotated for S288c in SGD. Triangles indicate full-length Ty2 elements identified in the sequence of RM11. Red peaks correspond to potential Ty1 or Ty2 elements present in S288c, while green peaks correspond to potential Ty1 or Ty2 peaks present in RM11. (B) Comparison of location of Ty1 full-length elements (green) and Ty2 full-length elements present in S288c. Symbols are as above. Numbers above various peaks refer to the following: 1, false-negative S288c elements obscured by overlapping elements in RM11; 2, false-negative S288c elements located in regions that are poorly represented by features on the array; 3, false-negative S288c elements that missed the criteria for calling a peak; 4, unannotated Ty1 full-length elements in S288c confirmed in this study; 5, false-positive peaks due to borderline elevated differential hybridization; 6, false-positive peaks corresponding to non-Ty repetitive elements in the genome. Grey horizontal lines above and below the central line for each chromosome correspond to a 3-fold difference in normalized ratio of <t>Cy5</t> and Cy3 signal intensity.
    Cy5, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 4382 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cy5/product/Agilent technologies
    Average 94 stars, based on 4382 article reviews
    Price from $9.99 to $1999.99
    cy5 - by Bioz Stars, 2020-12
    94/100 stars
      Buy from Supplier

    95
    GE Healthcare cy5 dctp
    Validation of Whole Genome Transposon Analysis Using Two Sequenced Strains of S. cerevisiae (A) Whole genome comparison of full-length Ty1 and Ty2 elements from yeast strains RM11 and S288c after hybridization to the same Agilent yeast whole genome microarray. Black circles indicate the position of Ty1 or Ty2 full-length elements annotated for S288c in SGD. Triangles indicate full-length Ty2 elements identified in the sequence of RM11. Red peaks correspond to potential Ty1 or Ty2 elements present in S288c, while green peaks correspond to potential Ty1 or Ty2 peaks present in RM11. (B) Comparison of location of Ty1 full-length elements (green) and Ty2 full-length elements present in S288c. Symbols are as above. Numbers above various peaks refer to the following: 1, false-negative S288c elements obscured by overlapping elements in RM11; 2, false-negative S288c elements located in regions that are poorly represented by features on the array; 3, false-negative S288c elements that missed the criteria for calling a peak; 4, unannotated Ty1 full-length elements in S288c confirmed in this study; 5, false-positive peaks due to borderline elevated differential hybridization; 6, false-positive peaks corresponding to non-Ty repetitive elements in the genome. Grey horizontal lines above and below the central line for each chromosome correspond to a 3-fold difference in normalized ratio of <t>Cy5</t> and Cy3 signal intensity.
    Cy5 Dctp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 95/100, based on 2453 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cy5 dctp/product/GE Healthcare
    Average 95 stars, based on 2453 article reviews
    Price from $9.99 to $1999.99
    cy5 dctp - by Bioz Stars, 2020-12
    95/100 stars
      Buy from Supplier

    96
    GE Healthcare cy5 dutp
    Validation of Whole Genome Transposon Analysis Using Two Sequenced Strains of S. cerevisiae (A) Whole genome comparison of full-length Ty1 and Ty2 elements from yeast strains RM11 and S288c after hybridization to the same Agilent yeast whole genome microarray. Black circles indicate the position of Ty1 or Ty2 full-length elements annotated for S288c in SGD. Triangles indicate full-length Ty2 elements identified in the sequence of RM11. Red peaks correspond to potential Ty1 or Ty2 elements present in S288c, while green peaks correspond to potential Ty1 or Ty2 peaks present in RM11. (B) Comparison of location of Ty1 full-length elements (green) and Ty2 full-length elements present in S288c. Symbols are as above. Numbers above various peaks refer to the following: 1, false-negative S288c elements obscured by overlapping elements in RM11; 2, false-negative S288c elements located in regions that are poorly represented by features on the array; 3, false-negative S288c elements that missed the criteria for calling a peak; 4, unannotated Ty1 full-length elements in S288c confirmed in this study; 5, false-positive peaks due to borderline elevated differential hybridization; 6, false-positive peaks corresponding to non-Ty repetitive elements in the genome. Grey horizontal lines above and below the central line for each chromosome correspond to a 3-fold difference in normalized ratio of <t>Cy5</t> and Cy3 signal intensity.
    Cy5 Dutp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 96/100, based on 2444 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cy5 dutp/product/GE Healthcare
    Average 96 stars, based on 2444 article reviews
    Price from $9.99 to $1999.99
    cy5 dutp - by Bioz Stars, 2020-12
    96/100 stars
      Buy from Supplier

    94
    Becton Dickinson percp cy5 5
    Validation of Whole Genome Transposon Analysis Using Two Sequenced Strains of S. cerevisiae (A) Whole genome comparison of full-length Ty1 and Ty2 elements from yeast strains RM11 and S288c after hybridization to the same Agilent yeast whole genome microarray. Black circles indicate the position of Ty1 or Ty2 full-length elements annotated for S288c in SGD. Triangles indicate full-length Ty2 elements identified in the sequence of RM11. Red peaks correspond to potential Ty1 or Ty2 elements present in S288c, while green peaks correspond to potential Ty1 or Ty2 peaks present in RM11. (B) Comparison of location of Ty1 full-length elements (green) and Ty2 full-length elements present in S288c. Symbols are as above. Numbers above various peaks refer to the following: 1, false-negative S288c elements obscured by overlapping elements in RM11; 2, false-negative S288c elements located in regions that are poorly represented by features on the array; 3, false-negative S288c elements that missed the criteria for calling a peak; 4, unannotated Ty1 full-length elements in S288c confirmed in this study; 5, false-positive peaks due to borderline elevated differential hybridization; 6, false-positive peaks corresponding to non-Ty repetitive elements in the genome. Grey horizontal lines above and below the central line for each chromosome correspond to a 3-fold difference in normalized ratio of <t>Cy5</t> and Cy3 signal intensity.
    Percp Cy5 5, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 2511 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/percp cy5 5/product/Becton Dickinson
    Average 94 stars, based on 2511 article reviews
    Price from $9.99 to $1999.99
    percp cy5 5 - by Bioz Stars, 2020-12
    94/100 stars
      Buy from Supplier

    94
    Becton Dickinson cd4 percp cy5 5
    HIV-specific TGF-β-positive <t>CD4</t> + T cells are CTLA-4 negative. PBMCs ( n = 6) were stimulated with HIV peptides and then stained with anti-TGF-β PE, anti-CD3 AmCyan, anti-CD4 <t>PerCP</t> <t>Cy5.5,</t> anti-CD8 PE Cy7, and anti-CTLA-4 APC, and analyzed by flow cytometry. Gating on the CTLA-4-positive cells was performed using the fluorescence-minus-one (FMO) control for CTLA-4. ( A ) Representative plots of samples that were first gated on the CD3 + /CD4 + and CD3 + /CD8 + lymphocyte population and then the percentages of CTLA-4-positive cells were determined. ( B ) Representative plots of samples that were first gated on the CD3 + /CD4 + lymphocyte population and then the percent of TGF-β-positive cells that expressed CTLA-4 was determined after subtraction of the background values. The values marked with an asterisk represent the fraction of TGF-β-positive cells that expresses CTLA-4 over the total number of TGF-β-positive cells (equivalent to 100%).
    Cd4 Percp Cy5 5, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 1250 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cd4 percp cy5 5/product/Becton Dickinson
    Average 94 stars, based on 1250 article reviews
    Price from $9.99 to $1999.99
    cd4 percp cy5 5 - by Bioz Stars, 2020-12
    94/100 stars
      Buy from Supplier

    94
    Becton Dickinson cd3 percp cy5 5
    HIV-specific TGF-β-positive <t>CD4</t> + T cells are CTLA-4 negative. PBMCs ( n = 6) were stimulated with HIV peptides and then stained with anti-TGF-β PE, anti-CD3 AmCyan, anti-CD4 <t>PerCP</t> <t>Cy5.5,</t> anti-CD8 PE Cy7, and anti-CTLA-4 APC, and analyzed by flow cytometry. Gating on the CTLA-4-positive cells was performed using the fluorescence-minus-one (FMO) control for CTLA-4. ( A ) Representative plots of samples that were first gated on the CD3 + /CD4 + and CD3 + /CD8 + lymphocyte population and then the percentages of CTLA-4-positive cells were determined. ( B ) Representative plots of samples that were first gated on the CD3 + /CD4 + lymphocyte population and then the percent of TGF-β-positive cells that expressed CTLA-4 was determined after subtraction of the background values. The values marked with an asterisk represent the fraction of TGF-β-positive cells that expresses CTLA-4 over the total number of TGF-β-positive cells (equivalent to 100%).
    Cd3 Percp Cy5 5, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 949 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cd3 percp cy5 5/product/Becton Dickinson
    Average 94 stars, based on 949 article reviews
    Price from $9.99 to $1999.99
    cd3 percp cy5 5 - by Bioz Stars, 2020-12
    94/100 stars
      Buy from Supplier

    92
    The Jackson Laboratory cy5
    Magnetic activation is not dependent upon the targeting moiety. Stably transfected HeLa cells expressing chimeric ACP-EGFR, enzymatically modified by biotin CoA and carrying bound Strv-SPIONs. (a) 30 seconds, (b) 180 seconds of magnetization, (c) no magnetization. Green channel, Strv-SPION-biocytin-Alexa546; red channel, phosphorylated-EGFR (pY-EGFR 1068 and <t>GARIG-CY5),</t> overlay of green and red channels. Scale bar, 10 µm.
    Cy5, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 92/100, based on 687 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cy5/product/The Jackson Laboratory
    Average 92 stars, based on 687 article reviews
    Price from $9.99 to $1999.99
    cy5 - by Bioz Stars, 2020-12
    92/100 stars
      Buy from Supplier

    92
    Becton Dickinson cd8 percp cy5 5
    Magnetic activation is not dependent upon the targeting moiety. Stably transfected HeLa cells expressing chimeric ACP-EGFR, enzymatically modified by biotin CoA and carrying bound Strv-SPIONs. (a) 30 seconds, (b) 180 seconds of magnetization, (c) no magnetization. Green channel, Strv-SPION-biocytin-Alexa546; red channel, phosphorylated-EGFR (pY-EGFR 1068 and <t>GARIG-CY5),</t> overlay of green and red channels. Scale bar, 10 µm.
    Cd8 Percp Cy5 5, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 764 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cd8 percp cy5 5/product/Becton Dickinson
    Average 92 stars, based on 764 article reviews
    Price from $9.99 to $1999.99
    cd8 percp cy5 5 - by Bioz Stars, 2020-12
    92/100 stars
      Buy from Supplier

    99
    Becton Dickinson anti cd4 percp cy5 5
    Magnetic activation is not dependent upon the targeting moiety. Stably transfected HeLa cells expressing chimeric ACP-EGFR, enzymatically modified by biotin CoA and carrying bound Strv-SPIONs. (a) 30 seconds, (b) 180 seconds of magnetization, (c) no magnetization. Green channel, Strv-SPION-biocytin-Alexa546; red channel, phosphorylated-EGFR (pY-EGFR 1068 and <t>GARIG-CY5),</t> overlay of green and red channels. Scale bar, 10 µm.
    Anti Cd4 Percp Cy5 5, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 736 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti cd4 percp cy5 5/product/Becton Dickinson
    Average 99 stars, based on 736 article reviews
    Price from $9.99 to $1999.99
    anti cd4 percp cy5 5 - by Bioz Stars, 2020-12
    99/100 stars
      Buy from Supplier

    94
    Becton Dickinson pe cy5
    Magnetic activation is not dependent upon the targeting moiety. Stably transfected HeLa cells expressing chimeric ACP-EGFR, enzymatically modified by biotin CoA and carrying bound Strv-SPIONs. (a) 30 seconds, (b) 180 seconds of magnetization, (c) no magnetization. Green channel, Strv-SPION-biocytin-Alexa546; red channel, phosphorylated-EGFR (pY-EGFR 1068 and <t>GARIG-CY5),</t> overlay of green and red channels. Scale bar, 10 µm.
    Pe Cy5, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 1009 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pe cy5/product/Becton Dickinson
    Average 94 stars, based on 1009 article reviews
    Price from $9.99 to $1999.99
    pe cy5 - by Bioz Stars, 2020-12
    94/100 stars
      Buy from Supplier

    99
    GE Healthcare cy5 nhs ester
    Magnetic activation is not dependent upon the targeting moiety. Stably transfected HeLa cells expressing chimeric ACP-EGFR, enzymatically modified by biotin CoA and carrying bound Strv-SPIONs. (a) 30 seconds, (b) 180 seconds of magnetization, (c) no magnetization. Green channel, Strv-SPION-biocytin-Alexa546; red channel, phosphorylated-EGFR (pY-EGFR 1068 and <t>GARIG-CY5),</t> overlay of green and red channels. Scale bar, 10 µm.
    Cy5 Nhs Ester, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 657 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cy5 nhs ester/product/GE Healthcare
    Average 99 stars, based on 657 article reviews
    Price from $9.99 to $1999.99
    cy5 nhs ester - by Bioz Stars, 2020-12
    99/100 stars
      Buy from Supplier

    91
    Agilent technologies cy5 labeled crna
    Magnetic activation is not dependent upon the targeting moiety. Stably transfected HeLa cells expressing chimeric ACP-EGFR, enzymatically modified by biotin CoA and carrying bound Strv-SPIONs. (a) 30 seconds, (b) 180 seconds of magnetization, (c) no magnetization. Green channel, Strv-SPION-biocytin-Alexa546; red channel, phosphorylated-EGFR (pY-EGFR 1068 and <t>GARIG-CY5),</t> overlay of green and red channels. Scale bar, 10 µm.
    Cy5 Labeled Crna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 91/100, based on 787 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cy5 labeled crna/product/Agilent technologies
    Average 91 stars, based on 787 article reviews
    Price from $9.99 to $1999.99
    cy5 labeled crna - by Bioz Stars, 2020-12
    91/100 stars
      Buy from Supplier

    99
    Thermo Fisher streptavidin cy5
    Magnetic activation is not dependent upon the targeting moiety. Stably transfected HeLa cells expressing chimeric ACP-EGFR, enzymatically modified by biotin CoA and carrying bound Strv-SPIONs. (a) 30 seconds, (b) 180 seconds of magnetization, (c) no magnetization. Green channel, Strv-SPION-biocytin-Alexa546; red channel, phosphorylated-EGFR (pY-EGFR 1068 and <t>GARIG-CY5),</t> overlay of green and red channels. Scale bar, 10 µm.
    Streptavidin Cy5, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 627 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin cy5/product/Thermo Fisher
    Average 99 stars, based on 627 article reviews
    Price from $9.99 to $1999.99
    streptavidin cy5 - by Bioz Stars, 2020-12
    99/100 stars
      Buy from Supplier

    91
    Becton Dickinson anti cd8 percp cy5 5
    Magnetic activation is not dependent upon the targeting moiety. Stably transfected HeLa cells expressing chimeric ACP-EGFR, enzymatically modified by biotin CoA and carrying bound Strv-SPIONs. (a) 30 seconds, (b) 180 seconds of magnetization, (c) no magnetization. Green channel, Strv-SPION-biocytin-Alexa546; red channel, phosphorylated-EGFR (pY-EGFR 1068 and <t>GARIG-CY5),</t> overlay of green and red channels. Scale bar, 10 µm.
    Anti Cd8 Percp Cy5 5, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 548 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti cd8 percp cy5 5/product/Becton Dickinson
    Average 91 stars, based on 548 article reviews
    Price from $9.99 to $1999.99
    anti cd8 percp cy5 5 - by Bioz Stars, 2020-12
    91/100 stars
      Buy from Supplier

    91
    Becton Dickinson anti cd3 percp cy5 5
    Heat shock protein 60 ( HSP 60) induces dendritic cell ( DC ) activation/maturation; 1 of 3 individual experiments is presented here. DC s at the concentration of 2×10 6 /mL were cultured with or without HSP 60. A, Expression of CD 86, CD 83, CD 40, and HLA ‐ II was analyzed by flow cytometry after 24 h of stimulation with 5 μg/mL of HSP 60. Figure in histogram shows higher induction of these markers by HSP 60. Percentage of histogram shift or mean fluorescence intensity was calculated from triplicate samples and P values are CD 86 ≤0.001, CD 83 ≤0.001, HLA ‐ II ≤0.05, and CD 40 ≤0.001. B, Cytokine profile of DC against HSP 60 is listed. DC s were stimulated as mentioned, cultured for 24 h, and cell supernatant was collected for measurement of cytokines. Mostly pro‐inflammatory cytokines are highly increased by HSP 60. C, In similar condition, DC s activation was observed by HSP 90 (5 μg/mL). P value from triplicates samples CD 86 ≤0.0001, CD 83 ≤0.001, CD 40 ≤0.01, and HLA ‐ II ≤0.001. D, HSP 90 induced‐ DC s were cocultured with T cells but no activation of T cells was observed. APC indicates antigen‐presenting cells; FITC, fluorescein isothiocyanate; IL‐6, interleukin‐6; <t>Percp‐Cy5.5,</t> Peridinin Chlorophyll Protein‐Cyanine 5.5; TGF‐β1, transforming growth factor‐β1.
    Anti Cd3 Percp Cy5 5, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 471 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti cd3 percp cy5 5/product/Becton Dickinson
    Average 91 stars, based on 471 article reviews
    Price from $9.99 to $1999.99
    anti cd3 percp cy5 5 - by Bioz Stars, 2020-12
    91/100 stars
      Buy from Supplier

    97
    Thermo Fisher pe cy5
    Heat shock protein 60 ( HSP 60) induces dendritic cell ( DC ) activation/maturation; 1 of 3 individual experiments is presented here. DC s at the concentration of 2×10 6 /mL were cultured with or without HSP 60. A, Expression of CD 86, CD 83, CD 40, and HLA ‐ II was analyzed by flow cytometry after 24 h of stimulation with 5 μg/mL of HSP 60. Figure in histogram shows higher induction of these markers by HSP 60. Percentage of histogram shift or mean fluorescence intensity was calculated from triplicate samples and P values are CD 86 ≤0.001, CD 83 ≤0.001, HLA ‐ II ≤0.05, and CD 40 ≤0.001. B, Cytokine profile of DC against HSP 60 is listed. DC s were stimulated as mentioned, cultured for 24 h, and cell supernatant was collected for measurement of cytokines. Mostly pro‐inflammatory cytokines are highly increased by HSP 60. C, In similar condition, DC s activation was observed by HSP 90 (5 μg/mL). P value from triplicates samples CD 86 ≤0.0001, CD 83 ≤0.001, CD 40 ≤0.01, and HLA ‐ II ≤0.001. D, HSP 90 induced‐ DC s were cocultured with T cells but no activation of T cells was observed. APC indicates antigen‐presenting cells; FITC, fluorescein isothiocyanate; IL‐6, interleukin‐6; <t>Percp‐Cy5.5,</t> Peridinin Chlorophyll Protein‐Cyanine 5.5; TGF‐β1, transforming growth factor‐β1.
    Pe Cy5, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 771 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pe cy5/product/Thermo Fisher
    Average 97 stars, based on 771 article reviews
    Price from $9.99 to $1999.99
    pe cy5 - by Bioz Stars, 2020-12
    97/100 stars
      Buy from Supplier

    99
    Jackson Immuno cy5 affinipure donkey anti mouse igg
    Heat shock protein 60 ( HSP 60) induces dendritic cell ( DC ) activation/maturation; 1 of 3 individual experiments is presented here. DC s at the concentration of 2×10 6 /mL were cultured with or without HSP 60. A, Expression of CD 86, CD 83, CD 40, and HLA ‐ II was analyzed by flow cytometry after 24 h of stimulation with 5 μg/mL of HSP 60. Figure in histogram shows higher induction of these markers by HSP 60. Percentage of histogram shift or mean fluorescence intensity was calculated from triplicate samples and P values are CD 86 ≤0.001, CD 83 ≤0.001, HLA ‐ II ≤0.05, and CD 40 ≤0.001. B, Cytokine profile of DC against HSP 60 is listed. DC s were stimulated as mentioned, cultured for 24 h, and cell supernatant was collected for measurement of cytokines. Mostly pro‐inflammatory cytokines are highly increased by HSP 60. C, In similar condition, DC s activation was observed by HSP 90 (5 μg/mL). P value from triplicates samples CD 86 ≤0.0001, CD 83 ≤0.001, CD 40 ≤0.01, and HLA ‐ II ≤0.001. D, HSP 90 induced‐ DC s were cocultured with T cells but no activation of T cells was observed. APC indicates antigen‐presenting cells; FITC, fluorescein isothiocyanate; IL‐6, interleukin‐6; <t>Percp‐Cy5.5,</t> Peridinin Chlorophyll Protein‐Cyanine 5.5; TGF‐β1, transforming growth factor‐β1.
    Cy5 Affinipure Donkey Anti Mouse Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 99/100, based on 339 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cy5 affinipure donkey anti mouse igg/product/Jackson Immuno
    Average 99 stars, based on 339 article reviews
    Price from $9.99 to $1999.99
    cy5 affinipure donkey anti mouse igg - by Bioz Stars, 2020-12
    99/100 stars
      Buy from Supplier

    99
    Jackson Immuno cy5 affinipure donkey anti rabbit igg
    Heat shock protein 60 ( HSP 60) induces dendritic cell ( DC ) activation/maturation; 1 of 3 individual experiments is presented here. DC s at the concentration of 2×10 6 /mL were cultured with or without HSP 60. A, Expression of CD 86, CD 83, CD 40, and HLA ‐ II was analyzed by flow cytometry after 24 h of stimulation with 5 μg/mL of HSP 60. Figure in histogram shows higher induction of these markers by HSP 60. Percentage of histogram shift or mean fluorescence intensity was calculated from triplicate samples and P values are CD 86 ≤0.001, CD 83 ≤0.001, HLA ‐ II ≤0.05, and CD 40 ≤0.001. B, Cytokine profile of DC against HSP 60 is listed. DC s were stimulated as mentioned, cultured for 24 h, and cell supernatant was collected for measurement of cytokines. Mostly pro‐inflammatory cytokines are highly increased by HSP 60. C, In similar condition, DC s activation was observed by HSP 90 (5 μg/mL). P value from triplicates samples CD 86 ≤0.0001, CD 83 ≤0.001, CD 40 ≤0.01, and HLA ‐ II ≤0.001. D, HSP 90 induced‐ DC s were cocultured with T cells but no activation of T cells was observed. APC indicates antigen‐presenting cells; FITC, fluorescein isothiocyanate; IL‐6, interleukin‐6; <t>Percp‐Cy5.5,</t> Peridinin Chlorophyll Protein‐Cyanine 5.5; TGF‐β1, transforming growth factor‐β1.
    Cy5 Affinipure Donkey Anti Rabbit Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 99/100, based on 292 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cy5 affinipure donkey anti rabbit igg/product/Jackson Immuno
    Average 99 stars, based on 292 article reviews
    Price from $9.99 to $1999.99
    cy5 affinipure donkey anti rabbit igg - by Bioz Stars, 2020-12
    99/100 stars
      Buy from Supplier

    99
    BioLegend percp cy5 5
    Heat shock protein 60 ( HSP 60) induces dendritic cell ( DC ) activation/maturation; 1 of 3 individual experiments is presented here. DC s at the concentration of 2×10 6 /mL were cultured with or without HSP 60. A, Expression of CD 86, CD 83, CD 40, and HLA ‐ II was analyzed by flow cytometry after 24 h of stimulation with 5 μg/mL of HSP 60. Figure in histogram shows higher induction of these markers by HSP 60. Percentage of histogram shift or mean fluorescence intensity was calculated from triplicate samples and P values are CD 86 ≤0.001, CD 83 ≤0.001, HLA ‐ II ≤0.05, and CD 40 ≤0.001. B, Cytokine profile of DC against HSP 60 is listed. DC s were stimulated as mentioned, cultured for 24 h, and cell supernatant was collected for measurement of cytokines. Mostly pro‐inflammatory cytokines are highly increased by HSP 60. C, In similar condition, DC s activation was observed by HSP 90 (5 μg/mL). P value from triplicates samples CD 86 ≤0.0001, CD 83 ≤0.001, CD 40 ≤0.01, and HLA ‐ II ≤0.001. D, HSP 90 induced‐ DC s were cocultured with T cells but no activation of T cells was observed. APC indicates antigen‐presenting cells; FITC, fluorescein isothiocyanate; IL‐6, interleukin‐6; <t>Percp‐Cy5.5,</t> Peridinin Chlorophyll Protein‐Cyanine 5.5; TGF‐β1, transforming growth factor‐β1.
    Percp Cy5 5, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 2232 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/percp cy5 5/product/BioLegend
    Average 99 stars, based on 2232 article reviews
    Price from $9.99 to $1999.99
    percp cy5 5 - by Bioz Stars, 2020-12
    99/100 stars
      Buy from Supplier

    cy5  (Dianova)
    93
    Dianova cy5
    Flotillin-2 knockdown inhibits APP endocytosis in N2a cells and primary hippocampal neurons. A , CFP–swAPP-transfected N2a cells were labeled at 4°C with anti-GFP antibody, washed, and incubated at 37°C for 5, 20, and 45 min to allow internalization of antibodies bound to CFP–swAPP. To visualize cell-surface retained APP, cells were labeled with <t>Cy5-conjugated</t> secondary antibody at 4°C (pseudocolored in blue). After fixing, cells were permeabilized and stained with a Cy3-conjugated secondary antibody to resolve internalized APP (red). The cell surface was identified on confocal sections by the Cy5 label, and APP endocytosis was measured by calculating the intensity ratio of (cell interior Cy3)/(surface Cy5) labeled CFP–swAPP. The graph shows the increase in internalized APP over the time course of 45 min, the corresponding confocal images display the increasing amount of internalized APP (Cy3), whereas Cy5-labeled surface APP decreases over time ( n > 95 cells, values represent means + SEM; * p
    Cy5, supplied by Dianova, used in various techniques. Bioz Stars score: 93/100, based on 408 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cy5/product/Dianova
    Average 93 stars, based on 408 article reviews
    Price from $9.99 to $1999.99
    cy5 - by Bioz Stars, 2020-12
    93/100 stars
      Buy from Supplier

    99
    BioLegend percp cy5 5 anti mouse cd45
    Flotillin-2 knockdown inhibits APP endocytosis in N2a cells and primary hippocampal neurons. A , CFP–swAPP-transfected N2a cells were labeled at 4°C with anti-GFP antibody, washed, and incubated at 37°C for 5, 20, and 45 min to allow internalization of antibodies bound to CFP–swAPP. To visualize cell-surface retained APP, cells were labeled with <t>Cy5-conjugated</t> secondary antibody at 4°C (pseudocolored in blue). After fixing, cells were permeabilized and stained with a Cy3-conjugated secondary antibody to resolve internalized APP (red). The cell surface was identified on confocal sections by the Cy5 label, and APP endocytosis was measured by calculating the intensity ratio of (cell interior Cy3)/(surface Cy5) labeled CFP–swAPP. The graph shows the increase in internalized APP over the time course of 45 min, the corresponding confocal images display the increasing amount of internalized APP (Cy3), whereas Cy5-labeled surface APP decreases over time ( n > 95 cells, values represent means + SEM; * p
    Percp Cy5 5 Anti Mouse Cd45, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 300 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/percp cy5 5 anti mouse cd45/product/BioLegend
    Average 99 stars, based on 300 article reviews
    Price from $9.99 to $1999.99
    percp cy5 5 anti mouse cd45 - by Bioz Stars, 2020-12
    99/100 stars
      Buy from Supplier

    94
    GE Healthcare cy5 5
    Flotillin-2 knockdown inhibits APP endocytosis in N2a cells and primary hippocampal neurons. A , CFP–swAPP-transfected N2a cells were labeled at 4°C with anti-GFP antibody, washed, and incubated at 37°C for 5, 20, and 45 min to allow internalization of antibodies bound to CFP–swAPP. To visualize cell-surface retained APP, cells were labeled with <t>Cy5-conjugated</t> secondary antibody at 4°C (pseudocolored in blue). After fixing, cells were permeabilized and stained with a Cy3-conjugated secondary antibody to resolve internalized APP (red). The cell surface was identified on confocal sections by the Cy5 label, and APP endocytosis was measured by calculating the intensity ratio of (cell interior Cy3)/(surface Cy5) labeled CFP–swAPP. The graph shows the increase in internalized APP over the time course of 45 min, the corresponding confocal images display the increasing amount of internalized APP (Cy3), whereas Cy5-labeled surface APP decreases over time ( n > 95 cells, values represent means + SEM; * p
    Cy5 5, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 469 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cy5 5/product/GE Healthcare
    Average 94 stars, based on 469 article reviews
    Price from $9.99 to $1999.99
    cy5 5 - by Bioz Stars, 2020-12
    94/100 stars
      Buy from Supplier

    N/A
    Cyanine 5 Bihexanoic Acid Dye Potassium Salt is a water soluble amine reactive fluorescent reagent Suggested to be used for protein and peptide labeling
      Buy from Supplier

    N/A
    A fluorescent dye that is thiol reactive and water soluble
      Buy from Supplier

    N/A
    Cyanine 5 Monofunctional Hexanoic Acid Dye Potassium Salt is a water soluble thiol reactive fluorescent reagent Suggested to be used for protein and peptide labeling
      Buy from Supplier

    N/A
    Cyanine 5 Monofunctional MTSEA Dye Potassium Salt is a water soluble thiol reactive fluorescent reagent Suggested to be used for protein and peptide labeling
      Buy from Supplier

    Image Search Results


    Comparative analyses of biomolecular activities on the DT20 and PEG surfaces. (a) Detection of the dynamics of Holliday junction through FRET. (b) The FRET histograms of single Holliday junctions obtained at 80 mM Mg 2+ . (c) The transition rates of Holliday junction decreases as the Mg 2+ concentration increases. Error bars indicate s.d. (d) PcrA reeling causes the looping of 5’ ssDNA tail. (e) Representative asymmetric sawtooth-shaped intensity and FRET-time traces of 5’ ssDNA tail looping (green and magenta for Cy3 and Cy5 signals, respectively). (f) ∆t histograms obtained at different ATP concentrations, 10 µM ATP (top) and 500 µM ATP (bottom). (g) The FRET signal distinguishes PCNA bound DinB from non-specifically bound DinB. A Holliday junction structure is used to prevent PCNA from sliding off the ssDNA region. 14 (h) Fluorescence images of DinB-PCNA interaction showing the overlay of the Cy3 (green) and Cy5 (magenta) channels for both surfaces. The scale bar indicates 4.5 µm.

    Journal: Nature methods

    Article Title: An Improved Surface Passivation Method for Single-Molecule Studies

    doi: 10.1038/nmeth.3143

    Figure Lengend Snippet: Comparative analyses of biomolecular activities on the DT20 and PEG surfaces. (a) Detection of the dynamics of Holliday junction through FRET. (b) The FRET histograms of single Holliday junctions obtained at 80 mM Mg 2+ . (c) The transition rates of Holliday junction decreases as the Mg 2+ concentration increases. Error bars indicate s.d. (d) PcrA reeling causes the looping of 5’ ssDNA tail. (e) Representative asymmetric sawtooth-shaped intensity and FRET-time traces of 5’ ssDNA tail looping (green and magenta for Cy3 and Cy5 signals, respectively). (f) ∆t histograms obtained at different ATP concentrations, 10 µM ATP (top) and 500 µM ATP (bottom). (g) The FRET signal distinguishes PCNA bound DinB from non-specifically bound DinB. A Holliday junction structure is used to prevent PCNA from sliding off the ssDNA region. 14 (h) Fluorescence images of DinB-PCNA interaction showing the overlay of the Cy3 (green) and Cy5 (magenta) channels for both surfaces. The scale bar indicates 4.5 µm.

    Article Snippet: Purified proteins were pre-incubated in 1 ml of reaction buffer (80 mM K-Hepes, pH 7.6, 1 M KCl, 1 mM TCEP, 3 M urea) at 20 °C for 30 min, then reacted with a 6-fold molar excess of maleimide-linked Cy3 or Cy5 (GE Healthcare) at 20 °C for another 2 hours.

    Techniques: Concentration Assay, Fluorescence

    The DT20 surface and its improved passivation capacity. (a) The schematics of the DT20 surface. (b) Non-specific binding of Cy5-labeled DinB on the DT20 and PEG surfaces, with and without NeutrAvidin, measured by the average surface spot counts of DinB over an imaging area of 2500 µm 2 at different concentrations of DinB. (c) The surface spot counts over an imaging area of 2500 µm 2 for non-specific binding of seven different proteins. Rep was labeled with Cy5 and tested at pH 8.0, four ribosomal proteins (S4, S16, S17 and S20) were labeled with Cy3 and tested at pH 7.6, IgG # 1 (611-701-127, Rockland) and IgG # 2 (A-11004, Invitrogen) were labeled with Alexa 647 and Alexa 568, respectively and tested at pH 8.0. (d) Fluorescence images of the surface (DT20) immobilized streptavidin-Alexa 594 (SA-A594) by confocal and STED microscope in the presence of 100 nM diffusing SA-A594 (top). Line profiles of single SA-A594 marked by white arrows (bottom). (e) Non-specific binding tests of the DT20 and PEG surfaces incubated with 100 nM SA-A594 by STED microscope. (f) Non-specific binding of Cy3-labeled ssDNA at different concentrations, measured at pH 8.0 and pH 7.1. (g) The surface spot counts over an imaging area of 2500 µm 2 for non-specific binding of 500 nM ssDNA (18 nt) labeled with three different fluorophores. All error bars in Fig. 1 b, c, f, g indicate standard deviation (s.d.).

    Journal: Nature methods

    Article Title: An Improved Surface Passivation Method for Single-Molecule Studies

    doi: 10.1038/nmeth.3143

    Figure Lengend Snippet: The DT20 surface and its improved passivation capacity. (a) The schematics of the DT20 surface. (b) Non-specific binding of Cy5-labeled DinB on the DT20 and PEG surfaces, with and without NeutrAvidin, measured by the average surface spot counts of DinB over an imaging area of 2500 µm 2 at different concentrations of DinB. (c) The surface spot counts over an imaging area of 2500 µm 2 for non-specific binding of seven different proteins. Rep was labeled with Cy5 and tested at pH 8.0, four ribosomal proteins (S4, S16, S17 and S20) were labeled with Cy3 and tested at pH 7.6, IgG # 1 (611-701-127, Rockland) and IgG # 2 (A-11004, Invitrogen) were labeled with Alexa 647 and Alexa 568, respectively and tested at pH 8.0. (d) Fluorescence images of the surface (DT20) immobilized streptavidin-Alexa 594 (SA-A594) by confocal and STED microscope in the presence of 100 nM diffusing SA-A594 (top). Line profiles of single SA-A594 marked by white arrows (bottom). (e) Non-specific binding tests of the DT20 and PEG surfaces incubated with 100 nM SA-A594 by STED microscope. (f) Non-specific binding of Cy3-labeled ssDNA at different concentrations, measured at pH 8.0 and pH 7.1. (g) The surface spot counts over an imaging area of 2500 µm 2 for non-specific binding of 500 nM ssDNA (18 nt) labeled with three different fluorophores. All error bars in Fig. 1 b, c, f, g indicate standard deviation (s.d.).

    Article Snippet: Purified proteins were pre-incubated in 1 ml of reaction buffer (80 mM K-Hepes, pH 7.6, 1 M KCl, 1 mM TCEP, 3 M urea) at 20 °C for 30 min, then reacted with a 6-fold molar excess of maleimide-linked Cy3 or Cy5 (GE Healthcare) at 20 °C for another 2 hours.

    Techniques: Binding Assay, Labeling, Imaging, Fluorescence, Microscopy, Incubation, Standard Deviation

    Biochemical preparation and DNA cleavage activity of dye-labeled Cas9 a , Size exclusion chromatograms of Cy3/Cy5-labeling reactions with cysteine-free Cas9 (C80S/C574S) or the two double-cysteine Cas9 variants used to generate Cas9 hinge and Cas9 HNH-1 . Reactions contained 10 μM Cas9 and 200 μM Cy3- and Cy5-maleimide, and were separated on a Superdex 200 10/300 column (GE Healthcare). Cysteine-free Cas9 was unreactive. b , Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of unlabeled and dye-labeled Cas9 variants. The gel was scanned for Cy3 and Cy5 fluorescence (right) before being stained with Coommassie blue (left). For gel source data, see Supplementary Figure 1 . c, Representative radiolabeled DNA cleavage assay with wild-type (WT) Cas9 and doubly-labeled Cas9 variants used in this study, resolved by denaturing PAGE (left); quantified data and exponential fits are shown on the right. S, substrate; NT, cleaved non-target strand; T, cleaved target strand. Error bars represent the standard deviation from three experiments.

    Journal: Nature

    Article Title: Conformational control of DNA target cleavage by CRISPR–Cas9

    doi: 10.1038/nature15544

    Figure Lengend Snippet: Biochemical preparation and DNA cleavage activity of dye-labeled Cas9 a , Size exclusion chromatograms of Cy3/Cy5-labeling reactions with cysteine-free Cas9 (C80S/C574S) or the two double-cysteine Cas9 variants used to generate Cas9 hinge and Cas9 HNH-1 . Reactions contained 10 μM Cas9 and 200 μM Cy3- and Cy5-maleimide, and were separated on a Superdex 200 10/300 column (GE Healthcare). Cysteine-free Cas9 was unreactive. b , Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of unlabeled and dye-labeled Cas9 variants. The gel was scanned for Cy3 and Cy5 fluorescence (right) before being stained with Coommassie blue (left). For gel source data, see Supplementary Figure 1 . c, Representative radiolabeled DNA cleavage assay with wild-type (WT) Cas9 and doubly-labeled Cas9 variants used in this study, resolved by denaturing PAGE (left); quantified data and exponential fits are shown on the right. S, substrate; NT, cleaved non-target strand; T, cleaved target strand. Error bars represent the standard deviation from three experiments.

    Article Snippet: Preparation of dye-labeled Cas9 Labeling reactions were conducted in Cas9 gel filtration buffer (20 mM Tris-Cl pH 7.5, 200 mM KCl, 5% glycerol, 1 mM TCEP) and contained 10 μM Cas9 and 200 μM Cy3- and Cy5-maleimide (GE Healthcare).

    Techniques: Activity Assay, Labeling, Polyacrylamide Gel Electrophoresis, SDS Page, Fluorescence, Staining, DNA Cleavage Assay, Standard Deviation

    Validation of Whole Genome Transposon Analysis Using Two Sequenced Strains of S. cerevisiae (A) Whole genome comparison of full-length Ty1 and Ty2 elements from yeast strains RM11 and S288c after hybridization to the same Agilent yeast whole genome microarray. Black circles indicate the position of Ty1 or Ty2 full-length elements annotated for S288c in SGD. Triangles indicate full-length Ty2 elements identified in the sequence of RM11. Red peaks correspond to potential Ty1 or Ty2 elements present in S288c, while green peaks correspond to potential Ty1 or Ty2 peaks present in RM11. (B) Comparison of location of Ty1 full-length elements (green) and Ty2 full-length elements present in S288c. Symbols are as above. Numbers above various peaks refer to the following: 1, false-negative S288c elements obscured by overlapping elements in RM11; 2, false-negative S288c elements located in regions that are poorly represented by features on the array; 3, false-negative S288c elements that missed the criteria for calling a peak; 4, unannotated Ty1 full-length elements in S288c confirmed in this study; 5, false-positive peaks due to borderline elevated differential hybridization; 6, false-positive peaks corresponding to non-Ty repetitive elements in the genome. Grey horizontal lines above and below the central line for each chromosome correspond to a 3-fold difference in normalized ratio of Cy5 and Cy3 signal intensity.

    Journal: PLoS Genetics

    Article Title: Global Mapping of Transposon Location

    doi: 10.1371/journal.pgen.0020212

    Figure Lengend Snippet: Validation of Whole Genome Transposon Analysis Using Two Sequenced Strains of S. cerevisiae (A) Whole genome comparison of full-length Ty1 and Ty2 elements from yeast strains RM11 and S288c after hybridization to the same Agilent yeast whole genome microarray. Black circles indicate the position of Ty1 or Ty2 full-length elements annotated for S288c in SGD. Triangles indicate full-length Ty2 elements identified in the sequence of RM11. Red peaks correspond to potential Ty1 or Ty2 elements present in S288c, while green peaks correspond to potential Ty1 or Ty2 peaks present in RM11. (B) Comparison of location of Ty1 full-length elements (green) and Ty2 full-length elements present in S288c. Symbols are as above. Numbers above various peaks refer to the following: 1, false-negative S288c elements obscured by overlapping elements in RM11; 2, false-negative S288c elements located in regions that are poorly represented by features on the array; 3, false-negative S288c elements that missed the criteria for calling a peak; 4, unannotated Ty1 full-length elements in S288c confirmed in this study; 5, false-positive peaks due to borderline elevated differential hybridization; 6, false-positive peaks corresponding to non-Ty repetitive elements in the genome. Grey horizontal lines above and below the central line for each chromosome correspond to a 3-fold difference in normalized ratio of Cy5 and Cy3 signal intensity.

    Article Snippet: The extracted DNA samples were labeled with Cy3 (5′ flanking) or Cy5 (3′ flanking), and hybridized to an Agilent yeast whole genome microarray.

    Techniques: Hybridization, Microarray, Sequencing

    A General Schematic Diagram of the Steps Involved in Extracting, Labeling, and Identifying the Position of Transposons within a Genome Step 1. Genomic DNA is digested with a restriction endonuclease containing a cut site (triangle) within the transposon (red box). This results in multiple restriction fragments, including ones containing transposon and contiguous flanking DNA. Step 2. Digested DNA (which may be pooled from multiple separate digests) is mixed with oligonucleotide probes that have been designed to anneal to specific sequences within the transposon. Separate probe sets anneal to different transposons (as shown), or separate genomic DNA samples are used to compare transposon content from different sources. Step 3. After heat denaturation and reannealing, the mixture is incubated in the presence of a DNA polymerase and dNTPs, one of which is biotinylated (stick with star atop it). This allows specific extension from the annealed 3′ probe termini. Step 4. Extended probes and their annealed templates are purified away from the mixture using magnetic streptavidin-coated beads (star labeled with Fe +3 ). Step 5. The extracted templates are released by heating. Step 6. The templates are labeled using Cy3- or Cy5-labeled nucleotides (green and red lollipops, respectively) in the presence of random primers and a DNA polymerase. Step 7. Differentially labeled DNAs are hybridized to a microarray slide with features distributed throughout the genome. After washing, the array is scanned to identify features (circles) that are common to both DNA sources (yellow circles) or that have been differentially extracted (green or red circles). Step 8. The log 2 ratio of signal intensity for the two dyes is quantitated and graphically represented along each chromosome to identify contiguous segments of differential signal that correspond to the DNA flanking the original transposons.

    Journal: PLoS Genetics

    Article Title: Global Mapping of Transposon Location

    doi: 10.1371/journal.pgen.0020212

    Figure Lengend Snippet: A General Schematic Diagram of the Steps Involved in Extracting, Labeling, and Identifying the Position of Transposons within a Genome Step 1. Genomic DNA is digested with a restriction endonuclease containing a cut site (triangle) within the transposon (red box). This results in multiple restriction fragments, including ones containing transposon and contiguous flanking DNA. Step 2. Digested DNA (which may be pooled from multiple separate digests) is mixed with oligonucleotide probes that have been designed to anneal to specific sequences within the transposon. Separate probe sets anneal to different transposons (as shown), or separate genomic DNA samples are used to compare transposon content from different sources. Step 3. After heat denaturation and reannealing, the mixture is incubated in the presence of a DNA polymerase and dNTPs, one of which is biotinylated (stick with star atop it). This allows specific extension from the annealed 3′ probe termini. Step 4. Extended probes and their annealed templates are purified away from the mixture using magnetic streptavidin-coated beads (star labeled with Fe +3 ). Step 5. The extracted templates are released by heating. Step 6. The templates are labeled using Cy3- or Cy5-labeled nucleotides (green and red lollipops, respectively) in the presence of random primers and a DNA polymerase. Step 7. Differentially labeled DNAs are hybridized to a microarray slide with features distributed throughout the genome. After washing, the array is scanned to identify features (circles) that are common to both DNA sources (yellow circles) or that have been differentially extracted (green or red circles). Step 8. The log 2 ratio of signal intensity for the two dyes is quantitated and graphically represented along each chromosome to identify contiguous segments of differential signal that correspond to the DNA flanking the original transposons.

    Article Snippet: The extracted DNA samples were labeled with Cy3 (5′ flanking) or Cy5 (3′ flanking), and hybridized to an Agilent yeast whole genome microarray.

    Techniques: Labeling, Incubation, Purification, Microarray

    Identifying a Unique Ty1 Element in Otherwise Isogenic Strains (A) Two isogenic yeast strains (FY5 and FY2) differ only by the presence of a Ty1 insertion in Chromosome V within the URA3 gene in FY2. After labeling transposon extracted DNA from FY2 with Cy3 (green) and transposon extracted DNA from FY5 with Cy5 (red), the labeled DNA was hybridized to an Agilent yeast whole genome microarray with > 40,000 unique features (yeast repetitive DNA was avoided during array construction). Log 2 ratio of hybridization for each feature along each chromosome is shown plotted in genome order using the TreeView Karyoscope function. The one region of significant differential hybridization is marked with an arrow. The grey horizontal lines above and below each chromosome correspond to 3-fold differential hybridization intensity. (B) Zoom view of a portion of Chromosome V and the peak of differential hybridization corresponding to the ∼8 kb surrounding URA3 (red box). The positions of nearby restriction sites for the enzymes used initially to digest genomic DNA are shown based on a GBrowse view of the region from SGD.

    Journal: PLoS Genetics

    Article Title: Global Mapping of Transposon Location

    doi: 10.1371/journal.pgen.0020212

    Figure Lengend Snippet: Identifying a Unique Ty1 Element in Otherwise Isogenic Strains (A) Two isogenic yeast strains (FY5 and FY2) differ only by the presence of a Ty1 insertion in Chromosome V within the URA3 gene in FY2. After labeling transposon extracted DNA from FY2 with Cy3 (green) and transposon extracted DNA from FY5 with Cy5 (red), the labeled DNA was hybridized to an Agilent yeast whole genome microarray with > 40,000 unique features (yeast repetitive DNA was avoided during array construction). Log 2 ratio of hybridization for each feature along each chromosome is shown plotted in genome order using the TreeView Karyoscope function. The one region of significant differential hybridization is marked with an arrow. The grey horizontal lines above and below each chromosome correspond to 3-fold differential hybridization intensity. (B) Zoom view of a portion of Chromosome V and the peak of differential hybridization corresponding to the ∼8 kb surrounding URA3 (red box). The positions of nearby restriction sites for the enzymes used initially to digest genomic DNA are shown based on a GBrowse view of the region from SGD.

    Article Snippet: The extracted DNA samples were labeled with Cy3 (5′ flanking) or Cy5 (3′ flanking), and hybridized to an Agilent yeast whole genome microarray.

    Techniques: Labeling, Microarray, Hybridization

    Analysis of Modified Bacterial (“Artificial”) Transposon Insertions in the S. cerevisiae Genome (A) Positions of five independent pooled artificial transposons from a yeast insertion library were determined after extracting StuI-digested yeast genomic DNA with probes designed to correspond to either strand at the 5′ or 3′ end of URA3, labeling with Cy3 and Cy5, respectively, and hybridizing to an Agilent yeast whole genome microarray. Arrows signify locations of significant differential hybridization. “URA3” indicates the actual URA3 locus on Chromosome V. The asterisk indicates an insertion on Chromosome XVI in which only the flanking region 3′ to the transposon is detected. Vertical lines above and below the horizontal for each chromosome represent the log 2 ratio of hybridization intensity for Cy5 versus Cy3 at each feature along the Agilent yeast whole genome microarray. For each insertion, the actual insertion site, determined by sequencing, and the position of the first significant flanking features are as follows: Chromosome IV, 368020, and 367656–367715 and 368589–368648; Chromosome IX, 55576, and 55291–55350 and 55808–55867; Chromosome XI, 613654, and 612706–612765 and 614005–614064; Chromosome XII, 387226, and 386346–386405 and 387248–387307; and Chromosome XVI, 296609, and 296350–296409 to 297592–297651. (B) An enlargement of the region detected on Chromosome XI, showing the structure of the artificial transposon, its unique StuI site, the bases covered by the oligonucleotides in the features on either side of the transition from significant differential Cy5 labeling to Cy3 labeling, and the position of the actual insertion. The map of the region from GBrowse of SGD shows the position of StuI restriction sites in the region. Grey horizontal lines above and below the central line for each chromosome correspond to a 3-fold ratio of signal intensity. Note that the transposon inserted in opposite orientation relative to the chromosome numbering, and is therefore flipped in the figure.

    Journal: PLoS Genetics

    Article Title: Global Mapping of Transposon Location

    doi: 10.1371/journal.pgen.0020212

    Figure Lengend Snippet: Analysis of Modified Bacterial (“Artificial”) Transposon Insertions in the S. cerevisiae Genome (A) Positions of five independent pooled artificial transposons from a yeast insertion library were determined after extracting StuI-digested yeast genomic DNA with probes designed to correspond to either strand at the 5′ or 3′ end of URA3, labeling with Cy3 and Cy5, respectively, and hybridizing to an Agilent yeast whole genome microarray. Arrows signify locations of significant differential hybridization. “URA3” indicates the actual URA3 locus on Chromosome V. The asterisk indicates an insertion on Chromosome XVI in which only the flanking region 3′ to the transposon is detected. Vertical lines above and below the horizontal for each chromosome represent the log 2 ratio of hybridization intensity for Cy5 versus Cy3 at each feature along the Agilent yeast whole genome microarray. For each insertion, the actual insertion site, determined by sequencing, and the position of the first significant flanking features are as follows: Chromosome IV, 368020, and 367656–367715 and 368589–368648; Chromosome IX, 55576, and 55291–55350 and 55808–55867; Chromosome XI, 613654, and 612706–612765 and 614005–614064; Chromosome XII, 387226, and 386346–386405 and 387248–387307; and Chromosome XVI, 296609, and 296350–296409 to 297592–297651. (B) An enlargement of the region detected on Chromosome XI, showing the structure of the artificial transposon, its unique StuI site, the bases covered by the oligonucleotides in the features on either side of the transition from significant differential Cy5 labeling to Cy3 labeling, and the position of the actual insertion. The map of the region from GBrowse of SGD shows the position of StuI restriction sites in the region. Grey horizontal lines above and below the central line for each chromosome correspond to a 3-fold ratio of signal intensity. Note that the transposon inserted in opposite orientation relative to the chromosome numbering, and is therefore flipped in the figure.

    Article Snippet: The extracted DNA samples were labeled with Cy3 (5′ flanking) or Cy5 (3′ flanking), and hybridized to an Agilent yeast whole genome microarray.

    Techniques: Modification, Labeling, Microarray, Hybridization, Sequencing

    HIV-specific TGF-β-positive CD4 + T cells are CTLA-4 negative. PBMCs ( n = 6) were stimulated with HIV peptides and then stained with anti-TGF-β PE, anti-CD3 AmCyan, anti-CD4 PerCP Cy5.5, anti-CD8 PE Cy7, and anti-CTLA-4 APC, and analyzed by flow cytometry. Gating on the CTLA-4-positive cells was performed using the fluorescence-minus-one (FMO) control for CTLA-4. ( A ) Representative plots of samples that were first gated on the CD3 + /CD4 + and CD3 + /CD8 + lymphocyte population and then the percentages of CTLA-4-positive cells were determined. ( B ) Representative plots of samples that were first gated on the CD3 + /CD4 + lymphocyte population and then the percent of TGF-β-positive cells that expressed CTLA-4 was determined after subtraction of the background values. The values marked with an asterisk represent the fraction of TGF-β-positive cells that expresses CTLA-4 over the total number of TGF-β-positive cells (equivalent to 100%).

    Journal: AIDS Research and Human Retroviruses

    Article Title: HIV-Specific TGF-?-Positive CD4+ T Cells Do Not Express Regulatory Surface Markers and Are Regulated by CTLA-4

    doi: 10.1089/aid.2009.0149

    Figure Lengend Snippet: HIV-specific TGF-β-positive CD4 + T cells are CTLA-4 negative. PBMCs ( n = 6) were stimulated with HIV peptides and then stained with anti-TGF-β PE, anti-CD3 AmCyan, anti-CD4 PerCP Cy5.5, anti-CD8 PE Cy7, and anti-CTLA-4 APC, and analyzed by flow cytometry. Gating on the CTLA-4-positive cells was performed using the fluorescence-minus-one (FMO) control for CTLA-4. ( A ) Representative plots of samples that were first gated on the CD3 + /CD4 + and CD3 + /CD8 + lymphocyte population and then the percentages of CTLA-4-positive cells were determined. ( B ) Representative plots of samples that were first gated on the CD3 + /CD4 + lymphocyte population and then the percent of TGF-β-positive cells that expressed CTLA-4 was determined after subtraction of the background values. The values marked with an asterisk represent the fraction of TGF-β-positive cells that expresses CTLA-4 over the total number of TGF-β-positive cells (equivalent to 100%).

    Article Snippet: Cells were then stained with specific combinations of the following antibodies: CD4 PerCP CY5.5, CD127 Alexa Fluor 647, CD8 PE CY7, and CD3 AmCyan (BD Pharmingen).

    Techniques: Staining, Flow Cytometry, Cytometry, Fluorescence

    HIV-specific TGF-β-positive CD4 + T cells do not produce IL-10 or IFN-γ. PBMCs were stimulated with HIV peptides and then stained with anti-IFN-γ FITC, anti-TGF-β PE, anti-IL-10 APC, anti-CD3 AmCyan, anti-CD4 PerCP CY5.5, and anti-CD8 PE CY7, and analyzed by flow cytometry. Samples were first gated on the CD3 + /CD4 + lymphocyte population and then the percent of TGF-β, IL-10, and IFN-γ-positive CD4 + T cells was determined. Representative plots of the number of TGF-β, IL-10, and IFN-γ-positive CD4 + T cells after subtraction of the background values.

    Journal: AIDS Research and Human Retroviruses

    Article Title: HIV-Specific TGF-?-Positive CD4+ T Cells Do Not Express Regulatory Surface Markers and Are Regulated by CTLA-4

    doi: 10.1089/aid.2009.0149

    Figure Lengend Snippet: HIV-specific TGF-β-positive CD4 + T cells do not produce IL-10 or IFN-γ. PBMCs were stimulated with HIV peptides and then stained with anti-IFN-γ FITC, anti-TGF-β PE, anti-IL-10 APC, anti-CD3 AmCyan, anti-CD4 PerCP CY5.5, and anti-CD8 PE CY7, and analyzed by flow cytometry. Samples were first gated on the CD3 + /CD4 + lymphocyte population and then the percent of TGF-β, IL-10, and IFN-γ-positive CD4 + T cells was determined. Representative plots of the number of TGF-β, IL-10, and IFN-γ-positive CD4 + T cells after subtraction of the background values.

    Article Snippet: Cells were then stained with specific combinations of the following antibodies: CD4 PerCP CY5.5, CD127 Alexa Fluor 647, CD8 PE CY7, and CD3 AmCyan (BD Pharmingen).

    Techniques: Staining, Flow Cytometry, Cytometry

    HIV-specific CD4 + T cells produce TGF-β. PBMCs were stimulated with HIV peptides then stained with anti-TGF-β PE, anti-CD3 AmCyan, anti-CD4 PerCP CY5.5, and anti-CD8 PE CY7, and analyzed by flow cytometry. Samples were first gated on the CD3 + /CD4 + lymphocyte population and then the percent of TGF-β-positive CD4 + T cells was determined. Representative plots of HIV-specific CD4 + T cells expressing TGF-β after subtraction of the background values.

    Journal: AIDS Research and Human Retroviruses

    Article Title: HIV-Specific TGF-?-Positive CD4+ T Cells Do Not Express Regulatory Surface Markers and Are Regulated by CTLA-4

    doi: 10.1089/aid.2009.0149

    Figure Lengend Snippet: HIV-specific CD4 + T cells produce TGF-β. PBMCs were stimulated with HIV peptides then stained with anti-TGF-β PE, anti-CD3 AmCyan, anti-CD4 PerCP CY5.5, and anti-CD8 PE CY7, and analyzed by flow cytometry. Samples were first gated on the CD3 + /CD4 + lymphocyte population and then the percent of TGF-β-positive CD4 + T cells was determined. Representative plots of HIV-specific CD4 + T cells expressing TGF-β after subtraction of the background values.

    Article Snippet: Cells were then stained with specific combinations of the following antibodies: CD4 PerCP CY5.5, CD127 Alexa Fluor 647, CD8 PE CY7, and CD3 AmCyan (BD Pharmingen).

    Techniques: Staining, Flow Cytometry, Cytometry, Expressing

    TGF-β receptor II blockade increases IFN-γ expression by HIV-specific T cells. PBMCs were stimulated with Gag peptides in the presence of anti-TGF-β R II (or isotype control). PBMCs were then stained with anti-IFN-γ FITC, anti-CD3 AmCyan, anti-CD4 PerCP Cy5.5, and anti-CD8 PE Cy7 and analyzed by flow cytometry. Samples were first gated on the CD3 + /CD4 + and CD3 + /CD8 + lymphocyte populations and then the percent of IFN-γ-positive cells was determined after subtraction of the background values. Plots are from three independent experiments yielding similar results.

    Journal: AIDS Research and Human Retroviruses

    Article Title: HIV-Specific TGF-?-Positive CD4+ T Cells Do Not Express Regulatory Surface Markers and Are Regulated by CTLA-4

    doi: 10.1089/aid.2009.0149

    Figure Lengend Snippet: TGF-β receptor II blockade increases IFN-γ expression by HIV-specific T cells. PBMCs were stimulated with Gag peptides in the presence of anti-TGF-β R II (or isotype control). PBMCs were then stained with anti-IFN-γ FITC, anti-CD3 AmCyan, anti-CD4 PerCP Cy5.5, and anti-CD8 PE Cy7 and analyzed by flow cytometry. Samples were first gated on the CD3 + /CD4 + and CD3 + /CD8 + lymphocyte populations and then the percent of IFN-γ-positive cells was determined after subtraction of the background values. Plots are from three independent experiments yielding similar results.

    Article Snippet: Cells were then stained with specific combinations of the following antibodies: CD4 PerCP CY5.5, CD127 Alexa Fluor 647, CD8 PE CY7, and CD3 AmCyan (BD Pharmingen).

    Techniques: Expressing, Staining, Flow Cytometry, Cytometry

    CTLA-4 blockade decreases TGF-β expression by HIV-specific CD4 + T Cells. PBMCs ( n = 6) were stimulated with Gag peptides in the presence of anti-CTLA4 (or isotype control), then stained with anti-IFN-γ FITC, anti-TGF-β PE, anti-CD3 AmCyan, anti-CD4 PerCP CY5.5, and anti-CD8 PE CY7, and analyzed by flow cytometry. Samples were first gated on the CD3 + /CD4 + lymphocyte population and then the percent of TGF-β- and IFN-γ-positive cells was determined. Results were expressed as percent of Gag-specific CD4 + T cells expressing TGF-β or IFN-γ after subtraction of the background values. ( A ) Representative plots of Gag-specific CD4 + T cells expressing TGF-β or IFN-γ in the presence or absence of anti-CTLA-4. ( B ) Dashed line represents the cutoff for significant cytokine expression. Percentages in parentheses are median values. The two dots joined by a line represent the values obtained from the same individual and analysis was performed by paired t test.

    Journal: AIDS Research and Human Retroviruses

    Article Title: HIV-Specific TGF-?-Positive CD4+ T Cells Do Not Express Regulatory Surface Markers and Are Regulated by CTLA-4

    doi: 10.1089/aid.2009.0149

    Figure Lengend Snippet: CTLA-4 blockade decreases TGF-β expression by HIV-specific CD4 + T Cells. PBMCs ( n = 6) were stimulated with Gag peptides in the presence of anti-CTLA4 (or isotype control), then stained with anti-IFN-γ FITC, anti-TGF-β PE, anti-CD3 AmCyan, anti-CD4 PerCP CY5.5, and anti-CD8 PE CY7, and analyzed by flow cytometry. Samples were first gated on the CD3 + /CD4 + lymphocyte population and then the percent of TGF-β- and IFN-γ-positive cells was determined. Results were expressed as percent of Gag-specific CD4 + T cells expressing TGF-β or IFN-γ after subtraction of the background values. ( A ) Representative plots of Gag-specific CD4 + T cells expressing TGF-β or IFN-γ in the presence or absence of anti-CTLA-4. ( B ) Dashed line represents the cutoff for significant cytokine expression. Percentages in parentheses are median values. The two dots joined by a line represent the values obtained from the same individual and analysis was performed by paired t test.

    Article Snippet: Cells were then stained with specific combinations of the following antibodies: CD4 PerCP CY5.5, CD127 Alexa Fluor 647, CD8 PE CY7, and CD3 AmCyan (BD Pharmingen).

    Techniques: Expressing, Staining, Flow Cytometry, Cytometry

    CTLA-4 blockade increases IFN-γ expression by CMV-specific CD4 + T cells. PBMCs ( n = 6) were stimulated with CMV PP65 peptides in the presence of anti-CTLA4 (or isotype control), then stained with anti-IFN-γ FITC, anti-TGF-β PE, anti-CD3 AmCyan, anti-CD4 PerCP CY5.5, and anti-CD8 PE CY7, and analyzed by flow cytometry. Samples were first gated on the CD3 + /CD4 + lymphocyte population and the percent of IFN-γ-positive cells was determined. Results were expressed as percent of CMV-specific CD4 + T cells expressing IFN-γ after subtraction of the background values. The dashed line represents the cutoff for significant cytokine expression. Percentages in parentheses are median values. The two dots joined by a line represent the values obtained from the same individual and analysis was performed by paired t test.

    Journal: AIDS Research and Human Retroviruses

    Article Title: HIV-Specific TGF-?-Positive CD4+ T Cells Do Not Express Regulatory Surface Markers and Are Regulated by CTLA-4

    doi: 10.1089/aid.2009.0149

    Figure Lengend Snippet: CTLA-4 blockade increases IFN-γ expression by CMV-specific CD4 + T cells. PBMCs ( n = 6) were stimulated with CMV PP65 peptides in the presence of anti-CTLA4 (or isotype control), then stained with anti-IFN-γ FITC, anti-TGF-β PE, anti-CD3 AmCyan, anti-CD4 PerCP CY5.5, and anti-CD8 PE CY7, and analyzed by flow cytometry. Samples were first gated on the CD3 + /CD4 + lymphocyte population and the percent of IFN-γ-positive cells was determined. Results were expressed as percent of CMV-specific CD4 + T cells expressing IFN-γ after subtraction of the background values. The dashed line represents the cutoff for significant cytokine expression. Percentages in parentheses are median values. The two dots joined by a line represent the values obtained from the same individual and analysis was performed by paired t test.

    Article Snippet: Cells were then stained with specific combinations of the following antibodies: CD4 PerCP CY5.5, CD127 Alexa Fluor 647, CD8 PE CY7, and CD3 AmCyan (BD Pharmingen).

    Techniques: Expressing, Staining, Flow Cytometry, Cytometry

    Magnetic activation is not dependent upon the targeting moiety. Stably transfected HeLa cells expressing chimeric ACP-EGFR, enzymatically modified by biotin CoA and carrying bound Strv-SPIONs. (a) 30 seconds, (b) 180 seconds of magnetization, (c) no magnetization. Green channel, Strv-SPION-biocytin-Alexa546; red channel, phosphorylated-EGFR (pY-EGFR 1068 and GARIG-CY5), overlay of green and red channels. Scale bar, 10 µm.

    Journal: PLoS ONE

    Article Title: Magnetic Nanoparticles as Mediators of Ligand-Free Activation of EGFR Signaling

    doi: 10.1371/journal.pone.0068879

    Figure Lengend Snippet: Magnetic activation is not dependent upon the targeting moiety. Stably transfected HeLa cells expressing chimeric ACP-EGFR, enzymatically modified by biotin CoA and carrying bound Strv-SPIONs. (a) 30 seconds, (b) 180 seconds of magnetization, (c) no magnetization. Green channel, Strv-SPION-biocytin-Alexa546; red channel, phosphorylated-EGFR (pY-EGFR 1068 and GARIG-CY5), overlay of green and red channels. Scale bar, 10 µm.

    Article Snippet: Primary antibodies were detected using secondary F(ab)2 fragments of goat anti-rabbit IgG (GARIG) labeled with Cy3 or Cy5 (Jackson Labs) at 1 µg/ml.

    Techniques: Activation Assay, Stable Transfection, Transfection, Expressing, Modification

    Shc activation as a result of magnetic activation of EGFR. Confocal immunofluorescence images of cells incubated for 15 min at 37°C after 0 sec (a) or after 30 sec (b) or 180 sec (c) magnetic field activation. Image columns left to right: MS Alexa-488 biocytin (green); indirect immunofluorescence of MAb against pY-317 Shc protein and GARIG-CY5 (red); overlay of the first 2 columns; two-dimensional colocalization histograms of MS and pY317-Shc fluorescence signals after deconvolution of 50 optical sections using SVI Huygens software.

    Journal: PLoS ONE

    Article Title: Magnetic Nanoparticles as Mediators of Ligand-Free Activation of EGFR Signaling

    doi: 10.1371/journal.pone.0068879

    Figure Lengend Snippet: Shc activation as a result of magnetic activation of EGFR. Confocal immunofluorescence images of cells incubated for 15 min at 37°C after 0 sec (a) or after 30 sec (b) or 180 sec (c) magnetic field activation. Image columns left to right: MS Alexa-488 biocytin (green); indirect immunofluorescence of MAb against pY-317 Shc protein and GARIG-CY5 (red); overlay of the first 2 columns; two-dimensional colocalization histograms of MS and pY317-Shc fluorescence signals after deconvolution of 50 optical sections using SVI Huygens software.

    Article Snippet: Primary antibodies were detected using secondary F(ab)2 fragments of goat anti-rabbit IgG (GARIG) labeled with Cy3 or Cy5 (Jackson Labs) at 1 µg/ml.

    Techniques: Activation Assay, Immunofluorescence, Incubation, Size-exclusion Chromatography, Mass Spectrometry, Fluorescence, Software

    Effect of magnetization time on the level of EGFR phosphorylation. (a, a′, a′′) The time axis shown vertically. Confocal images of MS 488Alexa biocytin signal (green, left panels) and of anti-pY-EGFR 1068 and GARIG-Cy5 (rainbow intensity scale, middle panels) on A431 cells as a function of the applied magnetic field for time intervals of 30, 60 and 180 s. Overlay images are depicted with green/red LUTs, Alexa488-biocytin/GARIG-Cy5 respectively (right panels). Scale bar, 10 µm. (b) Fluorescence intensity ratio of pY-EGFR to MS signals as a function of magnetization. (c) Mean pixel intensity of the pY-EGFR signal from 5 images for each time point as a function of MS magnetization time. (e) Western blot analysis of A431 cell extracts for pY-EGFR 1148. Lane 1, sample obtained from A431 cells incubated with MS in the absence of a magnetic field. Lanes 2–4, pY-EGFR signals for 30, 60 or 180 s of MS magnetization. Lane 5 and 6, pY-EGFR signals after treatment with 30 nM (saturating) or 100 pM EGF. Lane 7, extract of untreated cells. (e) Signal intensities of the positive pY-EGFR lanes in (d) relative to the lane from 30 nM EGF treated cells.

    Journal: PLoS ONE

    Article Title: Magnetic Nanoparticles as Mediators of Ligand-Free Activation of EGFR Signaling

    doi: 10.1371/journal.pone.0068879

    Figure Lengend Snippet: Effect of magnetization time on the level of EGFR phosphorylation. (a, a′, a′′) The time axis shown vertically. Confocal images of MS 488Alexa biocytin signal (green, left panels) and of anti-pY-EGFR 1068 and GARIG-Cy5 (rainbow intensity scale, middle panels) on A431 cells as a function of the applied magnetic field for time intervals of 30, 60 and 180 s. Overlay images are depicted with green/red LUTs, Alexa488-biocytin/GARIG-Cy5 respectively (right panels). Scale bar, 10 µm. (b) Fluorescence intensity ratio of pY-EGFR to MS signals as a function of magnetization. (c) Mean pixel intensity of the pY-EGFR signal from 5 images for each time point as a function of MS magnetization time. (e) Western blot analysis of A431 cell extracts for pY-EGFR 1148. Lane 1, sample obtained from A431 cells incubated with MS in the absence of a magnetic field. Lanes 2–4, pY-EGFR signals for 30, 60 or 180 s of MS magnetization. Lane 5 and 6, pY-EGFR signals after treatment with 30 nM (saturating) or 100 pM EGF. Lane 7, extract of untreated cells. (e) Signal intensities of the positive pY-EGFR lanes in (d) relative to the lane from 30 nM EGF treated cells.

    Article Snippet: Primary antibodies were detected using secondary F(ab)2 fragments of goat anti-rabbit IgG (GARIG) labeled with Cy3 or Cy5 (Jackson Labs) at 1 µg/ml.

    Techniques: Mass Spectrometry, Fluorescence, Western Blot, Incubation

    Heat shock protein 60 ( HSP 60) induces dendritic cell ( DC ) activation/maturation; 1 of 3 individual experiments is presented here. DC s at the concentration of 2×10 6 /mL were cultured with or without HSP 60. A, Expression of CD 86, CD 83, CD 40, and HLA ‐ II was analyzed by flow cytometry after 24 h of stimulation with 5 μg/mL of HSP 60. Figure in histogram shows higher induction of these markers by HSP 60. Percentage of histogram shift or mean fluorescence intensity was calculated from triplicate samples and P values are CD 86 ≤0.001, CD 83 ≤0.001, HLA ‐ II ≤0.05, and CD 40 ≤0.001. B, Cytokine profile of DC against HSP 60 is listed. DC s were stimulated as mentioned, cultured for 24 h, and cell supernatant was collected for measurement of cytokines. Mostly pro‐inflammatory cytokines are highly increased by HSP 60. C, In similar condition, DC s activation was observed by HSP 90 (5 μg/mL). P value from triplicates samples CD 86 ≤0.0001, CD 83 ≤0.001, CD 40 ≤0.01, and HLA ‐ II ≤0.001. D, HSP 90 induced‐ DC s were cocultured with T cells but no activation of T cells was observed. APC indicates antigen‐presenting cells; FITC, fluorescein isothiocyanate; IL‐6, interleukin‐6; Percp‐Cy5.5, Peridinin Chlorophyll Protein‐Cyanine 5.5; TGF‐β1, transforming growth factor‐β1.

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Induction of Dendritic Cell–Mediated Activation of T Cells From Atherosclerotic Plaques by Human Heat Shock Protein 60

    doi: 10.1161/JAHA.117.006778

    Figure Lengend Snippet: Heat shock protein 60 ( HSP 60) induces dendritic cell ( DC ) activation/maturation; 1 of 3 individual experiments is presented here. DC s at the concentration of 2×10 6 /mL were cultured with or without HSP 60. A, Expression of CD 86, CD 83, CD 40, and HLA ‐ II was analyzed by flow cytometry after 24 h of stimulation with 5 μg/mL of HSP 60. Figure in histogram shows higher induction of these markers by HSP 60. Percentage of histogram shift or mean fluorescence intensity was calculated from triplicate samples and P values are CD 86 ≤0.001, CD 83 ≤0.001, HLA ‐ II ≤0.05, and CD 40 ≤0.001. B, Cytokine profile of DC against HSP 60 is listed. DC s were stimulated as mentioned, cultured for 24 h, and cell supernatant was collected for measurement of cytokines. Mostly pro‐inflammatory cytokines are highly increased by HSP 60. C, In similar condition, DC s activation was observed by HSP 90 (5 μg/mL). P value from triplicates samples CD 86 ≤0.0001, CD 83 ≤0.001, CD 40 ≤0.01, and HLA ‐ II ≤0.001. D, HSP 90 induced‐ DC s were cocultured with T cells but no activation of T cells was observed. APC indicates antigen‐presenting cells; FITC, fluorescein isothiocyanate; IL‐6, interleukin‐6; Percp‐Cy5.5, Peridinin Chlorophyll Protein‐Cyanine 5.5; TGF‐β1, transforming growth factor‐β1.

    Article Snippet: Cells were collected and stained with anti‐CD3‐Percp/Cy5.5, anti‐CD 25‐PE/fluorescein isothiocyanate, anti‐CD69‐APC/fluorescein isothiocyanate and anti‐CD71‐BV421 (BD Bioscience).

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

    T‐cell activation and proliferation in dendritic cell ( DC )+T‐cell coculture. A, DC s were stimulated with heat shock protein 60 ( HSP 60) at the concentration of 2.5, 5, or 10 μg/mL. After overnight incubation, autologous T cells 4×10 5 were cocultured with 1×10 5 DC s. All the concentrations of HSP 60 induced T‐cell activation, where 5 or 10 μg/mL were a little stronger, which was tested by CD 25 expression in CD 3 T cells. B, One representative of minimum 3 experiments of T‐cell activation, which was determined by CD 69 early activation, CD 25 and CD 71 intermediate/late activation markers. DC s were stimulated with 5 μg/mL of HSP 60 and cocultured with CD 3 + T cells. For analysis, CD 3 + cells were gated, then percentage of CD 3+ CD 69/ CD 25/ CD 71 + cells was shown in the upper right of each gate. HSP 60‐induced DC s activated all of 3 activation markers in CD 3 + T cells, P ≤0.0001 from triplicate samples. C, In response to HSP 60, DC +T cells show a high proliferation rate; 1 representative of 3 individual experiments is shown here. APC Allophycocyanine; BrDu, 5‐brom‐2‐deoxiuridin; OD, Optical density; Percp‐Cy5.5, Peridinin Chlorophyll Protein‐Cyanine 5.5. * P ≤0.05; *** P ≤0.0001.

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Induction of Dendritic Cell–Mediated Activation of T Cells From Atherosclerotic Plaques by Human Heat Shock Protein 60

    doi: 10.1161/JAHA.117.006778

    Figure Lengend Snippet: T‐cell activation and proliferation in dendritic cell ( DC )+T‐cell coculture. A, DC s were stimulated with heat shock protein 60 ( HSP 60) at the concentration of 2.5, 5, or 10 μg/mL. After overnight incubation, autologous T cells 4×10 5 were cocultured with 1×10 5 DC s. All the concentrations of HSP 60 induced T‐cell activation, where 5 or 10 μg/mL were a little stronger, which was tested by CD 25 expression in CD 3 T cells. B, One representative of minimum 3 experiments of T‐cell activation, which was determined by CD 69 early activation, CD 25 and CD 71 intermediate/late activation markers. DC s were stimulated with 5 μg/mL of HSP 60 and cocultured with CD 3 + T cells. For analysis, CD 3 + cells were gated, then percentage of CD 3+ CD 69/ CD 25/ CD 71 + cells was shown in the upper right of each gate. HSP 60‐induced DC s activated all of 3 activation markers in CD 3 + T cells, P ≤0.0001 from triplicate samples. C, In response to HSP 60, DC +T cells show a high proliferation rate; 1 representative of 3 individual experiments is shown here. APC Allophycocyanine; BrDu, 5‐brom‐2‐deoxiuridin; OD, Optical density; Percp‐Cy5.5, Peridinin Chlorophyll Protein‐Cyanine 5.5. * P ≤0.05; *** P ≤0.0001.

    Article Snippet: Cells were collected and stained with anti‐CD3‐Percp/Cy5.5, anti‐CD 25‐PE/fluorescein isothiocyanate, anti‐CD69‐APC/fluorescein isothiocyanate and anti‐CD71‐BV421 (BD Bioscience).

    Techniques: Activation Assay, Concentration Assay, Incubation, Expressing

    Flotillin-2 knockdown inhibits APP endocytosis in N2a cells and primary hippocampal neurons. A , CFP–swAPP-transfected N2a cells were labeled at 4°C with anti-GFP antibody, washed, and incubated at 37°C for 5, 20, and 45 min to allow internalization of antibodies bound to CFP–swAPP. To visualize cell-surface retained APP, cells were labeled with Cy5-conjugated secondary antibody at 4°C (pseudocolored in blue). After fixing, cells were permeabilized and stained with a Cy3-conjugated secondary antibody to resolve internalized APP (red). The cell surface was identified on confocal sections by the Cy5 label, and APP endocytosis was measured by calculating the intensity ratio of (cell interior Cy3)/(surface Cy5) labeled CFP–swAPP. The graph shows the increase in internalized APP over the time course of 45 min, the corresponding confocal images display the increasing amount of internalized APP (Cy3), whereas Cy5-labeled surface APP decreases over time ( n > 95 cells, values represent means + SEM; * p

    Journal: The Journal of Neuroscience

    Article Title: Flotillin-Dependent Clustering of the Amyloid Precursor Protein Regulates Its Endocytosis and Amyloidogenic Processing in Neurons

    doi: 10.1523/JNEUROSCI.5345-07.2008

    Figure Lengend Snippet: Flotillin-2 knockdown inhibits APP endocytosis in N2a cells and primary hippocampal neurons. A , CFP–swAPP-transfected N2a cells were labeled at 4°C with anti-GFP antibody, washed, and incubated at 37°C for 5, 20, and 45 min to allow internalization of antibodies bound to CFP–swAPP. To visualize cell-surface retained APP, cells were labeled with Cy5-conjugated secondary antibody at 4°C (pseudocolored in blue). After fixing, cells were permeabilized and stained with a Cy3-conjugated secondary antibody to resolve internalized APP (red). The cell surface was identified on confocal sections by the Cy5 label, and APP endocytosis was measured by calculating the intensity ratio of (cell interior Cy3)/(surface Cy5) labeled CFP–swAPP. The graph shows the increase in internalized APP over the time course of 45 min, the corresponding confocal images display the increasing amount of internalized APP (Cy3), whereas Cy5-labeled surface APP decreases over time ( n > 95 cells, values represent means + SEM; * p

    Article Snippet: The fluorophore-conjugated secondary antibodies Alexa Fluor 488, cyanine 3 (Cy3)–IgG, and Cy5 were purchased from Dianova (Hamburg, Germany), Alexa Fluor 488-f(ab)-fragment was from Invitrogen, and Cy3-f(ab)-fragment was from Jackson ImmunoResearch (Newmarket, UK).

    Techniques: Transfection, Labeling, Incubation, Staining

    APP endocytosis occurs in a distinct clathrin pathway independent of epsin1 and is regulated by cholesterol. N2a cells were cotransfected with CFP–swAPP and siRNA against AP-2 or control siRNA or plasmids encoding for the dominant-negative (dn) mutant myc–AP180–C terminus, the wild-type control AP180–GFP, dominant-negative dynaminK44A–GFP, wild-type dynamin–GFP, dominant-negative myc–epsin1, or wild-type epsin1–GFP. Expression time of all constructs was below 12 h, and cells did not display any signs of toxicity. APP endocytosis was determined by an antibody uptake assay with anti-GFP antibody; clathrin-dependent endocytosis was assessed by transferrin–rhodamine uptake. Internalization was quantified by analysis of confocal images (Cy3/Cy5 ratio for APP, rhodamine intensity for transferrin). Results are shown as means + SEM ( n > 100 cells for each condition, * p

    Journal: The Journal of Neuroscience

    Article Title: Flotillin-Dependent Clustering of the Amyloid Precursor Protein Regulates Its Endocytosis and Amyloidogenic Processing in Neurons

    doi: 10.1523/JNEUROSCI.5345-07.2008

    Figure Lengend Snippet: APP endocytosis occurs in a distinct clathrin pathway independent of epsin1 and is regulated by cholesterol. N2a cells were cotransfected with CFP–swAPP and siRNA against AP-2 or control siRNA or plasmids encoding for the dominant-negative (dn) mutant myc–AP180–C terminus, the wild-type control AP180–GFP, dominant-negative dynaminK44A–GFP, wild-type dynamin–GFP, dominant-negative myc–epsin1, or wild-type epsin1–GFP. Expression time of all constructs was below 12 h, and cells did not display any signs of toxicity. APP endocytosis was determined by an antibody uptake assay with anti-GFP antibody; clathrin-dependent endocytosis was assessed by transferrin–rhodamine uptake. Internalization was quantified by analysis of confocal images (Cy3/Cy5 ratio for APP, rhodamine intensity for transferrin). Results are shown as means + SEM ( n > 100 cells for each condition, * p

    Article Snippet: The fluorophore-conjugated secondary antibodies Alexa Fluor 488, cyanine 3 (Cy3)–IgG, and Cy5 were purchased from Dianova (Hamburg, Germany), Alexa Fluor 488-f(ab)-fragment was from Invitrogen, and Cy3-f(ab)-fragment was from Jackson ImmunoResearch (Newmarket, UK).

    Techniques: Dominant Negative Mutation, Mutagenesis, Expressing, Construct