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  • 99
    Thermo Fisher streptavidin alexa fluor 647
    Streptavidin Alexa Fluor 647, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 469 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend streptavidin alexa fluor 647
    Streptavidin Alexa Fluor 647, supplied by BioLegend, used in various techniques. Bioz Stars score: 89/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jackson Immuno alexa fluor 647 streptavidin
    Alexa Fluor 647 Streptavidin, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 91/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 647 streptavidin
    Alexa Fluor 647 Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare streptavidin alexa fluor 647
    Streptavidin Alexa Fluor 647, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jackson Immuno streptavidin alexa fluor 647
    Streptavidin Alexa Fluor 647, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 96/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin alexa fluor 647 conjugate
    Overexpression of GnT-III in MDA-MB-231 cells significantly inhibited the α2,3-sialylation, but not α2,6-sialylation, at a post-transcriptional level. A , cell lysates from control and GnT-III transfected MDA-MB-231 cells were immunoblotted with E4-PHA and DSA lectins or immunoprecipitated ( IP ) with ConA-agarose and blotted with ConA, MAA, and SNA lectins. B , to confirm the effect of GnT-III expression on cell sialylation, cells transfected with or without GnT-III were incubated with ( bold line ) or without ( gray shading ) biotin-conjugated MAA (recognizing α2,3-sialylated proteins), followed by incubation with <t>streptavidin</t> <t>Alexa</t> <t>Fluor</t> 647 conjugate and subjected to FACS analysis. C , to determine the changes in the N -glycans on specific proteins after GnT-III overexpression, the cell lysates from control and GnT-III transfected MDA-MB-231 cells were also immunoprecipitated by ConA, E4-PHA, MAM (recognizing α2,3-sialylated proteins), and S. sieboldiana (recognizing α2,6-sialylated proteins) agaroses and probed with antibodies against α3 integrin, αv integrin, and β1 integrin separately. D , PA oligosaccharides treated with or without glycosidases from those cells were analyzed by reversed phase HPLC. E , PA oligosaccharides treated with or without neuraminidases from the control, GnT-III transfected MDA-MB-231 cells, as well as their ST6GAL1 knock-out counterparts, were analyzed by anion exchange HPLC to quantify the amount of sialylated N -glycans. F , RT-PCR using total RNA extracted from the control and GnT-III transfected MDA-MB-231 cells was carried out to examine the expression levels of genes involved in protein sialylation. The expression level of Gapdh was used as a loading control. SCT , sialic acid transporter; St3gal , β-galactoside α2,3-sialyltransferase; St6galnac , α- N -acetylgalactosaminide α2,6-sialyltransferase; NEU , neuraminidase. Con , control (DOX-inducible GnT-III-overexpressing cells without DOX treatment); OE , DOX inducible GnT-III-overexpressing cells treated with DOX; ST6GAL1 KO , ST6GAL1 knock-out cells.
    Streptavidin Alexa Fluor 647 Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 464 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin alexa fluor 647 r phycoerythrin conjugate
    Overexpression of GnT-III in MDA-MB-231 cells significantly inhibited the α2,3-sialylation, but not α2,6-sialylation, at a post-transcriptional level. A , cell lysates from control and GnT-III transfected MDA-MB-231 cells were immunoblotted with E4-PHA and DSA lectins or immunoprecipitated ( IP ) with ConA-agarose and blotted with ConA, MAA, and SNA lectins. B , to confirm the effect of GnT-III expression on cell sialylation, cells transfected with or without GnT-III were incubated with ( bold line ) or without ( gray shading ) biotin-conjugated MAA (recognizing α2,3-sialylated proteins), followed by incubation with <t>streptavidin</t> <t>Alexa</t> <t>Fluor</t> 647 conjugate and subjected to FACS analysis. C , to determine the changes in the N -glycans on specific proteins after GnT-III overexpression, the cell lysates from control and GnT-III transfected MDA-MB-231 cells were also immunoprecipitated by ConA, E4-PHA, MAM (recognizing α2,3-sialylated proteins), and S. sieboldiana (recognizing α2,6-sialylated proteins) agaroses and probed with antibodies against α3 integrin, αv integrin, and β1 integrin separately. D , PA oligosaccharides treated with or without glycosidases from those cells were analyzed by reversed phase HPLC. E , PA oligosaccharides treated with or without neuraminidases from the control, GnT-III transfected MDA-MB-231 cells, as well as their ST6GAL1 knock-out counterparts, were analyzed by anion exchange HPLC to quantify the amount of sialylated N -glycans. F , RT-PCR using total RNA extracted from the control and GnT-III transfected MDA-MB-231 cells was carried out to examine the expression levels of genes involved in protein sialylation. The expression level of Gapdh was used as a loading control. SCT , sialic acid transporter; St3gal , β-galactoside α2,3-sialyltransferase; St6galnac , α- N -acetylgalactosaminide α2,6-sialyltransferase; NEU , neuraminidase. Con , control (DOX-inducible GnT-III-overexpressing cells without DOX treatment); OE , DOX inducible GnT-III-overexpressing cells treated with DOX; ST6GAL1 KO , ST6GAL1 knock-out cells.
    Streptavidin Alexa Fluor 647 R Phycoerythrin Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 647 conjugated streptavidin
    Overexpression of GnT-III in MDA-MB-231 cells significantly inhibited the α2,3-sialylation, but not α2,6-sialylation, at a post-transcriptional level. A , cell lysates from control and GnT-III transfected MDA-MB-231 cells were immunoblotted with E4-PHA and DSA lectins or immunoprecipitated ( IP ) with ConA-agarose and blotted with ConA, MAA, and SNA lectins. B , to confirm the effect of GnT-III expression on cell sialylation, cells transfected with or without GnT-III were incubated with ( bold line ) or without ( gray shading ) biotin-conjugated MAA (recognizing α2,3-sialylated proteins), followed by incubation with <t>streptavidin</t> <t>Alexa</t> <t>Fluor</t> 647 conjugate and subjected to FACS analysis. C , to determine the changes in the N -glycans on specific proteins after GnT-III overexpression, the cell lysates from control and GnT-III transfected MDA-MB-231 cells were also immunoprecipitated by ConA, E4-PHA, MAM (recognizing α2,3-sialylated proteins), and S. sieboldiana (recognizing α2,6-sialylated proteins) agaroses and probed with antibodies against α3 integrin, αv integrin, and β1 integrin separately. D , PA oligosaccharides treated with or without glycosidases from those cells were analyzed by reversed phase HPLC. E , PA oligosaccharides treated with or without neuraminidases from the control, GnT-III transfected MDA-MB-231 cells, as well as their ST6GAL1 knock-out counterparts, were analyzed by anion exchange HPLC to quantify the amount of sialylated N -glycans. F , RT-PCR using total RNA extracted from the control and GnT-III transfected MDA-MB-231 cells was carried out to examine the expression levels of genes involved in protein sialylation. The expression level of Gapdh was used as a loading control. SCT , sialic acid transporter; St3gal , β-galactoside α2,3-sialyltransferase; St6galnac , α- N -acetylgalactosaminide α2,6-sialyltransferase; NEU , neuraminidase. Con , control (DOX-inducible GnT-III-overexpressing cells without DOX treatment); OE , DOX inducible GnT-III-overexpressing cells treated with DOX; ST6GAL1 KO , ST6GAL1 knock-out cells.
    Alexa Fluor 647 Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 300 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin conjugated alexa fluor 647
    Overexpression of GnT-III in MDA-MB-231 cells significantly inhibited the α2,3-sialylation, but not α2,6-sialylation, at a post-transcriptional level. A , cell lysates from control and GnT-III transfected MDA-MB-231 cells were immunoblotted with E4-PHA and DSA lectins or immunoprecipitated ( IP ) with ConA-agarose and blotted with ConA, MAA, and SNA lectins. B , to confirm the effect of GnT-III expression on cell sialylation, cells transfected with or without GnT-III were incubated with ( bold line ) or without ( gray shading ) biotin-conjugated MAA (recognizing α2,3-sialylated proteins), followed by incubation with <t>streptavidin</t> <t>Alexa</t> <t>Fluor</t> 647 conjugate and subjected to FACS analysis. C , to determine the changes in the N -glycans on specific proteins after GnT-III overexpression, the cell lysates from control and GnT-III transfected MDA-MB-231 cells were also immunoprecipitated by ConA, E4-PHA, MAM (recognizing α2,3-sialylated proteins), and S. sieboldiana (recognizing α2,6-sialylated proteins) agaroses and probed with antibodies against α3 integrin, αv integrin, and β1 integrin separately. D , PA oligosaccharides treated with or without glycosidases from those cells were analyzed by reversed phase HPLC. E , PA oligosaccharides treated with or without neuraminidases from the control, GnT-III transfected MDA-MB-231 cells, as well as their ST6GAL1 knock-out counterparts, were analyzed by anion exchange HPLC to quantify the amount of sialylated N -glycans. F , RT-PCR using total RNA extracted from the control and GnT-III transfected MDA-MB-231 cells was carried out to examine the expression levels of genes involved in protein sialylation. The expression level of Gapdh was used as a loading control. SCT , sialic acid transporter; St3gal , β-galactoside α2,3-sialyltransferase; St6galnac , α- N -acetylgalactosaminide α2,6-sialyltransferase; NEU , neuraminidase. Con , control (DOX-inducible GnT-III-overexpressing cells without DOX treatment); OE , DOX inducible GnT-III-overexpressing cells treated with DOX; ST6GAL1 KO , ST6GAL1 knock-out cells.
    Streptavidin Conjugated Alexa Fluor 647, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 124 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The Jackson Laboratory alexa fluor 647 conjugated streptavidin
    Overexpression of GnT-III in MDA-MB-231 cells significantly inhibited the α2,3-sialylation, but not α2,6-sialylation, at a post-transcriptional level. A , cell lysates from control and GnT-III transfected MDA-MB-231 cells were immunoblotted with E4-PHA and DSA lectins or immunoprecipitated ( IP ) with ConA-agarose and blotted with ConA, MAA, and SNA lectins. B , to confirm the effect of GnT-III expression on cell sialylation, cells transfected with or without GnT-III were incubated with ( bold line ) or without ( gray shading ) biotin-conjugated MAA (recognizing α2,3-sialylated proteins), followed by incubation with <t>streptavidin</t> <t>Alexa</t> <t>Fluor</t> 647 conjugate and subjected to FACS analysis. C , to determine the changes in the N -glycans on specific proteins after GnT-III overexpression, the cell lysates from control and GnT-III transfected MDA-MB-231 cells were also immunoprecipitated by ConA, E4-PHA, MAM (recognizing α2,3-sialylated proteins), and S. sieboldiana (recognizing α2,6-sialylated proteins) agaroses and probed with antibodies against α3 integrin, αv integrin, and β1 integrin separately. D , PA oligosaccharides treated with or without glycosidases from those cells were analyzed by reversed phase HPLC. E , PA oligosaccharides treated with or without neuraminidases from the control, GnT-III transfected MDA-MB-231 cells, as well as their ST6GAL1 knock-out counterparts, were analyzed by anion exchange HPLC to quantify the amount of sialylated N -glycans. F , RT-PCR using total RNA extracted from the control and GnT-III transfected MDA-MB-231 cells was carried out to examine the expression levels of genes involved in protein sialylation. The expression level of Gapdh was used as a loading control. SCT , sialic acid transporter; St3gal , β-galactoside α2,3-sialyltransferase; St6galnac , α- N -acetylgalactosaminide α2,6-sialyltransferase; NEU , neuraminidase. Con , control (DOX-inducible GnT-III-overexpressing cells without DOX treatment); OE , DOX inducible GnT-III-overexpressing cells treated with DOX; ST6GAL1 KO , ST6GAL1 knock-out cells.
    Alexa Fluor 647 Conjugated Streptavidin, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 94/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jackson Immuno alexa fluor 647 conjugated streptavidin
    Chitin deposition and β-glucan unmasking result from an active fungal process. (A-B) <t>Streptavidin-Alexa</t> 647 labeled SC5314-GFP hyphae were either UV inactivated or not before being incubated with neutrophils or alone for the amount of time indicated. (A) Representative images of cells from each group at the three hour timepoint are shown. (B) Quantitation of cells for the phenotype indicated over the timecourse. Cells were scored for the indicated phenotype and results are presented as the percent of total cells for each time point. The data represents the mean ± SEM of three independent experiments. (C-D) Streptavidin-Alexa 647-labeled SC5314-GFP was UV inactivated and incubated with or without neutrophils. (C) Results represent the pooled average MFI at sites from individual frames in timelapses, as in Fig 1D . (D) Representative images of the T(-1) and T(+2) timepoints from timelapses of UV inactivated C . albicans . Scale bars represent 10 μm. * p-value of
    Alexa Fluor 647 Conjugated Streptavidin, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 94/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin alexa fluor 488 647
    Chitin deposition and β-glucan unmasking result from an active fungal process. (A-B) <t>Streptavidin-Alexa</t> 647 labeled SC5314-GFP hyphae were either UV inactivated or not before being incubated with neutrophils or alone for the amount of time indicated. (A) Representative images of cells from each group at the three hour timepoint are shown. (B) Quantitation of cells for the phenotype indicated over the timecourse. Cells were scored for the indicated phenotype and results are presented as the percent of total cells for each time point. The data represents the mean ± SEM of three independent experiments. (C-D) Streptavidin-Alexa 647-labeled SC5314-GFP was UV inactivated and incubated with or without neutrophils. (C) Results represent the pooled average MFI at sites from individual frames in timelapses, as in Fig 1D . (D) Representative images of the T(-1) and T(+2) timepoints from timelapses of UV inactivated C . albicans . Scale bars represent 10 μm. * p-value of
    Streptavidin Alexa Fluor 488 647, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 488 555 647 streptavidin
    Chitin deposition and β-glucan unmasking result from an active fungal process. (A-B) <t>Streptavidin-Alexa</t> 647 labeled SC5314-GFP hyphae were either UV inactivated or not before being incubated with neutrophils or alone for the amount of time indicated. (A) Representative images of cells from each group at the three hour timepoint are shown. (B) Quantitation of cells for the phenotype indicated over the timecourse. Cells were scored for the indicated phenotype and results are presented as the percent of total cells for each time point. The data represents the mean ± SEM of three independent experiments. (C-D) Streptavidin-Alexa 647-labeled SC5314-GFP was UV inactivated and incubated with or without neutrophils. (C) Results represent the pooled average MFI at sites from individual frames in timelapses, as in Fig 1D . (D) Representative images of the T(-1) and T(+2) timepoints from timelapses of UV inactivated C . albicans . Scale bars represent 10 μm. * p-value of
    Alexa Fluor 488 555 647 Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hrp streptavidin alexa fluor 647 tyramide
    Chitin deposition and β-glucan unmasking result from an active fungal process. (A-B) <t>Streptavidin-Alexa</t> 647 labeled SC5314-GFP hyphae were either UV inactivated or not before being incubated with neutrophils or alone for the amount of time indicated. (A) Representative images of cells from each group at the three hour timepoint are shown. (B) Quantitation of cells for the phenotype indicated over the timecourse. Cells were scored for the indicated phenotype and results are presented as the percent of total cells for each time point. The data represents the mean ± SEM of three independent experiments. (C-D) Streptavidin-Alexa 647-labeled SC5314-GFP was UV inactivated and incubated with or without neutrophils. (C) Results represent the pooled average MFI at sites from individual frames in timelapses, as in Fig 1D . (D) Representative images of the T(-1) and T(+2) timepoints from timelapses of UV inactivated C . albicans . Scale bars represent 10 μm. * p-value of
    Hrp Streptavidin Alexa Fluor 647 Tyramide, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher proteins alexa fluor 647 streptavidin
    Chitin deposition and β-glucan unmasking result from an active fungal process. (A-B) <t>Streptavidin-Alexa</t> 647 labeled SC5314-GFP hyphae were either UV inactivated or not before being incubated with neutrophils or alone for the amount of time indicated. (A) Representative images of cells from each group at the three hour timepoint are shown. (B) Quantitation of cells for the phenotype indicated over the timecourse. Cells were scored for the indicated phenotype and results are presented as the percent of total cells for each time point. The data represents the mean ± SEM of three independent experiments. (C-D) Streptavidin-Alexa 647-labeled SC5314-GFP was UV inactivated and incubated with or without neutrophils. (C) Results represent the pooled average MFI at sites from individual frames in timelapses, as in Fig 1D . (D) Representative images of the T(-1) and T(+2) timepoints from timelapses of UV inactivated C . albicans . Scale bars represent 10 μm. * p-value of
    Proteins Alexa Fluor 647 Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 647 conjugated streptavidin sa af647
    Chitin deposition and β-glucan unmasking result from an active fungal process. (A-B) <t>Streptavidin-Alexa</t> 647 labeled SC5314-GFP hyphae were either UV inactivated or not before being incubated with neutrophils or alone for the amount of time indicated. (A) Representative images of cells from each group at the three hour timepoint are shown. (B) Quantitation of cells for the phenotype indicated over the timecourse. Cells were scored for the indicated phenotype and results are presented as the percent of total cells for each time point. The data represents the mean ± SEM of three independent experiments. (C-D) Streptavidin-Alexa 647-labeled SC5314-GFP was UV inactivated and incubated with or without neutrophils. (C) Results represent the pooled average MFI at sites from individual frames in timelapses, as in Fig 1D . (D) Representative images of the T(-1) and T(+2) timepoints from timelapses of UV inactivated C . albicans . Scale bars represent 10 μm. * p-value of
    Alexa Fluor 647 Conjugated Streptavidin Sa Af647, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin alexa647
    Chitin deposition and β-glucan unmasking result from an active fungal process. (A-B) <t>Streptavidin-Alexa</t> 647 labeled SC5314-GFP hyphae were either UV inactivated or not before being incubated with neutrophils or alone for the amount of time indicated. (A) Representative images of cells from each group at the three hour timepoint are shown. (B) Quantitation of cells for the phenotype indicated over the timecourse. Cells were scored for the indicated phenotype and results are presented as the percent of total cells for each time point. The data represents the mean ± SEM of three independent experiments. (C-D) Streptavidin-Alexa 647-labeled SC5314-GFP was UV inactivated and incubated with or without neutrophils. (C) Results represent the pooled average MFI at sites from individual frames in timelapses, as in Fig 1D . (D) Representative images of the T(-1) and T(+2) timepoints from timelapses of UV inactivated C . albicans . Scale bars represent 10 μm. * p-value of
    Streptavidin Alexa647, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 137 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson streptavidin alexa647
    Chitin deposition and β-glucan unmasking result from an active fungal process. (A-B) <t>Streptavidin-Alexa</t> 647 labeled SC5314-GFP hyphae were either UV inactivated or not before being incubated with neutrophils or alone for the amount of time indicated. (A) Representative images of cells from each group at the three hour timepoint are shown. (B) Quantitation of cells for the phenotype indicated over the timecourse. Cells were scored for the indicated phenotype and results are presented as the percent of total cells for each time point. The data represents the mean ± SEM of three independent experiments. (C-D) Streptavidin-Alexa 647-labeled SC5314-GFP was UV inactivated and incubated with or without neutrophils. (C) Results represent the pooled average MFI at sites from individual frames in timelapses, as in Fig 1D . (D) Representative images of the T(-1) and T(+2) timepoints from timelapses of UV inactivated C . albicans . Scale bars represent 10 μm. * p-value of
    Streptavidin Alexa647, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 79/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin alexa
    Chitin deposition and β-glucan unmasking result from an active fungal process. (A-B) <t>Streptavidin-Alexa</t> 647 labeled SC5314-GFP hyphae were either UV inactivated or not before being incubated with neutrophils or alone for the amount of time indicated. (A) Representative images of cells from each group at the three hour timepoint are shown. (B) Quantitation of cells for the phenotype indicated over the timecourse. Cells were scored for the indicated phenotype and results are presented as the percent of total cells for each time point. The data represents the mean ± SEM of three independent experiments. (C-D) Streptavidin-Alexa 647-labeled SC5314-GFP was UV inactivated and incubated with or without neutrophils. (C) Results represent the pooled average MFI at sites from individual frames in timelapses, as in Fig 1D . (D) Representative images of the T(-1) and T(+2) timepoints from timelapses of UV inactivated C . albicans . Scale bars represent 10 μm. * p-value of
    Streptavidin Alexa, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Overexpression of GnT-III in MDA-MB-231 cells significantly inhibited the α2,3-sialylation, but not α2,6-sialylation, at a post-transcriptional level. A , cell lysates from control and GnT-III transfected MDA-MB-231 cells were immunoblotted with E4-PHA and DSA lectins or immunoprecipitated ( IP ) with ConA-agarose and blotted with ConA, MAA, and SNA lectins. B , to confirm the effect of GnT-III expression on cell sialylation, cells transfected with or without GnT-III were incubated with ( bold line ) or without ( gray shading ) biotin-conjugated MAA (recognizing α2,3-sialylated proteins), followed by incubation with streptavidin Alexa Fluor 647 conjugate and subjected to FACS analysis. C , to determine the changes in the N -glycans on specific proteins after GnT-III overexpression, the cell lysates from control and GnT-III transfected MDA-MB-231 cells were also immunoprecipitated by ConA, E4-PHA, MAM (recognizing α2,3-sialylated proteins), and S. sieboldiana (recognizing α2,6-sialylated proteins) agaroses and probed with antibodies against α3 integrin, αv integrin, and β1 integrin separately. D , PA oligosaccharides treated with or without glycosidases from those cells were analyzed by reversed phase HPLC. E , PA oligosaccharides treated with or without neuraminidases from the control, GnT-III transfected MDA-MB-231 cells, as well as their ST6GAL1 knock-out counterparts, were analyzed by anion exchange HPLC to quantify the amount of sialylated N -glycans. F , RT-PCR using total RNA extracted from the control and GnT-III transfected MDA-MB-231 cells was carried out to examine the expression levels of genes involved in protein sialylation. The expression level of Gapdh was used as a loading control. SCT , sialic acid transporter; St3gal , β-galactoside α2,3-sialyltransferase; St6galnac , α- N -acetylgalactosaminide α2,6-sialyltransferase; NEU , neuraminidase. Con , control (DOX-inducible GnT-III-overexpressing cells without DOX treatment); OE , DOX inducible GnT-III-overexpressing cells treated with DOX; ST6GAL1 KO , ST6GAL1 knock-out cells.

    Journal: The Journal of Biological Chemistry

    Article Title: Expression of N-Acetylglucosaminyltransferase III Suppresses α2,3-Sialylation, and Its Distinctive Functions in Cell Migration Are Attributed to α2,6-Sialylation Levels *

    doi: 10.1074/jbc.M115.712836

    Figure Lengend Snippet: Overexpression of GnT-III in MDA-MB-231 cells significantly inhibited the α2,3-sialylation, but not α2,6-sialylation, at a post-transcriptional level. A , cell lysates from control and GnT-III transfected MDA-MB-231 cells were immunoblotted with E4-PHA and DSA lectins or immunoprecipitated ( IP ) with ConA-agarose and blotted with ConA, MAA, and SNA lectins. B , to confirm the effect of GnT-III expression on cell sialylation, cells transfected with or without GnT-III were incubated with ( bold line ) or without ( gray shading ) biotin-conjugated MAA (recognizing α2,3-sialylated proteins), followed by incubation with streptavidin Alexa Fluor 647 conjugate and subjected to FACS analysis. C , to determine the changes in the N -glycans on specific proteins after GnT-III overexpression, the cell lysates from control and GnT-III transfected MDA-MB-231 cells were also immunoprecipitated by ConA, E4-PHA, MAM (recognizing α2,3-sialylated proteins), and S. sieboldiana (recognizing α2,6-sialylated proteins) agaroses and probed with antibodies against α3 integrin, αv integrin, and β1 integrin separately. D , PA oligosaccharides treated with or without glycosidases from those cells were analyzed by reversed phase HPLC. E , PA oligosaccharides treated with or without neuraminidases from the control, GnT-III transfected MDA-MB-231 cells, as well as their ST6GAL1 knock-out counterparts, were analyzed by anion exchange HPLC to quantify the amount of sialylated N -glycans. F , RT-PCR using total RNA extracted from the control and GnT-III transfected MDA-MB-231 cells was carried out to examine the expression levels of genes involved in protein sialylation. The expression level of Gapdh was used as a loading control. SCT , sialic acid transporter; St3gal , β-galactoside α2,3-sialyltransferase; St6galnac , α- N -acetylgalactosaminide α2,6-sialyltransferase; NEU , neuraminidase. Con , control (DOX-inducible GnT-III-overexpressing cells without DOX treatment); OE , DOX inducible GnT-III-overexpressing cells treated with DOX; ST6GAL1 KO , ST6GAL1 knock-out cells.

    Article Snippet: Then cells were stained with anti-β1 integrin antibody (P5D2) or 10 μg/ml biotinylated SNA or MAA for 30 min on ice, followed by incubation with streptavidin conjugate Alexa Fluor 647 (Invitrogen) for 30 min on ice.

    Techniques: Over Expression, Multiple Displacement Amplification, Transfection, Immunoprecipitation, Expressing, Incubation, FACS, High Performance Liquid Chromatography, Knock-Out, Reverse Transcription Polymerase Chain Reaction

    Knock-out of ST6GAL1 endowed GnT-III with an anti-migratory role in MDA-MB-231 cells. The MDA-MB-231 derivative cell lines as indicated were incubated with ( bold line ) or without ( gray shading ) biotin-conjugated MAA, biotin-conjugated SNA, or β1 integrin antibody followed by incubation with appropriate Alexa Fluor 647 conjugate and subjected to FACS analysis ( A , D , and G ). Their cell migration toward FN was determined by Transwell assay. Cells that migrated through the Transwell membrane were stained with 0.5% crystal violet. Shown are representative examples recorded by phase contrast microscopy ( B , E , and H ). Scale bar , 200 μm. The migrated cells were counted under a microscope. The quantitative data were obtained from three independent experiments ( C , F , and I ). The p values were calculated using one-tail unpaired t test. Error bars indicate standard derivation. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Expression of N-Acetylglucosaminyltransferase III Suppresses α2,3-Sialylation, and Its Distinctive Functions in Cell Migration Are Attributed to α2,6-Sialylation Levels *

    doi: 10.1074/jbc.M115.712836

    Figure Lengend Snippet: Knock-out of ST6GAL1 endowed GnT-III with an anti-migratory role in MDA-MB-231 cells. The MDA-MB-231 derivative cell lines as indicated were incubated with ( bold line ) or without ( gray shading ) biotin-conjugated MAA, biotin-conjugated SNA, or β1 integrin antibody followed by incubation with appropriate Alexa Fluor 647 conjugate and subjected to FACS analysis ( A , D , and G ). Their cell migration toward FN was determined by Transwell assay. Cells that migrated through the Transwell membrane were stained with 0.5% crystal violet. Shown are representative examples recorded by phase contrast microscopy ( B , E , and H ). Scale bar , 200 μm. The migrated cells were counted under a microscope. The quantitative data were obtained from three independent experiments ( C , F , and I ). The p values were calculated using one-tail unpaired t test. Error bars indicate standard derivation. *, p

    Article Snippet: Then cells were stained with anti-β1 integrin antibody (P5D2) or 10 μg/ml biotinylated SNA or MAA for 30 min on ice, followed by incubation with streptavidin conjugate Alexa Fluor 647 (Invitrogen) for 30 min on ice.

    Techniques: Knock-Out, Multiple Displacement Amplification, Incubation, FACS, Migration, Transwell Assay, Staining, Microscopy

    GnT-III overexpression in HepG2 cells significantly increased cell migration, but knock-out of ST6GAL1 in this cell line conferred GnT-III with the opposite effect on cell motility. Overexpression of ST6GAL1 was able to neutralize the anti-migratory role of GnT-III in HeLa cells. A , cell lysates from the control and GnT-III-overexpressing HepG2 cells were immunoblotted with E4-PHA and DSA lectins to check the overexpression efficiency of GnT-III. B , their cell migration toward FN was determined by Transwell assay. Cells that migrated through the Transwell membrane were stained with 0.5% crystal violet. Shown is a representative example recorded by phase contrast microscopy. Scale bar , 100 μm. C , the migrated cells were counted under a microscope. The quantitative data were obtained from three independent experiments. D , ST6GAL1 knock-out HepG2 cells were incubated with ( bold line ) or without ( gray shading ) biotin-conjugated SNA, followed by incubation with streptavidin Alexa Fluor 647 conjugate and subjected to FACS analysis. E , the migratory ability of cells indicated toward FN was determined by Transwell assay. Cells that migrated through the Transwell membrane were stained with 0.5% crystal violet. Shown is a representative example recorded by phase contrast microscopy. Scale bar , 100 μm. F , the migrated cells were counted under a microscope. The quantitative data were obtained from three independent experiments. The p values were calculated using one-tail unpaired t test. Error bars indicate standard derivation. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Expression of N-Acetylglucosaminyltransferase III Suppresses α2,3-Sialylation, and Its Distinctive Functions in Cell Migration Are Attributed to α2,6-Sialylation Levels *

    doi: 10.1074/jbc.M115.712836

    Figure Lengend Snippet: GnT-III overexpression in HepG2 cells significantly increased cell migration, but knock-out of ST6GAL1 in this cell line conferred GnT-III with the opposite effect on cell motility. Overexpression of ST6GAL1 was able to neutralize the anti-migratory role of GnT-III in HeLa cells. A , cell lysates from the control and GnT-III-overexpressing HepG2 cells were immunoblotted with E4-PHA and DSA lectins to check the overexpression efficiency of GnT-III. B , their cell migration toward FN was determined by Transwell assay. Cells that migrated through the Transwell membrane were stained with 0.5% crystal violet. Shown is a representative example recorded by phase contrast microscopy. Scale bar , 100 μm. C , the migrated cells were counted under a microscope. The quantitative data were obtained from three independent experiments. D , ST6GAL1 knock-out HepG2 cells were incubated with ( bold line ) or without ( gray shading ) biotin-conjugated SNA, followed by incubation with streptavidin Alexa Fluor 647 conjugate and subjected to FACS analysis. E , the migratory ability of cells indicated toward FN was determined by Transwell assay. Cells that migrated through the Transwell membrane were stained with 0.5% crystal violet. Shown is a representative example recorded by phase contrast microscopy. Scale bar , 100 μm. F , the migrated cells were counted under a microscope. The quantitative data were obtained from three independent experiments. The p values were calculated using one-tail unpaired t test. Error bars indicate standard derivation. *, p

    Article Snippet: Then cells were stained with anti-β1 integrin antibody (P5D2) or 10 μg/ml biotinylated SNA or MAA for 30 min on ice, followed by incubation with streptavidin conjugate Alexa Fluor 647 (Invitrogen) for 30 min on ice.

    Techniques: Over Expression, Migration, Knock-Out, Transwell Assay, Staining, Microscopy, Incubation, FACS

    Overexpression of ST6GAL1 was able to neutralize the anti-migratory role of GnT-III in HeLa cells. A , cell lysates from the control and GnT-III-overexpressing HeLa cells were immunoblotted with E4-PHA and DSA lectins to check the overexpression efficiency of GnT-III. B , these two cells together with ST6GAL1 and GnT-III-overexpressing HeLa cells were incubated with ( bold line ) or without ( gray shading ) biotin-conjugated MAA or biotin-conjugated SNA, followed by incubation with streptavidin Alexa Fluor 647 conjugate and subjected to FACS analysis. C , their cell migration toward FN was determined by Transwell assay. Cells that migrated through the Transwell membrane were stained with 0.5% crystal violet. Shown is a representative example recorded by phase contrast microscopy. Scale bar , 200 μm. The migrated cells were counted under a microscope. The quantitative data were obtained from three independent experiments. The p values were calculated using one-tail unpaired t test. Error bars indicate standard derivation. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Expression of N-Acetylglucosaminyltransferase III Suppresses α2,3-Sialylation, and Its Distinctive Functions in Cell Migration Are Attributed to α2,6-Sialylation Levels *

    doi: 10.1074/jbc.M115.712836

    Figure Lengend Snippet: Overexpression of ST6GAL1 was able to neutralize the anti-migratory role of GnT-III in HeLa cells. A , cell lysates from the control and GnT-III-overexpressing HeLa cells were immunoblotted with E4-PHA and DSA lectins to check the overexpression efficiency of GnT-III. B , these two cells together with ST6GAL1 and GnT-III-overexpressing HeLa cells were incubated with ( bold line ) or without ( gray shading ) biotin-conjugated MAA or biotin-conjugated SNA, followed by incubation with streptavidin Alexa Fluor 647 conjugate and subjected to FACS analysis. C , their cell migration toward FN was determined by Transwell assay. Cells that migrated through the Transwell membrane were stained with 0.5% crystal violet. Shown is a representative example recorded by phase contrast microscopy. Scale bar , 200 μm. The migrated cells were counted under a microscope. The quantitative data were obtained from three independent experiments. The p values were calculated using one-tail unpaired t test. Error bars indicate standard derivation. *, p

    Article Snippet: Then cells were stained with anti-β1 integrin antibody (P5D2) or 10 μg/ml biotinylated SNA or MAA for 30 min on ice, followed by incubation with streptavidin conjugate Alexa Fluor 647 (Invitrogen) for 30 min on ice.

    Techniques: Over Expression, Incubation, FACS, Migration, Transwell Assay, Staining, Microscopy

    Chitin deposition and β-glucan unmasking result from an active fungal process. (A-B) Streptavidin-Alexa 647 labeled SC5314-GFP hyphae were either UV inactivated or not before being incubated with neutrophils or alone for the amount of time indicated. (A) Representative images of cells from each group at the three hour timepoint are shown. (B) Quantitation of cells for the phenotype indicated over the timecourse. Cells were scored for the indicated phenotype and results are presented as the percent of total cells for each time point. The data represents the mean ± SEM of three independent experiments. (C-D) Streptavidin-Alexa 647-labeled SC5314-GFP was UV inactivated and incubated with or without neutrophils. (C) Results represent the pooled average MFI at sites from individual frames in timelapses, as in Fig 1D . (D) Representative images of the T(-1) and T(+2) timepoints from timelapses of UV inactivated C . albicans . Scale bars represent 10 μm. * p-value of

    Journal: PLoS Pathogens

    Article Title: Neutrophil Attack Triggers Extracellular Trap-Dependent Candida Cell Wall Remodeling and Altered Immune Recognition

    doi: 10.1371/journal.ppat.1005644

    Figure Lengend Snippet: Chitin deposition and β-glucan unmasking result from an active fungal process. (A-B) Streptavidin-Alexa 647 labeled SC5314-GFP hyphae were either UV inactivated or not before being incubated with neutrophils or alone for the amount of time indicated. (A) Representative images of cells from each group at the three hour timepoint are shown. (B) Quantitation of cells for the phenotype indicated over the timecourse. Cells were scored for the indicated phenotype and results are presented as the percent of total cells for each time point. The data represents the mean ± SEM of three independent experiments. (C-D) Streptavidin-Alexa 647-labeled SC5314-GFP was UV inactivated and incubated with or without neutrophils. (C) Results represent the pooled average MFI at sites from individual frames in timelapses, as in Fig 1D . (D) Representative images of the T(-1) and T(+2) timepoints from timelapses of UV inactivated C . albicans . Scale bars represent 10 μm. * p-value of

    Article Snippet: Cells were then labeled with Alexa Fluor 647-conjugated Streptavidin (Jackson Immunoresearch; 36 μg/mL).

    Techniques: Labeling, Incubation, Quantitation Assay

    Neutrophil attachment results in rapid cell wall damage to C . albicans . (A-D) Streptavidin-Alexa 647-labeled SC5314-GFP was incubated with or without neutrophils. (A-C) Representative timelapse images at one minute intervals for (A) attacked, (B) unattacked and (C) control hyphal segments. (D) Relative streptavidin mean fluorescence intensity (MFI) at attacked and unattacked sites. Data represents the pooled average of thirteen cells measured in three experiments ± SEM. Scale bar = 10 μm. (E-I). Streptavidin-Alexa 647-labeled Hwp1-GFP fungi were incubated with or without neutrophils. (E-G) Representative time-lapse images for (E) attacked, (F) unattacked and (G) control hyphal segments. (H) Relative streptavidin intensity for twenty three cells and Hwp1-GFP intensity for thirty three cells imaged in three independent experiments was measured and the pooled average ± SEM is shown. (I) Relative intensity at the 2 minute post-attachment (T+2) time point is shown for the pooled data as box plots to illustrate the full dataset. Box plot whiskers represent the 1.5 interquartile range either below or above the lower or upper quartile ** p ≤0.01 and *** p≤0.001 (one-way ANOVA with Tukey’s post-test).

    Journal: PLoS Pathogens

    Article Title: Neutrophil Attack Triggers Extracellular Trap-Dependent Candida Cell Wall Remodeling and Altered Immune Recognition

    doi: 10.1371/journal.ppat.1005644

    Figure Lengend Snippet: Neutrophil attachment results in rapid cell wall damage to C . albicans . (A-D) Streptavidin-Alexa 647-labeled SC5314-GFP was incubated with or without neutrophils. (A-C) Representative timelapse images at one minute intervals for (A) attacked, (B) unattacked and (C) control hyphal segments. (D) Relative streptavidin mean fluorescence intensity (MFI) at attacked and unattacked sites. Data represents the pooled average of thirteen cells measured in three experiments ± SEM. Scale bar = 10 μm. (E-I). Streptavidin-Alexa 647-labeled Hwp1-GFP fungi were incubated with or without neutrophils. (E-G) Representative time-lapse images for (E) attacked, (F) unattacked and (G) control hyphal segments. (H) Relative streptavidin intensity for twenty three cells and Hwp1-GFP intensity for thirty three cells imaged in three independent experiments was measured and the pooled average ± SEM is shown. (I) Relative intensity at the 2 minute post-attachment (T+2) time point is shown for the pooled data as box plots to illustrate the full dataset. Box plot whiskers represent the 1.5 interquartile range either below or above the lower or upper quartile ** p ≤0.01 and *** p≤0.001 (one-way ANOVA with Tukey’s post-test).

    Article Snippet: Cells were then labeled with Alexa Fluor 647-conjugated Streptavidin (Jackson Immunoresearch; 36 μg/mL).

    Techniques: Labeling, Incubation, Fluorescence

    NETs are critical for immune attack to cause β-glucan unmasking. (A-B) Neutrophils from C57BL/6J mice were incubated with WT-FarRed670 hyphae. Neutrophils were not lysed and samples were stained for MPO or citrullinated H3. Representative images of MPO (A) and citrullinated H3 staining (B). Scale bar represents 20 μm. (C-D) Neutrophils were pretreated with DNase I, DPI or ABAH and then incubated with WT-FarRed670 for 2.5 hours. Neutrophils were not lysed and then samples were stained with CFW, sDectin-1-Fc and Sytox Green. Images were analyzed by scoring all viable cell segments for the presence of the indicated phenotypes and results are presented as the percentage of total cell segments counted which displayed the phenotype for each group. Data is presented as the mean ± SEM from three experiments. Cell viability was determined using characteristic cytoplasmic far red expression (see Methods ). ‡ p-value ≤ 0.01 when comparing the sDectin-1-Fc group from WT or DMSO treated to other groups. ¥ p-value ≤ 0.01 when comparing the chitin deposition group from WT or DMSO treated to other groups. * p-value ≤ 0.01 when comparing the overlap group from WT or DMSO treated to other groups. Comparisons done with one way ANOVA and Tukey’s post-test. (E-F) Neutrophils from C57BL/6J or gp91 phox-/- mice were incubated with Streptavidin-Alexa 647 labelled SC5314-GFP hyphae. Neutrophils were lysed and fungi were stained with sDectin-1-Fc and Calcofluor White. (E) A representative set of images are shown for each group. (F) Images were analyzed by scoring all viable cell segments for the presence of the indicated phenotypes and results are presented as the percentage of total cell segments counted which displayed the phenotype for each group. Data is presented as the mean ± SEM from three experiments. Cell viability was determined using characteristic cytoplasmic GFP expression (see Methods ). ‡ p-value ≤ 0.01 when comparing the sDectin-1-Fc group from WT to either gp91 phox-/- or no neutrophil groups. ¥ p-value ≤ 0.01 when comparing the chitin deposition group from WT to either gp91 phox-/- or no neutrophil groups. * p-value ≤ 0.01 when comparing the overlap group from WT to either gp91 phox-/- or no neutrophil groups. Comparisons done with one way ANOVA and Tukey’s post-test. (G-K) C57BL/6J or gp91 phox-/- mice were injected in the tail vein with SC5314-GFP and kidneys were harvested on day 5 post infection. (G-H) Representative images of kidney homogenates stained with sDectin-1-Fc and CFW Bottom panels show homogenates treated with secondary antibody only as a control. (I) Representative images of an overnight culture of SC5314-GFP grown in RPMI and then stained with sDectin-1-Fc and CFW. (J-K) Quantification of sDectin-1-Fc staining (J) and chitin staining (K). Data is presented as the mean ± SEM from two pooled experiments, except for the RPMI group which represents a single experiment. *** p-value ≤0.001 (Kruskal-Wallis with Dunn’s post-test). n.s means non-significant. Scale bar represents 10 μm.

    Journal: PLoS Pathogens

    Article Title: Neutrophil Attack Triggers Extracellular Trap-Dependent Candida Cell Wall Remodeling and Altered Immune Recognition

    doi: 10.1371/journal.ppat.1005644

    Figure Lengend Snippet: NETs are critical for immune attack to cause β-glucan unmasking. (A-B) Neutrophils from C57BL/6J mice were incubated with WT-FarRed670 hyphae. Neutrophils were not lysed and samples were stained for MPO or citrullinated H3. Representative images of MPO (A) and citrullinated H3 staining (B). Scale bar represents 20 μm. (C-D) Neutrophils were pretreated with DNase I, DPI or ABAH and then incubated with WT-FarRed670 for 2.5 hours. Neutrophils were not lysed and then samples were stained with CFW, sDectin-1-Fc and Sytox Green. Images were analyzed by scoring all viable cell segments for the presence of the indicated phenotypes and results are presented as the percentage of total cell segments counted which displayed the phenotype for each group. Data is presented as the mean ± SEM from three experiments. Cell viability was determined using characteristic cytoplasmic far red expression (see Methods ). ‡ p-value ≤ 0.01 when comparing the sDectin-1-Fc group from WT or DMSO treated to other groups. ¥ p-value ≤ 0.01 when comparing the chitin deposition group from WT or DMSO treated to other groups. * p-value ≤ 0.01 when comparing the overlap group from WT or DMSO treated to other groups. Comparisons done with one way ANOVA and Tukey’s post-test. (E-F) Neutrophils from C57BL/6J or gp91 phox-/- mice were incubated with Streptavidin-Alexa 647 labelled SC5314-GFP hyphae. Neutrophils were lysed and fungi were stained with sDectin-1-Fc and Calcofluor White. (E) A representative set of images are shown for each group. (F) Images were analyzed by scoring all viable cell segments for the presence of the indicated phenotypes and results are presented as the percentage of total cell segments counted which displayed the phenotype for each group. Data is presented as the mean ± SEM from three experiments. Cell viability was determined using characteristic cytoplasmic GFP expression (see Methods ). ‡ p-value ≤ 0.01 when comparing the sDectin-1-Fc group from WT to either gp91 phox-/- or no neutrophil groups. ¥ p-value ≤ 0.01 when comparing the chitin deposition group from WT to either gp91 phox-/- or no neutrophil groups. * p-value ≤ 0.01 when comparing the overlap group from WT to either gp91 phox-/- or no neutrophil groups. Comparisons done with one way ANOVA and Tukey’s post-test. (G-K) C57BL/6J or gp91 phox-/- mice were injected in the tail vein with SC5314-GFP and kidneys were harvested on day 5 post infection. (G-H) Representative images of kidney homogenates stained with sDectin-1-Fc and CFW Bottom panels show homogenates treated with secondary antibody only as a control. (I) Representative images of an overnight culture of SC5314-GFP grown in RPMI and then stained with sDectin-1-Fc and CFW. (J-K) Quantification of sDectin-1-Fc staining (J) and chitin staining (K). Data is presented as the mean ± SEM from two pooled experiments, except for the RPMI group which represents a single experiment. *** p-value ≤0.001 (Kruskal-Wallis with Dunn’s post-test). n.s means non-significant. Scale bar represents 10 μm.

    Article Snippet: Cells were then labeled with Alexa Fluor 647-conjugated Streptavidin (Jackson Immunoresearch; 36 μg/mL).

    Techniques: Mouse Assay, Incubation, Staining, Expressing, Injection, Infection

    Cell wall integrity sensing and remodeling are involved in the fungal response to neutrophil attack. (A-D) Streptavidin-Alexa 647 labeled C . albicans hyphae of the indicated strains were incubated with neutrophils or alone. Neutrophils were not lysed and samples were stained with CFW and sDectin-1-Fc. (A) Representative images of the HOG1 strain set. Images were analyzed by scoring cells for localized chitin deposition, sDectin-1-Fc staining and the overlap of the two phenotypes. (B) The scoring data was normalized based on the frequency of cell wall changes in the wildtype strain plus neutrophils and represents the mean ± SEM from three independent experiments. (C) Representative images of the chitin synthase strain set. Images were analyzed by scoring cells for increased chitin deposition, sDectin-1-Fc staining and the overlap of the two phenotypes. (D) Data is presented as the percent of total cells with the phenotypes and represents the mean ± SEM from three independent experiments. The Chs3-YFP strain was incubated with neutrophils in an imaging dish and timelapses were taken. (E) Panels taken from a timelapse of the Chs3-YFP strain after neutrophil attack. (F) The JC94-2 strain was incubated with neutrophils for the indicated time in an imaging dish before being imaged. Representative images from the 3 hour timepoint. The red dotted line represents the outline of a neutrophil. (G) Images were analyzed by obtaining the mean fluorescent intensity of GFP at sites with or without chitin deposition and the data is presented as the mean MFI ± SEM at sites from three pooled experiments except the 1h timepoint, which represents two pooled experiments. (H-I) The Sur7-GFP strain was incubated with neutrophils. Following incubation, neutrophils were lysed and fungi were stained with sDectin-1-Fc and CFW. Data represents the MFI of GFP or Cy3 staining at sites with or without chitin deposition or from the no neutrophil control and is presented as the mean MFI ± SEM from three pooled experiments. * p-value of ≤0.05, ** p-value of ≤0.01 and *** p-value of ≤0.001. For I, * p≤0.05 comparing Sur7-GFP at chitin deposition sites vs no chitin deposition or no PMN control. (¥) p≤0.001 comparing the WT and hog1/HOG1 plus neutrophil groups to the no neutrophil control groups. (≠) p≤ 0.01 comparing the hog1 plus neutrophils group to the no neutrophil control groups. (‡) p≤ 0.01 comparing sDectin-1-Fc at chitin deposition sites vs no chitin deposition sites or no neutrophil controls. (Student’s T-test for D and G or one way ANOVA with Tukey’s post-test for B and I). n.s. means non-significant. Scale bar represents 10 μm.

    Journal: PLoS Pathogens

    Article Title: Neutrophil Attack Triggers Extracellular Trap-Dependent Candida Cell Wall Remodeling and Altered Immune Recognition

    doi: 10.1371/journal.ppat.1005644

    Figure Lengend Snippet: Cell wall integrity sensing and remodeling are involved in the fungal response to neutrophil attack. (A-D) Streptavidin-Alexa 647 labeled C . albicans hyphae of the indicated strains were incubated with neutrophils or alone. Neutrophils were not lysed and samples were stained with CFW and sDectin-1-Fc. (A) Representative images of the HOG1 strain set. Images were analyzed by scoring cells for localized chitin deposition, sDectin-1-Fc staining and the overlap of the two phenotypes. (B) The scoring data was normalized based on the frequency of cell wall changes in the wildtype strain plus neutrophils and represents the mean ± SEM from three independent experiments. (C) Representative images of the chitin synthase strain set. Images were analyzed by scoring cells for increased chitin deposition, sDectin-1-Fc staining and the overlap of the two phenotypes. (D) Data is presented as the percent of total cells with the phenotypes and represents the mean ± SEM from three independent experiments. The Chs3-YFP strain was incubated with neutrophils in an imaging dish and timelapses were taken. (E) Panels taken from a timelapse of the Chs3-YFP strain after neutrophil attack. (F) The JC94-2 strain was incubated with neutrophils for the indicated time in an imaging dish before being imaged. Representative images from the 3 hour timepoint. The red dotted line represents the outline of a neutrophil. (G) Images were analyzed by obtaining the mean fluorescent intensity of GFP at sites with or without chitin deposition and the data is presented as the mean MFI ± SEM at sites from three pooled experiments except the 1h timepoint, which represents two pooled experiments. (H-I) The Sur7-GFP strain was incubated with neutrophils. Following incubation, neutrophils were lysed and fungi were stained with sDectin-1-Fc and CFW. Data represents the MFI of GFP or Cy3 staining at sites with or without chitin deposition or from the no neutrophil control and is presented as the mean MFI ± SEM from three pooled experiments. * p-value of ≤0.05, ** p-value of ≤0.01 and *** p-value of ≤0.001. For I, * p≤0.05 comparing Sur7-GFP at chitin deposition sites vs no chitin deposition or no PMN control. (¥) p≤0.001 comparing the WT and hog1/HOG1 plus neutrophil groups to the no neutrophil control groups. (≠) p≤ 0.01 comparing the hog1 plus neutrophils group to the no neutrophil control groups. (‡) p≤ 0.01 comparing sDectin-1-Fc at chitin deposition sites vs no chitin deposition sites or no neutrophil controls. (Student’s T-test for D and G or one way ANOVA with Tukey’s post-test for B and I). n.s. means non-significant. Scale bar represents 10 μm.

    Article Snippet: Cells were then labeled with Alexa Fluor 647-conjugated Streptavidin (Jackson Immunoresearch; 36 μg/mL).

    Techniques: Labeling, Incubation, Staining, Imaging

    mBirA expression and selected gold-standard INTACT (INT) lines. ( a ) Immunostaining of a late globular INT line (INT4; pIQD15-NTF , pWOX2-mBirA-3xMyc ) embryo showing whole embryo mBirA expression (Anti-Myc Tag and Alexa Fluor 488 antibody; green) and NTF biotinylation in the vascular tissue precursors (Strepdavidin conjugated with Alexa Fluor 647; red). Counterstaining by DAPI (blue). Insert: negative control (INT4). GFP signal (green) from the pIQD15-NTF is visible in the vascular tissue precursors after fixation. ( b ) Expression of pSCR-mBirA-mCherry in hypophysis at early globular stage (insert) and in QC and ground tissue precursors at late globular stage. ( c - j ) INT lines where NTF is expressed in the whole embryo (INT0; c ), suspensor (INT17; d ), protoderm (INT41; e ), hypophysis (INT40; f ), QC precursor (INT7b/a; g ), and precursor cells (INT55; h ) of the vascular (INT4; i ) and ground tissue (INT39; j ) initials. Scale bar represent 10 μm in all panels.

    Journal: Nature plants

    Article Title: Transcriptome dynamics revealed by a gene expression atlas of the early Arabidopsis embryo

    doi: 10.1038/s41477-017-0035-3

    Figure Lengend Snippet: mBirA expression and selected gold-standard INTACT (INT) lines. ( a ) Immunostaining of a late globular INT line (INT4; pIQD15-NTF , pWOX2-mBirA-3xMyc ) embryo showing whole embryo mBirA expression (Anti-Myc Tag and Alexa Fluor 488 antibody; green) and NTF biotinylation in the vascular tissue precursors (Strepdavidin conjugated with Alexa Fluor 647; red). Counterstaining by DAPI (blue). Insert: negative control (INT4). GFP signal (green) from the pIQD15-NTF is visible in the vascular tissue precursors after fixation. ( b ) Expression of pSCR-mBirA-mCherry in hypophysis at early globular stage (insert) and in QC and ground tissue precursors at late globular stage. ( c - j ) INT lines where NTF is expressed in the whole embryo (INT0; c ), suspensor (INT17; d ), protoderm (INT41; e ), hypophysis (INT40; f ), QC precursor (INT7b/a; g ), and precursor cells (INT55; h ) of the vascular (INT4; i ) and ground tissue (INT39; j ) initials. Scale bar represent 10 μm in all panels.

    Article Snippet: The Anti-Myc Tag antibody (clone 9E10) (Merck Millipore), and a secondary Anti-Mouse Alexa Fluor 488 (RRID:AB_141514) and/or Strepdavidin conjugated with Alexa Fluor 647 antibody (RRID:AB_2336066) (Thermo Fisher Scientific, OR, USA) were used (at 1:400 dilution).

    Techniques: Expressing, Immunostaining, Negative Control