automated image-analysis pipeline Search Results


image analysis software imaris  (Oxford Instruments)
99
Oxford Instruments image analysis software imaris
CFIm KD increases the activity of miRNAs. ( A ) Genome browser tracks showing the coverage of DICER1 TE (bottom track) by RNA-seq reads from two replicate experiments for each condition, with the two PAS that were quantified for this gene marked by black lines. The conditions are color-coded (as in Figure ) and also indicated on the y-axis. Y-axis shows the smoothened number of reads mapping along the TE, calculated by the GViz R package. ( B ) RNA fluorescence in situ imaging of DICER1 isoforms in Control and CFIm25 KD HEK293 cells with probes corresponding to the common region of the long and short 3′ UTRs (red) or to the region between the proximal and distal cleavage sites, thus present exclusively in the long 3′ UTR (green). Nuclei are marked with DAPI. Zoom-ins of the regions marked with dashed boxes are further shown both with the individual and merged channels. A snapshot of a digital representation of the actual <t>image</t> as processed in <t>IMARIS</t> is also depicted for reference. ( C ) Quantification of the copy number of the long and short 3′ UTR isoforms of DICER1 in the nucleus (left plot) and cytoplasm (right plot) of Control, CFIm25 and CFIm68 KD cells. Colocalization of the red and green signals reveals the presence of the long 3′ UTR isoform (yellow) whereas the signal from the red probe only reveals the presence of the shorter 3′ UTR isoform. mRNA copy numbers were estimated separately from the nucleus (overlapping with DAPI) and cytosol. Segregation of the signal was performed with IMARIS (see Methods). ( D ) Representative western blot showing the DICER1 expression in the Control, CFIm25 and CFIm68 KD cells. The quantification is relative to GAPDH. ( E ) qPCR measurements of let-7, miR-92a, miR-16 and miR-19b expression in CFIm25/68 KD cells relative to Control. ΔΔct values were calculated relative to U6 snRNA and then relative to the Control cells (where the ratio was set to 1). ( F ) Normalized Renilla luciferase expression of reporter mRNAs carrying binding sites for miR-16 and miR-92a in their 3′ UTRs, in Control, CFIm25 and CFIm68 KD cells, respectively. The Firefly luciferase expressed from the same construct was used as normalization control.
Image Analysis Software Imaris, supplied by Oxford Instruments, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/automated+image-analysis+pipeline/pmc08989530-156-12-15?v=Oxford+Instruments
Average 99 stars, based on 1 article reviews
image analysis software imaris - by Bioz Stars, 2026-07
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harmony software  (Revvity)
94
Revvity harmony software
CFIm KD increases the activity of miRNAs. ( A ) Genome browser tracks showing the coverage of DICER1 TE (bottom track) by RNA-seq reads from two replicate experiments for each condition, with the two PAS that were quantified for this gene marked by black lines. The conditions are color-coded (as in Figure ) and also indicated on the y-axis. Y-axis shows the smoothened number of reads mapping along the TE, calculated by the GViz R package. ( B ) RNA fluorescence in situ imaging of DICER1 isoforms in Control and CFIm25 KD HEK293 cells with probes corresponding to the common region of the long and short 3′ UTRs (red) or to the region between the proximal and distal cleavage sites, thus present exclusively in the long 3′ UTR (green). Nuclei are marked with DAPI. Zoom-ins of the regions marked with dashed boxes are further shown both with the individual and merged channels. A snapshot of a digital representation of the actual <t>image</t> as processed in <t>IMARIS</t> is also depicted for reference. ( C ) Quantification of the copy number of the long and short 3′ UTR isoforms of DICER1 in the nucleus (left plot) and cytoplasm (right plot) of Control, CFIm25 and CFIm68 KD cells. Colocalization of the red and green signals reveals the presence of the long 3′ UTR isoform (yellow) whereas the signal from the red probe only reveals the presence of the shorter 3′ UTR isoform. mRNA copy numbers were estimated separately from the nucleus (overlapping with DAPI) and cytosol. Segregation of the signal was performed with IMARIS (see Methods). ( D ) Representative western blot showing the DICER1 expression in the Control, CFIm25 and CFIm68 KD cells. The quantification is relative to GAPDH. ( E ) qPCR measurements of let-7, miR-92a, miR-16 and miR-19b expression in CFIm25/68 KD cells relative to Control. ΔΔct values were calculated relative to U6 snRNA and then relative to the Control cells (where the ratio was set to 1). ( F ) Normalized Renilla luciferase expression of reporter mRNAs carrying binding sites for miR-16 and miR-92a in their 3′ UTRs, in Control, CFIm25 and CFIm68 KD cells, respectively. The Firefly luciferase expressed from the same construct was used as normalization control.
Harmony Software, supplied by Revvity, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/automated+image-analysis+pipeline/bio_rxiv__2023__06__01__543323-232-11-13?v=Revvity
Average 94 stars, based on 1 article reviews
harmony software - by Bioz Stars, 2026-07
94/100 stars
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knime software  (KNIME GmbH)
90
KNIME GmbH knime software
CFIm KD increases the activity of miRNAs. ( A ) Genome browser tracks showing the coverage of DICER1 TE (bottom track) by RNA-seq reads from two replicate experiments for each condition, with the two PAS that were quantified for this gene marked by black lines. The conditions are color-coded (as in Figure ) and also indicated on the y-axis. Y-axis shows the smoothened number of reads mapping along the TE, calculated by the GViz R package. ( B ) RNA fluorescence in situ imaging of DICER1 isoforms in Control and CFIm25 KD HEK293 cells with probes corresponding to the common region of the long and short 3′ UTRs (red) or to the region between the proximal and distal cleavage sites, thus present exclusively in the long 3′ UTR (green). Nuclei are marked with DAPI. Zoom-ins of the regions marked with dashed boxes are further shown both with the individual and merged channels. A snapshot of a digital representation of the actual <t>image</t> as processed in <t>IMARIS</t> is also depicted for reference. ( C ) Quantification of the copy number of the long and short 3′ UTR isoforms of DICER1 in the nucleus (left plot) and cytoplasm (right plot) of Control, CFIm25 and CFIm68 KD cells. Colocalization of the red and green signals reveals the presence of the long 3′ UTR isoform (yellow) whereas the signal from the red probe only reveals the presence of the shorter 3′ UTR isoform. mRNA copy numbers were estimated separately from the nucleus (overlapping with DAPI) and cytosol. Segregation of the signal was performed with IMARIS (see Methods). ( D ) Representative western blot showing the DICER1 expression in the Control, CFIm25 and CFIm68 KD cells. The quantification is relative to GAPDH. ( E ) qPCR measurements of let-7, miR-92a, miR-16 and miR-19b expression in CFIm25/68 KD cells relative to Control. ΔΔct values were calculated relative to U6 snRNA and then relative to the Control cells (where the ratio was set to 1). ( F ) Normalized Renilla luciferase expression of reporter mRNAs carrying binding sites for miR-16 and miR-92a in their 3′ UTRs, in Control, CFIm25 and CFIm68 KD cells, respectively. The Firefly luciferase expressed from the same construct was used as normalization control.
Knime Software, supplied by KNIME GmbH, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/automated+image-analysis+pipeline/pm34717173-223-17-31?v=KNIME+GmbH
Average 90 stars, based on 1 article reviews
knime software - by Bioz Stars, 2026-07
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cellprofiler automated image analysis software  (Broad Institute Inc)
90
Broad Institute Inc cellprofiler automated image analysis software
CFIm KD increases the activity of miRNAs. ( A ) Genome browser tracks showing the coverage of DICER1 TE (bottom track) by RNA-seq reads from two replicate experiments for each condition, with the two PAS that were quantified for this gene marked by black lines. The conditions are color-coded (as in Figure ) and also indicated on the y-axis. Y-axis shows the smoothened number of reads mapping along the TE, calculated by the GViz R package. ( B ) RNA fluorescence in situ imaging of DICER1 isoforms in Control and CFIm25 KD HEK293 cells with probes corresponding to the common region of the long and short 3′ UTRs (red) or to the region between the proximal and distal cleavage sites, thus present exclusively in the long 3′ UTR (green). Nuclei are marked with DAPI. Zoom-ins of the regions marked with dashed boxes are further shown both with the individual and merged channels. A snapshot of a digital representation of the actual <t>image</t> as processed in <t>IMARIS</t> is also depicted for reference. ( C ) Quantification of the copy number of the long and short 3′ UTR isoforms of DICER1 in the nucleus (left plot) and cytoplasm (right plot) of Control, CFIm25 and CFIm68 KD cells. Colocalization of the red and green signals reveals the presence of the long 3′ UTR isoform (yellow) whereas the signal from the red probe only reveals the presence of the shorter 3′ UTR isoform. mRNA copy numbers were estimated separately from the nucleus (overlapping with DAPI) and cytosol. Segregation of the signal was performed with IMARIS (see Methods). ( D ) Representative western blot showing the DICER1 expression in the Control, CFIm25 and CFIm68 KD cells. The quantification is relative to GAPDH. ( E ) qPCR measurements of let-7, miR-92a, miR-16 and miR-19b expression in CFIm25/68 KD cells relative to Control. ΔΔct values were calculated relative to U6 snRNA and then relative to the Control cells (where the ratio was set to 1). ( F ) Normalized Renilla luciferase expression of reporter mRNAs carrying binding sites for miR-16 and miR-92a in their 3′ UTRs, in Control, CFIm25 and CFIm68 KD cells, respectively. The Firefly luciferase expressed from the same construct was used as normalization control.
Cellprofiler Automated Image Analysis Software, supplied by Broad Institute Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/automated+image-analysis+pipeline/pm24012606-108-23-30?v=Broad+Institute+Inc
Average 90 stars, based on 1 article reviews
cellprofiler automated image analysis software - by Bioz Stars, 2026-07
90/100 stars
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cellprofilertm  (Broad Institute Inc)
90
Broad Institute Inc cellprofilertm
CFIm KD increases the activity of miRNAs. ( A ) Genome browser tracks showing the coverage of DICER1 TE (bottom track) by RNA-seq reads from two replicate experiments for each condition, with the two PAS that were quantified for this gene marked by black lines. The conditions are color-coded (as in Figure ) and also indicated on the y-axis. Y-axis shows the smoothened number of reads mapping along the TE, calculated by the GViz R package. ( B ) RNA fluorescence in situ imaging of DICER1 isoforms in Control and CFIm25 KD HEK293 cells with probes corresponding to the common region of the long and short 3′ UTRs (red) or to the region between the proximal and distal cleavage sites, thus present exclusively in the long 3′ UTR (green). Nuclei are marked with DAPI. Zoom-ins of the regions marked with dashed boxes are further shown both with the individual and merged channels. A snapshot of a digital representation of the actual <t>image</t> as processed in <t>IMARIS</t> is also depicted for reference. ( C ) Quantification of the copy number of the long and short 3′ UTR isoforms of DICER1 in the nucleus (left plot) and cytoplasm (right plot) of Control, CFIm25 and CFIm68 KD cells. Colocalization of the red and green signals reveals the presence of the long 3′ UTR isoform (yellow) whereas the signal from the red probe only reveals the presence of the shorter 3′ UTR isoform. mRNA copy numbers were estimated separately from the nucleus (overlapping with DAPI) and cytosol. Segregation of the signal was performed with IMARIS (see Methods). ( D ) Representative western blot showing the DICER1 expression in the Control, CFIm25 and CFIm68 KD cells. The quantification is relative to GAPDH. ( E ) qPCR measurements of let-7, miR-92a, miR-16 and miR-19b expression in CFIm25/68 KD cells relative to Control. ΔΔct values were calculated relative to U6 snRNA and then relative to the Control cells (where the ratio was set to 1). ( F ) Normalized Renilla luciferase expression of reporter mRNAs carrying binding sites for miR-16 and miR-92a in their 3′ UTRs, in Control, CFIm25 and CFIm68 KD cells, respectively. The Firefly luciferase expressed from the same construct was used as normalization control.
Cellprofilertm, supplied by Broad Institute Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/automated+image-analysis+pipeline/pmc07184676-87-56-62?v=Broad+Institute+Inc
Average 90 stars, based on 1 article reviews
cellprofilertm - by Bioz Stars, 2026-07
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custom image-analysis pipeline in cellprofiler  (Broad Institute Inc)
90
Broad Institute Inc custom image-analysis pipeline in cellprofiler
CFIm KD increases the activity of miRNAs. ( A ) Genome browser tracks showing the coverage of DICER1 TE (bottom track) by RNA-seq reads from two replicate experiments for each condition, with the two PAS that were quantified for this gene marked by black lines. The conditions are color-coded (as in Figure ) and also indicated on the y-axis. Y-axis shows the smoothened number of reads mapping along the TE, calculated by the GViz R package. ( B ) RNA fluorescence in situ imaging of DICER1 isoforms in Control and CFIm25 KD HEK293 cells with probes corresponding to the common region of the long and short 3′ UTRs (red) or to the region between the proximal and distal cleavage sites, thus present exclusively in the long 3′ UTR (green). Nuclei are marked with DAPI. Zoom-ins of the regions marked with dashed boxes are further shown both with the individual and merged channels. A snapshot of a digital representation of the actual <t>image</t> as processed in <t>IMARIS</t> is also depicted for reference. ( C ) Quantification of the copy number of the long and short 3′ UTR isoforms of DICER1 in the nucleus (left plot) and cytoplasm (right plot) of Control, CFIm25 and CFIm68 KD cells. Colocalization of the red and green signals reveals the presence of the long 3′ UTR isoform (yellow) whereas the signal from the red probe only reveals the presence of the shorter 3′ UTR isoform. mRNA copy numbers were estimated separately from the nucleus (overlapping with DAPI) and cytosol. Segregation of the signal was performed with IMARIS (see Methods). ( D ) Representative western blot showing the DICER1 expression in the Control, CFIm25 and CFIm68 KD cells. The quantification is relative to GAPDH. ( E ) qPCR measurements of let-7, miR-92a, miR-16 and miR-19b expression in CFIm25/68 KD cells relative to Control. ΔΔct values were calculated relative to U6 snRNA and then relative to the Control cells (where the ratio was set to 1). ( F ) Normalized Renilla luciferase expression of reporter mRNAs carrying binding sites for miR-16 and miR-92a in their 3′ UTRs, in Control, CFIm25 and CFIm68 KD cells, respectively. The Firefly luciferase expressed from the same construct was used as normalization control.
Custom Image Analysis Pipeline In Cellprofiler, supplied by Broad Institute Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/automated+image-analysis+pipeline/pm32330008__nn9b08689_si_001-105-5-12?v=Broad+Institute+Inc
Average 90 stars, based on 1 article reviews
custom image-analysis pipeline in cellprofiler - by Bioz Stars, 2026-07
90/100 stars
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solexa automated pipeline  (Solexa)
86
Solexa solexa automated pipeline
CFIm KD increases the activity of miRNAs. ( A ) Genome browser tracks showing the coverage of DICER1 TE (bottom track) by RNA-seq reads from two replicate experiments for each condition, with the two PAS that were quantified for this gene marked by black lines. The conditions are color-coded (as in Figure ) and also indicated on the y-axis. Y-axis shows the smoothened number of reads mapping along the TE, calculated by the GViz R package. ( B ) RNA fluorescence in situ imaging of DICER1 isoforms in Control and CFIm25 KD HEK293 cells with probes corresponding to the common region of the long and short 3′ UTRs (red) or to the region between the proximal and distal cleavage sites, thus present exclusively in the long 3′ UTR (green). Nuclei are marked with DAPI. Zoom-ins of the regions marked with dashed boxes are further shown both with the individual and merged channels. A snapshot of a digital representation of the actual <t>image</t> as processed in <t>IMARIS</t> is also depicted for reference. ( C ) Quantification of the copy number of the long and short 3′ UTR isoforms of DICER1 in the nucleus (left plot) and cytoplasm (right plot) of Control, CFIm25 and CFIm68 KD cells. Colocalization of the red and green signals reveals the presence of the long 3′ UTR isoform (yellow) whereas the signal from the red probe only reveals the presence of the shorter 3′ UTR isoform. mRNA copy numbers were estimated separately from the nucleus (overlapping with DAPI) and cytosol. Segregation of the signal was performed with IMARIS (see Methods). ( D ) Representative western blot showing the DICER1 expression in the Control, CFIm25 and CFIm68 KD cells. The quantification is relative to GAPDH. ( E ) qPCR measurements of let-7, miR-92a, miR-16 and miR-19b expression in CFIm25/68 KD cells relative to Control. ΔΔct values were calculated relative to U6 snRNA and then relative to the Control cells (where the ratio was set to 1). ( F ) Normalized Renilla luciferase expression of reporter mRNAs carrying binding sites for miR-16 and miR-92a in their 3′ UTRs, in Control, CFIm25 and CFIm68 KD cells, respectively. The Firefly luciferase expressed from the same construct was used as normalization control.
Solexa Automated Pipeline, supplied by Solexa, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/automated+image-analysis+pipeline/pm20828606-193-11-11?v=Solexa
Average 86 stars, based on 1 article reviews
solexa automated pipeline - by Bioz Stars, 2026-07
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colony immunoblot assay coloblot  (SourceForge net)
90
SourceForge net colony immunoblot assay coloblot
Single cell level vs. colony level phenotypic detection of mutants. For a narrow distribution of the wild-type expression of a phenotype, single cell level analysis (e.g., by flow cytometry) and colony level screening could both detect no-expression mutants. However, if only 100 clones can be screened on one membrane, mutants present at lower frequency than about 1% are difficult to detect by <t>immunoblot.</t> Thousands of cells can, however, be screened by flow cytometry which considerably lower the detection limit. Nevertheless, the average expression measured on colonies by immunoblot allows detecting no-expression mutants among wild-type clones in which the expression of the phenotype is bimodal. Single cell level screening that could not be used as no-expression mutants would be included in the subpopulation of wild-type cells that transiently do not express the phenotype.
Colony Immunoblot Assay Coloblot, supplied by SourceForge net, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/automated+image-analysis+pipeline/pmc05655795-49-8-27?v=SourceForge+net
Average 90 stars, based on 1 article reviews
colony immunoblot assay coloblot - by Bioz Stars, 2026-07
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customised pipelines on cellprofiler 4.2.6  (Broad Institute Inc)
90
Broad Institute Inc customised pipelines on cellprofiler 4.2.6
Single cell level vs. colony level phenotypic detection of mutants. For a narrow distribution of the wild-type expression of a phenotype, single cell level analysis (e.g., by flow cytometry) and colony level screening could both detect no-expression mutants. However, if only 100 clones can be screened on one membrane, mutants present at lower frequency than about 1% are difficult to detect by <t>immunoblot.</t> Thousands of cells can, however, be screened by flow cytometry which considerably lower the detection limit. Nevertheless, the average expression measured on colonies by immunoblot allows detecting no-expression mutants among wild-type clones in which the expression of the phenotype is bimodal. Single cell level screening that could not be used as no-expression mutants would be included in the subpopulation of wild-type cells that transiently do not express the phenotype.
Customised Pipelines On Cellprofiler 4.2.6, supplied by Broad Institute Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/automated+image-analysis+pipeline/bio_rxiv__2025__03__27__645710-54-3-11?v=Broad+Institute+Inc
Average 90 stars, based on 1 article reviews
customised pipelines on cellprofiler 4.2.6 - by Bioz Stars, 2026-07
90/100 stars
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arivis  (Carl Zeiss)
90
Carl Zeiss arivis
Single cell level vs. colony level phenotypic detection of mutants. For a narrow distribution of the wild-type expression of a phenotype, single cell level analysis (e.g., by flow cytometry) and colony level screening could both detect no-expression mutants. However, if only 100 clones can be screened on one membrane, mutants present at lower frequency than about 1% are difficult to detect by <t>immunoblot.</t> Thousands of cells can, however, be screened by flow cytometry which considerably lower the detection limit. Nevertheless, the average expression measured on colonies by immunoblot allows detecting no-expression mutants among wild-type clones in which the expression of the phenotype is bimodal. Single cell level screening that could not be used as no-expression mutants would be included in the subpopulation of wild-type cells that transiently do not express the phenotype.
Arivis, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/automated+image-analysis+pipeline/bio_rxiv__2024__05__30__596647-229-12-13?v=Carl+Zeiss
Average 90 stars, based on 1 article reviews
arivis - by Bioz Stars, 2026-07
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fish foci  (Nikon)
99
Nikon fish foci
Single cell level vs. colony level phenotypic detection of mutants. For a narrow distribution of the wild-type expression of a phenotype, single cell level analysis (e.g., by flow cytometry) and colony level screening could both detect no-expression mutants. However, if only 100 clones can be screened on one membrane, mutants present at lower frequency than about 1% are difficult to detect by <t>immunoblot.</t> Thousands of cells can, however, be screened by flow cytometry which considerably lower the detection limit. Nevertheless, the average expression measured on colonies by immunoblot allows detecting no-expression mutants among wild-type clones in which the expression of the phenotype is bimodal. Single cell level screening that could not be used as no-expression mutants would be included in the subpopulation of wild-type cells that transiently do not express the phenotype.
Fish Foci, supplied by Nikon, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/automated+image-analysis+pipeline/pm37689780-327-0-15?v=Nikon
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fish foci - by Bioz Stars, 2026-07
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7t mri scanner  (Siemens AG)
90
Siemens AG 7t mri scanner
Single cell level vs. colony level phenotypic detection of mutants. For a narrow distribution of the wild-type expression of a phenotype, single cell level analysis (e.g., by flow cytometry) and colony level screening could both detect no-expression mutants. However, if only 100 clones can be screened on one membrane, mutants present at lower frequency than about 1% are difficult to detect by <t>immunoblot.</t> Thousands of cells can, however, be screened by flow cytometry which considerably lower the detection limit. Nevertheless, the average expression measured on colonies by immunoblot allows detecting no-expression mutants among wild-type clones in which the expression of the phenotype is bimodal. Single cell level screening that could not be used as no-expression mutants would be included in the subpopulation of wild-type cells that transiently do not express the phenotype.
7t Mri Scanner, supplied by Siemens AG, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/automated+image-analysis+pipeline/pmc08413808-9-5-5?v=Siemens+AG
Average 90 stars, based on 1 article reviews
7t mri scanner - by Bioz Stars, 2026-07
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Image Search Results


CFIm KD increases the activity of miRNAs. ( A ) Genome browser tracks showing the coverage of DICER1 TE (bottom track) by RNA-seq reads from two replicate experiments for each condition, with the two PAS that were quantified for this gene marked by black lines. The conditions are color-coded (as in Figure ) and also indicated on the y-axis. Y-axis shows the smoothened number of reads mapping along the TE, calculated by the GViz R package. ( B ) RNA fluorescence in situ imaging of DICER1 isoforms in Control and CFIm25 KD HEK293 cells with probes corresponding to the common region of the long and short 3′ UTRs (red) or to the region between the proximal and distal cleavage sites, thus present exclusively in the long 3′ UTR (green). Nuclei are marked with DAPI. Zoom-ins of the regions marked with dashed boxes are further shown both with the individual and merged channels. A snapshot of a digital representation of the actual image as processed in IMARIS is also depicted for reference. ( C ) Quantification of the copy number of the long and short 3′ UTR isoforms of DICER1 in the nucleus (left plot) and cytoplasm (right plot) of Control, CFIm25 and CFIm68 KD cells. Colocalization of the red and green signals reveals the presence of the long 3′ UTR isoform (yellow) whereas the signal from the red probe only reveals the presence of the shorter 3′ UTR isoform. mRNA copy numbers were estimated separately from the nucleus (overlapping with DAPI) and cytosol. Segregation of the signal was performed with IMARIS (see Methods). ( D ) Representative western blot showing the DICER1 expression in the Control, CFIm25 and CFIm68 KD cells. The quantification is relative to GAPDH. ( E ) qPCR measurements of let-7, miR-92a, miR-16 and miR-19b expression in CFIm25/68 KD cells relative to Control. ΔΔct values were calculated relative to U6 snRNA and then relative to the Control cells (where the ratio was set to 1). ( F ) Normalized Renilla luciferase expression of reporter mRNAs carrying binding sites for miR-16 and miR-92a in their 3′ UTRs, in Control, CFIm25 and CFIm68 KD cells, respectively. The Firefly luciferase expressed from the same construct was used as normalization control.

Journal: Nucleic Acids Research

Article Title: CFIm-mediated alternative polyadenylation remodels cellular signaling and miRNA biogenesis

doi: 10.1093/nar/gkac114

Figure Lengend Snippet: CFIm KD increases the activity of miRNAs. ( A ) Genome browser tracks showing the coverage of DICER1 TE (bottom track) by RNA-seq reads from two replicate experiments for each condition, with the two PAS that were quantified for this gene marked by black lines. The conditions are color-coded (as in Figure ) and also indicated on the y-axis. Y-axis shows the smoothened number of reads mapping along the TE, calculated by the GViz R package. ( B ) RNA fluorescence in situ imaging of DICER1 isoforms in Control and CFIm25 KD HEK293 cells with probes corresponding to the common region of the long and short 3′ UTRs (red) or to the region between the proximal and distal cleavage sites, thus present exclusively in the long 3′ UTR (green). Nuclei are marked with DAPI. Zoom-ins of the regions marked with dashed boxes are further shown both with the individual and merged channels. A snapshot of a digital representation of the actual image as processed in IMARIS is also depicted for reference. ( C ) Quantification of the copy number of the long and short 3′ UTR isoforms of DICER1 in the nucleus (left plot) and cytoplasm (right plot) of Control, CFIm25 and CFIm68 KD cells. Colocalization of the red and green signals reveals the presence of the long 3′ UTR isoform (yellow) whereas the signal from the red probe only reveals the presence of the shorter 3′ UTR isoform. mRNA copy numbers were estimated separately from the nucleus (overlapping with DAPI) and cytosol. Segregation of the signal was performed with IMARIS (see Methods). ( D ) Representative western blot showing the DICER1 expression in the Control, CFIm25 and CFIm68 KD cells. The quantification is relative to GAPDH. ( E ) qPCR measurements of let-7, miR-92a, miR-16 and miR-19b expression in CFIm25/68 KD cells relative to Control. ΔΔct values were calculated relative to U6 snRNA and then relative to the Control cells (where the ratio was set to 1). ( F ) Normalized Renilla luciferase expression of reporter mRNAs carrying binding sites for miR-16 and miR-92a in their 3′ UTRs, in Control, CFIm25 and CFIm68 KD cells, respectively. The Firefly luciferase expressed from the same construct was used as normalization control.

Article Snippet: Detection and analysis of spots were performed using automated pipelines developed in image analysis software IMARIS (BITPLANE).

Techniques: Activity Assay, RNA Sequencing, Fluorescence, In Situ, Imaging, Control, Western Blot, Expressing, Luciferase, Binding Assay, Construct

Single cell level vs. colony level phenotypic detection of mutants. For a narrow distribution of the wild-type expression of a phenotype, single cell level analysis (e.g., by flow cytometry) and colony level screening could both detect no-expression mutants. However, if only 100 clones can be screened on one membrane, mutants present at lower frequency than about 1% are difficult to detect by immunoblot. Thousands of cells can, however, be screened by flow cytometry which considerably lower the detection limit. Nevertheless, the average expression measured on colonies by immunoblot allows detecting no-expression mutants among wild-type clones in which the expression of the phenotype is bimodal. Single cell level screening that could not be used as no-expression mutants would be included in the subpopulation of wild-type cells that transiently do not express the phenotype.

Journal: Frontiers in Microbiology

Article Title: Detection of Mutations Affecting Heterogeneously Expressed Phenotypes by Colony Immunoblot and Dedicated Semi-Automated Image Analysis Pipeline

doi: 10.3389/fmicb.2017.02044

Figure Lengend Snippet: Single cell level vs. colony level phenotypic detection of mutants. For a narrow distribution of the wild-type expression of a phenotype, single cell level analysis (e.g., by flow cytometry) and colony level screening could both detect no-expression mutants. However, if only 100 clones can be screened on one membrane, mutants present at lower frequency than about 1% are difficult to detect by immunoblot. Thousands of cells can, however, be screened by flow cytometry which considerably lower the detection limit. Nevertheless, the average expression measured on colonies by immunoblot allows detecting no-expression mutants among wild-type clones in which the expression of the phenotype is bimodal. Single cell level screening that could not be used as no-expression mutants would be included in the subpopulation of wild-type cells that transiently do not express the phenotype.

Article Snippet: Here, we present an optimized version of the colony immunoblot assay (ColoBlot) and a dedicated semi-automated open-source image analysis pipeline [Colony Immunoblot Image Analysis (CIIA)] available at https://sourceforge.net/projects/coloblot-image-analysis/files/ .

Techniques: Expressing, Flow Cytometry, Clone Assay, Membrane, Western Blot

Schematic outline of the ColoBlot procedure. (I) Colonies grown on the master plate are transferred on a nitrocellulose membrane. (II) The membrane is then placed colony side up on a non-selective agar plate and incubated overnight. (III) The membrane is passaged over different buffer soaked Whatman papers to lyse colonies and bind cellular material to the membrane. (IV) The membrane is washed and excess cellular debris is removed by lightly scraping the membrane with Whatman paper. (V) Another round of washing is followed by blocking in TBS buffer containing 3% BSA. (VI) Protein-specific primary antibodies are added to the membrane in blocking solution. (VII) After three rounds of washing, HRP-conjugated secondary antibodies are added in blocking solution. Another three rounds of washing removes antibody in excess. (VIII) The staining was performed by exposing the membrane to 4-chloro-1-naphthol in presence of H 2 O 2 . This reaction is terminated after 10 min by washing with dH 2 O. (IX) A non-specific counter stain is performed by incubating the membrane in the presence of a Ponceau S solution. Destaining (to increase the signal-to-background ratio) is achieved with sequential washing with dH 2 O. (X) After drying, the membrane is ready to be scanned as an RGB TIFF image for CIIA.

Journal: Frontiers in Microbiology

Article Title: Detection of Mutations Affecting Heterogeneously Expressed Phenotypes by Colony Immunoblot and Dedicated Semi-Automated Image Analysis Pipeline

doi: 10.3389/fmicb.2017.02044

Figure Lengend Snippet: Schematic outline of the ColoBlot procedure. (I) Colonies grown on the master plate are transferred on a nitrocellulose membrane. (II) The membrane is then placed colony side up on a non-selective agar plate and incubated overnight. (III) The membrane is passaged over different buffer soaked Whatman papers to lyse colonies and bind cellular material to the membrane. (IV) The membrane is washed and excess cellular debris is removed by lightly scraping the membrane with Whatman paper. (V) Another round of washing is followed by blocking in TBS buffer containing 3% BSA. (VI) Protein-specific primary antibodies are added to the membrane in blocking solution. (VII) After three rounds of washing, HRP-conjugated secondary antibodies are added in blocking solution. Another three rounds of washing removes antibody in excess. (VIII) The staining was performed by exposing the membrane to 4-chloro-1-naphthol in presence of H 2 O 2 . This reaction is terminated after 10 min by washing with dH 2 O. (IX) A non-specific counter stain is performed by incubating the membrane in the presence of a Ponceau S solution. Destaining (to increase the signal-to-background ratio) is achieved with sequential washing with dH 2 O. (X) After drying, the membrane is ready to be scanned as an RGB TIFF image for CIIA.

Article Snippet: Here, we present an optimized version of the colony immunoblot assay (ColoBlot) and a dedicated semi-automated open-source image analysis pipeline [Colony Immunoblot Image Analysis (CIIA)] available at https://sourceforge.net/projects/coloblot-image-analysis/files/ .

Techniques: Membrane, Incubation, Blocking Assay, Staining

Salmonella Typhimurium and S . Enteritidis can be differentiated based on their O serotype using the ColoBlot analysis. (A) The ColoBlot procedure was performed on mixtures of S. Typhimurium (SL1344) and S . Enteritidis using a commercial anti-O5 serum as primary antibody. A representative image is shown stained with Ponceau S. (B) CIIA was performed on images from the ColoBlot procedure (see A ) and a representative histogram is shown. A maximum intensity threshold (35) was determined based on the local minimum of the histogram. The colonies identified and classified by CIIA are shown in the inset (red indicates colonies below the threshold and green indicates colonies at or above the threshold). (C) Regression analysis comparing the CIIA output to selective replica plating from eight membranes containing mixtures of S. Typhimurium (SL1344; Cm R ) and S. Enterica at different ratios. Slope = 0.8156 (extra sum-of-squares F -test compared to slope = 1; p = 0.0162); origin = 7.607; R 2 = 0.9728.

Journal: Frontiers in Microbiology

Article Title: Detection of Mutations Affecting Heterogeneously Expressed Phenotypes by Colony Immunoblot and Dedicated Semi-Automated Image Analysis Pipeline

doi: 10.3389/fmicb.2017.02044

Figure Lengend Snippet: Salmonella Typhimurium and S . Enteritidis can be differentiated based on their O serotype using the ColoBlot analysis. (A) The ColoBlot procedure was performed on mixtures of S. Typhimurium (SL1344) and S . Enteritidis using a commercial anti-O5 serum as primary antibody. A representative image is shown stained with Ponceau S. (B) CIIA was performed on images from the ColoBlot procedure (see A ) and a representative histogram is shown. A maximum intensity threshold (35) was determined based on the local minimum of the histogram. The colonies identified and classified by CIIA are shown in the inset (red indicates colonies below the threshold and green indicates colonies at or above the threshold). (C) Regression analysis comparing the CIIA output to selective replica plating from eight membranes containing mixtures of S. Typhimurium (SL1344; Cm R ) and S. Enterica at different ratios. Slope = 0.8156 (extra sum-of-squares F -test compared to slope = 1; p = 0.0162); origin = 7.607; R 2 = 0.9728.

Article Snippet: Here, we present an optimized version of the colony immunoblot assay (ColoBlot) and a dedicated semi-automated open-source image analysis pipeline [Colony Immunoblot Image Analysis (CIIA)] available at https://sourceforge.net/projects/coloblot-image-analysis/files/ .

Techniques: Staining

The CIIA pipeline. (I) The ColoBlot procedure was performed on a plated mixture of SL1344 (wild-type) and SL1344 Δ hilD using an anti-SipC primary antibody. (II) Colonies that did not express SipC, and are therefore not stained by 4-chloro-1-naphthol, are made visible by the non-specific Ponceau S staining. (III) The image (from II ) is split into its color component images [left membrane: red color component image (SipC stain); right membrane: green color component image (Ponceau S stain)]. (IV) After thresholding (colony detection threshold = 61), binary masking, and binary operations, ROIs are identified by the Ponceau S stain based on parameters inputted by the user (Analyze Particles). (V) The image of the SipC staining is blurred and denoised, and the ROIs (detected in IV) are overlain. (VI) The maximum intensity is measured for each ROI and plotted as a histogram (shown histogram was produced with GraphPad Prism version 7 for windows using maximum intensity data saved in the XLS file by CIIA). A threshold (here 30) is manually selected based on local minima in the histogram. (VII) ROIs are classified and highlighted on the output image by CIIA, based on the threshold definition (identified in VI ).

Journal: Frontiers in Microbiology

Article Title: Detection of Mutations Affecting Heterogeneously Expressed Phenotypes by Colony Immunoblot and Dedicated Semi-Automated Image Analysis Pipeline

doi: 10.3389/fmicb.2017.02044

Figure Lengend Snippet: The CIIA pipeline. (I) The ColoBlot procedure was performed on a plated mixture of SL1344 (wild-type) and SL1344 Δ hilD using an anti-SipC primary antibody. (II) Colonies that did not express SipC, and are therefore not stained by 4-chloro-1-naphthol, are made visible by the non-specific Ponceau S staining. (III) The image (from II ) is split into its color component images [left membrane: red color component image (SipC stain); right membrane: green color component image (Ponceau S stain)]. (IV) After thresholding (colony detection threshold = 61), binary masking, and binary operations, ROIs are identified by the Ponceau S stain based on parameters inputted by the user (Analyze Particles). (V) The image of the SipC staining is blurred and denoised, and the ROIs (detected in IV) are overlain. (VI) The maximum intensity is measured for each ROI and plotted as a histogram (shown histogram was produced with GraphPad Prism version 7 for windows using maximum intensity data saved in the XLS file by CIIA). A threshold (here 30) is manually selected based on local minima in the histogram. (VII) ROIs are classified and highlighted on the output image by CIIA, based on the threshold definition (identified in VI ).

Article Snippet: Here, we present an optimized version of the colony immunoblot assay (ColoBlot) and a dedicated semi-automated open-source image analysis pipeline [Colony Immunoblot Image Analysis (CIIA)] available at https://sourceforge.net/projects/coloblot-image-analysis/files/ .

Techniques: Staining, Membrane, Produced

The ColoBlot analysis can be used to follow changes during within-host growth. (A) MacConkey master plates with 50–150 colonies containing only SL1344 wild-type (left histograms), SL1344 Δ hilC (middle histograms), or SL1344 Δ hilD (right histogram) were analyzed with the CIIA. Stacked histograms show maximum intensity measurements of three independent membranes for each population. The total number of ROIs in each histogram is indicated. (B,C) SL1344 wild-type (Cm R ), SL1344 Δ hilD (Cm R ; Kan R ), and SL1344 Δ hilC (Kan R ) were mixed at different ratios and plated on MacConkey agar to yield 50–250 colonies. The CIIA was used to quantify expression of SipC. (B) A representative histogram of one membrane is shown. The thresholds are manually determined to fit the local minima in the histogram (shown as solid purple lines; numbers indicate the threshold value) and the resulting populations on the membrane are shown (right panel; colored ROIs correspond to colored bars in the maximum intensity histogram). (C) Replica plating was performed in parallel to CIIA and a correlation analysis was performed. Linear regression best-fit equations and R 2 values are shown in the panel inset. Wild-type: slope = 0.9978 [extra sum-of-squares F -test compared to slope = 1 not significant ( p = 0.9756)], origin = 4.951, R 2 = 0.9637; Δ hilC : slope = 0.9723 [extra sum-of-squares F -test compared to slope = 1 not significant ( p = 0.7956)], origin = 6.595, R 2 = 0.9172; Δ hilD : slope = 0.8734 [extra sum-of-squares F -test compared to slope = 1 not significant ( p = 0.0500)], origin = –6.285, R 2 = 0.9693. Each individual membrane was adjusted to correct the threshold for inter-membrane maximum intensity variation (thresholds shown in a table; the mean is calculated for each threshold and is used for subsequent analysis). (D,E) Streptomycin-pretreated 129 Sv/Ev mice were orally infected with mixtures of SL1344 wild-type (Cm R ), SL1344 Δ hilD (Cm R ; Kan R ), and SL1344 Δ hilC (Kan R ) at a 1:1:1 ratio. Population sizes were followed by plating resuspending fecal pellets on MacConkey plates supplemented with streptomycin and by performing replica plating in parallel to ColoBlot and CIIA (thresholds used are the mean values determined through threshold calibration in C ). (D) The histogram from the inoculum (1:1:1 ratio) is shown with indicated thresholds (shown as purple lines; numbers indicate threshold values). (E) Percentages of clones of each genotype (indicated by the legend; green = wild-type; yellow = Δ hilC ; red = Δ hilD ) detected after performing CIIA (solid lines) or replica plating (dashed lines) on plated resuspended fecal pellets (50–250 colonies). Fecal pellet population composition from two representative mice is shown.

Journal: Frontiers in Microbiology

Article Title: Detection of Mutations Affecting Heterogeneously Expressed Phenotypes by Colony Immunoblot and Dedicated Semi-Automated Image Analysis Pipeline

doi: 10.3389/fmicb.2017.02044

Figure Lengend Snippet: The ColoBlot analysis can be used to follow changes during within-host growth. (A) MacConkey master plates with 50–150 colonies containing only SL1344 wild-type (left histograms), SL1344 Δ hilC (middle histograms), or SL1344 Δ hilD (right histogram) were analyzed with the CIIA. Stacked histograms show maximum intensity measurements of three independent membranes for each population. The total number of ROIs in each histogram is indicated. (B,C) SL1344 wild-type (Cm R ), SL1344 Δ hilD (Cm R ; Kan R ), and SL1344 Δ hilC (Kan R ) were mixed at different ratios and plated on MacConkey agar to yield 50–250 colonies. The CIIA was used to quantify expression of SipC. (B) A representative histogram of one membrane is shown. The thresholds are manually determined to fit the local minima in the histogram (shown as solid purple lines; numbers indicate the threshold value) and the resulting populations on the membrane are shown (right panel; colored ROIs correspond to colored bars in the maximum intensity histogram). (C) Replica plating was performed in parallel to CIIA and a correlation analysis was performed. Linear regression best-fit equations and R 2 values are shown in the panel inset. Wild-type: slope = 0.9978 [extra sum-of-squares F -test compared to slope = 1 not significant ( p = 0.9756)], origin = 4.951, R 2 = 0.9637; Δ hilC : slope = 0.9723 [extra sum-of-squares F -test compared to slope = 1 not significant ( p = 0.7956)], origin = 6.595, R 2 = 0.9172; Δ hilD : slope = 0.8734 [extra sum-of-squares F -test compared to slope = 1 not significant ( p = 0.0500)], origin = –6.285, R 2 = 0.9693. Each individual membrane was adjusted to correct the threshold for inter-membrane maximum intensity variation (thresholds shown in a table; the mean is calculated for each threshold and is used for subsequent analysis). (D,E) Streptomycin-pretreated 129 Sv/Ev mice were orally infected with mixtures of SL1344 wild-type (Cm R ), SL1344 Δ hilD (Cm R ; Kan R ), and SL1344 Δ hilC (Kan R ) at a 1:1:1 ratio. Population sizes were followed by plating resuspending fecal pellets on MacConkey plates supplemented with streptomycin and by performing replica plating in parallel to ColoBlot and CIIA (thresholds used are the mean values determined through threshold calibration in C ). (D) The histogram from the inoculum (1:1:1 ratio) is shown with indicated thresholds (shown as purple lines; numbers indicate threshold values). (E) Percentages of clones of each genotype (indicated by the legend; green = wild-type; yellow = Δ hilC ; red = Δ hilD ) detected after performing CIIA (solid lines) or replica plating (dashed lines) on plated resuspended fecal pellets (50–250 colonies). Fecal pellet population composition from two representative mice is shown.

Article Snippet: Here, we present an optimized version of the colony immunoblot assay (ColoBlot) and a dedicated semi-automated open-source image analysis pipeline [Colony Immunoblot Image Analysis (CIIA)] available at https://sourceforge.net/projects/coloblot-image-analysis/files/ .

Techniques: Expressing, Membrane, Infection, Clone Assay

The ColoBlot analysis identifies TTSS-1 expression mutants emerging during within-host evolution. (A–C) M556 (Δ sseD S. Typhimurium SL1344; TTSS-1 expressing) were allowed to evolve for 10 days in C57BL/6 mice and fecal pellets were plated to yield 50–250 colonies per plate. The ancestral population (M556; plated after 4 days in C57BL/6) served as reference for SipC expression. An anti-SipC ColoBlot, followed by Ponceau S staining, was performed on the ancestral population (day 4 p.i.) and the evolved population (day 10 p.i.) [seen as insets in (A,B) , respectively]. (A) Maximum intensity histogram determined by CIIA of the ancestral population. The red line indicates the threshold corresponding to the upper value (47.0) of the first 5% of maximum intensity measurements (calculated by taking the maximum intensity value of ROI “ n ” that corresponds to the total ROI number multiplied by 0.05). (B) Maximum intensity histogram of the evolved population determined by CIIA. The 5% threshold of the ancestral population (determined in A ) is overlaid. ROIs with high (+; green arrow), medium (+/–; yellow arrow), or low (–; red arrow) maximum intensity were selected based on ROIs above the threshold (expected ancestral genotype; green arrow), and ROIs at the upper (yellow arrow) and lower (red arrow) bounds of the maximum intensity distribution below the threshold. (C) Output image of the evolved population by CIIA, with ROIs below the threshold shown in green (left image). SipC color component image (right image). Clones expressing high amount (+; green arrow), low amount (+/–; yellow arrow), or no (–; red arrow) SipC are indicated (as identified in B ). (D) Clones identified from (B) , and isolated from the master plate (membrane shown in C ), were streaked onto a MacConkey plate and an anti-SipC ColoBlot was performed to confirm their respective SipC expression level. Upper image shows the anti-SipC staining. The lower image shows the same membrane counterstained with Ponceau S. (E) Reconstructed strains carrying mutations as identified in the evolved clones by sequencing and corresponding to high (+; wild-type), low (+/–; Δ hilC ), or no (–; Δ hilD ) SipC expression. The strains were streaked onto a MacConkey plate and an anti-SipC ColoBlot was performed (upper image) and subsequently Ponceau S stained (lower image). (F) A gfp reporter cassette for TTSS-1 expression (P prgH :: gfp ) was inserted by P22 phage transduction into clones identified in (C) and the reconstructed strains shown in (E) , and analyzed using flow cytometry. Bacterial cells were identified by side-scatter. Percentages of GFP-positive events were calculated for each plot by defining a threshold according to the basal fluorescence level detected in a Δ hilD mutant.

Journal: Frontiers in Microbiology

Article Title: Detection of Mutations Affecting Heterogeneously Expressed Phenotypes by Colony Immunoblot and Dedicated Semi-Automated Image Analysis Pipeline

doi: 10.3389/fmicb.2017.02044

Figure Lengend Snippet: The ColoBlot analysis identifies TTSS-1 expression mutants emerging during within-host evolution. (A–C) M556 (Δ sseD S. Typhimurium SL1344; TTSS-1 expressing) were allowed to evolve for 10 days in C57BL/6 mice and fecal pellets were plated to yield 50–250 colonies per plate. The ancestral population (M556; plated after 4 days in C57BL/6) served as reference for SipC expression. An anti-SipC ColoBlot, followed by Ponceau S staining, was performed on the ancestral population (day 4 p.i.) and the evolved population (day 10 p.i.) [seen as insets in (A,B) , respectively]. (A) Maximum intensity histogram determined by CIIA of the ancestral population. The red line indicates the threshold corresponding to the upper value (47.0) of the first 5% of maximum intensity measurements (calculated by taking the maximum intensity value of ROI “ n ” that corresponds to the total ROI number multiplied by 0.05). (B) Maximum intensity histogram of the evolved population determined by CIIA. The 5% threshold of the ancestral population (determined in A ) is overlaid. ROIs with high (+; green arrow), medium (+/–; yellow arrow), or low (–; red arrow) maximum intensity were selected based on ROIs above the threshold (expected ancestral genotype; green arrow), and ROIs at the upper (yellow arrow) and lower (red arrow) bounds of the maximum intensity distribution below the threshold. (C) Output image of the evolved population by CIIA, with ROIs below the threshold shown in green (left image). SipC color component image (right image). Clones expressing high amount (+; green arrow), low amount (+/–; yellow arrow), or no (–; red arrow) SipC are indicated (as identified in B ). (D) Clones identified from (B) , and isolated from the master plate (membrane shown in C ), were streaked onto a MacConkey plate and an anti-SipC ColoBlot was performed to confirm their respective SipC expression level. Upper image shows the anti-SipC staining. The lower image shows the same membrane counterstained with Ponceau S. (E) Reconstructed strains carrying mutations as identified in the evolved clones by sequencing and corresponding to high (+; wild-type), low (+/–; Δ hilC ), or no (–; Δ hilD ) SipC expression. The strains were streaked onto a MacConkey plate and an anti-SipC ColoBlot was performed (upper image) and subsequently Ponceau S stained (lower image). (F) A gfp reporter cassette for TTSS-1 expression (P prgH :: gfp ) was inserted by P22 phage transduction into clones identified in (C) and the reconstructed strains shown in (E) , and analyzed using flow cytometry. Bacterial cells were identified by side-scatter. Percentages of GFP-positive events were calculated for each plot by defining a threshold according to the basal fluorescence level detected in a Δ hilD mutant.

Article Snippet: Here, we present an optimized version of the colony immunoblot assay (ColoBlot) and a dedicated semi-automated open-source image analysis pipeline [Colony Immunoblot Image Analysis (CIIA)] available at https://sourceforge.net/projects/coloblot-image-analysis/files/ .

Techniques: Expressing, Staining, Clone Assay, Isolation, Membrane, Sequencing, Transduction, Flow Cytometry, Fluorescence, Mutagenesis

Optimization of the incubation time with the chromogenic substrate. (A) Equal mixtures of SL1344 Δ hilC and SL1344 wild-type were plated on MacConkey agar to obtain approximately 50–250 colonies. An anti-SipC ColoBlot was performed. Prior to exposure of the membrane to the 4-chloro-1-naphthol reagent mixture, the membrane was cut into four equal parts (representative images for each quadrant are shown divided by a black line). Each quadrant was exposed to 4-chloro-1-naphthol in the presence of H 2 O 2 for 10 s, 30 s, 2 min, or 10 min (as indicated). (B) CIIA was performed on each quadrant independently and the resulting maximum intensity histograms are shown (representative histograms of the membrane shown in A ). (C) The mean maximum intensity for each ROI (i.e., the mean maximum intensity for each histogram in B ) in each quadrant is plotted as a function of time. Three membranes are plotted and hyperbolic curves are fit ( R 2 = 0.9119, 0.9703, and 0.931).

Journal: Frontiers in Microbiology

Article Title: Detection of Mutations Affecting Heterogeneously Expressed Phenotypes by Colony Immunoblot and Dedicated Semi-Automated Image Analysis Pipeline

doi: 10.3389/fmicb.2017.02044

Figure Lengend Snippet: Optimization of the incubation time with the chromogenic substrate. (A) Equal mixtures of SL1344 Δ hilC and SL1344 wild-type were plated on MacConkey agar to obtain approximately 50–250 colonies. An anti-SipC ColoBlot was performed. Prior to exposure of the membrane to the 4-chloro-1-naphthol reagent mixture, the membrane was cut into four equal parts (representative images for each quadrant are shown divided by a black line). Each quadrant was exposed to 4-chloro-1-naphthol in the presence of H 2 O 2 for 10 s, 30 s, 2 min, or 10 min (as indicated). (B) CIIA was performed on each quadrant independently and the resulting maximum intensity histograms are shown (representative histograms of the membrane shown in A ). (C) The mean maximum intensity for each ROI (i.e., the mean maximum intensity for each histogram in B ) in each quadrant is plotted as a function of time. Three membranes are plotted and hyperbolic curves are fit ( R 2 = 0.9119, 0.9703, and 0.931).

Article Snippet: Here, we present an optimized version of the colony immunoblot assay (ColoBlot) and a dedicated semi-automated open-source image analysis pipeline [Colony Immunoblot Image Analysis (CIIA)] available at https://sourceforge.net/projects/coloblot-image-analysis/files/ .

Techniques: Incubation, Membrane