BlobFinder, a tool for fluorescence microscopy image cytometry

Allalou, Amin; Wählby, Carolina

doi:10.1016/j.cmpb.2008.08.006

Cited by 117 publications

(111 citation statements)

References 13 publications

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“…The quantification of the dot-like PLA products was performed using BlobFinder V3.2. 42 and washed in PBS with 0.05 mg/ml CaCl 2 and 3% BSA. Cells were incubated for 1 h with the extracellular primary antibody against E-cadherin, HECD1 (Zymed Laboratories) at 1:50 dilution.…”

Section: Proximity Ligation Assaymentioning

confidence: 99%

The importance of E-cadherin binding partners to evaluate the pathogenicity of E-cadherin missense mutations associated to HDGC

et al. 2012

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In hereditary diffuse gastric cancer (HDGC), CDH1 germline gene alterations are causative events in 30% of the cases. In 20% of HDGC families, CDH1 germline mutations are of the missense type and the mutation carriers constitute a problem in terms of genetic counseling and surveillance. To access the pathogenic relevance of missense mutations, we have previously developed an in vitro method to functionally characterize them. Pathogenic E-cadherin missense mutants fail to aggregate and become more invasive, in comparison with cells expressing the wild-type (WT) protein. Herein, our aim was to develop a complementary method to unravel the pathogenic significance of E-cadherin missense mutations. We used cells stably expressing WT E-cadherin and seven HDGC-associated mutations (five intracellular and two extracellular) and studied by proximity ligation assays (PLA) how these mutants bind to fundamental regulators of E-cadherin function and trafficking. We focused our attention on the interaction with: p120, b-catenin, PIPKIc and Hakai. We showed that cytoplasmic E-cadherin mutations affect the interaction of one or more binding partners, compromising the E-cadherin stability at the plasma membrane and likely affecting the adhesion complex competence. In the present work, we demonstrated that the study of the interplay between E-cadherin and its binding partners, using PLA, is an easy, rapid, quantitative and highly reproducible technique that can be applied in routine labs to verify the pathogenicity of E-cadherin missense mutants for HDGC diagnosis, especially those located in the intracellular domain of the protein. Keywords: HDGC; E-cadherin; CDH1 mutations; E-cadherin trafficking; E-cadherin binding partners; diagnostic method INTRODUCTIONHereditary diffuse gastric cancer (HDGC) is an autosomal dominant cancer syndrome characterized by a high risk of developing diffuse gastric cancer 1-3 and lobular breast cancer 4-6 during life-time. CDH1 germline gene alterations (mutations or deletions), resulting in E-cadherin inactivation, are the only causative events described till now and were identified in approximately 30% of HDGC cases. 2,3,7 To date, 122 different germline mutations have been described in these families, 8 being the majority of them of the nonsense type, leading to alternative premature termination codons. 3 This type of CDH1 mutant transcripts is commonly downregulated by nonsensemediated decay leading to E-cadherin loss of function 9 and these patients are considered high-risk carriers and are counseled to perform prophylactic total gastrectomy. 10 In about 20% of HDGC families, carriers show CDH1 germline missense mutations 11 and, in contrast to truncating mutations, their pathogenic significance is not straightforward, therefore constituting a problem in terms of genetic counseling and surveillance.In 2004, Fitzgerald and Caldas 10 suggested that the significance of CDH1 missense mutations should be assessed in at least four affected members within a HDGC family in combination with functional and

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Section: Proximity Ligation Assaymentioning

confidence: 99%

The importance of E-cadherin binding partners to evaluate the pathogenicity of E-cadherin missense mutations associated to HDGC

et al. 2012

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“…Images were acquired with an AxioCam MRm camera (Zeiss, Stockholm, Sweden). Fluorescence microscopy signals were quantified with the BlobFinder software, 50 a freely available image analysis tool for image cytometry. Cell nuclei and point-source fluorescence signals were defined and quantified by shape and fluorescence intensity according to the standard procedures for this software tool.…”

Section: Proximity Ligation Assaymentioning

confidence: 99%

Oncolytic adenovirus modified with somatostatin motifs for selective infection of neuroendocrine tumor cells

Leja

Nilsson

et al. 2011

Gene Ther

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We have previously described the oncolytic adenovirus, Ad(CgA-E1A-miR122), herein denoted Ad5(CgA-E1A-miR122) that selectively replicates in and kills neuroendocrine cells, including freshly isolated midgut carcinoid cells from liver metastases. Ad5(CgA-E1A-miR122) is based on human adenovirus serotype 5 (Ad5) and infects target cells by binding to the coxsackieadenovirus receptor (CAR) and integrins on the cell surface. Some neuroendocrine tumor (NET) and neuroblastoma cells express low levels of CAR and are therefore poorly transduced by Ad5. However, they often express high levels of somatostatin receptors (SSTRs). Therefore, we introduced cyclic peptides, which contain four amino acids (FWKT) and mimic the binding site for SSTRs in the virus fiber knob. We show that FWKT-modified Ad5 binds to SSTR 2 on NET cells and transduces midgut carcinoid cells from liver metastases about 3-4 times better than non-modified Ad5. Moreover, FWKT-modified Ad5 overcomes neutralization in an ex vivo human blood loop model to greater extent than Ad5, indicating that fiber knob modification may prolong the systemic circulation time. We conclude that modification of adenovirus with the FWKT motif may be beneficial for NET therapy.

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“…The existing techniques based on digital microscopy [13][14][15][16] , however, are time-consuming and resource-demanding, as images are typically captured for the entire sample area, or even through three-dimensional space [17][18][19][20] , followed by stitching and processing to identify and quantitate targets of interest. Their quantification is also less accurate, because different types of noise and background emission interfere in the measurement of absolute intensities, and targets that are randomly located at the periphery of the field-of-view (FOV) have large variation in excitation and detection efficiencies [21][22][23] .…”

Section: Introductionmentioning

confidence: 99%

High-Precision Pinpointing of Luminescent Targets in Encoder-Assisted Scanning Microscopy Allowing High-Speed Quantitative Analysis

Zheng

Zhao

et al. 2015

Anal. Chem.

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The new agreement specifically addresses what authors can do with different versions of their manuscript -e.g. use in theses and collections, teaching and training, conference presentations, sharing with colleagues, and posting on websites and repositories. The terms under which these uses can occur are clearly identified to prevent misunderstandings that could jeopardize final publication of a manuscript (Section II, Permitted Uses by Authors). Easy Reference User Guide Posting Submitted Works on Websites and Repositories:A digital file of the unedited manuscript version of a Submitted Work may be made publicly available on websites or repositories (e.g. the Author's personal website, preprint servers, university networks or primary employer's institutional websites, third party institutional or subject-based repositories, and conference websites that feature presentations by the Author(s) based on the Submitted Work) under the following conditions:• The posting must be for non-commercial purposes and not violate the ACS' "Ethical Guidelines to Publication of Chemical Research" (see http://pubs.acs.org/ethics).• If the Submitted Work is accepted for publication in an ACS journal, then the following notice should be included at the time of posting, or the posting amended as appropriate: "This document is the unedited Author's version of a Submitted Work that was subsequently accepted for publication in [JournalTitle], copyright © American Chemical Society after peer review. To access the final edited and published work see [insert ACS Articles on Request author-directed link to Published Work, see http://pubs.acs.org/page/policy/articlesonrequest/index.html]." Note: It is the responsibility of the Author(s) to confirm with the appropriate ACS journal editor that the timing of the posting of the Submitted Work does not conflict with journal prior publication/embargo policies (see http://pubs.acs.org/page/policy/prior/index.html)If any prospective posting of the Submitted Work, whether voluntary or mandated by the Author(s)' funding agency, primary employer, or, in the case of Author(s) employed in academia, university administration, would violate any of the above conditions, the Submitted Work may not be posted. In these cases, Author(s) may either sponsor the immediate public availability of the final Published Work through participation in the feebased ACS AuthorChoice program (for information about this program see http://pubs.acs.org/page/policy/authorchoice/index.html) or, if applicable, seek a waiver from the relevant institutional policy. July, 2016 ABSTRACTCompared with routine microscopy imaging of a few analytes at a time, rapid scanning through the whole sample area of a microscope slide to locate every single target object offers many advantages in terms of simplicity, speed, throughput, and potential for robust quantitative analysis. Existing techniques that accommodate solid-phase samples incorporating individual micron-sized targets generally rely on digital microscopy and image analysis, with in...

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BlobFinder, a tool for fluorescence microscopy image cytometry

Cited by 117 publications

References 13 publications

The importance of E-cadherin binding partners to evaluate the pathogenicity of E-cadherin missense mutations associated to HDGC

The importance of E-cadherin binding partners to evaluate the pathogenicity of E-cadherin missense mutations associated to HDGC

Oncolytic adenovirus modified with somatostatin motifs for selective infection of neuroendocrine tumor cells

High-Precision Pinpointing of Luminescent Targets in Encoder-Assisted Scanning Microscopy Allowing High-Speed Quantitative Analysis

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