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Measurement of apoptosis by SCAN^©, a system for counting and analysis of fluorescently labelled nuclei.

ABSTRACT: Apoptosis-like programmed cell death (A-PCD) is a universal process common to all types of eukaryotic organisms. Because A-PCD-associated processes are conserved, it is possible to define A-PCD by a standard set of markers. Many of the popular methods to measure A-PCD make use of fluorescent ligands that change in intensity or cellular localization during A-PCD. In single cell organisms, it is possible to quantify levels of A-PCD by scoring the number of apoptotic cells using flow cytometry instruments. In a multicellular organism, quantification of A-PCD is more problematic due to the complex nature of the tissue. The situation is further complicated in filamentous fungi, in which nuclei are divided between compartments, each containing a number of nuclei, which can also migrate between the compartments. We developed SCAN^©, a System for Counting and Analysis of Nuclei, and used it to measure A-PCD according to two markers - chromatin condensation and DNA strand breaks. The package includes three modules designed for counting the number of nuclei in multi-nucleated domains, scoring the relative number of nuclei with condensed chromatin, and calculating the relative number of nuclei with DNA strand breaks. The method provides equal or better results compared with manual counting, the analysis is fast and can be applied on large data sets. While we demonstrated the utility of the software for measurement of A-PCD in fungi, the method is readily adopted for measurement of A-PCD in other types of multicellular specimens.

SUBMITTER: Shlezinger N

PROVIDER: S-EPMC5349136 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Measurement of apoptosis by SCAN<sup>©</sup>, a system for counting and analysis of fluorescently labelled nuclei.

Shlezinger Neta N Eizner Elad E Dubinchik Stas S Minz-Dub Anna A Tetroashvili Rachel R Reider Adi A Sharon Amir A

Microbial cell (Graz, Austria) 20141126 12

Apoptosis-like programmed cell death (A-PCD) is a universal process common to all types of eukaryotic organisms. Because A-PCD-associated processes are conserved, it is possible to define A-PCD by a standard set of markers. Many of the popular methods to measure A-PCD make use of fluorescent ligands that change in intensity or cellular localization during A-PCD. In single cell organisms, it is possible to quantify levels of A-PCD by scoring the number of apoptotic cells using flow cytometry inst ...[more]

PMID: 28357220

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Project description:PurposeWith the increase of especially hospital-acquired infections, timely and accurate diagnosis of bacterial infections is crucial for effective patient care. Molecular imaging has the potential for specific and sensitive detection of infections. Siderophores are iron-specific chelators recognized by specific bacterial transporters, representing one of few fundamental differences between bacterial and mammalian cells. Replacing iron by gallium-68 without loss of bioactivity is possible allowing molecular imaging by positron emission tomography (PET). Here, we report on the preclinical evaluation of the clinically used siderophore, desferrioxamine-B (Desferal®, DFO-B), radiolabelled with 68Ga for imaging of bacterial infections.MethodsIn vitro characterization of [68Ga]Ga-DFO-B included partition coefficient, protein binding and stability determination. Specific uptake of [68Ga]Ga-DFO-B was tested in vitro in different microbial cultures. In vivo biodistribution was studied in healthy mice and dosimetric estimation for human setting performed. PET/CT imaging was carried out in animal infection models, representing the most common pathogens.ResultsDFO-B was labelled with 68Ga with high radiochemical purity and displayed hydrophilic properties, low protein binding and high stability in human serum and PBS. The high in vitro uptake of [68Ga]Ga-DFO-B in selected strains of Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus agalactiae could be blocked with an excess of iron-DFO-B. [68Ga]Ga-DFO-B showed rapid renal excretion and minimal retention in blood and other organs in healthy mice. Estimated human absorbed dose was 0.02 mSv/MBq. PET/CT images of animal infection models displayed high and specific accumulation of [68Ga]Ga-DFO-B in both P. aeruginosa and S. aureus infections with excellent image contrast. No uptake was found in sterile inflammation, heat-inactivated P. aeruginosa or S. aureus and Escherichia coli lacking DFO-B transporters.ConclusionDFO-B can be easily radiolabelled with 68Ga and displayed suitable in vitro characteristics and excellent pharmacokinetics in mice. The high and specific uptake of [68Ga]Ga-DFO-B by P. aeruginosa and S. aureus was confirmed both in vitro and in vivo, proving the potential of [68Ga]Ga-DFO-B for specific imaging of bacterial infections. As DFO-B is used in clinic for many years and the estimated radiation dose is lower than for other 68Ga-labelled radiopharmaceuticals, we believe that [68Ga]Ga-DFO-B has a great potential for clinical translation.

Dataset Information

Measurement of apoptosis by SCAN©, a system for counting and analysis of fluorescently labelled nuclei.

Publications

Measurement of apoptosis by SCAN<sup>©</sup>, a system for counting and analysis of fluorescently labelled nuclei.

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Measurement of apoptosis by SCAN^©, a system for counting and analysis of fluorescently labelled nuclei.