Name: automated quantification and analysis of cell counting procedures using imagej plugins
Uploaded: 2016-11-17T15:00:00.000+00:00
Duration: 11 min 1 s
Description: Watch this Scientific Journal Video about Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins at JoVE.com

This work was supported by the Canadian Institute of Health Research to CP (OR 142730 and OR 89931). We would like to thank Jelena Brkic for her initial idea of binary particle analysis in ImageJ.

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+and+motility+assays.">Invasion and motility assays.</a> Cancer Cell Culture: Methods and Protocols. , 225-229 (2004).</li><li>Justus, C. R., Leffler, N., Ruiz-Echevarria, M., Yang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+Cell+Migration+and+Invasion+Assays.">In vitro Cell Migration and Invasion Assays.</a> J. Vis. Exp. (88), e51046 (2014).</li><li>Luo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-378a-5p+promotes+trophoblast+cell+survival,+migration+and+invasion+by+targeting+Nodal.">MicroRNA-378a-5p promotes trophoblast cell survival, migration and invasion by targeting Nodal.</a> J. 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Cancer. 130, 1036-1045 (2012).</li><li>Kramer, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al.+In+vitro+cell+migration+and+invasion+assays.">et al. In vitro cell migration and invasion assays.</a> Mutat. Res./Rev. Mutat. 752, 10-24 (2013).</li><li>Al-Khazraji, B. K., Medeiros, P. J., Novielli, N. M., Jackson, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+cell-counting+algorithm+for+fluorescently-stained+cells+in+migration+assays.">An automated cell-counting algorithm for fluorescently-stained cells in migration assays.</a> Biol. Proced. Online. 13, 1-6 (2011).</li><li>Abramoff, M. D., Magalhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+processing+with+ImageJ.">Image processing with ImageJ.</a> Biophotonics Intern. 11, 36-42 (2004).</li><li>Grishagin, I. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+cell+counting+with+ImageJ.">Automatic cell counting with ImageJ.</a> Anal. Biochem. 473, 63-65 (2015).</li><li>Thomas, C. R., Paul, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+image+analysis+in+cell+technology.">Applications of image analysis in cell technology.</a> Curr. Opin. 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The authors declare that they have no competing financial interests.

Kritische Schritte, Fehlerbehebung und Einschränkungen Das Wesen der automatisierten Rechenverfahren, insbesondere die der Partikelanalyse erfordert die mathematische Fähigkeit, diese Partikel zu definieren. Folglich ist die Genauigkeit der beiden Zellkonzentrationsrechner und Migrationsassay Zähler majorly abhängig von Bildwiedergabetreue, das heißt, wie genau das übernommene Bild das Membranzellprobe oder Migrationstest ähnelt. Es ist daher von größter Bedeutung Mikroskop und zugehö...

In - vitro - Zellzählung ist eine wichtige Grundtechnik in einem weiten Bereich von Gewebekulturexperimenten. Exakt die Anzahl der Zellen in einer Kultur , die Bestimmung ist von wesentlicher Bedeutung für die experimentelle Reproduzierbarkeit und Standardisierung 1,2. Zellzählung kann ein Hämozytometer manuell über als auch unter Verwendung einer Vielzahl von automatisierten Verfahren, jede mit ihren eigenen Vorteilen und Nachteilen 3,4,5 ausgeführt werden. Die meisten der automatisierten Verfahren zur Zellzählung gehören zu einer von zwei Klassen, diejenigen, die das Coulter-Prinzip verwenden oder Durchflusszytometrie. Coulter Zähler nutzen Sie Zellen elektrische Widerstand der Zellzahl und Größe zu bestimmen. Sie sind schnell, präzise und kostengünstiger als Durchflusszytometer. Jedoch werden sie selten für nur Zellzählung aufgrund ihrer beträchtlichen Kosten im Vergleich zu manuellen Zählung 3 verwendet. Durchflusszytometer, auf der anderen Seite, sind teuer, aber sie haben viele Anwendungen, wie beispielsweise Zellzählung, Analyse der Zellen Form, structure und Mess interne Zellmarker 4,5. Maschinen, die eine dieser beiden Prinzipien verwenden, sind von vielen Herstellern erhältlich. Manuelle Zählen ist erschwinglich , aber zeitaufwendig und unterliegt Bias , während die automatisierten Verfahren mit einem Bruchteil der Zeit kommen für die manuelle Zählung erforderlich , aber mit teuren Maschinen 6. Andere häufige Zellkulturverfahren sind in - vitro - Zellbeweglichkeit Assays, nämlich die Zellmigration und Invasion 7. Migration und Invasion Assays werden üblicherweise zur Untersuchung der Zellbeweglichkeit und Invasivität in Antwort auf eine chemotaktische Reaktion verwendet. Darüber hinaus sind sie häufig zu studieren Embryonalentwicklung, Differenzierung, Entzündungsreaktion, und die Metastasierung von mehreren Zelltypen 7-11 verwendet. Zellen, die durch die poröse Membran aus einem Migrationstest migriert oder eingedrungen kann auf zwei verschiedene Weisen quantifiziert werden. Erstens, indem die Zellen mit einem fluoreszierenden Farbstoff Anfärben Dissoziation from die Membran und Quantifizierung eines Fluoreszenzleser 12. Eine Einschränkung dieser Methode der Quantifizierung ist , dass keine Aufzeichnung der Membranen zurückgehalten werden kann , und es besteht keine Möglichkeit zur weiteren Analyse 13. Die zweite Quantifizierungsverfahren wird für Zellen migrierten / eingedrungen mit Fluoreszenzfarbstoff oder häufiger fixiert und gefärbt werden, wobei zytologischen Farbstoffe wie Kristallviolett, Toluidinblau oder Hämatoxylin-Farbstoff; dann werden die Zellen manuell mit invertierten Mikroskopbilder dieser Membranen quantifiziert , die eine sehr zeitraubende Aufgabe ist 12,13. Um die Nachteile der manuellen Zellzählung, zwei zuverlässigen und genauen automatischen Zellzähler für Zellkonzentration und für den Migrationstest überwunden wurden entwickelt. Diese automatisierten Zellzähler Algorithmen wurden für ImageJ als Plugin unter Verwendung von Oracle Java-Computer entwickelt. ImageJ ist ein öffentlicher und weit verbreiteten Bildbearbeitungswerkzeug durch das National Institute of Hea entwickeltlth (NIH) 14,15; Somit erleichtert diese Plugins für ImageJ Schreiben eine einfache Integration in die biologische Gemeinschaft. Automatisierung der Zellzählung sorgt für einen hohen Durchsatz und Reproduzierbarkeit im Vergleich zu manuellen Zählung. Obwohl andere verfügbare Software und Plugins verwendet werden kann , die Zellkonzentration durch Bildanalyse 5,16,17, Zellkonzentration Calculator Plugin ist schnell und kann auch behandeln Verdünnungen von Zellen und Behandlungen zu berechnen. Darüber hinaus können alle Ergebnisse und Berechnungen aus diesen beiden Zähler gespeichert und exportiert werden. Die beiden Plugins in diesem Dokument beschrieben sind, für die Verwendung eines Phasenkontrastmikroskop für Live Cell Imaging und großes Sichtfeld (gesamte Membran capture) Bildgebung für Migration Assay-Membranen durch die Verwendung eines Binokular optimiert. Die Plugins sind frei zum Download zur Verfügung mit Installationsanweisungen aus: http://peng.lab.yorku.ca/imagej-plugins.

<table><tbody><tr><td>HyClone Classical Liquid Media: RPMI 1640 - With L-Glutamine</td><td>Fisher Scientific</td><td>SH3002702</td><td>Cell culturing media&amp;nbsp;</td></tr><tr><td>Fetal bovian serum (FBS)</td><td>GIBCO BRL</td><td>P00015</td><td>Media suppliment</td></tr><tr><td>HTR8/SVneo trophoblast cell line</td><td>Cells were obtained from Dr. Charles Graham (Queen&amp;rsquo;s University, Kingston, Canada)</td><td></td><td>Software is designed to work with any cell line.</td></tr><tr><td>Trypsin</td><td>GIBCO BRL</td><td>27250-018</td><td>Prepared as 0.20% (w/v) in 10 &amp;micro;M EDTA 1x PBS</td></tr><tr><td>Accutase</td><td>Innovative Cell Technologies</td><td>AT104</td><td></td></tr><tr><td>10 cm cell culture plates</td><td>SARSTEDT</td><td>833902</td><td>Any tissue culture treated plates will be suitable</td></tr><tr><td>Transwell Polyester Membrane Inserts - 8.0&amp;nbsp;&amp;micro;m Pore size</td><td>Costar 3422 ordered from Fisher Scientific</td><td>7200150</td><td>For 24-well plates; Pore size: 8.0 &amp;micro;m; 6.5 mm diameter; 0.33 cm&lt;sup&gt;2&lt;/sup&gt; growth area</td></tr><tr><td>HARLECO Hematology Stains and Reagents, EMD Millipore - Soluntions 1, 2 &amp;amp; 3</td><td>EMD Millipore and ordered from VWR</td><td>65044A, B, &amp;amp; C</td><td>Hemacolor stain set consists of three 500 ml (16.9 oz.) poly bottles &amp;amp; includes a methanol fixative (Solution 1), an eosin or acid stain (Solution 2), and a methylene blue or basic stain (Solution 3)</td></tr><tr><td>Cotton Tipped Applicator</td><td>Puritan Medical</td><td>806-WC</td><td></td></tr><tr><td>Single-edge industrial razor blades</td><td>VWR</td><td>55411 - 055</td><td>Thickness: 0.30 mm (0.012&quot;)</td></tr><tr><td>Microscope Slides - Precleaned/Plain</td><td>Fisher Scientific</td><td>12550A3</td><td>Dimentions: 25 mm x 75 mm x 1.0 mm</td></tr><tr><td>Fisherbrand Cover Glasses - Rectangles no. 1</td><td>Fisher Scientific</td><td>12-545E</td><td>Thickness: 0.13 to 0.17 mm; Size: 50 mm x 22 mm</td></tr><tr><td>Fisher Chemical Permount Mounting Medium</td><td>Fisher Scientific</td><td>SP15-500</td><td></td></tr><tr><td>Leica Stereo dissecting microscope</td><td>Leica Microsystems</td><td></td><td>The microsope is equipped with Leica microscope camera Model MC170 HD &amp;amp; camera software is Leica App. Suite (LAS E2) Version 3.1.1 [Build: 490]. Microscope parts:&amp;nbsp; LED3000 Spot Light Illumination Model: MEB126, Leica M80 Optic Carrier Model M80, Objective achromat 1.0X, WD=90&amp;nbsp;mm Model: MOB306 &amp;amp; Objective achromat 0.32X, WD=303&amp;nbsp;mm Model: MOB315, Video Objective 0.5X Model: MTU-293</td></tr><tr><td>Hemacytometer</td><td>Assistant Germany&amp;nbsp;</td><td></td><td>0.100 mm Depth - 0.0025 mm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Olympus inverted light microscope</td><td>Olympus Corporation</td><td>CKX41SF</td><td>The microsope is equipped with Lumenera Infinity 1-2&lt;br /&gt;2.0 Megapixel CMOS Color Camera &amp;amp; camera software is Infinity analyze Version 6.5.2</td></tr><tr><td>Laminar flow cabinet 1300 Series A2</td><td>Thermo Scientific&amp;nbsp;</td><td>Model: 1375</td><td>Any laminar flow cabinet for cell culture work will be suitable&amp;nbsp;</td></tr><tr><td>Cell culture incubator</td><td>Thermo Scientific&amp;nbsp;</td><td>Model: 370</td><td>Any cell culture incubator will be suitable - Cells were cultured under humidefied environment, 5% CO&lt;sub&gt;2&lt;/sub&gt;, 37 &amp;deg;C&amp;nbsp;</td></tr><tr><td>ImageJ</td><td>NIH</td><td>Version 1.50e</td><td>Minimum version required</td></tr><tr><td>Java Runtime Environment</td><td>Oracle</td><td>Version 1.8.0_66</td><td>Minimum version required</td></tr></tbody></table>

automated quantification and analysis of cell counting procedures using imagej plugins

Das National Institute of Health ImageJ ist ein leistungsfähiges, frei verfügbaren Bildverarbeitungs-Software-Suite. ImageJ hat umfassende Partikelanalyse-Algorithmen, die effektiv genutzt werden kann, verschiedene biologische Partikel zu zählen. Wenn eine große Anzahl von Zellproben zu zählen, stellt der Zählkammer einen Engpass in Bezug auf die Zeit. Ebenso Membranen von Migration / Invasion Assays mit dem ImageJ Plug-Zellzähler, obwohl eine genaue Zählung, ist außerordentlich arbeitsintensiv, subjektiv und berüchtigt für verursachen Schmerzen am Handgelenk. Um diesem Bedarf zu begegnen, haben wir zwei Plugins innerhalb ImageJ für die einzige Aufgabe der automatisierten Hämozytometer (oder bekannten Volumen) und Migration / invasion Zellzählung. Beide Plugins verlassen sich auf die Fähigkeit, qualitativ hochwertige Mikroaufnahmen mit minimalen Hintergrund zu erfassen. Sie sind einfach für den schnellen Zählen und Analyse großer Probengrößen mit integrierten Analysetools zu verwenden und optimierte Kalibrierung von Zählungen zu helfen. Durch die Kombination der Kernprinzips von Zellzähler mit einem automatisierten Zählalgorithmus und post-counting Analyse Dies erhöht die Leichtigkeit, mit der Migrationsassays kann ohne Verlust an Genauigkeit bearbeitet werden.

Zellkonzentration Rechner Abbildung 1 zeigt den Gesamtprozess der CCC - Kalibrierung und zählbar Bildaufnahme. 1A und 1B zeigen die P-Quadrat Kalibrierungsbild und Berechnung von P-Quadrat Länge in Pixeln. CCC bestimmt Zellkonzentration in einem bestimmten Volumen nach der Formel: <img alt="Gleichung 1" src="/files/ftp_upload/54719/54719eq1.jpg" /></...

Watch this Scientific Journal Video about Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins at JoVE.com

Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins

This paper describes the quantification of hemocytometer and migration/invasion micrographs through two new open-source ImageJ plugins Cell Concentration Calculator and migration assay Counter. Furthermore, it describes image acquisition and calibration protocols as well as discusses in detail the input requirements of the plugins.

Automatische Quantifizierung und Analyse von Zellzählung Schritte bei der Verwendung ImageJ Plugins

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis ...

automated-quantification-analysis-cell-counting-procedures-using

Research

JoVE Journal

Biology

46.8K Aufrufe.  York University. This paper describes the quantification of hemocytometer and migration/invasion micrographs through two new open-source ImageJ plugins Cell Concentration Calculator and migration assay Counter. Furthermore, it describes image acquisition and calibration protocols as well as discusses in detail the input requirements of the plugins.

Serie: Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins

A Method of Targeted Cell Isolation via Glass Surface Functionalization

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual nuances in expression from patient to patient. Current methodologies that try to separate cells to fill this niche result in disruption of physiological expression, making the separation technique useless as a diagnostic tool. In this protocol, we describe the functionalization and optimization of a surface for the cellular capture and release. This functionalized surface integrates biotinylated antibodies with a glass surface functionalized with an aminosilane (APTES), desthiobiotin and streptavidin. Cell release is facilitated through the introduction of biotin, allowing the recollection and purification of cells captured by the surface. This release is done through the targeting of the secondary moiety desthiobiotin, which results in a much more gentle release paradigm. This reduction in harsh reagents and shear forces reduces changes in cellular expression. The functionalized surface captures up to 80% of cells in a single cell mixture and has demonstrated 50% capture in a dual-cell mixture. Applications of this technology to xenografts and cancer separation studies are investigated. Quantification techniques for surface verification such as plate reader and ImageJ analyses are described as well.

This protocol describes customizable surface functionalization of the desthiobiotin, streptavidin, and APTES system in order to isolate specific cell types of interest. In addition, this manuscript covers the applications, optimization, and verification of this process.

Ein Verfahren zur gezielten Zellisolierung über Glas Oberflächenfunktionalisierung

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual ...

Forschung

Krebsforschung

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Verbesserte Visualisierung und quantitative Analyse von Arzneimittelwirkungen mit mikrostrukturierten Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Biotechnik

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Automatisierte Quantifizierung Hämatopoetische Zell - Stromal Cell Interactions in histologische Bilder unentkalkte Knochen

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Entwicklungsbiologie

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

Quantitative Immunfluoreszenz Maßnahme Global lokalisierte Übersetzung

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Biochemie

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

Überwachung der Proteinaggregationskinetik in vivo mittels automatisierter Einschlusszählung bei Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts 

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

Quantitative Messung der Invadopodia-vermittelte extrazellulären Matrix Proteolyse in Einzel-und Multizelluläre Contexts

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Biologie

Area-based Image Analysis Algorithm for Quantification of Macrophage-fibroblast Cocultures

Quantification of cells is necessary for a wide range of biological and biochemical studies. Conventional image analysis of cells typically employs either fluorescence detection approaches, such as immunofluorescent staining or transfection with fluorescent proteins or edge detection techniques, which are often error-prone due to noise and other non-idealities in the image background. 
We designed a new algorithm that could accurately count and distinguish macrophages and fibroblasts, cells of different phenotypes that often colocalize during tissue regeneration. MATLAB was used to implement the algorithm, which differentiated distinct cell types based on differences in height from the background. A primary algorithm was developed using an area-based method to account for variations in cell size/structure and high-density seeding conditions. 
Non-idealities in cell structures were accounted for with a secondary, iterative algorithm utilizing internal parameters such as cell coverage computed using experimental data for a given cell type. Finally, an analysis of coculture environments was carried out using an isolation algorithm in which various cell types were selectively excluded based on the evaluation of relative height differences within the image. This approach was found to accurately count cells within a 5% error margin for monocultured cells and within a 10% error margin for cocultured cells.

We present a method, which utilizes a generalizable area-based image analysis approach to identify cell counts. Analysis of different cell populations exploited the significant cell height and structure differences between distinct cell types within an adaptive algorithm.

Area-based Image Analysis Algorithm zur Quantifizierung von Makrophagen-Fibroblasten-Kokulturen

Quantification of cells is necessary for a wide range of biological and biochemical studies. Conventional image analysis of cells typically employs either ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

Die Untersuchung der Proteinqualitätskontrolle System der<em&gt; D. discoideum</em&gt; Verwendung von Temperaturgesteuerte Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to the substrate as a function of the number and size of focal adhesions, and cell shape, characterized by the cell shape index and other shape descriptors. Focal adhesions were visualized by paxillin staining and cell-cell borders were marked by junction plakoglobin and actin. The methods for cell culture and staining were standard; images represent single focal planes; image analysis was performed using publicly available image processing software. The presented protocols are used to quantify the number and size of focal adhesions and the differences in cell shape distribution in the monolayers, but they can be repurposed for the quantification of the size and shape of any other distinct cellular structure that can be stained (e.g., mitochondria or nuclei). Assessing these parameters is important in the characterization of the dynamic forces in adherent cell layer, including cell adhesion and actomyosin contractility that affects cell shape.

The article describes quantification of 1) the size and number of focal adhesions and 2) cell shape index and its distribution from confocal images of the confluent monolayers of MCF7 cells.

Quantifizierung von Zell-Substrat-Adhäsionsflächen und Zellformverteilungen in MCF7-Zell-Monolayern

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to ...

Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and cell debris, through bulk endocytosis. This pathway plays an essential role in a variety of cellular processes, including the regulation of immune responses and cancer cell metabolism. Given this importance in biological function, examining cell culture conditions can provide valuable information by identifying regulators of this pathway and optimizing conditions to be employed in the discovery of novel therapeutic approaches. The study describes an automated imaging and analysis technique using standard laboratory equipment and a cell imaging multi-mode plate reader for the rapid quantification of the macropinocytic index in adherent cells. The automated method is based on the uptake of high molecular weight fluorescent dextran and can be applied to 96-well microplates to facilitate assessments of multiple conditions in one experiment or fixed samples mounted onto glass coverslips. This approach is aimed at maximizing reproducibility and reducing experimental variation while being both time-saving and cost-effective.

Automated assays using multi-well microplates are advantageous approaches for identifying pathway regulators by allowing the assessment of a multitude of conditions in a single experiment. Here, we have adapted the well-established macropinosome imaging and quantification protocol to a 96-well microplate format and provide a comprehensive outline for automation using a multi-mode plate reader.

Automatisierte Bildgebung und Analyse zur Quantifizierung fluoreszenzmarkierter Makropinosomen

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and ...