Diese Arbeit wurde von der National Science Foundation (DBI 0227541) unterstützt.

<ol>
	<li>Brewer, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+controlled+vocabulary+and+software+application+to+analyze+fruit+shape+variation+in+tomato+and+other+plant+species.">Development of a controlled vocabulary and software application to analyze fruit shape variation in tomato and other plant species.</a> Plant Physiol. 141 (1), 15-25 (2006).</li><li>Gonzalo, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomato+Fruit+Shape+Analysis+Using+Morphometric+and+Morphology+Attributes+Implemented+in+Tomato+Analyzer+Software+Program.">Tomato Fruit Shape Analysis Using Morphometric and Morphology Attributes Implemented in Tomato Analyzer Software Program.</a> Journal of the American Society for Horticultural Science. 134 (1), 77-87 (2009).</li><li>Darrigues, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomato+analyzer-color+test:+A+new+tool+for+efficient+digital+phenotyping.">Tomato analyzer-color test: A new tool for efficient digital phenotyping.</a> Journal of the American Society for Horticultural Science. 133 (4), 579-586 (2008).</li><li>Brewer, M. T., Moyseenko, J. B., Monforte, A. J., van der Knaap, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+variation+in+tomato:+a+comprehensive+study+of+quantitative+trait+loci+controlling+fruit+shape+and+development.">Morphological variation in tomato: a comprehensive study of quantitative trait loci controlling fruit shape and development.</a> J Exp Bot. 58 (6), 1339-1349 (2007).</li><li>Gonzalo, M. J., Knaap, E. v. a. n. d. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+analysis+into+the+genetic+bases+of+morphology+in+tomato+varieties+exhibiting+elongated+fruit+shape.">A comparative analysis into the genetic bases of morphology in tomato varieties exhibiting elongated fruit shape.</a> Theor Appl Genet. 116 (5), 647-656 (2008).</li><li>Rodr&#237;guez, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+SUN,+OVATE+and+FAS+in+the+tomato+germplasm+and+their+effect+on+fruit+morphology+(in+preparation.">Diversity of SUN, OVATE and FAS in the tomato germplasm and their effect on fruit morphology (in preparation.</a> , .</li><li>Wu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Masters Thesis. , (2009).</li><li>Orsi, C. H., Tanksley, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+variation+in+an+ABC+transporter+gene+associated+with+seed+size+evolution+in+tomato+species.">Natural variation in an ABC transporter gene associated with seed size evolution in tomato species.</a> PLoS Genet. 5 (1), e1000347-e1000347 (2009).</li><li>Yue, B., Cai, X., Yuan, W., Vick, B., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+quantitative+trait+loci+(QTL)+controlling+seed+morphology+and+disk+diameter+in+sunflower+(Helianthus+annuus+L).">Mapping quantitative trait loci (QTL) controlling seed morphology and disk diameter in sunflower (Helianthus annuus L).</a> Helia. 32, 17-35 (2009).</li><li>Zeng, Z. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+Basis+for+Separation+of+Multiple+Linked+Gene+Effects+in+Mapping+Quantitative+Trait+Loci.">Theoretical Basis for Separation of Multiple Linked Gene Effects in Mapping Quantitative Trait Loci.</a> Proceedings of the National Academy of Sciences of the United States of America. 90 (23), 10972-10976 (1993).</li><li>Zeng, Z. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+Mapping+of+Quantitative+Trait+Loci.">Precision Mapping of Quantitative Trait Loci.</a> Genetics. 136 (4), 1457-1468 (1994).</li><li>Jiang, C. J., Zeng, Z. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple-Trait+Analysis+of+Genetic-Mapping+for+Quantitative+Trait+Loci.">Multiple-Trait Analysis of Genetic-Mapping for Quantitative Trait Loci.</a> Genetics. 140 (3), 1111-1127 (1995).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> IPGRI Descriptors for Tomato (Lycopersicon spp.). , 47-47 (1996).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> UPOV Guidelines for the Conduct of Tests for Distinctness, Uniformity, and Stability. TOMATO (Lycopersicon Lycopersicum (L.) Karsten Ex Farw.). , 51-51 (2001).</li><li>Depypere, L., Chaerle, P., Mijnsbrugge, K. V., Goetghebeur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stony+endocarp+dimension+and+shape+variation+in+Prunus+section+Prunus.">Stony endocarp dimension and shape variation in Prunus section Prunus.</a> Annals of Botany. 100 (7), 1585-1597 (2007).</li><li>Depypere, L., Chaerle, P., Breyne, P., Mijnsbrugge, K. V., Goetghebeur, P., P, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combined+morphometric+and+AFLP+based+diversity+study+challenges+the+taxonomy+of+the+European+members+of+the+complex+Prunus+L.+section+Prunus.+.">A combined morphometric and AFLP based diversity study challenges the taxonomy of the European members of the complex Prunus L. section Prunus. .</a> Plant systematics and evolution. 279 (1-4), 219-231 (2009).</li><li>Xiao, H., Jiang, N., Schaffner, E., Stockinger, E. J., van der Knaap, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+retrotransposon-mediated+gene+duplication+underlies+morphological+variation+of+tomato+fruit.">A retrotransposon-mediated gene duplication underlies morphological variation of tomato fruit.</a> Science. 319 (5869), 1527-1530 (2008).</li></ol>

High-throughput und genaue Auswertung der Pflanze Orgel Morphologie und Farbe ist wegen seiner quantitativer Natur und oft subjektiven Quantifizierung schwierig. TA bietet objektive und genaue Messungen von mehreren Früchten morphologischen und kolorimetrischen Eigenschaften in einem High-Throughput-und semi-automatische Weise. Der Nutzen der TA hat in Studien, die zur Identifizierung von Tomatenfrucht Form QTL 2,4,5 führte nachgewiesen. Darüber hinaus hat die Einstufung von cultivars6 und die Taxonomie der eine Gruppe von Arten wurden anhand der Ausgabe von TA 15,16. Hohe Ausdruck der Tomatenfrucht Form Gen so ergibt sich längliche Früchte 17. Mit TA-, Blatt-und cotyledon Form wurde in den Zeilen auszudrücken Sonne zu einem hohen Niveau 7 bestimmt, dass der Einsatz effektiv genutzt werden kann, um Morphologie der anderen Pflanzenorgane sowie zu messen. Darüber hinaus hat TA wurde von entscheidender Bedeutung bei der Identifizierung der genetischen Grundlagen der Größe der Samen in Tomaten und Sonnenblumen 8,9. Schließlich ist die Color Test-Modul in TA implementiert genauer und präziser und günstiger als andere Methoden, um Obst Farbe in Tomaten zu analysieren. Zusammenfassend hat TA zu einem wichtigen Werkzeug für die objektive und zuverlässige Beurteilung der Morphologie und Farbvariationen der Pflanzenorgane.

tomato analyzer: a useful software application to collect accurate and detailed morphological and colorimetric data from two-dimensional objects

Mess-Obst Morphologie und Farbe Züge Gemüse-und Obstkulturen in eine objektive und reproduzierbare Weise ist für detaillierte phänotypische Analysen dieser Merkmale wichtig. Tomato Analyzer (TA) ist ein Software-Programm, dass 37 Attribute im Zusammenhang mit zweidimensionalen Form in eine semi-automatische und reproduzierbare Weise 1,2 Maßnahmen. Viele dieser Attribute, wie Winkel am distalen und proximalen Enden der Frucht und Bereichen Einzug, sind schwer zu manuell zu quantifizieren. Grundlegende Mess-, Obst-Shape-Index, Blockiness, Homogenität, Proximale Fruit End Shape, Distal Fruit End Shape, Asymmetrie, Internal Exzentrizität, Breiten-Section und Morphometrie: Die Attribute werden in zehn Kategorien innerhalb der Software organisiert. Die letzte Kategorie erfordert weder Vorkenntnisse noch vorgegebenen Vorstellungen von der Gestalt Attribute, bietet so morphometrische Analyse eine unvoreingenommene Option, die besser an High-Throughput-Analysen können als Attribut Analyse angepasst. TA bietet auch die Color Test-Anwendung, die entworfen, um Farbmessungen aus gescannten Bildern zu sammeln und erlauben das Scannen Geräte kalibriert werden mit Farbstandards 3 war. TA bietet verschiedene Optionen für den Export und analysieren Formattribut, morphometrische und Farbdaten. Die Daten können in eine Excel-Datei im Batch-Modus (mehr als 100 Bilder auf einmal) exportiert werden oder exportiert als einzelne Bilder. Der Anwender kann zwischen Ausgang, dass die durchschnittliche zeigt für jedes Attribut für die Objekte in jedem Bild (einschließlich Standardabweichung), oder eine Ausgabe, die die Werte des Attributs zeigt für jedes Objekt auf dem Bild zu wählen. TA hat sich als wertvolles und effektives Werkzeug für indentifying und Bestätigung Tomatenfrucht Form Quantitative Trait Loci (QTL), sowie die Durchführung von eingehenden Analysen der Wirkung der wichtigsten Obst-Form Gene auf Pflanzenmorphologie worden. Außerdem können TA verwendet werden, um objektiv zu klassifizieren Frucht in verschiedener Form Kategorien. Schließlich können Früchte in Form und Farbe Züge in anderen Pflanzenarten sowie andere Pflanzenorgane wie Blätter und Samen mit TA ausgewertet werden.

Watch this Scientific Journal Video about Tomato Analyzer: A Useful Software Application to Collect Accurate and Detailed Morphological and Colorimetric Data from Two-dimensional Objects at JoVE.com

Tomato Analyzer: A Useful Software Application to Collect Accurate and Detailed Morphological and Colorimetric Data from Two-dimensional Objects

Tomato Analyzer (TA) quantifiziert Attribute zweidimensionale Formen und Farben in eine reproduzierbare und genaue Weise. Eine Schritt-für-Schritt-Verfahren zur Erlangung hoher Qualität digitalisierten Bilder von Tomatenfrucht, morphologische und Farbe analysiert diese Bilder und mehrere Anwendungen unter Verwendung der Daten durch die Software erzeugt werden beschrieben.

Tomato Analyzer: A Useful Software-Anwendung, um genaue und detaillierte morphologische und farbmetrischen Daten von zweidimensionalen Objekten sammeln

Measuring fruit morphology and color traits of vegetable and fruit crops in an objective and reproducible way is important for detailed phenotypic ...

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Research

JoVE Journal

Biology

26.2K Aufrufe.  The Ohio State University. Tomato Analyzer (TA) quantifiziert Attribute zweidimensionale Formen und Farben in eine reproduzierbare und genaue Weise. Eine Schritt-für-Schritt-Verfahren zur Erlangung hoher Qualität digitalisierten Bilder von Tomatenfrucht, morphologische und Farbe analysiert diese Bilder und mehrere Anwendungen unter Verwendung der Daten durch die Software erzeugt werden beschrieben.

Serie: Tomato Analyzer: A Useful Software Application to Collect Accurate and Detailed Morphological and Colorimetric Data from Two-dimensional Objects

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

Die Beurteilung Zweidimensionale Kristallisation Trials of Small Membranproteinen für Strukturbiologie Studien Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Forschung

Biologie

Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters

We demonstrate a microscopic instrument that can measure subcellular texture arising from organelle morphology and organization within unstained living cells. The proposed instrument extends the sensitivity of label-free optical microscopy to nanoscale changes in organelle size and shape and can be used to accelerate the study of the structure-function relationship pertaining to organelle dynamics underlying fundamental biological processes, such as programmed cell death or cellular differentiation. The microscope can be easily implemented on existing microscopy platforms, and can therefore be disseminated to individual laboratories, where scientists can implement and use the proposed methods with unrestricted access.
The proposed technique is able to characterize subcellular structure by observing the cell through two-dimensional optical Gabor filters. These filters can be tuned to sense with nanoscale (10's of nm) sensitivity, specific morphological attributes pertaining to the size and orientation of non-spherical subcellular organelles. While based on contrast generated by elastic scattering, the technique does not rely on a detailed inverse scattering model or on Mie theory to extract morphometric measurements. This technique is therefore applicable to non-spherical organelles for which a precise theoretical scatter description is not easily given, and provides distinctive morphometric parameters that can be obtained within unstained living cells to assess their function. The technique is advantageous compared with digital image processing in that it operates directly on the object's field transform rather than the discretized object's intensity. It does not rely on high image sampling rates and can therefore be used to rapidly screen morphological activity within hundreds of cells at a time, thus greatly facilitating the study of organelle structure beyond individual organelle segmentation and reconstruction by fluorescence confocal microscopy of highly magnified digital images of limited fields of view.
In this demonstration we show data from a marine diatom to illustrate the methodology. We also show preliminary data collected from living cells to give an idea of how the method may be applied in a relevant biological context.

We demonstrate a dark-field microscopy method based on Gabor-like filtering to measure subcellular dynamics within single living cells. The technique is sensitive to alterations in the structure of organelles, such as mitochondrial fragmentation.

Optical Microscopy Scatter auf zweidimensionalen Gabor-Filtern

We demonstrate a microscopic instrument that can measure subcellular texture arising from organelle morphology and organization within unstained living ...

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these organisms form networks of interconnected hyphae, known as pellets, which are heterogeneous in size. Here we describe a method to analyze and sort mycelial pellets using a Complex Object Parametric Analyzer and Sorter (COPAS). Detailed instructions are given for the use of the instrument and the basic statistical analysis of the data. We furthermore describe how pellets can be sorted according to user-defined settings, which enables downstream processing such as the analysis of the RNA or protein content. Using this methodology the mechanism underlying heterogeneous growth can be tackled. This will be instrumental for improving streptomycetes as a cell factory, considering the fact that productivity correlates with pellet size.

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

Liquid-grown Streptomyces cultures are characterized by mycelial pellets that are heterogeneous in size. We here describe a method to analyze and sort such pellets in a high-throughput manner. These pellets can be used for further analyses, which will provide leads to understand and control growth heterogeneity.

Sortierung der<em&gt; Streptomyces</em&gt; Zell Pellets mit einem komplexen Objekt Parametric Analyzer und Sorter

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these ...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

Anwendung von Retinsäure zu Osteozyten Kulturen aus primären Maus-Osteoblasten erhalten

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

Hochdurchsatz-Analyse von Bild Tumorsphäroide: Eine benutzerfreundliche Software-Anwendung, um die Größe der Sphäroide automatisch und präzise messen

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators.
Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software1&#160;to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.

Presented is a flexible informatics workflow enabling multiplexed image-based analysis of fluorescently labeled cells. The workflow quantifies nuclear and cytoplasmic markers and computes marker translocation between these compartments. Procedures are provided for perturbation of cells using siRNA and reliable methodology for marker detection by indirect immunofluorescence in 96-well formats.

Workflow für Hoch Inhalt, einzelne Zell Quantifizierung der Fluoreszenzmarker von Universal Mikroskop Daten, Unterstützung von Open Source Software

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Komplette Workflow zur Analyse von Histon-post-translationale Modifikationen Mit Bottom-up-Massenspektrometrie: Von Histone Extraktion in Datenanalyse

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

Fisch Sperma Bewertung mit Software und Kühlgeräte

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...

Diagonal Method to Measure Synergy Among Any Number of Drugs

A synergistic drug combination has a higher efficacy compared to the effects of individual drugs. Checkerboard assays, where drugs are combined in many doses, allow sensitive measurement of drug interactions. However, these assays are costly and do not scale well for measuring interaction among many drugs. Several recent studies have reported drug interaction measurements using a diagonal sampling of the traditional checkerboard assay. This alternative methodology greatly decreases the cost of drug interaction experiments and allows interaction measurement for combinations with many drugs. Here, we describe a protocol to measure the three pairwise interactions and one three-way interaction among three antibiotics in duplicate, in five days, using only three 96-well microplates and standard laboratory equipment. We present representative results showing that the three-antibiotic combination of Levofloxacin + Nalidixic Acid + Penicillin G is synergistic. Our protocol scales up to measure interactions among many drugs and in other biological contexts, allowing for efficient screens for multi-drug synergies against pathogens and tumors.

In this protocol, we describe how to make Loewe additivity-based drug interaction measurements for pairwise and three-way drug combinations.

Diagonale Methode zur Maßnahme Synergie zwischen beliebig vieler Medikamente

A synergistic drug combination has a higher efficacy compared to the effects of individual drugs. Checkerboard assays, where drugs are combined in many ...

Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, Rhizoctonia bataticola. In the literature, comprehensive and detailed step-by-step protocols on disease assays are sparse. This article provides complete details on the steps involved in setting up a blotting paper technique for quickly screening genotypes for resistance to DRR. The blotting paper technique is easy and less expensive. Another method, based on the sick pot approach, is a mimic of natural infection and can be applied to study the interacting components&#8212;plant, pathogen, and environment&#8212;involved in the disease triangle.
Moreover, in nature, DRR occurs mostly in rainfed chickpea cultivation areas, where soil moisture recedes as crop growth advances. Drought stress is known to predispose chickpea plants to DRR disease. Pathomorphological and molecular understanding of plant-pathogen interaction under drought stress can pave the way for the identification of elite DRR-resistant varieties from the chickpea germplasm pool. This article provides a stepwise methodology for the preparation of a sick pot and subsequent disease assay. Overall, the information presented herein will help researchers prepare R. bataticola fungal inoculum, maintain this pathogen, set up the blotting paper technique, prepare sick culture and sick pot, and assess pathogen infection in chickpea plants.

This study presents methodologies to study the pathomorphological and molecular mechanisms underlying chickpea&#8211;Rhizoctonia bataticola interaction. The blotting paper method is useful to rapidly study chickpea genotype responses, while the sick pot-based method can be used to simultaneously impose drought and R. bataticola infection and screen for tolerant genotypes.

Dry Root Rot Disease Assays in Chickpea: eine detaillierte Methodik

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, ...