La ricerca descritta in questo documento è stata eseguita presso la struttura BMIT presso la Canadian Light Source, che è supportata dal Canada Foundation for Innovation, Natural Sciences and Engineering Research Council of Canada, dall'Università del Saskatchewan, dal Governo del Saskatchewan, dalla Western Economic Diversification Canada, dal National Research Council Canada e dal Canadian Institutes of Health Research. Gli autori ringraziano gli scienziati beamline presso la Canadian Light Source, in particolare Adam Webb, Denise Miller, Sergey Gasilov e Ning Zu per l'assistenza nella configurazione e risoluzione dei problemi dei sistemi di microscopio SkyScan SRμCT e white beam. Desideriamo anche ringraziare Beth Dalzell dell'University of Toledo College of Medicine and Life Sciences e il Dr. Jeffrey Wenstrup della Northeast Ohio Medical University per l'accesso a campioni cadaverici per questo studio. JM Andronowski è supportato attraverso fondi di ricerca di start-up forniti dall'Università di Akron e da una sovvenzione del National Institute of Justice Research and Development in Forensic Science for Criminal Justice Purposes (2018-DU-BX-0188). RA Davis è supportato da un assistente di laurea fornito dall'Università di Akron. Le attrezzature e le forniture utilizzate per il coring e la segatura sono state acquistate con fondi di start-up forniti dall'Università di Akron e la sovvenzione NSF EAR-1624242 a CW Holyoke.

<ol>
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Gli autori non hanno nulla da rivelare.

Non esiste un protocollo completo e standardizzato per l'acquisto di campioni di nucleo osseo corticale uniforme e cilindrico per l'imaging SRμCT ad alta risoluzione con configurazioni FOV limitate. Il protocollo qui dettagliato riempie quel vuoto fornendo un tutorial completo su come procurarsi campioni di nucleo osseo corticale di dimensioni costanti per l'imaging SRμCT e la successiva visualizzazione accurata e l'estrazione di dati microarchitettotechi. Abbiamo dimostrato che il nostro protocollo fornisce un metodo ...

Con i recenti progressi nella tecnologia di imaging, è ora possibile acquisire dati di imaging a raggi X con una risoluzione molto elevata. I sistemi micro-CT desktop (μCT) sono lo standard attuale per l'imaging dell'osso cancello a causa della loro natura non distruttiva1. Quando si imagingno caratteristiche microstrutturali dell'osso corticale, tuttavia, l'uso di μCT è stato più limitato. A causa di vincoli di risoluzione, i sistemi desktop non possono raggiungere la risoluzione richiesta per l'immagine di caratteristiche microstrutturali più piccole dei pori corticali, come le lacune osteocite. Per questa applicazione, SRμCT è ideale grazie alla maggiore risoluzione di questi sistemi1. Ad esempio, esperimenti presso la Canadian Light Source (CLS) sulle linee di fascio BMIT (BioMedical Imaging and Therapy)2 hanno prodotto immagini con voxel di dimensioni fino a 0,9 μm. Studi precedenti1,3,4,5 hanno utilizzato questa risoluzione per acquisire proiezioni e successivi rendering tridimensionali (3D) da campioni ossei corticali da ossa lunghe umane ( Figura1) per quantificare la densità lacunare degli osteociti4,6,7,8,9 e la variazione della forma lacunare e della dimensione3 nella durata della vita umana e tra i sessi. Ulteriori studi hanno dimostrato la presenza di bande osteoniche nell'uomo10, un fenomeno precedentemente riconosciuto come associato solo a mammiferi non umani nella letteratura antropologica forense.
Per ottenere una risoluzione eccezionale, il fascio di raggi X deve essere finemente focalizzato all'interno del campo visivo (FOV), che spesso limita la dimensione massima del campione a pochi millimetri di diametro. Attualmente, non ci sono state procedure complete e standardizzate descritte nella letteratura che delineano l'approvvigionamento di campioni ossei che soddisfano queste restrizioni. Centrare i campioni all'interno del FOV è fondamentale per garantire che 1) il campione rimanga centrato mentre ruota di 180 ° durante l'imaging e 2) gli artefatti di scansione siano limitati poiché non vi è troncamento dell'immagine. In altre parole, nessuna porzione del campione al di fuori del FOV interferisce con il fascio che entra nel suo punto focale all'interno del FOV. In questo caso, l'algoritmo di ricostruzione viene privato di alcuni dei dati di attenuazione necessari per una ricostruzione completamente corretta. Vale inoltre la pena notare che le scansioni a 360° (rotazione completa) riducono al minimo gli effetti dell'indurimento del fascio ma aumentano gli artefatti causati dal disallineamento e dal movimento del campione durante l'imaging. Pertanto, mentre una scansione a 360 ° genererà in genere dati più puliti, il tempo di imaging viene raddoppiato e quindi è necessario affrontare un compromesso tra costi sperimentali e qualità dei dati.
Un aspetto importante e spesso trascurato degli esperimenti di imaging osseo è la tecnica accurata e replicabile di preparazione dei campioni eseguita prima della scansione. Gli studi che incorporano metodi SRμCT nei loro esperimenti menzionano brevemente il loro protocollo di campionamento, ma gli autori forniscono pochi o nessun dettaglio riguardo alla particolare metodologia utilizzata per raccogliere i loro campioni. Molti di questi studi menzionano il taglio di blocchi ossei rettilinei di dimensioni arbitrarie, ma generalmente non forniscono ulteriori informazioni sugli utensili o sui materiali di incorporamentoutilizzati 3,4,10,11,12,13,14. Alcuni ricercatori usano comunemente strumenti rotativi portatili (ad esempio, Dremel) per rimuovere blocchi rettilinei di osso da una regione di interesse (ROI)3,4,10,11,12,13,14. Questo metodo si traduce in campioni di dimensioni non uniforme che possono essere più grandi del FOV, aumentando la probabilità di artefatti di scansione e troncamento delle immagini. Tali campioni spesso richiedono un'ulteriore raffinazione utilizzando una sega a wafer di diamante di precisione (ad esempio, Buehler Isomet). L'approvvigionamento di campioni con dimensioni coerenti (fino ai due centesimi/mm) è fondamentale per garantire che i set di dati acquisiti siano della massima qualità e che i risultati successivi siano replicabili.
La segnalazione limitata della metodologia di approvvigionamento del campione aggiunge un ulteriore livello di difficoltà quando si tenta di utilizzare e/o convalidare metodi eseguiti in uno studio precedente. Attualmente, i ricercatori devono contattare direttamente gli autori per ulteriori dettagli sulle loro procedure di campionamento. Il protocollo qui dettagliato fornisce ai ricercatori biomedici una tecnica di campionamento accuratamente documentata, replicabile ed economica. L'obiettivo principale di questo articolo è quello di fornire un tutorial completo su come procurarsi campioni di nucleo osseo corticale di dimensioni costanti utilizzando una pressa per fresatura e una punta di coring a diamante per la visualizzazione accurata e l'estrazione di dati microarchitettotechi. Questo metodo viene modificato dalle procedure utilizzate per raccogliere regolarmente cilindri uniformi di piccolo diametro (1-5 mm) da blocchi di materiali duri in meccanica delle rocce adalta pressione 15,16,17,18,19.

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			<td height="42" style="height:42px;width:215px;">1-1/8&#34; plunge cutting carbide for composites</td>
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			<td height="21" style="height:21px;width:215px;">70% Ethanol</td>
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			<td height="21" style="height:21px;width:215px;">Centrifuge tubes</td>
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			<td height="21" style="height:21px;width:215px;">Crystalbond 509-3 Epoxy</td>
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			<td height="21" style="height:21px;width:215px;">CTAnalyser</td>
			<td style="width:120px;">Bruker microCT</td>
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			<td>Download and install at https://www.bruker.com/products/microtomography/micro-ct-software/3dsuite.html</td>
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			<td height="42" style="height:42px;width:215px;">Diamond wafering saw blade for composite material</td>
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			<td height="42" style="height:42px;width:215px;">Fixturing clamps for XY machine table for mill/drill</td>
			<td style="width:120px;">MSC Industrial Supply</td>
			<td style="width:133px;">#04804571</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass microscope slides</td>
			<td style="width:120px;">Ted Pella</td>
			<td style="width:133px;">26005</td>
			<td>75x50mm slides, 1mm thick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass slide chuck</td>
			<td style="width:120px;">Buehler</td>
			<td style="width:133px;">#112488</td>
			<td>Large enough to hold 75x50mm glass slides</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Hot plate capable of reaching 140 &#176;C</td>
			<td style="width:120px;">ThermoScientific</td>
			<td style="width:133px;">HP88850105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Incubator</td>
			<td style="width:120px;">NAPCO</td>
			<td>Model 4200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Isocut Fluid</td>
			<td style="width:120px;">Buehler</td>
			<td style="width:133px;">111193032</td>
			<td>Lubricant; 30mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Jeweler&#39;s diamond coring drill bit</td>
			<td style="width:120px;">Otto Frei</td>
			<td style="width:133px;">#119.050</td>
			<td>2mm inner diameter hollow stem coring bit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">NRecon</td>
			<td style="width:120px;">Bruker microCT</td>
			<td style="width:133px;">v.1.6.10.2</td>
			<td>Download and install at https://www.bruker.com/products/microtomography.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Oscillating saw</td>
			<td style="width:120px;">Harbor Freight</td>
			<td style="width:133px;">62866</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Oven-safe glass dishes</td>
			<td style="width:120px;">Pyrex</td>
			<td style="width:133px;">1117715</td>
			<td>Glass food storage container</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Precision slow-speed saw (Isomet 1000)</td>
			<td style="width:120px;">Buehler</td>
			<td style="width:133px;">111280160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Razor blades</td>
			<td style="width:120px;">Amazon</td>
			<td style="width:133px;">25181</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Shallow aluminum tins</td>
			<td style="width:120px;">Amazon</td>
			<td style="width:133px;">B01MRWLD0R</td>
			<td>~8cm diameter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Specimen cups</td>
			<td style="width:120px;">Amazon</td>
			<td style="width:133px;">616784425436 885334344729</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Tergazyme detergent</td>
			<td style="width:120px;">Alconox</td>
			<td style="width:133px;">1304-1</td>
			<td>1.8kg box</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Ultrasonic cleaner</td>
			<td style="width:120px;">MTI Corporation</td>
			<td style="width:133px;">KJ201508006</td>
			<td></td>
		</tr>
	</tbody>
</table>

a sectioning, coring, and image processing guide for high-throughput cortical bone sample procurement and analysis for synchrotron micro-ct

L'osso è un tessuto dinamico e meccanicamente attivo che cambia struttura nel corso della vita umana. I prodotti del processo di rimodellamento osseo sono stati studiati sostanzialmente utilizzando tecniche bidimensionali tradizionali. I recenti progressi nella tecnologia di imaging a raggi X attraverso la tomografia micro-computerizzata desktop (μCT) e la tomografia micro-computerizzata a radiazione di sincrotrone (SRμCT) hanno permesso l'acquisizione di scansioni tridimensionali (3D) ad alta risoluzione di un campo visivo più ampio (FOV) rispetto ad altre tecniche di imaging 3D (ad esempio, SEM) fornendo un quadro più completo delle strutture microscopiche all'interno dell'osso corticale umano. Il campione deve essere centrato accuratamente all'interno del FOV, tuttavia, per limitare la comparsa di artefatti striati noti per influire sull'analisi dei dati. Studi precedenti hanno riportato l'approvvigionamento di blocchi ossei rettilinei di forma irregolare che si traducono in artefatti di imaging dovuti a bordi irregolari o troncamento dell'immagine. Abbiamo applicato un protocollo di campionamento geologico (coring) per procurare campioni di nucleo osseo corticale di dimensioni costanti per esperimenti SRμCT dall'aspetto anteriore della femora umana. Questo metodo di coring è efficiente e minimamente distruttivo per i tessuti. Crea campioni cilindrici uniformi che riducono gli artefatti di imaging per natura di essere isometrici durante la rotazione e forniscono una lunghezza uniforme del percorso per i raggi X durante la scansione. L'elaborazione delle immagini dei dati tomografici a raggi X di campioni cored e di forma irregolare conferma il potenziale della tecnica per migliorare la visualizzazione e l'analisi della microarchitettura ossea corticale. Un obiettivo di questo protocollo è fornire un metodo affidabile e ripetibile per l'estrazione di nuclei ossei corticali adattabili per vari tipi di esperimenti di imaging osseo ad alta risoluzione. Un obiettivo generale del lavoro è quello di creare un approvvigionamento osseo corticale standardizzato per SRμCT che sia conveniente, coerente e diretto. Questa procedura può essere ulteriormente adattata dai ricercatori in campi correlati che comunemente valutano materiali compositi duri come in antropologia biologica, geoscienze o scienze dei materiali.

Il metodo descritto di campionamento di base si è rivelato altamente efficace ed efficiente. I campioni di coring che utilizzano questo protocollo hanno permesso l'approvvigionamento di campioni di dimensioni costanti >300 per esperimenti sulla beamline CLS BMIT-BM2, con un FOV di ~ 2 mm a 1,49 μm di dimensione voxel. Per convalidare la consistenza del diametro del nucleo, sono state effettuate tre misurazioni lungo la lunghezza (superiore, centrale, inferiore) di un sottoinsieme di nuclei femor...

Watch this Scientific Journal Video about A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT at JoVE.com

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

Abbiamo utilizzato un protocollo di campionamento geologico (coring) per procurare campioni ossei corticali di dimensioni uniformi per esperimenti SRμCT dall'aspetto anteriore della femora umana. Questo metodo è minimamente distruttivo, efficiente, si traduce in campioni cilindrici che riducono al minimo gli artefatti di imaging da forme campione irregolari e migliorano la visualizzazione e l'analisi microarchitettonica.

Guida all'elaborazione di sezioni, coring e immagini per l'approvvigionamento e l'analisi di campioni ossei corticali ad alta produttività per synchrotron Micro-CT

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been ...

a-sectioning-coring-image-processing-guide-for-high-throughput

Research

JoVE Journal

Biology

5.0K Views.  The University of Akron. Abbiamo utilizzato un protocollo di campionamento geologico (coring) per procurare campioni ossei corticali di dimensioni uniformi per esperimenti SRμCT dall'aspetto anteriore della femora umana. Questo metodo è minimamente distruttivo, efficiente, si traduce in campioni cilindrici che riducono al minimo gli artefatti di imaging da forme campione irregolari e migliorano la visualizzazione e l'analisi microarchitettonica.

Video: A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

Una schermata di High Throughput per Biomining attività Cellulase da librerie metagenomiche

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

Ricerca

Biologia

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

Preparazione di adulti Drosophila Occhi di sezionamento sottile e analisi microscopica

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

Una guida pratica per Phylogenetics per i non esperti

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Un saggio di legame High Throughput MHC II per l&#39;analisi quantitativa dei peptidi epitopi

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

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.

High-throughput Immagine Analisi di sferoidi tumorali: Un software applicativo di facile utilizzo per misurare le dimensioni del Spheroids automaticamente e con precisione

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

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recupero di Zebrafish adulti cuori per applicazioni ad alta velocità effettiva

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Screening ad alto rendimento per i modulatori chimici di Genes post-trascrizionale regolamentati

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

Analisi qPCRTag - A High Throughput, Real Time PCR Assay per Sc2.0 genotipizzazione

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

Preparazione del campione per l&#39;analisi di massa Citometria

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.

In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections of mineralized tissues.

-High Throughput, Multi-Image Cryohistology di mineralizzati tessuti

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious ...

Guida all'elaborazione di sezioni, coring e immagini per l'approvvigionamento e l'analisi di campioni ossei corticali ad alta produttività per synchrotron Micro-CT

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