<html> Qui discutiamo le procedure sperimentali che sono coinvolti nella LUCAS [1-3]. Per illustrare la prova del concetto di LUCAS verrà descritto il processo di imaging per un campione di sangue intero. A. Imaging Set-up La piattaforma LUCAS Imaging presenta notevoli vantaggi per fornire un&#39;alternativa conveniente e compatto agli attuali point-of-care citometria e strumenti di diagnostica medica, soprattutto per le risorse limitate. Invece di rilevare l&#39;immagine delle celle, LUCAS cattura invece ologrammi digitale delle cellule che vengono creati dalla interferenza della luce diffusa da ogni cellula con la luce dello sfondo. Attento controllo della coerenza parziale spaziale della luce è fondamentale per attivare la registrazione olografica. 1. Sensore Array digitale La piattaforma LUCAS utilizza una matrice di sensori optoelettronici per la registrazione digitale ologrammi singola cella. A tal fine, pagano due dispositivi (CCD; modelli di esempio: KAI-11002, KAF-39000, da Kodak) o Complementary Metal-Oxide Semiconductor chip (CMOS, Modello Esempio: MT9P031, Micron) possono essere utilizzati. Le dimensioni dei pixel per il Kodak carica dispositivi coppia, KAI-11002, KAF-39000, e sensori di immagini CMOS Micron sono 9 micron, 6,8 micron e 2,2 micron, con un FOV attivo di 10 cm2, 18 cm2, e il 24,4 mm2, rispettivamente. [1-2]. 2. Fonte di luce A differenza di molti altre modalità di imaging microscopico, LUCAS non necessita di un laser e quindi anche un semplice diodo ad emissione luminosa (LED) può essere utilizzato per l&#39;illuminazione. Al fine di consentire l&#39;illuminazione regolabili lunghezza d&#39;onda, possiamo anche utilizzare un monocromatore con una lampada allo xeno (Cornerstone T260, Newport Corp.) insieme ad uno standard di qualità in fibra di silice fusa che consiste in un fascio di fibre (77.564, Newport) e foro stenopeico di ~ diametro di 100 micron trova in ~ 5-10 cm sopra la superficie del sensore. Questo parametro sintonizzabile configurazione illuminazione lunghezza d&#39;onda fornisce una piattaforma flessibile in cui le firme olografica delle cellule può essere regolata, e ibridi firme digitali possono essere sintetizzati per migliorare il rapporto segnale-rumore per una precisione migliore caratterizzazione e la specificità. [3] B. Preparazione del campione e Imaging Un proof of concept di LUCAS basati su chip di imaging sarà dimostrata utilizzando una soluzione eterogenea come descritto di seguito. Un protocollo simile potrebbe essere applicata per vari altri tipi di cellule [1-3]. 1. Diluizione del sangue intero e preparazione della soluzione eterogenea <ol><li> Per iniziare il processo, ottenere un campione di sangue intero e microsfere di polistirolo di vari diametri (micron 5, 10, e 20. Duke scientifico), e portare tutti a temperatura ambiente. </li><li> Aggiungere 2 ml di RPMI 1640 liquidi classico (Fisher Scientific) come diluente in una sterile 5 ml tubo in polipropilene. </li><li> Dopo l&#39;incubazione del campione di sangue intero per 30 minuti, pipetta fino a 10 ml di eritrociti sedimentati campione e trasferirlo nella soluzione RPMI. </li><li> Aggiungere un volume totale di 20 microlitri microsfere di polistirolo in eritrociti diluizione RPMI, e agitare la soluzione delle cellule pipettando gentilmente o con agitatore magnetico con ancoretta. </li><li> Posizionare un volume totale di 5-15 ul di soluzione eterogenea su una copertina slip e un secondo posto, scivolare identica copertura rispetto alla soluzione utilizzando pinze. Il campione deve essere inserita tra i due coprioggetto in modo uniforme. </li><li> Quindi, utilizzando una penna a vuoto (NT57-636, Edmund Optics) caricare il campione attraverso la regione attiva della matrice di sensori per l&#39;imaging. </li></ol> 2. Sangue colorazione intero <ol><li> A partire dalla preparazione di una miscela di 0,1% tamponata eosina Y e una soluzione diluita di metilene New sfruttando l&#39;energia di tipo puro eosina Y (PM = 691,85, Acros Organics), senza zinco puro New colorante blu di metilene (PM = 347,90, Acros Organics), e Monoidrato di ossalato di potassio (ACS reagente 99,0%, Acros Organics), ogni soluzione è purificato con una dimensione di 0,45 micron pori Syringless filtro (Whatman) per acquosa colorazione dei globuli bianchi. </li><li> Poi, mescolare delicatamente un reagente sciolto colorazione acquosa con il campione di sangue intero con un rapporto 1:1 in volume di un bicchiere in polipropilene utilizzando un agitatore magnetico per 2 minuti e incubare per 10 minuti. </li><li> Trasferire una soluzione macchiato del sangue di 50 microlitri in un tubo di polipropilene 5 ml e diluire con RPMI 1640 classica soluzione liquida (Fisher Scientific) di 1,95 ml di acquisire un rapporto tra volume di 1:200. Poi, il vortice di diluizione per 30 secondi. Un tasso di diluizione diverse possono essere introdotti anche in questa fase. </li><li> Pipettare una soluzione macchiato cellula di 10-100 microlitri tra coprioggetto o all&#39;interno di un serbatoio microfluidica. Quindi, utilizzando una penna a vuoto (NT57-636, Edmund Optics), posto il campione sulla zona attiva di matrice di sensori. </li></ol> RAPPRESENTANTE RISULTATI ve_content &quot;&gt; <img src="/files/ftp_upload/1650/1650Fig1tmb.jpg" alt="Figura 1" border="0" /> Figura 1: LUCAS consente il conteggio accurato digitale e la differenziazione di cellule diverse e micro-oggetti in base alla loro firme 2D olografica senza l&#39;uso di lenti, laser o altri componenti ottici ingombranti. Caratteristica firme LUCAS di vari micro-oggetti sono illustrati in questa figura e sono confrontati con un microscopio convenzionale immagine ottenuta con una lente obiettivo 40X. Si prega di <a href="https://www.jove.com/files/ftp_upload/1650/1650Fig1.jpg">cliccare qui</a> per vedere una versione più grande della figura 1. <img src="/files/ftp_upload/1650/1650Fig2.jpg" alt="Figura 2" /> Figura 2: (a sinistra) l&#39;immagine raw di una miscela eterogenea contenente i globuli rossi, 10um, 5um e 3 particelle um. (Destra) completamente automatizzato LUCAS risultati di caratterizzazione per lo stesso campo di vista sono illustrati. Si noti che l&#39;algoritmo di decisione è solido nel caratterizzare le regioni ad alta densità così come particelle con basso rapporto segnale rumore, come le perline 3um. <img src="/files/ftp_upload/1650/1650Fig3tmb.jpg" alt="Figura 3" border="0" /> Figura 3: L&#39;interfaccia LUCAS personalizzato è illustrato. LUCAS software basato su Java permette ingressi per le varie condizioni sperimentali, quali le dimensioni dei pixel del sensore o la lunghezza d&#39;onda della luce. Selezione di uno specifico campo di vista l&#39;immagine può anche essere fatte e gli schemi delle cellule bersaglio può essere definita dall&#39;utente per costruire una biblioteca di statistica ombra delle cellule. L&#39;immagine acquisita LUCAS possono essere caratterizzati sulla base di questi dati di formazione (ad esempio, la biblioteca ombra delle cellule) e la marcata (contati) immagine viene visualizzata per l&#39;utente. Le statistiche dei risultati contano sono inoltre memorizzate in un file XML per ulteriori analisi.

<ol>
	<li>Seo, S., Su, T., Tseng, D. K., Erlinger, A., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Lensfree+Holographic+Imaging+for+On-Chip+Cytometry+and+Diagnostics".">"Lensfree Holographic Imaging for On-Chip Cytometry and Diagnostics".</a> Lab Chip. , (2009).</li><li>Su, T., Seo, S., Erlinger, A., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="High-throughput+Imaging+and+Characterization+of+a+Heterogeneous+Cell+Solution+on+a+Chip"+.">"High-throughput Imaging and Characterization of a Heterogeneous Cell Solution on a Chip" .</a> Biotechnology and Bioengineering. , (2008).</li><li>Su, T., Seo, S., Erlinger, A., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Multi-color+LUCAS:+Lensfree+on-chip+cytometry+using+tunable+monochromatic+illumination+and+digital+noise+reduction".">"Multi-color LUCAS: Lensfree on-chip cytometry using tunable monochromatic illumination and digital noise reduction".</a> Cellular and Molecular Bioengineering. , (2008).</li></ol>

Abbiamo mostrato che la piattaforma LUCAS può contare e identificare con precisione micro-objects/cells varie su un chip in base alla loro firma olografica, e fornisce uno strumento promettente per point-of-care diagnostica medica e high-throughput di biologia cellulare. Al fine di elaborare con precisione gli schemi registrato olografica, abbiamo implementato una custom-software sviluppato decisione LUCAS. Questo algoritmo, che utilizza un database di immagini di diffrazione statistica creata da formazione di immagini...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Charged couple device (CCD)</td>
<td>&nbsp;</td>
<td>KODAK</td>
<td>KAI-11002</td>
<td>&nbsp;</td>
<tr>
<td>Charged couple device (CCD)</td>
<td>&nbsp;</td>
<td>KODAK</td>
<td>KAF-39000</td>
<td>&nbsp;</td>
<tr>
<td>Complementary Metal-Oxide-Semiconductor (CMOS)</td>
<td>&nbsp;</td>
<td>Micron</td>
<td>MT9P031</td>
<td>&nbsp;</td>
<tr>
<td>Xenon Lamp</td>
<td>&nbsp;</td>
<td>Newport Corp.</td>
<td>Cornerstone T260</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum pen</td>
<td>&nbsp;</td>
<td>Edmund Optics</td>
<td>NT57-636</td>
<td>&nbsp;</td>
<tr>
<td>5, 10, and 20 &mu;m Microbeads</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>4000 Series</td>
<td>&nbsp;</td>
<tr>
<td>RPMI</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>1640</td>
<td>&nbsp;</td>
<tr>
<td>Pure Eosin Y</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>MW=691.85</td>
<td>&nbsp;</td>
<tr>
<td>New Methylene Blue(NMB) Dye</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>MW=347.90</td>
<td>&nbsp;</td>
<tr>
<td>Potassium Oxalate Monohydrate</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>99.0% Reagent ACS</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

lensless on-chip imaging of cells provides a new tool for high-throughput cell-biology and medical diagnostics

Tradizionali microscopi ottici celle di immagine mediante l&#39;uso di lenti dell&#39;obiettivo che lavorano insieme con altri obiettivi e componenti ottici. Anche se molto efficace, questo approccio classico ha alcune limitazioni per la miniaturizzazione della piattaforma di imaging per renderlo compatibile con lo stato avanzato dell&#39;arte in microfluidica. In questo rapporto, vi presentiamo i dettagli sperimentali di una senza lenti on-chip concetto di imaging chiamata LUCAS (<strong<em&gt; L</em</strong&gt; Ensless<strong<em&gt; U</em</strong&gt; LTRA-ampio campo di vista<strong<em&gt; C</em</strong&gt; Ell monitoraggio<strong<em&gt; A</em</strong&gt; Rray piattaforma basata su<strong<em&gt; S</em</strong&gt; Hadow imaging) che non richiede alcuna obiettivi microscopio o altri componenti ottici ingombranti per l&#39;immagine di una soluzione eterogenea di cellule nel corso ultra-ampio campo di vista che può estendersi a grandi come ~ 18 centimetri<sup&gt; 2</sup&gt;. Inoltre, a differenza dei microscopi tradizionali, LUCAS può immagine una soluzione eterogenea di cellule di interesse su una profondità di campo di ~ 5 mm senza la necessità di riorientare che corrisponde a un massimo di ~ 9 ml del campione. Questa piattaforma di imaging registra le ombre (cioè, senza lenti ologrammi digitali) di ogni cella di interesse all&#39;interno del suo campo visivo, e automatizzata elaborazione digitale di queste ombre delle cellule in grado di determinare il tipo, il numero e le posizioni relative delle celle all&#39;interno della soluzione. Perché non richiede alcun componente ingombranti optical o meccanico fasi di scansione offre una piattaforma di dimensioni decisamente miniaturizzato che al tempo stesso di ridurre i costi, il che è molto importante soprattutto per i punti di assistenza strumenti diagnostici. Inoltre, la velocità di imaging di questa piattaforma è ordini di grandezza maggiore rispetto ai tradizionali microscopi ottici, che potrebbe essere estremamente utile per high-throughput di biologia cellulare, esperimenti.

Watch this Scientific Journal Video about Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics at JoVE.com

Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics

Lensfree on-chip di imaging e caratterizzazione di cellule è illustrata. Questo approccio on-chip di imaging cellulare fornisce uno strumento compatto e conveniente per la diagnostica medica e applicazioni ad elevato throughput biologia cellulare, che lo rende particolarmente adatto per le impostazioni delle risorse poveri.

Senza lenti on-chip Imaging di celle fornisce un nuovo strumento per High-throughput Cell-Biologia e diagnostica medica

Conventional optical microscopes image cells by use of objective lenses that work together with other lenses and optical components. While quite ...

lensless-on-chip-imaging-cells-provides-new-tool-for-high-throughput

Research

JoVE Journal

Biology

11.9K Views.  University of California, Los Angeles. Lensfree on-chip di imaging e caratterizzazione di cellule è illustrata. Questo approccio on-chip di imaging cellulare fornisce uno strumento compatto e conveniente per la diagnostica medica e applicazioni ad elevato throughput biologia cellulare, che lo rende particolarmente adatto per le impostazioni delle risorse poveri.

Video: Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics

Tracheotomy: A Method for Transplantation of Stem Cells to the Lung

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell therapy may alleviate many of the symptoms associated with lung disease and induce alveolar repair in adults. Concurrent with the ongoing search for stem cells applicable for human treatment, precise delivery and homing (to the site of disease) must be reassured for successful therapy. Here, I report that stem cells can safely be instilled via the trachea opening a non-stop route to the lung. This method involves a skin incision, caudal insertion of a cannula into and along the tracheal lumen, and injection of a stem cell vehicle mixture into airways of the lung. A broad range of media solutions and stabilizers can be instilled via tracheotomy, resulting in the ability to deliver a wider range of cell types. With alveolar epithelium confining these cells to the lumen, lung expansion and negative pressure during inhalation may also assist in stem cell integration. Tracheal delivery of stem cells, with a quick uptake and the ability to handle a large range of treatments, could accelerate the development of cell-based therapies, opening new avenues for treatment of lung disease.

Significant breakthroughs in stem cell identification are continuously being made. To translate these discoveries, however, novel methods for cellular delivery must be devised. Here I report that the airways provide a safe route for stem cell transplantation to the lungs.

Tracheotomia: Un metodo per il trapianto di cellule staminali al polmone

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell ...

Ricerca

Biologia

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

Un gradiente di generazione del dispositivo Microfluidic di Biologia Cellulare

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

Biochimica e High Throughput valutazione microscopica di massa grassa in<em&gt; Caenorhabditis Elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Anti-Nuclear Antibody Screening Using HEp-2 Cells

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although IIF is an excellent screening test in expert hands, the technical difficulties of processing and reading IIF slides &#8211; such as the labor intensive slide processing, manual reading, the need for experienced, trained technologists and the use of dark room &#8211; make the IIF method difficult to fit in the workflow of modern, automated laboratories.
The first and crucial step towards high quality ANA screening is careful slide processing. This procedure is labor intensive, and requires full understanding of the process, as well as attention to details and experience.
Slide reading is performed by fluorescent microscopy in dark rooms, and is done by trained technologists who are familiar with the various patterns, in the context of cell cycle and the morphology of interphase and dividing cells. Provided that IIF is the first line screening tool for SARD, understanding the steps to correctly perform this technique is critical.
Recently, digital imaging systems have been developed for the automated reading of IIF slides. These systems, such as the NOVA View Automated Fluorescent Microscope, are designed to streamline the routine IIF workflow. NOVA View acquires and stores high resolution digital images of the wells, thereby separating image acquisition from interpretation; images are viewed an interpreted on high resolution computer monitors. It stores images for future reference and supports the operator&#8217;s interpretation by providing fluorescent light intensity data on the images. It also preliminarily categorizes results as positive or negative, and provides pattern recognition for positive samples. In summary, it eliminates the need for darkroom, and automates and streamlines the IIF reading/interpretation workflow. Most importantly, it increases consistency between readers and readings. Moreover, with the use of barcoded slides, transcription errors are eliminated by providing sample traceability and positive patient identification. This results in increased patient data integrity and safety.
The overall goal of this video is to demonstrate the IIF procedure, including slide processing, identification of common IIF patterns, and the introduction of new advancements to simplify and harmonize this technique.

Indirect immunofluorescent (IIF) assays have traditionally been used for the detection of antinuclear antibodies (ANA) in human serum. The presence of these antibodies can aid in the diagnosis of systemic autoimmune rheumatic diseases (SARD). This protocol demonstrates how to effectively perform the IIF technique to accurately detect these autoantibodies.

Anti-Nuclear Antibody screening utilizzando cellule Hep-2

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although ...

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 ...

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 ...

High-Throughput Metabolic Profiling for Model Refinements of Microalgae

Metabolic models are reconstructed based on an organism&#39;s available genome annotation and provide predictive tools to study metabolic processes at a systems-level. Genome-scale metabolic models may include gaps as well as reactions that are unverified experimentally. Reconstructed models of newly isolated microalgal species will result in weaknesses due to these gaps, as there is usually sparse biochemical evidence available for the metabolism of such isolates. The phenotype microarray (PM) technology is an effective, high-throughput method that functionally determines cellular metabolic activities&#160;in response to a wide array of entry metabolites. Combining the high throughput phenotypic assays with metabolic modeling can allow existing metabolic network models to be rapidly reconstructed or optimized by providing biochemical evidence to support and expand genomic evidence. This work will show the use of PM assays for the study of microalgae by using the green microalgal model species Chlamydomonas reinhardtii as an example. Experimental evidence for over 254 reactions obtained by PM was used in this study to expand and refine a genome-scale C. reinhardtii metabolic network model, iRC1080, by approximately 25 percent. The protocol created here can be used as a basis for functionally profiling the metabolism of other microalgae, including known microalgae mutants and new isolates.

This protocol demonstrates the use of a phenotype microarray (PM) technology platform to define metabolic requirements of Chlamydomonas reinhardtii, a green microalga, and refine an existing metabolic network model.

Profilazione metabolica ad alto rendimento per perfezionamenti del modello di microalghe

Metabolic models are reconstructed based on an organism&#39;s available genome annotation and provide predictive tools to study metabolic processes at a ...

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental inputs into stress tolerance, pathogenicity, and other key components of fitness. This manuscript describes a microscope-based assay that tracks approximately 105&#160;Saccharomyces cerevisiae microcolonies per experiment. After automated time-lapse imaging of yeast immobilized in a multiwell plate, microcolony growth rates are easily analyzed with custom image-analysis software. For each microcolony, expression and localization of fluorescent proteins and survival of acute stress can also be monitored. This assay allows precise estimation of strains&#39; average growth rates, as well as comprehensive measurement of heterogeneity in growth, gene expression, and stress tolerance within clonal populations.

Yeast growth phenotypes are precisely measured through highly parallel time-lapse imaging of immobilized cells growing into microcolonies. Simultaneously, stress tolerance, protein expression, and protein localization can be monitored, generating integrated datasets to study how environmental and genetic differences, as well as gene-expression heterogeneity among isogenic cells, modulate growth.

Imaging live ad alta produttività di microcolonie per misurare l'eterogeneità nella crescita e nell'espressione genica

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental ...

A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell function, and therefore DNA damage may play a critical role in the development of, conceivably, all major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging. Single-cell gel electrophoresis (i.e., the comet assay) is one of the most common and sensitive methods to study the formation and repair of a wide range of types of DNA damage (e.g., single- and double-strand breaks, alkali-labile sites, DNA-DNA crosslinks, and, in combination with certain repair enzymes, oxidized purines, and pyrimidines), in both in vitro and in vivo systems. However, the low sample throughput of the conventional assay and laborious sample workup are limiting factors to its widest possible application. With the "scoring" of comets increasingly automated, the limitation is now the ability to process significant numbers of comet slides. Here, a high-throughput (HTP) variant of the comet assay (HTP comet assay) has been developed, which significantly increases the number of samples analyzed, decreases assay run time, the number of individual slide manipulations, reagent requirements, and risk of physical damage to the gels. Furthermore, the footprint of the electrophoresis tank is significantly decreased due to the vertical orientation of the slides and integral cooling. Also reported here is a novel approach to chilling comet assay slides, which conveniently and efficiently facilitates the solidification of the comet gels. Here, the application of these devices to representative comet assay methods has been described. These simple innovations greatly support the use of the comet assay and its application to areas of study such as exposure biology, ecotoxicology, biomonitoring, toxicity screening/testing, together with understanding pathogenesis.

The comet assay is a popular means of detecting DNA damage. This study describes an approach to running slides in representative variants of the comet assay. This approach significantly increased the number of samples while decreasing assay run-time, the number of slide manipulations, and the risk of damage to gels.

Un approccio di analisi delle comete ad alto rendimento per la valutazione del danno al DNA cellulare

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell ...

A High-Throughput Platform for Culture and 3D Imaging of Organoids

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by standard imaging approaches. This protocol describes a way to prepare microfabricated organoid culture chips, which enable multiscale, 3D live imaging on a user-friendly instrument requiring minimal manipulations and capable of up to 300 organoids/h imaging throughput. These culture chips are compatible with both air and immersion objectives (air, water, oil, and silicone) and a wide range of common microscopes (e.g., spinning disk, point scanner confocal, wide field, and brightfield). Moreover, they can be used with light-sheet modalities such as the single-objective, single-plane illumination microscopy (SPIM) technology (soSPIM). 
The protocol described here gives detailed steps for the preparation of the microfabricated culture chips and the culture and staining of organoids. Only a short length of time is required to become familiar with, and consumables and equipment can be easily found in normal biolabs. Here, the 3D imaging capabilities will be demonstrated only with commercial standard microscopes (e.g., spinning disk for 3D reconstruction and wide field microscopy for routine monitoring).

This paper presents a fabrication protocol for a new type of culture substrate with hundreds of microcontainers per mm2, in which organoids can be cultured and observed using high-resolution microscopy. The cell seeding and immunostaining protocols are also detailed.

Una piattaforma ad alto rendimento per la coltura e l'imaging 3D di organoidi

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by ...