<html> Ici, nous discutons des procédures expérimentales qui sont impliqués dans LUCAS [1-3]. Pour illustrer la preuve de concept de LUCAS, nous allons décrire le processus d&#39;imagerie pour un échantillon de sang total. A. Mise en place d&#39;imagerie La plate-forme d&#39;imagerie LUCAS présente des avantages significatifs pour fournir une alternative rentable et compacte pour les systèmes point-of-care cytométrie et instruments de diagnostic médical, notamment pour les ressources limitées. Plutôt que de détecter l&#39;image des cellules, LUCAS capture au lieu d&#39;hologrammes numériques des cellules qui sont créés par l&#39;interférence de la lumière diffusée à partir de chaque cellule avec la lumière de fond. Un contrôle minutieux de la cohérence spatiale partielle de l&#39;éclairage est essentiel pour permettre l&#39;enregistrement holographique. 1. Réseau de capteurs numériques La plateforme utilise un tableau LUCAS capteurs optoélectroniques à enregistrer numériquement des hologrammes cellule individuelle. À cette fin, chargé des dispositifs couple (CCD; Sample Models: KAI-11002, KAF-39000, de Kodak) ou Complementary metal-oxyde-semiconducteur puces (CMOS, modèle de l&#39;échantillon: MT9P031, Micron) peuvent être utilisés. Les tailles des pixels pour les appareils Kodak chargée couple, KAI-11002, KAF-39000, et les capteurs d&#39;image CMOS Micron sont 9 um, um 6,8 et 2,2 um, avec un actif FOV de 10 cm2, 18 cm2, et 24,4 mm2, respectivement. [1-2]. 2. Source de lumière Contrairement à la plupart des autres modalités d&#39;imagerie microscopique, LUCAS n&#39;a pas besoin d&#39;un laser, et donc même une simple diode électroluminescente (DEL) peut être utilisé pour l&#39;éclairage. Afin de permettre l&#39;éclairage de longueur d&#39;onde accordable, nous pouvons aussi utiliser un monochromateur avec une lampe au xénon (Cornerstone T260, Newport Corp) avec une fibre en silice fondue de qualité standard qui se compose d&#39;un faisceau de fibres (77 564, Newport) et sténopé de ~ 100 pm de diamètre situé à ~ 5-10 cm au-dessus de la surface du capteur. Cette configuration longueur d&#39;onde accordable l&#39;éclairage fournit une plateforme flexible où les signatures holographique des cellules peut être ajusté, et les signatures numériques hybrides peuvent être synthétisés afin d&#39;améliorer le rapport signal sur bruit pour une meilleure précision de la caractérisation et la spécificité. [3] Préparation d&#39;échantillons B. et imagerie Une preuve de concept de LUCAS basée sur la puce d&#39;imagerie sera démontrée en utilisant une solution hétérogène tel que décrit ci-dessous. Un protocole similaire pourrait être appliquée pour divers autres types de cellules [1-3]. 1. Dilution de sang total et de préparation de la solution hétérogène <ol><li> Pour commencer le processus, d&#39;obtenir un échantillon de sang total et les microbilles de polystyrène de différents diamètres (um 5, 10 et 20. Duke Scientific), et amener tous à température ambiante. </li><li> Ajouter 2 mL de milieu RPMI 1640 liquides classiques (Fisher Scientific) comme diluant dans un tube en polypropylène stériles de 5 ml. </li><li> Après une incubation de l&#39;échantillon de sang total pendant 30 minutes, une pipette en hausse de 10 uL d&#39;échantillon des érythrocytes sédimentés et le transférer dans la solution RPMI. </li><li> Ajouter un volume total de 20 microbilles en polystyrène uL dans du RPMI de dilution des érythrocytes, et agiter la solution de cellules par pipetage douceur ou en utilisant un agitateur magnétique avec un agitateur. </li><li> Placer un volume total de 5-15 uL solution hétérogène sur une lamelle et placer une seconde lamelle identique sur la solution en utilisant une pince. L&#39;échantillon doit être pris en sandwich entre les deux lamelles uniformément. </li><li> Puis, en utilisant un stylo vide (NT57-636, Edmund Optics) charge l&#39;échantillon dans la région active du réseau de capteurs pour l&#39;imagerie. </li></ol> 2. Coloration du sang total <ol><li> En commençant par la préparation d&#39;un mélange de 0,1% tamponné éosine Y et une solution diluée de méthylène Nouveau en utilisant la puissance type pur éosine Y (MW = 691,85, Acros Organics), sans zinc pur Nouveau Bleu de Méthylène colorant (MW = 347,90, Acros Organics), et oxalate de potassium (ACS Réactif 99,0%, Acros Organics), chaque solution est purifiée avec une taille de pores 0,45 pm Syringless filtre (Whatman) pour coloration aqueuse de globules blancs. </li><li> Ensuite, incorporer délicatement un réactif dissous coloration aqueuse à l&#39;échantillon de sang total à un rapport de volume de 1:1 dans un bécher en polypropylène en utilisant un agitateur magnétique pendant 2 minutes et laisser incuber pendant 10 minutes. </li><li> Transfert d&#39;une solution tachés de sang de 50 uL dans un tube en polypropylène 5 ml et diluer avec du RPMI 1640 solution liquide classique (Fisher Scientific) de 1,95 mL d&#39;acquérir un rapport de volume de 1:200. Puis, la dilution de vortex pendant 30 secondes. Un taux de dilution différents peuvent également être introduits à ce stade. </li><li> Pipeter une solution cellulaire teinté de 10-100 uL entre les lamelles ou dans un réservoir microfluidique. Puis, en utilisant un stylo vide (NT57-636, Edmund Optics), placer l&#39;échantillon sur la zone active de la matrice de capteurs. </li></ol> Les résultats représentatifs ve_content &quot;&gt; <img src="/files/ftp_upload/1650/1650Fig1tmb.jpg" alt="Figure 1" border="0" /> Figure 1: LUCAS permet un comptage précis et la différenciation des cellules numériques diverses et les micro-objets en fonction de leurs signatures 2D holographique sans utiliser de lentilles, de lasers ou d&#39;autres composants optiques encombrants. Caractéristique signatures LUCAS de divers micro-objets sont illustrés dans cette figure et sont comparées à une image au microscope classique obtenu avec un objectif 40X. S&#39;il vous plaît <a href="https://www.jove.com/files/ftp_upload/1650/1650Fig1.jpg">cliquez ici</a> pour voir une version agrandie de la figure 1. <img src="/files/ftp_upload/1650/1650Fig2.jpg" alt="Figure 2" /> Figure 2: (gauche) Image brute d&#39;un mélange hétérogène contenant des globules rouges, 10um, 5um et 3 particules um. (Droite) entièrement automatisée des résultats de caractérisation LUCAS pour le même champ de vue sont illustrés. Notez que l&#39;algorithme de décision est robuste dans la caractérisation des régions à haute densité ainsi que des particules de faible rapport signal à bruit tel que les perles 3um. <img src="/files/ftp_upload/1650/1650Fig3tmb.jpg" alt="Figure 3" border="0" /> Figure 3: L&#39;interface personnalisée LUCAS est illustré. Basé sur Java LUCAS logiciel permet des entrées pour différentes conditions expérimentales telles que la taille des pixels du capteur ou de la longueur d&#39;onde de la lumière. Sélection d&#39;un champ de vue spécifique sur l&#39;image peut également être faite et les modèles de cellules cibles peuvent être définies par l&#39;utilisateur de construire une bibliothèque de cellules statistiques ombre. L&#39;image acquise LUCAS peut alors être caractérisée basées sur ces données de formation (par exemple, la bibliothèque de cellules d&#39;ombre) et la marque (comptés) l&#39;image est affichée à l&#39;utilisateur. Statistiques des résultats de comptage sont également stockés dans un fichier XML pour une analyse ultérieure.

<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>

Nous avons montré que la plate-forme LUCAS peut dénombrer et identifier précisément micro-objects/cells différentes sur une puce basée sur leurs signatures holographique, et fournit un outil prometteur pour le point-of-care diagnostic médical et à haut débit de biologie cellulaire. Afin de traiter avec exactitude les tendances enregistrées holographique, nous avons implémenté un logiciel personnalisé développé décision LUCAS. Cet algorithme, qui utilise une base de données de diffraction statistiques im...

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

Microscopes optiques conventionnelles cellules d&#39;image par l&#39;utilisation de lentilles de l&#39;objectif qui travaillent ensemble avec les autres lentilles et des composants optiques. Bien qu&#39;assez efficace, cette approche classique a certaines limites de la miniaturisation de la plateforme d&#39;imagerie pour le rendre compatible avec l&#39;état avancé de l&#39;art en microfluidique. Dans ce rapport, nous présentons les détails expérimentaux d&#39;un concept d&#39;imagerie sur puce sans lentille appelée LUCAS (<strong<em&gt; L</em</strong&gt; Ensless<strong<em&gt; U</em</strong&gt; Ltra-large champ de vision<strong<em&gt; C</em</strongSurveillance ell&gt;<strong<em&gt; Une</em</strong&gt; Plate-forme basée sur rray<strong<em&gt; S</em</strong&gt; Hadow imagerie) qui ne nécessite pas d&#39;objectifs de microscope ou d&#39;autres composants optiques encombrants à l&#39;image d&#39;une solution de cellules hétérogènes sur un champ ultra-large de vue qui peut durer aussi grand que ~ 18 cm<sup&gt; 2</sup&gt;. En outre, contrairement microscopes classiques, LUCAS peut l&#39;image d&#39;une solution cellulaire hétérogène d&#39;intérêts sur une profondeur de champ d&#39;environ 5 mm sans la nécessité d&#39;un recentrage qui correspond à à ~ 9 ml volume d&#39;échantillon. Cette plate-forme d&#39;imagerie enregistre les ombres (c&#39;est à dire, sans lentille hologrammes numériques) de chaque cellule d&#39;intérêt dans son champ de vision, et automatisé de traitement numérique de ces ombres de cellule peut déterminer le type, le comte et la position relative des cellules dans la solution. Parce qu&#39;il ne nécessite aucun composant encombrant optique ou mécanique phases de numérisation il offre une plate-forme de manière significative miniaturisés qui dans le même temps réduit les coûts, ce qui est assez important pour en particulier point de service des outils de diagnostic. Par ailleurs, le débit d&#39;imagerie de cette plateforme est de plusieurs ordres de grandeur meilleure que microscopes optiques conventionnels, ce qui pourrait être extrêmement précieux pour les haut-débit expériences de biologie cellulaire.

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 sur puce d&#39;imagerie et de caractérisation des cellules est illustré. Cette approche sur-puce d&#39;imagerie cellulaire fournit un outil compact et rentable pour le diagnostic médical et applications à haut débit biologie cellulaire, ce qui rend particulièrement adapté aux milieux pauvres en ressources.

Lensless sur puce imagerie de cellules fournit un nouvel outil pour le haut débit de biologie cellulaire et le diagnostic médical

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 vues.  University of California, Los Angeles. Lensfree sur puce d'imagerie et de caractérisation des cellules est illustré. Cette approche sur-puce d'imagerie cellulaire fournit un outil compact et rentable pour le diagnostic médical et applications à haut débit biologie cellulaire, ce qui rend particulièrement adapté aux milieux pauvres en ressources.

Vidéo: 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.

Trachéotomie: Une méthode pour la transplantation de cellules souches pour le poumon

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 ...

Recherche

Biologie

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 dispositif microfluidique Dégradé génératrices de biologie cellulaire

 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.

Biochimique et High Throughput évaluation microscopique de la masse grasse dans<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-nucléaire de dépistage des anticorps Utilisation cellules 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.

À haut débit d&#39;analyse de l&#39;image de Sphéroïdes tumorales: Une application logicielle conviviale pour mesurer la taille de Sphéroïdes automatiquement et avec précision

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.

Haut-débit criblage de modulateurs chimique des gènes post-transcriptionnelle réglementées

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.

Profilage métabolique à haut débit pour les raffinements de modèles de microalgues

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.

Imagerie en direct à haut débit des microcolonies pour mesurer l’hétérogénéité de la croissance et de l’expression des gènes

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.

Une approche de dosage de comète à haut débit pour évaluer les dommages à l’ADN cellulaire

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.

Une plateforme à haut débit pour la culture et l’imagerie 3D d’organoïdes

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