Le soutien de l&#39;Institut australien des sciences et génie nucléaires est reconnu. TCK a été récipiendaire du prix Ainse. Médecine de laboratoire épigénomique est soutenu par la National Health and Medical Research Council of Australia (566559). HR est pris en charge par un Australien d&#39;études supérieures et des prix BakerIDI étincelles lumineuses. Ce travail est financé par le CRC d&#39;Imagerie Biomédicale Development Ltd (CRC-BID), mis en place et soutenus dans le cadre de la recherche du gouvernement australien centres du programme de coopération. Le CO est le destinataire d&#39;un australien d&#39;études supérieures prix et une bourse supplémentaire CRC-BID.

1. Culture cellulaire et expérimental <ol><li> Kératinocytes humains sont maintenues sous forme de monocouches dans 75 cm 2 flacons de culture tissulaire contenant 15 ml de kératinocytes-SFM (K-SFM) (GIBCO, un milieu sans sérum) additionné de L-glutamine (2 mM), facteur de croissance épidermique (5 ng / mL), extrait pituitaire bovin (40 pg / ml) et 20 mg / mL de gentamicine. Les cellules sont cultivées dans une atmosphère humidifiée 2 5% l&#39;environnement à 37 ° C. </li><li> Les cellules sont ensemencées en 12 x 25 cm 2 flacons de culture tissulaire contenant 5 ml de milieu K-SFM. </li></ol> Suspensions monocellulaires sont préparés par traitement à la trypsine. Les cellules sont lavées avec du PBS et incubées avec un 0,05% de trypsine / EDTA solution pendant 5-10 minutes. Lorsque les cellules commencent à s&#39;arrondir et ~ 30% sont détachés, 3 volumes de Eagle modifié par Dulbecco milieu contenant 10% de sérum fœtal bovin est ajouté pour neutraliser la trypsine. Les cellules sont détachées par aspirationet vers le bas (20 fois). Les cellules sont comptées à l&#39;aide d&#39;un hémocytomètre. Le nombre de cellules appropriées sont ensemencées en fonction du temps de doublement de la ligne de cellules (environ 20 heures pour l&#39;homme FEP-1811 kératinocytes). L&#39;objectif est de parvenir à une confluence de 90% (~ 10 6 cellules par flacon) le jour de l&#39;expérience. Une expérience composé de 12 flacons est optimal pour un seul essai clonogénique (six témoins non irradiés et irradiés six flacons), qui peut être complété en environ quatre heures. 2. Traitement et irradiation <ol><li> Traiter les cellules pendant un temps approprié avec un rayonnement pertinentes composé modifiant et exposer les cellules aux rayonnements ionisants soit γ-irradiation ou rayons-X. </li></ol> Typiquement six flacons servir efficacité d&#39;étalement (non traité) et les commandes de médicaments seulement. Les six autres flacons sont irradiées. Dans cet exemple, les kératinocytes humains sonttraitées avec différentes concentrations de Cinnulin PF (PCF; nutraceutiques intégrité internationales, Spring Hill, Tennessee, États-Unis, des données représentatives de 20 pg / mL est indiqué ci-dessous), une formulation soluble dans l&#39;eau antioxydant naturel, pendant 1 heure à 37 ° C. Les cellules sont irradiées avec 4 Gy en utilisant une source de 137 Cs (Gammacell 1000 irradiateur Elite; Nordion International, ON, Canada, 1,6 Gy / min). 3. Placage <ol><li> Après le traitement, les suspensions cellulaires simples sont obtenus comme décrit précédemment. </li><li> Le nombre de cellules dans chaque échantillon sont comptés soigneusement à l&#39;aide d&#39;un hémocytomètre et dilué de sorte que le nombre de cellules appropriées sont ensemencées dans des boîtes de Pétri (cinq répétitions de chaque en 15 plats mm). L&#39;efficacité d&#39;étalement et / ou fraction survivante devrait être prévu au moment de décider du nombre de cellules de graines par plaque. L&#39;objectif est de parvenir à une fourchette comprise entre 20 à 150 colonies. </li></ol> Des boîtes de Pétri sont disposés dans un p humidifiéboîte de clonage lastic et incubées dans un environnement de CO 2 5% à 37 ° C pour la formation de colonies. Le temps d&#39;incubation pour la formation de colonies varie de 1-3 semaines pour les lignées cellulaires différentes, il est admis que le temps doit être équivalente à au moins six divisions cellulaires. Dans cet exemple, les plats de contrôle des kératinocytes humains besoin de huit jours pour former des clones suffisamment nombreuse composée de 50 cellules ou plus. 4. Fixation et coloration des colonies Effectuez les étapes suivantes dans une hotte. <ol><li> Retirer doucement le support de chacune des plaques par aspiration. </li><li> Laver chaque plaque avec 5 mL de solution saline à 0,9%. </li><li> Fixer les colonies avec 5 ml solution à 10% formol neutre tamponné pendant 15-30 minutes. </li><li> Coloration à 5 ml 0,01% (p / v) de cristal violet à dH 2 O pendant 30-60 minutes. </li><li> Laver cristal violet excès avec dH 2 O et permettre plats à sécher. </li></ol>5. Le comptage de colonies Stéréomicroscope <ol><li> Des colonies contenant plus de 50 cellules individuelles sont comptées à l&#39;aide d&#39;un stéréomicroscope. </li></ol> D&#39;imagerie numérique et de comptage en utilisant un logiciel d&#39;imagerie <ol><li> Les images numériques des colonies sont obtenues à l&#39;aide d&#39;un appareil photo ou le périphérique de numérisation </li><li> Les colonies sont comptées à l&#39;aide d&#39;imagerie paquets logiciels d&#39;analyse comme décrit ci-dessous. </li></ol> Le comptage cellulaire en utilisant ImageJ (Fidji Version 1.44a) <ol><li> Ouvrez le fichier image à Fidji, allez dans Fichier -&gt; Ouvrir. </li><li> Si nécessaire convertir l&#39;image en format 8 bits, allez dans Image -&gt; Réglages ... -&gt; Seuil. </li><li> Ajuster le seuil de réduire les niveaux de fond non spécifique de sorte que seules les colonies sont détectées. </li><li> Compter les colonies en utilisant le suivant: aller au Processus -&gt; Binaire -&gt; Trouver maxima. </li></ol> Pour ce format d&#39;image, la tolérance au bruit peut être réglé sur 0. Assurez-vous que l&#39;option est cochée fond clair etun aperçu de la détection des maxima de vérifier que toutes les colonies de cellules ont été correctement enregistré.

<ol>
	<li>Puck, T. T., Marcus, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+of+x-rays+on+mammalian+cells.">Action of x-rays on mammalian cells.</a> J Exp Med. 103, 653-666 (1956).</li><li>Hurlin, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+human+papillomavirus+type+18-immortalized+human+keratinocytes+to+a+malignant+phenotype.">Progression of human papillomavirus type 18-immortalized human keratinocytes to a malignant phenotype.</a> Proc Natl Acad Sci U S A. 88, 570-574 (1991).</li></ol>

Dans cet exemple, l&#39;homme FEP-1811 kératinocytes ont été traitées avec différentes concentrations allant jusqu&#39;à 100 mg / mL PCF; données sont présentées pour 20 pg / ml PCF pendant 1 heure à 37 ° C. Cellules de traitement suivants ont été irradiés avec 4 Gy en utilisant une source de 137 Cs (Gammacell 1000 irradiateur Elite; Nordion International, ON, Canada, 1,6 Gy / min). Pour le contrôle non traités et de drogues traitements à seulement 100 cellules ont été étalées dans chaque...

<!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> Nom du produit </th><th> Type </th><th> Entreprise </th><th> Numéro de catalogue </th><th> Commenter </th></tr></thead><tbody><tr><td> Kératinocytes milieu sans sérum </td><td> Le milieu de croissance </td><td> Invitrogen </td><td> 17005042 </td><td> Supplémenté avec 2 mM de L-glutamine, la gentamicine 20μg/ml, du facteur de croissance épidermique, extrait d&#39;hypophyse bovine. </td></tr><tr><td> Trypsine-EDTA </td><td></td><td> Invitrogen </td><td> 15400-054 </td><td> 0,05% de trypsine / EDTA pour détacher les cellules adhérentes; 10 en stock x dilué dans du PBS w / o Ca 2 + / Mg 2 +. </td></tr><tr><td> Dulbecco milieu modifié essentiel </td><td> Le milieu de croissance </td><td> Invitrogen </td><td> 11885-084 </td><td> Supplémenté avec 10% de FBS et 20μg/mlgentamicine; utilisé pour neutraliser la trypsine / EDTA. </td></tr><tr><td> Saline 0,09% </td><td> Solution </td><td></td><td></td><td> Utilisé pour laver les colonies. </td></tr><tr><td> 10% Neutre solution de formaline tamponnée </td><td> Solution </td><td> Sigma-Aldrich </td><td> HT501128 </td><td> ~ Formaldéhyde à 4%; utilisé pour fixer les colonies. </td></tr><tr><td> Cristal violet </td><td> Poudre </td><td> SPI-Chem </td><td> 02577-MB </td><td> 0,01% solution de cristal violet, établi en dH 2 O, utilisé pour colorer les colonies. </td></tr><tr><td> Gammacell 1000 irradiateur élite </td><td></td><td> Nordion International Inc </td><td></td><td></td><tr><td> Des boîtes de Pétri </td><td> 60 x 15 mm </td><td> Faucon </td><td> 353002 </td><td></td><tr><td> Hémocytomètre </td><td></td><td> Hawksley; médical et de laboratoire </td><td> AC1000 </td><td></td><tr><td> Boîte de clonage </td><td></td><td></td><td></td><td> Boîte en plastique avec des trous de poing au côté opposés de couvercle; pour stocker des boîtes de Pétri pendant l&#39;incubation prolongée pour la formation de colonies. </td></tr><tr><td> Stéréomicroscope </td><td> Type 102 </td><td> Nikon </td><td></td><td> Utilisé pour compter les colonies composées de 50 cellules&gt;. </td></tr></tbody></table></div>

clonogenic assay: adherent cells

Le clonogénique (ou formant colonie) test a été mis en place depuis plus de 50 ans, le document original décrivant la technique a été publié en 1956 1. En dehors de la documentation du procédé, l&#39;étude de référence initiale générée de la première courbe de réponse en rayonnement dose de rayons X irradiés mammifères (HeLa) des cellules en culture 1. Fondamentalement, le test clonogénique permet d&#39;évaluer les différences de viabilité de reproduction (capacité des cellules à produire une descendance; c&#39;est à dire une seule cellule pour former une colonie de 50 cellules ou plus) entre les cellules témoins non traitées et les cellules qui ont subi divers traitements tels que l&#39;exposition aux rayonnements ionisants, divers composés chimiques (par exemple des agents cytotoxiques) ou dans d&#39;autres cas de manipulation génétique. Le dosage est devenue la technique la plus largement acceptée en radiobiologie et a été largement utilisé pour évaluer la sensibilité aux rayonnements de différentes lignées cellulaires. En outre, le test clonogénique est couramment utilisé pour monitoring l&#39;efficacité des composés rayonnement modification et pour déterminer les effets des agents cytotoxiques et d&#39;autres thérapies anti-cancéreuses sur la capacité de formation de colonies, dans différentes lignées cellulaires. Une expérience de survie clonogénique typique utilisant des lignées de cellules adhérentes comporte trois composantes distinctes, 1) le traitement de la monocouche de cellules dans des flacons de culture tissulaire, 2) préparation des suspensions cellulaires simples et placage d&#39;un nombre approprié de cellules dans des boîtes de Pétri et 3) fixation et coloration des colonies après une période d&#39;incubation approprié, qui peut varier de 1-3 semaines, en fonction de la lignée cellulaire. Ici nous montrent le mode opératoire général pour la réalisation du test clonogénique avec des lignées de cellules adhérentes à l&#39;aide d&#39;une ligne cellulaire de kératinocytes humains immortalisés (FEP-1811) 2. En outre, nos objectifs sont de décrire les caractéristiques communes des essais clonogéniques y compris le calcul de l&#39;efficacité d&#39;étalement et des fractions de survie après exposition des cellules aux radiations, et d&#39;illustrer la modification desrayonnement de réponse à l&#39;utilisation d&#39;une formulation anti-oxydant naturel.

Watch this Scientific Journal Video about Clonogenic Assay: Adherent Cells at JoVE.com

Clonogenic Assay: Adherent Cells

L&#39;applicabilité du test clonogénique pour évaluer la viabilité de la reproduction a été mis en place depuis plus de 50 ans. Ici, nous démontrons la procédure générale pour réaliser le dosage clonogénique de cellules adhérentes.

Test clonogénique: cellules adhérentes

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Research

JoVE Journal

Biology

47.8K vues.  The Alfred Medical Research and Education Precinct. L'applicabilité du test clonogénique pour évaluer la viabilité de la reproduction a été mis en place depuis plus de 50 ans. Ici, nous démontrons la procédure générale pour réaliser le dosage clonogénique de cellules adhérentes.

Vidéo: Clonogenic Assay: Adherent Cells

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

Fabrication et fonctionnement d&#39;un insert d&#39;oxygène pour les adhérents des cultures cellulaires

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...

Recherche

Biologie

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

En fractionnement subcellulaire situ des adhérentes et non-adhérents des cellules de mammifères

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Un test basé sur Matrigel Tube Formation d&#39;évaluer l&#39;activité Vasculogenic de cellules tumorales

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

Isolement des cellules trophoblastiques primaires de souris et le test invasion trophoblastique

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

L&#39;identification des cibles de microARN humaine avec le système LightSwitch Luciferase Assay utilisant 3&#39;UTR-reporter Construit et un microARN dans les cellules adhérentes Mimic

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induction et analyse des épithéliale à mésenchymateuses transition

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

Une analyse fonctionnelle pour les jonctions lacunaires examen; Électroporation des cellules adhérentes sur Indium Tin Oxide-

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Néphrotoxine microinjection dans Zebrafish au Modèle lésion rénale aiguë

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Une interface utilisateur graphique pour le suivi assisté par logiciel de la concentration de protéines dans les protrusions cellulaires dynamiques

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

La microscopie vidéo sans lentille pour la dynamique et l’analyse Quantitative de la Culture de cellules adhérentes

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...