Die Unterstützung des Australian Institute of Nuclear Science and Engineering anerkannt wird. TCK war der Empfänger der AINSE Auszeichnungen. Epigenomischen Medicine Lab wird von der National Health and Medical Research Council of Australia (566559) unterstützt. HR wird von einer australischen Post-Graduate-und BakerIDI hellen Funken Auszeichnungen unterstützt. Diese Arbeit wird durch die CRC for Biomedical Imaging Development Ltd (CRC-BID), gegründet und unterstützt unter der australischen Regierung Cooperative Research Centres-Programm finanziert. CO ist der Empfänger einer australischen Post-Graduate-Award und eine CRC-BID zusätzlichen Stipendium.

1. Cell Culture and Experimental Set-up <ol><li> Menschliche Keratinozyten werden als Monolayer in 75 cm 2 Zellkulturflaschen mit 15 ml Keratinozyten-SFM (K-SFM) gepflegt Medium (Gibco, serum-freiem Medium) mit L-Glutamin (2 mM), epidermal growth factor (5 ng ergänzt / mL), Rinder-Hypophysen-Extrakt (40 ug / ml) und 20 mg / mL Gentamicin. Die Zellen werden in einer befeuchteten 5% CO 2-Umgebung bei 37 ° C. </li><li> Die Zellen werden in 12 x 25 cm 2 Zellkulturflaschen mit 5 ml K-SFM Medium ausgesät. </li></ol> Einzel-Zell-Suspensionen werden durch Trypsinierung vorbereitet. Die Zellen werden gewaschen mit Phosphat-gepufferter Salzlösung resuspendiert und mit einer 0,05% Trypsin / EDTA-Lösung für 5-10 Minuten. Wenn die Zellen zu starten gerundet und ~ 30% sind freistehend, 3 Bände Dulbecco modifiziertem Eagle-Medium mit 10% fötalem Rinderserum hinzugefügt, um das Trypsin zu neutralisieren. Die Zellen werden durch Auf-und Abpipettieren (20 mal) abgelöst. Die Zellen werden gezählt unter Verwendung einer Zählkammer. Entsprechende Zellzahlen werden nach der Verdopplungszeit der Zell-Linie (ca. 20 Stunden für den menschlichen FEP-1811 Keratinozyten) ausgesät. Ziel ist es, ~ 90% Konfluenz (~ 10 6 Zellen pro Flasche) am Tag des Experiments zu erreichen. Ein Experiment, bestehend aus 12 Flaschen ist optimal für ein einzelnes klonogene Test (sechs unbestrahlten Kontrolle und sechs bestrahlten Kolben), die in etwa vier Stunden abgeschlossen werden kann. 2. Behandlung und Bestrahlung <ol><li> Treat-Zellen für eine angemessene Zeit mit einer entsprechenden Strahlung modifizierenden Verbindung und setzen Zellen gegenüber ionisierender Strahlung entweder γ-Strahlung oder Röntgenstrahlung. </li></ol> In der Regel sechs Flaschen dienen als Platierungseffizienz (unbehandelt) und Medikament nur Kontrollen. Die anderen sechs Flaschen werden bestrahlt. , Eine wasserlösliche natürliche Antioxidans Formulierung, für 1 Stunde in diesem Beispiel humaner Keratinozyten mit verschiedenen Konzentrationen von Cinnulin PF (; Integrität Nutraceuticals International, Spring Hill, TN, USA repräsentative Daten für 20 ug / mL ist unten dargestellt CPF) behandelt bei 37 ° C. Die Zellen werden mit 4 Gy mit einer 137 Cs-Quelle (; Nordion International, ON, Canada, 1,6 Gy / min Gammacell 1000 Elite Strahler) bestrahlt. 3. Plating <ol><li> Nach der Behandlung werden einzelne Zellsuspensionen erhalten, wie oben beschrieben. </li><li> Die Anzahl der Zellen in jeder Probe gezählt werden vorsichtig mit einer Zählkammer und so verdünnt, dass geeignete Zellzahlen in Petrischalen ausgesät werden (fünf Wiederholungen der einzelnen in 15 mm Schalen). Die Platierungseffizienz und / oder überlebenden Anteil sollte erwarten, wenn die Entscheidung die Anzahl der Zellen, um Samen pro Platte werden. Das Ziel ist es, einen Bereich zwischen 20 erreichen - 150 Kolonien. </li> Petrischalen werden in einem befeuchteten Kunststoff Klonen Feld angeordnet und inkubiert in einer 5% CO 2-Umgebung bei 37 ° C für Kolonie-Bildung. </ol> Die Inkubationszeit für Koloniebildung variiert von 1-3 Wochen für die verschiedenen Zelllinien, es wird angenommen, dass die Zeit entsprechen müssen mindestens sechs Zellteilungen. In diesem Beispiel erfordern die Kontrolle Gerichte für die menschliche Keratinozyten acht Tage, um ausreichend große Klone, bestehend aus 50 oder mehr Zellen zu bilden. 4. Fixierung und Färbung Colonies Führen Sie die folgenden Schritte in einer Abzugshaube. <ol><li> Entfernen Sie vorsichtig das Medium aus jeder der Platten durch Aspiration. </li><li> Wash jede Platte mit 5 ml 0,9% iger Kochsalzlösung. </li><li> Fix die Kolonien mit 5 ml 10% neutral gepuffertem Formalin-Lösung für 15-30 Minuten. </li><li> Stain mit 5 ml 0,01% (w / v) Kristallviolett in dH 2 O für 30-60 Minuten. </li><li> Wash überschüssige Kristallviolett mit dH 2 O und ermöglichen Geschirr nicht richtig getrocknet. </li></ol> 5. Colony Counting Stereomikroskop <ol><li> Kolonien mit mehr als 50 einzelnen Zellen werden gezählt mit einem Stereomikroskop. </li></ol> Digital Imaging und Zählen mit Imaging-Software <ol><li> Digitale Bilder von den Kolonien werden unter Verwendung einer Kamera oder einem Scanner </li><li> Kolonien gezählt mit bildgebenden Analyse-Software-Pakete wie unten beschrieben. </li></ol> Zellzählung mit ImageJ (Fidschi Version 1.44a) <ol><li> Öffnen Sie die Bilddatei in Fiji, gehen Sie zu File -&gt; Open. </li><li> &gt; Anpassen ...-&gt; Threshold - Bei Bedarf konvertieren Sie das Bild in 8-Bit-Format, um Bild zu gehen. </li><li> Passen Schwelle auf ein Niveau von unspezifischen Hintergrund zu reduzieren, so dass nur die Kolonien erkannt werden. </li><li> Graf Kolonien mit den folgenden: go to Process -&gt; Binary -&gt; Find Maxima. </li></ol> Für dieses Bildformat, kann Rausch Toleranz auf 0 gesetzt werden. Stellen Sie sicher, dass hellem Hintergrund Option aktiviert ist und eine Vorschau der erkannt Maxima zu prüfen, ob alle Zellkolonien wurden korrekt registriert.

<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>

In diesem Beispiel menschliche FEP-1811 Keratinozyten wurden mit verschiedenen Konzentrationen von bis zu 100 pg / mL CPF behandelt; Daten werden für 20 ug / mL CPF für 1 Stunde bei 37 ° C gezeigt Nach der Behandlung wurden die Zellen mit 4 Gy mit einer 137 Cs-Quelle (; Nordion International, ON, Canada, 1,6 Gy / min Gammacell 1000 Elite Strahler) bestrahlt. Für die Kontrolle unbehandelt und Drogen nur Behandlungen 100 Zellen wurden in jeder Petrischale und 1000 Zellen pro Schale wurden die bestrahlten Pr...

<!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>Keratinocyte-serum free medium</td>
<td>Growth medium</td>
<td>Invitrogen</td>
<td>17005042</td>
<td>Supplemented with 2mM L-glutamine, 20&#x03BC;g/ml gentamicin, epidermal growth factor, bovine pituitary extract.
</td>
</tr>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15400-054</td>
<td>0.05% trypsin / EDTA to detach adherent cells; 10 x stock diluted in PBS w/o Ca2+/Mg2+.</td>
</tr>
<tr>
<td>Dulbecco&#x2019;s modified essential medium</td>
<td>Growth medium</td>
<td>Invitrogen</td>
<td>11885-084</td>
<td>Supplemented with 10% FBS and 20&mu;g/ml gentamicin; used to neutralise trypsin/EDTA.</td>
</tr>
<tr>
<td>0.09% Saline</td>
<td>Solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td> Used to wash colonies.</td>
</tr>
<tr>
<td>10% Neutral buffered formalin solution</td>
<td>Solution</td>
<td>Sigma-Aldrich</td>
<td>HT501128</td>
<td>~4% formaldehyde; used to fix colonies.</td>
</tr>
<tr>
<td>Crystal violet</td>
<td>Powder</td>
<td>SPI-Chem</td>
<td>02577-MB</td>
<td>0.01% crystal violet solution, prepared in dH2O, used to stain colonies.</td>
</tr>
<tr>
<td>Gammacell 1000 elite irradiator</td>
<td>&nbsp;</td>
<td>Nordion International Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Petri dishes</td>
<td>60 x 15 mm</td>
<td>Falcon</td>
<td>353002</td>
<td>&nbsp;</td>
<tr>
<td>Haemocytometer</td>
<td>&nbsp;</td>
<td>Hawksley; Medical and Laboratory Equipment</td>
<td>AC1000</td>
<td>&nbsp;</td>
<tr>
<td>Cloning box</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Plastic box with holes punched at opposite sides of lid; for storing petri dishes during prolonged incubation for colony formation.</td>
</tr>
<tr>
<td>Stereomicroscope</td>
<td>Type 102</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>Used to count colonies consisting of &gt;50 cells.</td>
</tr>		</tbody>
		</table>
		</div>

clonogenic assay: adherent cells

Die klonogenen (oder koloniebildende)-Assay wurde für mehr als 50 Jahren besteht; die ursprüngliche Papier beschreibt die Technik wurde in 1956 1 veröffentlicht. Abgesehen von der Dokumentation der Methode erzielte das erste Meilenstein-Studie die erste Strahlung Dosis-Wirkungs-Kurve für X-ray bestrahlt Säugetieren (HeLa) Zellen in Kultur 1. Grundsätzlich ermöglicht die klonogene Test eine Bewertung der Unterschiede in der reproduktiven Lebensfähigkeit (Kapazität der Zellen auf die Nachkommen zu produzieren, dh eine einzelne Zelle, um eine Kolonie von 50 oder mehr Zellen zu bilden) zwischen Kontrolle unbehandelte Zellen und Zellen, die verschiedene Behandlungen wie Belichtung unterzogen haben auf ionisierende Strahlung, verschiedene chemische Verbindungen (zB Zytostatika) oder in anderen Fällen genetische Manipulation. Der Test hat sich die am weitesten akzeptierte Methode in der Strahlenbiologie und wurde vielfach zur Beurteilung der Strahlenempfindlichkeit verschiedener Zelllinien verwendet. Weiterhin ist die klonogenen allgemein zur Überwachung der Wirksamkeit von Strahlung modifizierende Verbindungen und zur Bestimmung der Wirkung von Zytostatika und anderen Anti-Krebs-Therapeutika auf Basis der Kolonie-bildende Fähigkeit, in verschiedene Zelllinien verwendet. Ein typisches klonogenen Überleben Experiment mit adhärenten Zellen Zeilen umfasst drei Komponenten, 1) Behandlung der Zellrasen in Zellkulturflaschen, 2) Vorbereitung der einzelnen Zellsuspensionen und Beschichtung eine entsprechende Anzahl von Zellen in Petrischalen und 3) Fixierung und Färbung Kolonien nach einer entsprechenden Inkubationszeit könnte, die von 1-3 Wochen liegen, je nach Zelllinie. Hier zeigen wir das allgemeine Verfahren für die Durchführung der klonogene Test mit adhärenten Zelllinien mit dem Einsatz von einer immortalisierten humanen Keratinozyten-Zelllinie (FEP-1811) 2. Auch sind unsere Ziele zu gemeinsamen Merkmale der klonogenen Assays einschließlich Berechnung der Platierungseffizienz und Überleben Fraktionen nach Exposition von Zellen gegenüber Strahlung zu beschreiben, und Änderungen der Strahlung-Response mit dem Einsatz von ein natürliches Antioxidans Formulierung zu veranschaulichen.

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

Clonogenic Assay: Adherent Cells

Die Anwendbarkeit des klonogene Test zur Bewertung der reproduktiven Überlebensfähigkeit seit mehr als 50 Jahren etabliert. Hier zeigen wir das allgemeine Verfahren für die Durchführung der klonogene Test mit adhärenten Zellen.

Klonogene Test: adhärenter Zellen

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

47.9K Aufrufe.  The Alfred Medical Research and Education Precinct. Die Anwendbarkeit des klonogene Test zur Bewertung der reproduktiven Überlebensfähigkeit seit mehr als 50 Jahren etabliert. Hier zeigen wir das allgemeine Verfahren für die Durchführung der klonogene Test mit adhärenten Zellen.

Serie: 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.

Herstellung und Betrieb eines Oxygen Insert für adhärente Zellkulturen

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

Forschung

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.

In situ subzelluläre Fraktionierung von adhärenten und nicht adhärenten Säugetierzellen

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.

Ein Matrigel-Based Rohr Formation Assay zur vaskulären Aktivität von Tumorzellen Beurteilen

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.

Isolation of Primary Maus Trophoblastzellen und Trophoblastinvasion 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 ...

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.

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.

Identifizierung von Zielen für Menschenrechte microRNAs mit dem LightSwitch Luciferase Assay System mit 3&#39;UTR-Reporter-Konstrukte und eine microRNA Mimic in adhärenter Zellen

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.

Induktion und Analyse von epithelialen zu mesenchymalen 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.

Ein Funktionstest für Gap Junction-Prüfung; Elektroporation von adhärenten Zellen auf Indium-Zinn-Oxid

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.

Nephrotoxin Mikroinjektion in Zebrabärbling akuten Nierenschädigung zu Modell

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.

Eine grafische Benutzeroberfläche für die softwaregestützte Verfolgung der Proteinkonzentration in dynamischen zellulären Vorsprüngen

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.

Objektiv-kostenlose Videomikroskopie für die Dynamik und Quantitative Analyse der adhärente Zellkultur

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