Teil I: Die Strukturierung der Elektroden <ol><li> Saubere und dehydrierte ITO beschichtete Glasträger wurden mit positiven Fotolack beschichteten </li><li> Die Oberfläche wurde bei 100 ° C gebacken, um Lösungsmittel aus dem Lack entfernen </li><li> Nach dem Backen wurde die Oberfläche mit UV-Licht durch eine Photomaske mit dem Canon Mask Aligner ausgesetzt </li><li> Die belichteten Bereiche wurden in einer Entwicklerlösung entfernt </li><li> Die Oberfläche war hart gebacken, um alle verbleibenden Entwickler Lösung zu entfernen </li><li> Die ITO Regionen, die nicht durch Fotolack Strukturierung geschützt wurden entfernt in einem sauren Ätzmittel geätzt </li><li> Verbleibende Photoresist wurde im Ultraschallbad in Aceton auf die ITO-Elektroden Form entfernt </li></ol> Teil II: Oberflächenmodifizierung <ol><li> Geändert Elektroden sind in einer Plasmakammer gereinigt und modifiziert mit 2% PEG Silan in Toluol. Die Inkubation wird für 2 Stunden durchgeführt, gefolgt von 2 Stunden backen. </li></ol> Hinweis: Dieser Vorgang erfolgt in einem Handschuh-Tasche mit Stickstoff gefüllt, um Anwesenheit von Feuchtigkeit zu vermeiden durchgeführt, wie PEG Silan reaktiv gegenüber ist. Teil III: Elektrochemie <ol><li> Stahldrähte sind Elektroden angebracht. </li><li> Die PEG Silan modifizierte Elektroden sind in einer elektrochemischen Zelle in der Lage, elektrochemischen Experimente durchzuführen. </li><li> PEG Silan aus miteinander verbundenen Regionen abgezogen wird mit einem Drei-Elektroden-System in PBS. </li></ol> Teil IV: Cell Musterung <ol><li> Fibroblasten sind mit dem vor kurzem ausgezogen Substraten inkubiert. Nach Inkubation dieser Zellen, die PEG Silan beraubt Regionen messen, aber die Zellen sonst nirgends binden an der Oberfläche. </li><li> Gemusterte Zellen werden in frischem Medium inkubiert und visualisiert mit Hilfe eines Mikroskops. </li></ol>

<ol>
	<li>Lee, J. Y., Jones, C., Zern, M. A., Revzin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Local+Tissue-Specific+Gene+Expression+in+Cellular+Micropatterns.">Analysis of Local Tissue-Specific Gene Expression in Cellular Micropatterns.</a> Analytical Chemistry. 78, 8305-8312 (2006).</li></ol>

In diesem Video haben wir die Zelle Musterung auf optisch transparenten Indium-Zinn-Oxid-Elektroden nachgewiesen. Nach Herstellung der ITO-Elektroden mittels Photolithographie, wurden sie mit einem Zell-resistive Monoschicht von PEG Silan modifiziert. Diese Monoschicht wurde desorbiert mit Elektrochemie Wechsel der Oberfläche von Zellen-resistive, Zell-Kleber. Strukturierung von 3T3 Maus-Fibroblasten hat in seinem Video gezeigt worden. Wir haben auch Strukturierung Hepatozyten, primären Zellen und Sternzellen mit der gleichen Technik. Dar...

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<td>Indium Tin Oxide</td>
<td>Other</td>
<td>Delta Technologies</td>
<td>CB 40IN</td>
<td>&nbsp;</td>
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<td>PEG silane</td>
<td>Reagent</td>
<td>Gelest Inc.</td>
<td>SIM6492.66</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>320331</td>
<td>&nbsp;</td>
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<td>Nitric Acid</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>258121</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>459836</td>
<td>&nbsp;</td>
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<td>Acetone</td>
<td>Reagent</td>
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<td>650501</td>
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<td>C3867</td>
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<td>3T3 murine Fibroblasts</td>
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patterning cells on optically transparent indium tin oxide electrodes

Die Fähigkeit, präzise räumliche und zeitliche Kontrolle über Zell-Oberflächen-Wechselwirkungen Übung ist eine wichtige Voraussetzung für die Montage von mehrzelligen Konstrukte, die als in-vitro-Mimetika von nativen Geweben. In dieser Studie wurden Photolithographie und Nassätzen Techniken verwendet, um einzeln adressierbare Indium-Zinn-Oxid (ITO)-Elektroden auf Glassubstraten zu fertigen. Die Glassubstrate mit ITO-Mikroelektroden wurden mit Poly (ethylenglycol) (PEG) Silan, damit sie Protein-und Zell-resistive geändert. Präsenz von isolierenden PEG-Moleküle auf der Elektrodenoberfläche wurde durch Cyclovoltammetrie beschäftigt Kaliumferricyanid als Redox-Reporter-Molekül nachgewiesen. Wichtig ist, dass die Anwendung der reduktiven Potential Desorption der PEG-Schicht verursacht, was zu einer Regeneration des leitenden Elektrode Oberfläche und das Aussehen der typischen Ferricyanid Redoxpeaks. Die Anwendung der reduktiven Potential entsprach auch der ITO-Elektrode Eigenschaften Umschalten von Zell-Non-Klebstoff auf Zell-Kleber. Elektrochemisches Abtragen von PEG-Silan-Schicht aus ITO-Mikroelektroden für die Zelladhäsion erlaubt, in einem räumlich definierten Weise erfolgen, mit zellulären Mustern entsprechenden eng an die Elektrode Muster. Mikrostrukturierung von mehreren Zelltypen wurde auf diesen Substraten demonstriert. In Zukunft wird die Kontrolle der biointerfacial Eigenschaften durch diese Methode leisten kann, um zelluläre Mikroumgebung durch die Anordnung von drei oder mehr Zelltypen Ingenieur in eine präzise geometrische Konfiguration auf einem optisch transparenten Substrat.

Watch this Scientific Journal Video about Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes at JoVE.com

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

Non-Fouling-PEG Silan-Monoschicht wurde von einzeln adressierbaren ITO-Elektroden auf Glas durch Anlegen einer reduktiven Potential desorbiert. Elektrochemisches Abtragen von PEG-Silan-Schicht aus ITO-Mikroelektroden für die Zelladhäsion erlaubt, in einem räumlich definierten Weise erfolgen, mit zellulären Mustern entsprechenden eng an die Elektrode Muster.

Patterning Cells auf optisch transparenten Indium-Zinn-Oxid-Elektroden

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular ...

patterning-cells-on-optically-transparent-indium-tin-oxide-electrodes

Research

JoVE Journal

Biology

11.7K Aufrufe.  University of California, Davis. Non-Fouling-PEG Silan-Monoschicht wurde von einzeln adressierbaren ITO-Elektroden auf Glas durch Anlegen einer reduktiven Potential desorbiert. Elektrochemisches Abtragen von PEG-Silan-Schicht aus ITO-Mikroelektroden für die Zelladhäsion erlaubt, in einem räumlich definierten Weise erfolgen, mit zellulären Mustern entsprechenden eng an die Elektrode Muster.

Serie: Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

Strukturierung von embryonalen Stammzellen mit dem Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Forschung

Biologie

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades. Today, such pipettes are made from glass capillaries using a programmable puller. Such instruments heat the capillary using either a metal filament or a laser and draw out the glass using gravity, a motor or both. Pipettes for patch-clamp recording are formed using only heat and gravity, while sharp electrodes for intracellular recording use a combination of heat, gravity, and a motor. The procedure used to make intracellular recording pipettes is similar to that used to make injection needles for a variety of applications, including cRNA injection into Xenopus oocytes. In general, capillary glass &lt;1.2 mm in diameter is used to make pipettes for patch clamp recording, while narrower glass is used for intracellular recording (outer diameter = 1.0 mm). For each tool, the puller is programmed slightly differently. This video shows how to make both kinds of recording pipettes using pre-established puller programs.

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

Machen Patch-Pipetten und Sharp Elektroden mit einem programmierbaren Puller

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades.  Today, such pipettes are made from glass capillaries ...

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

Herstellung von amperometrische Elektroden

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

We present a procedure for forming a poly(ethylene glycol) (PEG) trimethoxysilane self-assembled monolayer (SAM) on a silicon substrate with gold microelectrodes. The PEG-SAM is formed in a single assembly step and prevents biofouling on silicon and gold surfaces. The SAM is used to coat microelectrodes patterned with standard, positive-tone lithography. Using the microtubule as an example, we apply a DC voltage to induce electrophoretic migration to the SAM-coated electrode in a reversible manner. A flow chamber is used for imaging the electrophoretic migration and microtubule patterning in situ using epifluorescence microscopy. This method is generally applicable to biomolecule patterning, as it employs electrophoresis to immobilize target molecules and thus does not require specific molecular interactions. Further, it avoids problems encountered when attempting to pattern the SAM molecules directly using lithographic techniques. The compatibility with electron beam lithography allows this method to be used to pattern biomolecules at the nanoscale.

We present a procedure for forming a poly(ethylene glycol) self-assembled monolayer (PEG-SAM) on a silicon substrate with gold microelectrodes. The PEG-SAM is formed in a single step and prevents biofouling on silicon and gold surfaces. Electrophoresis is then used for patterning biomolecules down to the nanoscale.

Antifouling Selbstorganisierte Monoschichten auf Mikroelektroden für die Strukturierung von Biomolekülen

We present a procedure for forming a poly(ethylene glycol) (PEG) trimethoxysilane self-assembled monolayer (SAM) on a silicon substrate with gold ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Genetic Studies of Human DNA-Reparatur-Proteinen unter Verwendung von Hefe als Modellsystem

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Die Analyse der Funktion von kleinen GTPasen durch Mikroinjektion von Plasmiden in polarisierten Epithelzellen

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

Nachweis von Stickstoffmonoxid und Superoxid-Radikalanion von paramagnetischer Resonanzspektroskopie von Zellen mit Spinfallen

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

Visualisierung der mitochondrialen Atmungskette Funktion mit Cytochrom C Oxidase / Succinat-Dehydrogenase (COX / SDH) Double-Kennzeichnung Histochemie

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

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. ...