Die Autoren bedanken sich bei Stem Cell Technology und Shahid Ghazi Tabriz Unternehmen bzw. anerkennen, für die Bereitstellung von DMEM ohne Phenolrot und steriles Wasser zur Injektion als Geschenk.

MCF-7 Aussaat in 96-Well-Platte: MCF-7-Zellen wurden in 25 t-Kolben auf ein Medium mit Dulbecco modifiziertem Eagle-Medium (DMEM), 10% FBS, 100 U / ml Penicillin und 100 ug / ml Streptomycin bei 37 ° C mit 5% CO 2, 95% Luft und Feuchtigkeit abgeschlossen. Einmal erreicht ~ 90% Konfluenz wurden sie getrennt mit 0,05% Trypsin / EDTA und gezählt mittels Trypanblau und Zählkammer. Diese Zellen wurden dann in einer Konzentration von 4 × 10 4 Zellen / cm 2 resuspendiert und auf 96-Well-Platte (dh 250 ul / well) von einem 8-Kanal-Pipette. Für Untergrundabsorption wurden einige Brunnen blieb zellfreien, dh als leere Kontrolle. Die Behandlung von Zellen mit unterschiedlichen nanopolyplexes: Bei 40-50% Konfluenz (48 Stunden nach der Aussaat) wurden die kultivierten Zellen mit nanostrukturierten starburst Polyamidoamin Dendrimere (dh Superfect ® und Polyfect ®) und ein neuer Test in Polymer nach der Transfektion Anweisung vom Lieferanten zur Verfügung gestellt behandelt. Die Zellen wurden auch mit EGFR behandelt und verschlüsselt antisense allein, und mit den drei verschiedenen nanopolyplexes dieser beiden Oligonukleotide und mit Polymeren (n = 4). Vier Brunnen wurden blieben als Kontrolle unbehandelt. Nach 4 Stunden der Behandlung Medien wurden entfernt und mit frischem Medien. MTT-Assay für die Bewertung die Lebensfähigkeit der Zellen: MTT-Assay wurde 24 Stunden nach der Transfektion durchgeführt. Zu diesem Zweck wurde MTT-Lösung bei 1mg/ml in PBS hergestellt und durch einen 0,2 um Filter filtriert. Dann wurden 50 ul MTT plus 200 ul DMEM ohne Phenolrot in jede Vertiefung gegeben, mit Ausnahme der zellfreien leeren Brunnen. Die Zellen wurden für 4 Stunden bei 37 ° C mit 5% CO 2, 95% Luft und vollständige Luftfeuchtigkeit. Nach 4 Stunden wurde der MTT-Lösung entfernt und ersetzt mit 200 ul DMSO und 25 &amp;mgr; l Sorenson Glycin-Puffer (0,1 M Glycin, NaCl 0,1 M, pH: 10,5 mit 0,1 NaOH). Die Platte wurde für weitere 5 min bei Raumtemperatur inkubiert und die optische Dichte (OD) der Brunnen wurde mit einem Platten-Lesegerät bei einem Test Wellenlänge von 570 nm und einer Referenz-Wellenlänge von 630 nm.

<ol>
	<li>Plumb, J. A., Milroy, R., Kaye, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+pH+Dependence+of+3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium+Bromide-Formazan+Absorption+on+Chemosensitivity+Determined+by+a+Novel+Tetrazolium-based+Assay.">Effects of the pH Dependence of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide-Formazan Absorption on Chemosensitivity Determined by a Novel Tetrazolium-based Assay.</a> Cancer Research. 49, 4435-4435 (1989).</li><li>Mosmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Colorimetric+Assay+for+Cellular+Growth+and+Survival:+Application+to+Proliferation+and+Cytotoxicity+Assays.">Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays.</a> Journal of Lmmunological Methods. 65, 55-55 (1983).</li><li>Yang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+and+Redox+States+in+the+C6+Glioma+Cells+Following+Acute+Exposure+to+Zn,+Se++4+,+and+Se++6+and+the+Correlation+with+Apoptosis.">Energy and Redox States in the C6 Glioma Cells Following Acute Exposure to Zn, Se +4 , and Se +6 and the Correlation with Apoptosis.</a> Toxicology Mechanisms and Methods. 16 (1), 13-13 (2006).</li><li>Spinner, D. M., Bartlett, J. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> MTT Growth Assays in Ovarian Cancer, in Ovarian Cancer: Methods and Protocols. , 175-177 (2000).</li></ol>

Der MTT-Test gilt als eine vielseitige Methode werden und dementsprechend die Lebensfähigkeit der Zellen könnte auf verschiedene Behandlungen ausgewertet werden. Die Produktion von resultierende Formazan zu sein scheint proportional zum Füllstand des Energiestoffwechsels in den Zellen. Daher ist es möglich, die metabolisch aktivierten Zellen auch in Abwesenheit der Zellproliferation zu messen. Die Höhe der Formazan ist proportional zur Höhe des MTT in das Inkubationsmedium. Während produzierte die Konzentration von MTT, die erforderlich, um maximale Menge an Formazan zu erreichen ist, kann unter Ausnutzung verschiedener Zelllinien zu ändern. Außerdem genutzt haben diesen Test konnte sehr kleine Zahl von lebenden Zellen erkannt werden und das Auftreten von Fehlern wäre minimal, da gibt es keine Notwendigkeit für Waschschritte. Die Absorption von Formazan variiert mit der Zellzahl sowie pH-Wert, mit Zugabe von Puffer bei pH 10,5 1 konnte überwunden werden. Die Farbe des Formazan wird für ein paar Stunden bei Raumtemperatur 2 stabil. Im Falle von mehr als einer Platte, sollten Kontrollen in anderen Platten enthalten als gut. Trotzdem leidet diese Methode von einigen geringfügigen Nachteile: a) metabolisch inaktiven Zellen können nicht mit toten Zellen 3, b diskriminiert werden) MTT-Lösung vor Licht geschützt werden sollen, obwohl es konnte bei 4 ° C für maximal 1 Monat 2 gespeichert werden, c) es versäumt, Drogen Stabilität in das Medium zu validieren, und d) Zellen für MTT verwendet kann nicht nachträglich für andere Assays verwendet werden. Es sollte hervorgerufen, dass Phenolrot bei 570 nm absorbiert werden. Ferner hat es zuvor berichtet, dass die Phenolrot Östrogen-Aktivität, die das Zellwachstum Muster innerhalb einiger östrogenempfindlichen Zellen, daraus ungenaue MTT Ergebnisse beeinflussen könnten, besitzt. Zur Vermeidung solcher Auswirkungen, haben wir DMEM ohne Phenolrot 4 verwendet.

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		<div>
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>25 t-flask</td>
<td>&nbsp;</td>
<td>Orange Scientific</td>
<td>5510100</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P3813</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s Modified Eagle&#x2019;s Medium - high glucose</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D5671</td>
<td>&nbsp;</td>
<tr>
<td>MTT</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M2128</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F2442</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin 200,000 u/ml</td>
<td>&nbsp;</td>
<td>CinnaGen</td>
<td>CR7913</td>
<td>&nbsp;</td>
<tr>
<td>Streptomycin 200 mg/ml</td>
<td>&nbsp;</td>
<td>CinnaGen</td>
<td>CR7912</td>
<td>&nbsp;</td>
<tr>
<td>trypsin/EDTA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T3924</td>
<td>&nbsp;</td>
<tr>
<td>trypan blue</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T8154</td>
<td>&nbsp;</td>
<tr>
<td>Superfect</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>301105</td>
<td>&nbsp;</td>
<tr>
<td>Polyfect</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>301305</td>
<td>&nbsp;</td>
<tr>
<td>EGFR antisense</td>
<td>&nbsp;</td>
<td>MWG biotech</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Scrambled antisense</td>
<td>&nbsp;</td>
<td>MWG biotech</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DMEM without phenol red</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11880</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G8898</td>
<td>&nbsp;</td>
<tr>
<td>hemocytometer</td>
<td>&nbsp;</td>
<td>HBG </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>8-channel pipette</td>
<td>&nbsp;</td>
<td>TreffLab Transferpette</td>
<td>96.9918.10.1</td>
<td>&nbsp;</td>
<tr>
<td>Disposable syringe filter </td>
<td>&nbsp;</td>
<td>IWAKI</td>
<td>2052-025</td>
<td>&nbsp;</td>
<tr>
<td>Plate reader equipped with 570 nm &630 nm filters </td>
<td>&nbsp;</td>
<td>Biotek ELx808</td>
<td>ELX808</td>
<td>&nbsp;</td>
<tr>
<td>96-well plate with lid (flat bottom)</td>
<td>&nbsp;</td>
<td>IWAKI</td>
<td>3860-096</td>
<td>&nbsp;</td>
<tr>
<td>Laminar flow hood</td>
<td>&nbsp;</td>
<td>Esco class II Biosafety Cabinet</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CO2 Incubator</td>
<td>&nbsp;</td>
<td>ASTEC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

cellular toxicity of nanogenomedicine in mcf-7 cell line: mtt assay

Zytotoxizität der futuristischen nanogenomedicine (zB short interfering RNA und antisense) behindern können ihre klinischen Entwicklung. Von diesen kann das Gen-basierten Medizin und / oder seinem Träger zu entlocken Zelltoxizität. Für die Beurteilung eines solchen Zytotoxizität, ist eine gemeinsame Methodik weitgehend abhängig von Nutzung der 3 - (4, 5-Dimethyl-2-Thiazolyl) -2, 5-diphenyl-2H-Tetrazolium Bromid (MTT) bestimmt, der weithin als genutzt eine kolorimetrische Ansatz bezogen auf die Aktivität der mitochondrialen Dehydrogenase-Enzyme in den Zellen. In dieser Untersuchung wurden MCF-7-Zellen in 96-Well-Platte angeimpft und bei 50% Konfluenz wurden sie mit verschiedenen nanopolyplexes behandelt und dem Test nach 24 Stunden MTT. Wasserlösliche gelben MTT wird durch die metabolisch aktive Zellen, die in Wasser unlöslichen violetten Formazan, die weiter in Dimethylsulfoxid und Sornson s-Puffer pH 10,5 gelöst metabolisiert. Das resultierende Produkt kann durch Spektrophotometrie mit einem Platten-Lesegerät bei 570 nm quantifiziert werden.

Watch this Scientific Journal Video about Cellular Toxicity of Nanogenomedicine in MCF-7 Cell Line: MTT assay at JoVE.com

Cellular Toxicity of Nanogenomedicine in MCF-7 Cell Line: MTT assay

Der MTT-Test ist eine einfache und reproduzierbare Farbtestverfahren zur Beurteilung der Lebensfähigkeit der Zellen basiert auf Reduktion der gelben MTT und Produktion von wasserunlöslichen Formazan lila. Hier hat sich die Rentabilität der MCF-7 Zellen nach Behandlung von nanogenomedicine ausgewertet worden.

Zelluläre Toxizität von Nanogenomedicine in MCF-7 Zelllinie: MTT-Assay

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based ...

cellular-toxicity-of-nanogenomedicine-in-mcf-7-cell-line-mtt-assay

Nanyang Technological University

Research

JoVE Journal

Biology

44.5K Aufrufe.  Tabriz University (Medical Sciences). Der MTT-Test ist eine einfache und reproduzierbare Farbtestverfahren zur Beurteilung der Lebensfähigkeit der Zellen basiert auf Reduktion der gelben MTT und Produktion von wasserunlöslichen Formazan lila. Hier hat sich die Rentabilität der MCF-7 Zellen nach Behandlung von nanogenomedicine ausgewertet worden.

Serie: Cellular Toxicity of Nanogenomedicine in MCF-7 Cell Line: MTT assay

Proboscis Extension Response (PER) Assay in Drosophila

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth (labellum), there are hair-like structures called sensilla which houses taste neurons. When an attractive substance makes contact to the labellum, the fly extends its proboscis to consume the material. Proboscis Extension Response (PER) assay measures this taste behavior response, and it is a useful method to learn about food preferences in a single fly. Solutions of various sugars, such as sucrose, glucose and fructose, are very attractive to the fly. The effect of aversive substances can also be tested as reduction of PER when mixed in a sweet solution.Despite the simplicity of the basic procedure, there are many things that can prevent it from working. One of the factors that requires attention is the fly's responsive state. The required starvation time to bring the fly to the proper responsive state varies drastically from 36 to 72 hours. We established a series of controls to evaluate the fly's state and which allows screening out of non-responsive or hyper-responsive individual animals. Another important factor is the impact level and the position of the contact to the labellum, which would be difficult to describe by words. This video presentation demonstrates all these together with several other improvements that would increase the reproducibility of this method.

Proboscis Extension Response PER Assay in Drosophila

Proboscis extension response or PER is a taste behavior assay that has been used in flies as well as in honeybees. When the proboscis makes contact with an attractive substance, the fly extends its proboscis to consume the substance. Solutions of various sugars are very attractive to the fly.

Proboscis Erweiterung Response (PER) Assay in Drosophila

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth ...

Forschung

Biologie

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5' amine modified to allow fixation to the membrane. Primers are 5' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours). 
The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified
simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific.
Published applications of mPCR/RLB include detection of antibiotic resistance genes1,2, typing of methicillin-resistant Staphylococcus aureus3-5 and Salmonella sp6, molecular serotyping of Streptococcus pneumoniae7,8, Streptococcus agalactiae9 and enteroviruses10,11, identification of Mycobacterium sp12, detection of genital13-15 and respiratory tract16 and other17 pathogens and detection and identification of mollicutes18. However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms.
The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane.

Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB

An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.

Multiplex PCR und Reverse-Line-Blot-Hybridisierung Assay (MPCR / RLB)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Langfristige letale Toxizität Test mit dem Krustentier Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

Messung zellulärer Chemotaxis mit ECIS / Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Mechanische Stimulation induzierte Calcium Wellenausbreitung in Zellmonoschichten: Das Beispiel Bovine Endothelzellen

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells and cultured neurons are great systems for imaging of motor-driven organelle transport in vivo. Here we describe detailed protocols for culturing both cell types, their imaging and analysis of transport.

Organell-Transport in kultivierten<em&gt; Drosophila</em&gt; Zellen: S2-Zelllinie und primären Neuronen.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

Ein Test für Laterallinie Regeneration im erwachsenen Zebrafisch

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Ein-Kanal-Cell-Attached-Patch-Clamp-Aufnahme

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Generating a &#34;Humanized&#34; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.

Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

This protocol describes how to generate a Drosophila S2 cell line that is sensitive to small molecule inhibitors of kinesin-5. The use of these cells in a cell-based error correction assay is also outlined.

Erzeugen eines &quot;humanisierte&quot;<em&gt; Drosophila</em&gt; S2-Zelllinie Sensitive pharmakologische Hemmung der Kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper ...

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ultimately kill. Such A/B toxins generally consist of an enzymatically active Asubunit (e.g., ricin toxin A (RTA)) and one or more cell binding Bsubunit(s), which are responsible for toxin binding to specific cell surface receptors. Our current knowledge of how A/B toxins are capable of efficiently intoxicating cells helped scientists to understand fundamental cellular mechanisms, like endocytosis and intracellular protein sorting in higher eukaryotic cells. From a medical point of view, it is likewise important to identify the major toxin trafficking routes to find adequate treatment solutions for patients or to eventually develop therapeutic toxin-based applications for cancer therapy.
Since genome-wide analyses of A/B toxin trafficking in mammalian cells is complex, time-consuming, and expensive, several studies on A/B toxin transport have been performed in the yeast model organism Saccharomyces cerevisiae. Despite being less complex, fundamental cellular processes in yeast and higher eukaryotic cells are similar and very often results obtained in yeast can be transferred to the mammalian situation.
Here, we describe a fast and easy to use reporter assay to analyze the intracellular trafficking of RTA in yeast. An essential advantage of the new assay is the opportunity to investigate not only RTA retro-translocation from the endoplasmic reticulum (ER) into the cytosol, but rather endocytosis and retrograde toxin transport from the plasma membrane into the ER. The assay makes use of a reporter plasmid that allows indirect measurement of RTA toxicity through fluorescence emission of the green fluorescent protein (GFP) after in vivo translation. Since RTA efficiently prevents the initiation of protein biosynthesis by 28S rRNA depurination, this assay allows the identification of host cell proteins involved in intracellular RTA transport through the detection of changes in fluorescence emission.

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

In the manuscript, we describe the use of a yeast-based fluorescence reporter assay to identify cellular components involved in the trafficking and killing processes of the cytotoxic A subunit of the plant toxin ricin (RTA).

Ein einfache Fluoreszenz basierende Reporter Assay zelluläre Komponenten identifizieren erforderlich für Ricin Toxin eine Kette (RTA) Handel mit Hefe

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...

Zelluläre Toxizität von Nanogenomedicine in MCF-7 Zelllinie: MTT-Assay

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