Os autores gostariam de agradecer Tecnologia de Células-Tronco e empresas Shahid Ghazi Tabriz, respectivamente, para a prestação de DMEM sem água vermelha e estéril para injeção de fenol como presentes.

MCF-7 semeadura em 96 poços da placa: MCF-7 células foram cultivadas em 25 t frasco em meio contendo Médio Modified Dulbecco Águia (DMEM), 10% FBS, 100 U / ml de penicilina e 100 estreptomicina mcg / ml a 37 ° C com 5% de CO 2, 95% ar e umidade completa. Uma vez alcançada a 90% confluência, eles foram destacados utilizando 0,05% de tripsina / EDTA e contadas por meio de azul de tripano e hemocitômetro. Essas células foram então ressuspendidas a uma concentração de 4 × 10 4 células / cm 2 e depois acrescentada a placa de 96 poços (ou seja, 250 mL / poço) por uma pipeta de 8 canais. Para a absorção de fundo, alguns poços foram permaneceu livre de células, ou seja, como controle em branco. Tratamento de células com nanopolyplexes diferentes: Na confluência de 40-50% (48 horas após a semeadura), as células cultivadas foram tratadas com dendrímeros starburst nanoestruturados poliamidoamina (ie, Superfect ® e Polyfect ®) e um polímero novo teste seguindo as instruções fornecidas pelo fornecedor de transfecção. Células também foram tratados com EGFR e mexidos antisense sozinho, e com os três nanopolyplexes diferentes destes dois oligonucleotídeos e com polímeros (n = 4). Quatro poços foram permaneceram sem tratamento como controle. Após 4 horas de tratamento da mídia foram removidos e repostos com a mídia fresco. MTT ensaio para avaliar a viabilidade das células: MTT foi realizada 24 horas após transfecção. Para este fim, a solução de MTT foi preparado em 1mg/ml em PBS e foi filtrado por um filtro de 0,2 mm. Então, 50 ul de MTT mais 200 mL de DMEM sem vermelho de fenol foram adicionados em cada poço, com exceção dos poços sem células em branco. Células foram incubadas durante 4 horas a 37 ° C com 5% de CO 2, 95% de ar e umidade completa. Após 4 horas, a solução MTT foi removido e substituído com 200 mL de DMSO e 25μl de Sorenson tampão glicina (glicina 0,1 M, NaCl 0,1 M, pH: 10,5 com 0,1 NaOH). A placa foi novamente incubada por 5 min à temperatura ambiente, ea densidade óptica (DO) dos poços foi determinada utilizando um leitor de placas em um teste de comprimento de onda de 570 nm e um comprimento de onda de referência de 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>

O ensaio de MTT é considerado um método versátil e, consequentemente, a viabilidade das células poderia ser avaliada após vários tratamentos. A produção de formazan resultante parece ser proporcional ao nível do metabolismo energético nas células. Portanto, é possível medir as células metabolicamente ativados, mesmo na ausência de proliferação celular. A quantidade de formazan produzido é proporcional à quantidade de MTT no meio de incubação. Enquanto, a concentração de MTT que é necessário para...

<!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>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

Citotoxicidade da nanogenomedicine futurista (eg, curtas de RNA interferente e antisense) pode prejudicar o seu desenvolvimento clínico. Desses, o medicamento baseadas em genes e / ou seu portador pode provocar toxicidade celular. Para avaliação de citotoxicidade, uma metodologia comum é em grande parte dependente de utilização de 3 - (4, 5-dimetil-2-tiazolil) -2, 5-difenil-2H-tetrazolium brometo de ensaio (MTT), que tem sido amplamente utilizado como uma abordagem colorimétrico baseado na atividade das enzimas desidrogenase mitocondrial nas células. Na presente investigação, MCF-7 foram inoculadas em células de 96 poços da placa e em 50% confluência foram tratados com nanopolyplexes diferentes e submetidos a ensaio de MTT após 24 horas. MTT solúvel em água amarela é metabolizado pelas células metabolicamente ativas para o formazan insolúvel em água roxa, que é mais dissolvido em dimetilsulfóxido pH e Sornson s tampão 10.5. O produto resultante pode ser quantificado por espectrofotometria utilizando um leitor de placas a 570 nm.

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

O MTT é um ensaio simples e reprodutível colorimétrico para avaliação de viabilidade celular com base na redução do MTT amarelo e produção de formazan insolúvel em água roxa. Aqui, a viabilidade de células MCF-7 sobre o tratamento de nanogenomedicine foi avaliada.

Toxicidade celular de Nanogenomedicine em MCF-7 Linhagem Celular: MTT

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

Research

JoVE Journal

Biology

44.4K visualizações.  Tabriz University (Medical Sciences). O MTT é um ensaio simples e reprodutível colorimétrico para avaliação de viabilidade celular com base na redução do MTT amarelo e produção de formazan insolúvel em água roxa. Aqui, a viabilidade de células MCF-7 sobre o tratamento de nanogenomedicine foi avaliada.

Vídeo: 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.

Extensão Proboscis Response Assay (PER) em 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 ...

Biologia

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.

A longo prazo de Teste de toxicidade letal com o crustáceo 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.

Medição de Quimiotaxia celulares com ECIS / Taxis

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

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.

Transporte Organelle em Cultivadas<em&gt; Drosophila</em&gt; Células: Linha celular S2 e primário neurônios.

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.

Um ensaio para a lateral de Regeneração de Linha em Zebrafish Adulto

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 ...

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 &quot;Humanized&quot; 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.

Gerando um &quot;humanizado&quot;<em&gt; Drosophila</em&gt; S2 linha de células sensíveis a inibição farmacológica de cinesina-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).

Um ensaio simples repórter baseado em fluorescência para identificar componentes celulares necessários para toxina ricina uma cadeia (RTA) o tráfico de levedura

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 ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Efeito de proteínas fluorescentes na fusão parceiros usando Polyglutamine ensaios de toxicidade em levedura

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.

Ensaio de Durotaxis de célula única para avaliar o controle mecânico do movimento celular e eventos de sinalização relacionados

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

Medição da Toxicidade do Peptídeo RAN em C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...