Este estudo foi apoiado pelo Desenvolvimento do Programa Acadêmico Prioritário das Instituições de Ensino Superior de Jiangsu (PAPD) e pelo Projeto de Pesquisa de Reforma do Ensino da Universidade Farmacêutica da China (2019XJYB18).

1. Preparo do meio semissólido
<ol><li>Ágar semissólido tradicional
	<ol><li>Preparar o ágar semissólido tradicional de acordo com a receita do meio teste de motilidade bacteriana utilizando os ingredientes básicos11. Dissolver 10 g de triptose, 15 g de NaCl, 4 g de ágar em água destilada suficiente, ajustar o pH para 7,2 ± 0,2 e perfazer o volume final para 1.000 mL.</li>
		<li>Autoclavar o ágar a 121 °C por 20 min e distribuí-lo em tubos de ensaio de 10 mL como meio semissólido de 3 cm de altura.</li>
	</ol></li>
	<li>Ágar semissólido tradicional com TTC
	<ol><li>Após a autoclavagem do meio semissólido convencional, resfriá-lo a 50 °C, adicionar 5 mL de solução TTC 1% estéril a 100 mL de meio, misturar e dispensá-lo em tubos de ensaio de 10 mL para formar um meio semissólido de 3 cm de altura.</li>
	</ol></li>
</ol>2. Cepas bacterianas
NOTA: Oitenta cepas foram isoladas do ambiente aquático e identificadas usando um instrumento automatizado de identificação bacteriana (ver Tabela de Materiais), incluindo Escherichia coli, Pseudomonas aeruginosa, Salmonella spp., Vibrio spp. , Klebsiella pneumoniae e Aeromonas hydrophila (Tabela 1). Staphylococcus aureus (ver Tabela de Materiais) foi utilizado como controle negativo não móvel; Escherichia coli, Pseudomonas aeruginosa e Salmonella typhimurium (ver Tabela de Materiais) foram utilizadas como cepas controle positivas.
<ol><li>Identificar cepas bacterianas de teste a serem usadas para análise de motilidade.</li>
	<li>Inclua controles negativos não móveis e cepas de controle positivo móveis.</li>
</ol>3. Observação da motilidade bacteriana reforçada pelo TTC
<ol><li>Seleccionar colónias únicas de bactérias de ensaio a partir de placas de ágar e inocular-as nos dois meios semissólidos acima referidos (passos 1.1.2 e 1.2.1) por punção utilizando agulhas inoculantes.</li>
	<li>Cultivar os tubos a 37 °C na incubadora por 24-48 h para observar os resultados.</li>
	<li>Observe o estado de crescimento: caracterize as bactérias como não móveis (-) se apenas a linha de punção for vermelha. Caracterizar as bactérias como móveis (+) se a cor vermelha se espalhar levemente para fora ao longo da linha de punção12.</li>
</ol>4. Efeito de diferentes concentrações de ágar sobre a motilidade bacteriana
<ol><li>Preparar meios semissólidos contendo 0,3%, 0,5% e 0,8% de ágar e inocular-os por punção, conforme descrito acima. Observe os resultados após 24-48 h de incubação.</li>
</ol>

<ol>
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Y., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explore+the+influencing+factors+of+bacterial+motility.">Explore the influencing factors of bacterial motility.</a> Health Care Today. 6, 50-51 (2018).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamentous+Phytophthora+pathogens+deploy+effectors+to+interfere+with+bacterial+growth+and+motility.">Filamentous Phytophthora pathogens deploy effectors to interfere with bacterial growth and motility.</a> Frontiers in Microbiology. 11, 581511 (2020).</li><li>K&#252;hn, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+arrangement+of+several+flagellins+within+bacterial+flagella+improves+motility+in+different+environments.">Spatial arrangement of several flagellins within bacterial flagella improves motility in different environments.</a> Nature Communication. 9 (1), 5369 (2018).</li><li>Mitchell, A. J., Wimpenny, J. W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+agar+concentration+on+the+growth+and+morphology+of+submerged+colonies+of+motile+and+non-motile+bacteria.">The effects of agar concentration on the growth and morphology of submerged colonies of motile and non-motile bacteria.</a> Journal of Applied Microbiology. 83 (1), 76-84 (2010).</li><li>Xu, A., Zhang, M., Du, W., Wang, D., Ma, L. 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Os autores não têm conflitos de interesse a declarar.

A detecção da motilidade bacteriana pelo método do meio semissólido é afetada por váriosfatores13,14. Condições de crescimento bacteriano, como oxigênio (aeróbio na superfície do ágar, não aeróbio no fundo do tubo com o meio semissólido), pH e temperatura, podem afetar a viabilidade dos flagelos bacterianos, o que pode levar à redução da motilidade ou mesmo à perda da motilidade15. Além disso, algumas bactérias do tipo...

A motilidade bacteriana desempenha um papel crítico na patogenicidade bacteriana, na formação de biofilme e na resistência a drogas1. A motilidade bacteriana está intimamente associada à patogenicidade e é necessária para a colonização bacteriana durante a infecção precoce das células dohospedeiro2. A formação de biofilme está intimamente relacionada à motilidade bacteriana, onde as bactérias aderem à superfície do meio sólido através da motilidade. A motilidade bacteriana tem sido considerada positivamente correlacionada com a formação de biofilme. Um alto grau de resistência bacteriana a drogas devido ao biofilme pode levar a infecções persistentes que são uma ameaça à saúde humana 3,4,5. Portanto, é importante detectar a motilidade bacteriana. O teste de motilidade bacteriana é usado principalmente para examinar a motilidade de diferentes formas de bactérias no estado vivo, que podem determinar indiretamente a presença ou ausência de flagelos e, portanto, tem um papel importante na identificação de bactérias.
Existem métodos diretos e indiretos para detectar a motilidade bacteriana6. Como as bactérias com flagelos apresentam motilidade, é possível detectar se as bactérias são móveis indiretamente, detectando a presença ou ausência de flagelos. Por exemplo, é possível detectar a motilidade indiretamente por microscopia eletrônica e coloração flagelar para indicar que as bactérias são móveis. Também é possível detectar por métodos diretos, como queda de suspensão e punção semissólida. 
O método de punção semissólido comumente utilizado em laboratórios de microbiologia de graduação para detectar motilidade bacteriana inocula a bactéria na punção em meio ágar semissólido contendo 0,4-0,8% de ágar, de acordo com a direção do crescimento bacteriano. Se a bactéria crescer ao longo da linha de punção para se espalhar, aparecem vestígios de crescimento semelhantes a nuvens (semelhantes a escovas), indicando a presença de flagelos e, portanto, motilidade. Se não houver vestígios de crescimento na linha de punção, a bactéria não é flagelada nem móvel. 
No entanto, este método tem suas desvantagens: as bactérias são incolores e transparentes, a atividade flagelar é afetada pelas características fisiológicas das bactérias vivas e outros fatores, e a concentração de ágar e o pequeno diâmetro do tubo de ensaio. Além disso, bactérias aeróbias são adequadas apenas para o crescimento na superfície do ágar, afetando a observação da motilidade bacteriana. Assim, para aprimorar este experimento, o cloreto de 2,3,5-trifeniltetrazólio (TTC) (incolor) foi adicionado ao meio para estabelecer um método mais confiável e intuitivo de determinação da motilidade bacteriana do que o método atual de punção direta, utilizando desidrogenases intracelulares para catalisar a formação de um produto vermelho do TTC 7,8,9,10.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bacto Agar</td>
			<td>Difco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli</td>
			<td>ATCC</td>
			<td>ATCC25922</td>
			<td>Positive control</td>
		</tr>
		<tr>
			<td>Pseudomonas aeruginosa</td>
			<td>ATCC</td>
			<td>ATCC27853</td>
			<td>Positive control</td>
		</tr>
		<tr>
			<td>Salmonella typhimurium</td>
			<td>ATCC</td>
			<td>ATCC14028</td>
			<td>Positive control</td>
		</tr>
		<tr>
			<td>Staphylococcus aureus</td>
			<td>ATCC</td>
			<td>ATCC25923</td>
			<td>Negative nonmotile control</td>
		</tr>
		<tr>
			<td>Tryptose</td>
			<td>&#160;OXOID</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TTC</td>
			<td>Sigma</td>
			<td>298-96-4</td>
			<td></td>
		</tr>
		<tr>
			<td>VITEK 2 automated microbial identification system</td>
			<td>Bio M&#233;rieux</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing bacterial motility based on a color reaction

A motilidade bacteriana é crucial para a patogenicidade bacteriana, formação de biofilme e resistência a drogas. A motilidade bacteriana é crucial para a invasão e/ou disseminação de muitas espécies patogênicas. Portanto, é importante detectar a motilidade bacteriana. Condições de crescimento bacteriano, como oxigênio, pH e temperatura, podem afetar o crescimento bacteriano e a expressão de flagelos bacterianos. Isso pode levar à redução da motilidade ou mesmo à perda da motilidade, resultando na avaliação imprecisa da motilidade bacteriana. Com base na reação de cor do cloreto de 2,3,5-trifenil tetrazólio (TTC) por desidrogenases intracelulares de bactérias vivas, o TTC foi adicionado ao ágar semissólido tradicional para detecção da motilidade bacteriana. Os resultados mostraram que este método de ágar semissólido TTC para detecção de motilidade bacteriana é simples, fácil de operar e não envolve instrumentos grandes e caros. Os resultados também mostraram que a maior motilidade foi observada em meio semissólido preparado com 0,3% de ágar. Em comparação com o meio semissólido tradicional, os resultados são mais fáceis de avaliar e mais precisos.

Tanto as cepas padrão quanto as isoladas foram comparadas para detecção da motilidade, e os resultados são mostrados na Tabela 1. Devido à ausência de flagelos, Staphylococcus aureus e Klebsiella pneumoniae cresceram apenas ao longo da linha inoculada em ambos os meios semissólido tradicional e TTC. Em contraste, Pseudomonas aeruginosa, Escherichia coli e Salmonella typhimurium apresentaram crescimento em todas as direções ao redor da linhagem inocula...

Watch this Scientific Journal Video about Visualizing Bacterial Motility Based on a Color Reaction at JoVE.com

Visualizing Bacterial Motility Based on a Color Reaction

Aqui, apresentamos um protocolo para detectar motilidade bacteriana baseado em uma reação de cor. As principais vantagens deste método são que é fácil de avaliar e mais preciso, e não requer equipamentos especializados.

Visualizando a motilidade bacteriana com base em uma reação de cor

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or ...

visualizing-bacterial-motility-based-on-a-color-reaction

Research

JoVE Journal

Biology

3.6K visualizações.  China Pharmaceutical University. Aqui, apresentamos um protocolo para detectar motilidade bacteriana baseado em uma reação de cor. As principais vantagens deste método são que é fácil de avaliar e mais preciso, e não requer equipamentos especializados.

Vídeo: Visualizing Bacterial Motility Based on a Color Reaction

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

Avaliar motilidade celular Neural Stem Usando um dispositivo Agarose Gel baseado microfluídicos

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Biologia

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Replicação de DNA visualizar Único molécula com microscopia de fluorescência

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

Visualizando Localização RNA em Xenopus Oócitos

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Gastrointestinal Motility Monitor (GIMM)

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility in isolated segments of guinea pig distal colon. The complete system consists of a computer, video camera, illuminated organ bath, peristaltic and heated water bath circulating pumps, and custom GIMM software to record and analyze data. Compared with traditional methods of monitoring colonic peristalsis, the GIMM system allows for continuous, quantitative evaluation of motility. The guinea pig distal colon is bathed in warmed, oxygenated Krebs solution, and fecal pellets inserted in the oral end are propelled along the segment of colon at a rate of about 2 mm/sec. Movies of the fecal pellet proceeding along the segment are captured, and the GIMM software can be used track the progress of the fecal pellet. Rates of propulsive motility can be obtained for the entire segment or for any particular region of interest. In addition to analysis of bolus-induced motility patterns, spatiotemporal maps can be constructed from captured video segments to assess spontaneous motor activity patterns. Applications of this system include pharmacological evaluation of the effects of receptor agonists and antagonists on propulsive motility, as well as assessment of changes that result from pathophysiological conditions, such as inflammation or stress. The guinea pig distal colon propulsive motility assay, using the GIMM system, is straightforward and simple to learn, and it provides a reliable and reproducible method of assessing propulsive motility.

Gastrointestinal Motility Monitor GIMM

Evaluation of colonic motility in the guinea pig distal colon with the Gastrointestinal Motility Monitor (GIMM) is a straightforward and simple to learn approach to quantitatively evaluate propulsive motility in the gastrointestinal tract.

Monitor de motilidade gastrointestinal (GIMM)

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

Preparação, Imaging, e quantificação de Motilidade Ensaios superfície da bactéria

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

Monitoramento retículo endoplasmático homeostase do cálcio Usando um<em&gt; Gaussia</em&gt; Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Medição e Análise de Produção de Ácido Extracelular determinar Glicosidases Classificação

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&amp;#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Ensaio de triagem à base de SA-β-galactosidase para a identificação de medicamentos Senoterapêuticos

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. Here, we present an easy and inexpensive method that would be instrumental for testing new drugs decreasing uterine contractility. This method utilizes the unique ability of the entire mouse reproductive tract to exhibit spontaneous motility when maintained ex vivo in a Petri dish containing oxygenated Krebs buffer. This spontaneous motility resembles the wave-like myometrial activity of the human uterus, referred to as endometrial waves. To demonstrate the effectiveness of the method, we employed a well-known uterine relaxant drug, epinephrine. We demonstrate that the spontaneous motility of the entire mouse reproductive tract can be quickly and reversibly inhibited by 1 &#181;M epinephrine in this Petri dish model. Documenting the changes of uterine motility can be easily done using an ordinary smart phone or a sophisticated digital camera. We developed a MATLAB-based algorithm allowing motion tracking to quantify spontaneous uterine motility changes by measuring the rate of uterine horn movements. A major advantage of this ex vivo approach is that the reproductive tract remains intact throughout the entire experiment, preserving all intrinsic intrauterine cellular interactions. The major limitation of this approach is that up to 10-20% of uteri may exhibit no spontaneous motility. Thus far, this is the first quantitative ex vivo method for assessing spontaneous uterine motility in a Petri dish model.

Uterine contractions are important for the well-being of females. However, pathologically increased contractility may result in dysmenorrhea, especially in younger females. Here, we describe a simple ex vivo preparation allowing quick assessment of the efficacy of smooth muscle relaxants that may be used for treating dysmenorrhea.

Método ex vivo para avaliar a motilidade espontânea do trato reprodutivo do mouse e um algoritmo de rastreamento de movimento do útero baseado em MATLAB para análise de dados

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. ...

A Capsule-Based Model for Immature Hard Tick Stages Infestation on Laboratory Mice

Ticks are obligatory blood feeding parasites at all stages of development (except eggs) and are recognized as vectors of various pathogens. The use of mouse models in tick research is critical for understanding their biology and tick-host-pathogen interactions. Here we demonstrate a non-laborious technique for the feeding of immature stages of hard ticks on laboratory mice. The benefit of the method is its simplicity, short duration, and the ability to monitor or collect ticks at different time points of an experiment. In addition, the technique allows attachment of two individual capsules on the same mouse, which is beneficial for a variety of experiments where two different groups of ticks are required to feed on the same animal. The non-irritating and flexible capsule is made from easily accessible materials and minimizes the discomfort of the experimental animals. Furthermore, euthanasia is not necessary, mice recover completely after the experiment and are available for re-use.

In this study, a feeding system for nymphal and larval stages of hard tick was developed using a capsule attached to laboratory mouse. The feeding capsule is made from flexible materials and remains firmly attached to the mouse for at least one week and allows comfortable monitoring of tick feeding.

Um modelo baseado em cápsulas para infestação de estágios de carrapato duro imaturo em ratos de laboratório

Ticks are obligatory blood feeding parasites at all stages of development (except eggs) and are recognized as vectors of various pathogens. The use of ...