Este trabajo es apoyado por el NIH R01 GM47607, CA163296 y NSF 1.334.493 de CET. Damos las gracias a los miembros del laboratorio Turner para la lectura crítica del manuscrito. Todos los datos que se muestran en este informe fueron reproducidas con permiso de Wormer et al. 2014 11.

1. Preparación de Durotaxis Cámaras <ol><li> Para preparar un solo 6 pocillos placa de cultivo de tejidos, tarar la balanza con un tubo cónico de 50 ml. Pesar aproximadamente 10 g de la solución de base de PDMS en el tubo de 50 ml (la solución es bastante viscoso). </li><li> Para un 90: 1 sustrato (Cumplimiento de ~ 1 kPa), se divide el peso medido de la solución de base de PDMS en el tubo por 90 para determinar la cantidad correcta de solución de agente de reticulación necesario. Añadir la cantidad calculada de la solución reticulante PDMS al mismo tubo. Ejemplo: 10 g / 90 = 0,11 g. Añadir 0,11 g de la solución de agente de reticulación PDMS a la solución base de PDMS. NOTA: La base y reticulantes soluciones son a la vez proporcionan en el kit de PDMS. </li><li> Mezclar vigorosamente la mezcla base / reticulante PDMS durante 5 min a RT usando una espátula pequeña. En esta etapa la mezcla contendrá un gran número de burbujas de aire. </li><li> Centrifugar el sustrato PDMS en una centrífuga de mesa durante 5 min a 50 xg a temperatura ambiente para eliminar el bubbles. Aún si hay burbujas después del 5 min, se centrifuga de nuevo. </li><li> Pipeta 1 ml de 90: 1 PDMS sustrato en cada pocillo del cultivo de tejidos tratados placa de 6 pocillos. Cualquier burbuja de aire restantes presentes en los PDMS pueden ser eliminados en esta etapa haciendo estallar ellos usando una aguja de 21 G. Permitir que el PDMS se extiendan durante 30 minutos en el pozo. </li><li> Hervir 12 mm de vidrio # 1 cubreobjetos en agua destilada durante 5 min. Repetir dos veces y almacenar los cubreobjetos en agua destilada. </li><li> Coloque uno, cubreobjetos se secó en cada pocillo de la placa de cultivo de tejidos tocando suavemente un lado del cubreobjetos en la solución de PDMS a continuación, colocar el cubreobjetos sobre los PDMS. A medida que el cubreobjetos se asienta, los PDMS comenzarán a invadir lo largo de los bordes del cubreobjetos, pero no cubren completamente. Esto generará una interfaz entre el PDMS y el vidrio después del curado (ver la Figura 1A, B). </li><li> Incubar la placa a 70 ° C en un horno durante 16 horas para curar (se endurecen) el PDMS. Coloque el plate en una campana de cultivo celular y UV esterilizar durante 10 minutos. NOTA: Lo mejor es hacer las placas en un par de días de uso. Sin embargo, las placas pueden ser envueltos en Parafilm sin ningún tipo de tampón y se almacenaron a 4 ° C durante un máximo de 2 semanas sin ninguna disminución notable en la calidad. </li></ol> 2. Celular Chapado NOTA: Si el estudio del efecto de la caída siRNA mediada, realice la caída con las instrucciones del fabricante o el protocolo optimizado para el tipo de célula de elección. <ol><li> Escudo durotaxis cada cámara con 1 ml de 10 mg / ml de fibronectina en PBS sin calcio y magnesio durante 1 hora a 37 ° C. Alternativamente, la fibronectina se puede aplicar el día antes del análisis mediante la incubación de los PDMS con 10 mg / ml de fibronectina en PBS durante 16 horas a 4 ° C. Asegúrese de que toda la superficie se sumerge en el fibronectina en solución PBS. </li><li> Preparar desnaturalizado por calor 1% de BSA. Pesar 0,5 g de BSA y se disuelven en 50 ml de PBS.Filtrar esterilizar la solución a través de un filtro y el calor de 0,22 micras a 80 ° C durante 12 min. Nota: Prepare esta solución el día antes de la célula de recubrimiento y se almacena a 4 ° C. </li><li> Aspirar la solución de fibronectina y lavar 3 veces con PBS. Añadir 1 ml de BSA desnaturalizado calor en PBS a cada pocillo e incubar durante 30 min a TA. </li><li> Trypsinize y contar las células de elección, mientras que las cámaras de durotaxis están bloqueando con la BSA desnaturalizado por calor. </li><li> Plate 1 x 10 5 células en un volumen de 2 ml en cada pocillo de la cámara durotaxis, utilizando los medios necesarios para el tipo de célula particular de elección. Permitir que las células se adhieran y se extendieron sobre el sustrato durante 4 horas en un incubador humidificado a 37 ° C con 5% de CO 2. NOTA: las células U2OS se mantienen rutinariamente en DMEM con 10% FBS, complementado con 2 mM L-glutamina, piruvato de sodio 1 mM, y 10 IU / ml de penicilina, 10 mg / ml de estreptomicina. NOTA: El número de células utilizado en este ejemplo está optimizado paraCélulas U2OS. Se puede requerir optimización para otros tipos de células. Esta densidad de las células da suficiente espacio para migrar sin interacciones significativas con otras células. </li></ol> Imaging 3. Células vivas <ol><li> Realizar imágenes de células vivas en un microscopio invertido, con contraste de fase con un objetivo de 10X. El microscopio debe estar equipado con una, cámara húmeda del medio ambiente cerrado, lo que permite el control de la temperatura a 37 ° C y 5% de CO 2 durante la exploración a largo plazo. </li><li> Después de que las células se han extendido durante aproximadamente 3,5 horas, montar la placa en la cámara microscopio. Permitir que la muestra (s) se equilibre en la cámara durante 30 min. </li><li> Establecer automatizado, multi-punto de visita en el microscopio si está disponible. Centrarse en la interfaz entre las 90: 1 PDMS y el cubreobjetos de vidrio y elegir los puntos a la imagen en todo el interfaz con un promedio de 40 puntos por durotaxis cámara. Imagen de las células cada 10 min durante un máximo de 16 horas. NOTA: La reregión de interés aparecerá como dos líneas, con la línea externa correspondiente al borde del cubreobjetos y la línea interior correspondiente a la interfaz real entre el PDMS y el cubreobjetos. Ver Figura 1B. </li></ol> Análisis 4. Datos <ol><li> Generar una hoja de cálculo como en la Tabla 1. </li><li> Cuente el número de eventos de cruce de los PDMS a la superficie de vidrio y viceversa de cada película generada. Anote el número de eventos de cruce en la columna correspondiente en la hoja de cálculo Excel. NOTA: Un evento de cruce se define como el núcleo de la célula que pasa sobre el límite entre el PDMS y productos de vidrio en cualquier dirección. </li><li> Para cuantificar múltiples cruces, contar el número de veces que el celular cruzó la interfaz. Ese número se debe registrar en la columna de excel correspondiente al sustrato sobre el que la célula se encuentra en el extremo de la película. Repita el análisis de todas las células que atraviesa la interfaz en tpelícula que él. Excluir las células que migran fuera del campo de visión durante la exploración. NOTA: También es importante verificar, por recuento de células al inicio del experimento, que las células son capaces de adherirse igual a los PDMS recubiertas de fibronectina y superficies cubreobjetos de vidrio. </li><li> Calcular el porcentaje de células que migró de PDMS a la superficie de vidrio (es decir, se sometió a durotaxis). Añadir el número de cruzar eventos de suave a duro y los múltiples eventos de cruce que terminaron en el disco y se dividen por el número total de eventos de cruce. </li><li> Calcular el porcentaje de múltiples cruces dividiendo el número de cruces de múltiples por el número total de cruces. </li></ol>

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The authors have no conflicts to disclose.

Aquí se describe un ensayo simple para estudiar durotaxis en la migración de las células. Una de las principales de este ensayo es la facilidad de preparación de las cámaras durotaxis utilizando PDMS. La rigidez de los sustratos puede ser manipulado fácilmente cambiando la relación de la solución de base de PDMS a reticulante para permitir el estudio de diversas rigideces en el ensayo. Sin embargo, una limitación potencial del sistema es que las células sólo están expuestos a un solo cambio en la rigidez del...

La matriz extracelular (ECM) se compone de una serie compleja de proteínas estructurales y de reticulación incluyendo el colágeno, fibronectina y laminina. Aunque está bien establecido que el ECM proporciona importante apoyo estructural a los tejidos celulares, cada vez hay más pruebas que indican que las células responden activamente a los cambios físicos en su entorno de ECM para regular diversos procesos celulares, incluyendo la supervivencia celular, la diferenciación y migración celular. Por ejemplo, las diferencias en la rigidez de la ECM pueden conducir las células madre mesenquimales hacia diferentes linajes, con sustratos blandos (~ 1 kPa) que promueven linajes neurogénicos mientras sustratos rígidos (~ 25 kPa) promueven la diferenciación osteogénica 1. Del mismo modo, un aumento en la rigidez de la matriz estromal ha sido demostrado para promover la tumorigénesis mamaria de las células epiteliales y la invasión en el tejido circundante 2,3. Un aspecto particularmente interesante de este mechanosignresultados de la actividad aling en un proceso conocido como durotaxis, en el que las células migran preferentemente hacia un sustrato más rígido 4,5. Las células constantemente sentido, las características físicas de su entorno extracelular a través de receptor de integrina de unión a la ECM. Esto, a su vez, promueve la acumulación de numerosas proteínas estructurales y de señalización a sus dominios citoplásmicos para impulsar la formación de estructuras adhesivas conocidas como adhesiones focales o contactos focales 6,7. Desde integrinas no tienen actividad enzimática intrínseca, las señales se transmiten desde el ECM a través de estas proteínas accesorias para coordinar la respuesta de la célula a su entorno cambiante 8. En consecuencia, la identificación y caracterización de las proteínas clave que intervienen en la regulación de mechanosignaling y durotaxis es un área importante de investigación. Varios sistemas de modelos se han desarrollado para estudiar durotaxis in vitro, pero la mayoría tienenutilizados sustratos revestidos con colágeno de poliacrilamida 4. Sin embargo, la preparación de los sustratos de poliacrilamida puede ser técnicamente difícil y el colágeno utilizado en estos ensayos debe ser reticulado químicamente al sustrato 9. Sustratos de polidimetilsiloxano (PDMS) se ha demostrado que exhiben propiedades mecánicas comparables a los sustratos de poliacrilamida al 10. Sin embargo, los sustratos de PDMS se preparan por simple mezcla de una proporción de la base a agente reticulante y estos sustratos pueden estar recubiertos con proteínas ECM sin la necesidad de reticulación química, haciendo así más fácil PDMS una herramienta para estudiar los efectos de rigidez en el comportamiento celular. En este documento, se describe cómo preparar una cámara durotaxis simple en el que un sustrato suave PDMS se integra con un cubreobjetos de vidrio rígido. El ensayo, como se indica a continuación, ofrece un método rápido y sencillo para estudiar durotaxis. Para este estudio se utilizaron células de osteosarcoma U2OS humanas combinadas con mediada por siRNAdesmontables de cdGAP para estudiar el papel de esta proteína de adhesión focal en durotaxis 11. Es importante destacar que este protocolo se puede adaptar fácilmente a las necesidades individuales. Otros tipos de células pueden ser sustituidos por las células U2OS y cualquier proteína pueden ser derribados o sobreexpresa para determinar los efectos sobre el comportamiento celular durante durotaxis. Además, este protocolo puede ser adaptado para incorporar proteínas etiquetadas con fluorescencia para analizar su dinámica y el comportamiento utilizando FRAP o FRET enfoques.

<table><tbody><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dow Corning</td><td>3097358-1004</td><td>Sylgard 184 Silicone Elastomer Kit</td></tr><tr><td>#1 Cover glass 12 mm</td><td>Fisher Scientific</td><td>12-545-82</td><td></td></tr><tr><td>6-well plate</td><td>Celltreat</td><td>229106</td><td></td></tr><tr><td>DMEM</td><td>Cellgro</td><td>15-017-CM</td><td></td></tr><tr><td>L-Glutamine</td><td>Cellgro</td><td>25-005-CI</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Fisher Scientific</td><td>BP356-100</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Cellgro</td><td>30-002-CI</td><td></td></tr><tr><td>Fibronectin</td><td>BD Biosciences</td><td>610077</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>21600-044</td><td></td></tr><tr><td>Falcon tubes</td><td>Celltreat</td><td>229456</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>U2OS cells</td><td>ATCC</td><td>HTB-96</td><td></td></tr></tbody></table>

a simplified system for evaluating cell mechanosensing and durotaxis in vitro

Las propiedades de la composición y mecánicas de la matriz extracelular son muy variables entre los tipos de tejidos. Esta diversidad estroma de tejido conectivo en gran medida el comportamiento celular impactos para regular los procesos normal y patológico incluyendo la proliferación celular, la diferenciación, la señalización de adhesión y migración direccional. En este sentido, la capacidad innata de ciertos tipos de células para migrar hacia un sustrato de matriz más rígida, o menos compatible que se conoce como durotaxis. Este fenómeno juega un papel importante durante el desarrollo embrionario, la reparación de heridas y la invasión de células de cáncer. A continuación, describimos un ensayo sencillo para estudiar durotaxis, in vitro, usando sustratos (PDMS) de polidimetilsiloxano. Preparación del descrito durotaxis cámaras crea una interfaz de rigidez entre el gel PDMS relativamente blando y un cubreobjetos de vidrio rígida. En el ejemplo, hemos utilizado estos durotaxis cámaras para demostrar un papel para el cdc42 / Rac1 GTPasa prote activaciónen, cdGAP, en mechanosensing y durotaxis regulación en células de osteosarcoma U2OS humanas. Este ensayo es fácilmente adaptable a otros tipos de células y / o desmontables de otras proteínas de interés, para explorar sus respectivas funciones en mechanosignaling y durotaxis.

Un diagrama esquemático de la cámara de durotaxis se muestra en la Figura 1A. Sustrato PDMS Soft (a 90: mezcla 1 de la base de PDMS a reticulante soluciones) se extiende en una placa de 6 pocillos y un cubreobjetos de vidrio se coloca en la parte superior de los PDMS, que luego cubre parcialmente la superficie superior del cubreobjetos, creando de este modo una interfaz entre los dos sustratos de diferente cumplimiento. La rigidez del sustrato PDMS blando es ~ 1 kPa, que es comparable con el cumplimie...

Watch this Scientific Journal Video about A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro at JoVE.com

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

Un sistema simplificado para la evaluación de la célula Mechanosensing y Durotaxis<em&gt; In Vitro</em

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

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Biology

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

8.2K vistas.  SUNY Upstate Medical University. Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

Video: A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which facilitate the functional assessment of the seeded cells. To that end, microscale cantilever technology offers a platform with which to measure the contractile functionality of a range of cell types, including skeletal, cardiac, and smooth muscle cells, through assessment of contraction induced substrate bending. Application of multiplexed cantilever arrays provides the means to develop moderate to high-throughput protocols for assessing drug efficacy and toxicity, disease phenotype and progression, as well as neuromuscular and other cell-cell interactions. This manuscript provides the details for fabricating reliable cantilever arrays for this purpose, and the methods required to successfully culture cells on these surfaces. Further description is provided on the steps necessary to perform functional analysis of contractile cell types maintained on such arrays using a novel laser and photo-detector system. The representative data provided highlights the precision and reproducible nature of the analysis of contractile function possible using this system, as well as the wide range of studies to which such technology can be applied. Successful widespread adoption of this system could provide investigators with the means to perform rapid, low cost functional studies in vitro, leading to more accurate predictions of tissue performance, disease development and response to novel therapeutic treatment.

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

This protocol describes the use of microscale silicon cantilevers as pliable culture surfaces for measuring the contractility of muscle cells in vitro. Cellular contraction causes cantilever bending, which can be measured, recorded, and converted into readouts of force, providing a non-invasive and scalable system for measuring contractile function in vitro.

Utilización de microescala silicio voladizos para evaluar la función contráctil celular<em&gt; In Vitro</em

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

Investigación

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Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts&#39; rheology.

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

We present a setup of optical tweezers coupled to a light sheet microscope, and its implementation to probe cell mechanics without beads in the Drosophila embryo.

Sondeo de la célula mecánica con pinzas ópticas libres de grano en el embrión de Drosophila

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to ...

Biología del Desarrollo

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

Mediciones directas de la fuerza de la mecánica subcelular en confinamiento utilizando pinzas ópticas

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Un ensayo de conducta para Mechanosensation de clones en MARCM-basa<em&gt; Drosophila melanogaster</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Neurociencias

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.

La medición de la quimiotaxis celular con ECIS / taxis

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

Biología

Mechano-Node-Pore Sensing: A Rapid, Label-Free Platform for Multi-Parameter Single-Cell Viscoelastic Measurements

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer metastasis. Conventional methods for measuring these properties, such as atomic force microscopy (AFM) and micropipette aspiration (MA), capture rich information, reflecting a cell's full viscoelastic response; however, these methods are limited by very low throughput. High-throughput approaches, such as real-time deformability cytometry (RT-DC), can only measure limited mechanical information, as they are often restricted to single-parameter readouts that only reflect a cell's elastic properties. In contrast to these methods, mechano-node-pore sensing (mechano-NPS) is a flexible, label-free microfluidic platform that bridges the gap in achieving multi-parameter viscoelastic measurements of a cell with moderate throughput. A direct current (DC) measurement is used to monitor cells as they transit a microfluidic channel, tracking their size and velocity before, during, and after they are forced through a narrow constriction. This information (i.e., size and velocity) is used to quantify each cell's transverse deformation, resistance to deformation, and recovery from deformation. In general, this electronics-based microfluidic platform provides multiple viscoelastic cell properties, and thus a more complete picture of a cell's mechanical state. Because it requires minimal sample preparation, utilizes a straightforward electronic measurement (in contrast to a high-speed camera), and takes advantage of standard soft lithography fabrication, the implementation of this platform is simple, accessible, and adaptable to downstream analysis. This platform's flexibility, utility, and sensitivity have provided unique mechanical information on a diverse range of cells, with the potential for many more applications in basic science and clinical diagnostics.

Presented here is a method to mechanically phenotype single cells using an electronics-based microfluidic platform called mechano-node-pore sensing (mechano-NPS). This platform maintains moderate throughput of 1-10 cells/s while measuring both the elastic and viscous biophysical properties of cells.

Detección de poro de nodo mecánico: una plataforma rápida y sin etiquetas para mediciones viscoelásticas unicelulares multiparamétricas

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer ...

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such as aspiration or coverslip compression have been used to mechanically stimulate whole embryos. These approaches limit experimental design as they are imprecise, require manual handling, and can process only a couple of embryos simultaneously. Microfluidic systems have great potential for automating such experimental tasks while increasing throughput and precision. This article describes a microfluidic system developed to precisely compress whole Drosophila melanogaster (fruit fly) embryos. This system features microchannels with pneumatically actuated deformable sidewalls and enables embryo alignment, immobilization, compression, and post-stimulation collection. By parallelizing these microchannels into seven lanes, steady or dynamic compression patterns can be applied to hundreds of Drosophila embryos simultaneously. Fabricating this system on a glass coverslip facilitates the simultaneous mechanical stimulation and imaging of samples with high-resolution microscopes. Moreover, the utilization of biocompatible materials, like PDMS, and the ability to flow fluid through the system make this device capable of long-term experiments with media-dependent samples. This approach also eliminates the requirement for manual mounting which mechanically stresses samples. Furthermore, the ability to quickly collect samples from the microchannels enables post-stimulation analyses, including -omics assays which require large sample numbers unattainable using traditional mechanical stimulation approaches. The geometry of this system is readily scalable to different biological systems, enabling numerous fields to benefit from the functional features described herein including high sample throughput, mechanical stimulation or immobilization, and automated alignment.

The present protocol describes the design, fabrication, and characterization of a microfluidic system capable of aligning, immobilizing, and precisely compressing hundreds of Drosophila melanogaster embryos with minimal user intervention. This system enables high-resolution imaging and recovery of samples for post-stimulation analysis and can be scaled to accommodate other multicellular biological systems.

Mecanoestimulación de organismos multicelulares mediante un sistema de compresión microfluídica de alto rendimiento

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such ...

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

El análisis de la superficie celular Adhesión Remodeling en respuesta a la tensión mecánica usando perlas magnéticas

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Evaluación de Dictyostelium discoideum respuesta a estimulación mecánica aguda

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like pressure or mechanical perturbations. Mechanoreceptors detect their stimuli and transmit signals via a great diversity of mechanisms. Some of the most common roles for mechanoreceptors are in neuronal responses, like touch and pain, or hair cells which function in balance and hearing. Mechanosensation is also important for cell types which are regularly exposed to shear stress such as endothelial cells, which line blood vessels, or blood cells which experience shear in normal circulation. Viscometers are devices that detect the viscosity of fluids. Rotational viscometers may also be used to apply a known shear force to fluids. The ability of these instruments to introduce uniform shear to fluids has been exploited to study many biological fluids including blood and plasma. Viscometry may also be used to apply shear to the cells in a solution, and to test the effects of shear on specific ligand-receptor pairs. Here, we utilize cone-plate viscometry to test the effects of endogenous levels of shear stress on platelets treated with antibodies against the platelet mechanosensory receptor complex GPIb-IX.

We describe an in-solution method to apply uniform shear to platelet surface receptors using cone-plate viscometry. This method may also be used more broadly to apply shear to other cell types and cell-fragments and need not target a specific ligand-receptor pair.

Un ensayo de cizalla uniforme para los receptores humanos de plaquetas y de superficie celular a través de Viscometry de placa cónica

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like ...

Un sistema simplificado para la evaluación de la célula Mechanosensing y Durotaxis

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