Agradecemos al profesor Lobo Frommer (Institución Carnegie para la Ciencia, la Universidad de Stanford) para la amabilidad de darnos la glucosa FRET plásmido y el Dr. Uwe Ludewig (Universidad de Hohenheim) por la amabilidad de suministro de la glutamina FRET construir en el vector de expresión vegetal pUTKan. AB está financiado por el programa de becas de Prioridad BBSRC apuntado. Tanto CK y KP son apoyados por RCUK Becas en Procesamiento biofarmacéuticos. CK También desea agradecer a Lonza biológicos por su apoyo financiero. El Centro para la Biología Sintética e Innovación es generosamente apoyada por el EPSRC.

1. Línea Celular Revival y mantenimiento <ol><li> Revive células CHO en 9 ml de medio de CD-CHO suplementado con 8 mM de L-glutamina y ml / L de suplemento de hipoxantina 100x / timidina 10 (medio de crecimiento completo). </li><li> Se centrifuga a 100 × g durante 5 min. </li><li> Resuspender las células en 10 ml de medio de crecimiento completo fresco. </li><li> Extraiga una muestra de 1 ml y determinar la concentración de células viables mediante microscopía óptica utilizando el método de exclusión del colorante azul de tripano en un hemocitómetro. </li><li> Iniciar un cultivo a una densidad de siembra de 3 x 10 5 células / ml en 125 ml agitar frascos. </li><li> Mantener el cultivo en una incubadora a 37 ° C, una atmósfera humidificada de 5% de CO 2, en una plataforma de agitación orbital que gira a 120 rpm. </li><li> Subcultura cada 3-4 días en el medio de crecimiento completo a una densidad de siembra de 2 x 10 5 células / ml. </li></ol> 2. La transfección de la línea celular con el plásmido Containing el biosensor Gene <ol><li> Preparar ADN plásmido usando un kit de purificación de plásmido a gran escala. </li><li> Mantener las células en las condiciones apropiadas (37 ° C, 5% de CO 2) 12-24 h antes de la transfección para asegurar las células se están dividiendo activamente en el momento de la transfección. </li><li> Contar las células usando el método de exclusión del colorante azul de tripano, y luego preparar un volumen de trabajo de 20 ml de cultivo de células en un matraz de agitación de 125 ml a una concentración de 1 x 10 6 células / ml. </li><li> Utilice un kit de transfección adecuado para la línea celular en uso. El ADN plásmido relación de reactivos de transfección a será dependiente de la línea celular y el vector. Se recomienda que este se optimiza mediante pruebas de un intervalo de relaciones dentro del rango especificado por el fabricante antes de realizar las transfecciones finales. También mantener al menos un cultivo de control negativo que contiene las células, pero sin ADN. </li><li> Incubar durante 4 días en modo estático y luego transferir a una Shaking plataforma que gira a 125 rpm. </li><li> Añadir el antibiótico apropiado para la selección del plásmido a una concentración adecuada como se determina por una curva de muerte. En este estudio, se añadió zeocina a una concentración final de 400 g / ml para la selección de las células transfectadas. </li><li> Cambie los medios en un intervalo de tiempo apropiado para la línea celular, añadiendo antibióticos cada vez, hasta que las células en el control así han muerto. </li><li> Utilice los pozos más confluentes de progresar a temblar culturas. </li><li> Establecer un banco de células mediante la congelación de las células en viales criogénicos que contienen 10 7 células viables en 1 ml de mezcla de congelación. En este trabajo, esta consiste en medio de crecimiento 92,5% y 7,5% de sulfóxido de dimetilo. </li></ol> 3. Lote y Fed-batch Curva de Crecimiento Celular <ol><li> Siga las instrucciones para el mantenimiento de células arriba para establecer cultivos de células por triplicado de las células CHO transfectadas en 250 ml de matraces de agitación con un volumen de trabajo de 50 ml. </li><li> Mantener la cultures en un incubador humidificado de células a 37 ° C, con 5% de CO 2, en una plataforma de agitación orbital que gira a 125 rpm, y retirar 4,1 ml muestras de cada cultivo en crecimiento a intervalos de 24 hr. </li><li> Utilice 100 l de la muestra para determinar la concentración celular y la viabilidad celular viable con el método de exclusión del colorante azul de tripano. </li><li> Repetir hasta que la concentración de células viables se reduce a cero. </li><li> Repetir para los cultivos de células de alimentación por lotes se completa con la cantidad apropiada de glucosa o glutamina en el día 6 para restaurar sus concentraciones a sus valores iniciales de 36 mM y 4 mM, respectivamente. Se recomienda que también se mantienen los cultivos de control adicional, que han de ser alimentados con el mismo volumen (12 ml en este caso) de agua pura. </li></ol> 4. FRET mediciones de la relación <ol><li> Tomar 2 ml de las muestras diarias retirados en el paso 3.2 anterior y se centrifuga a 100 × g durante 5 min a 4 ° C. </li><li> Resuspfinalizado el pellet de células en 2 ml de hielo frío tampón fosfato salino (PBS). Transfiera esta en una placa de 6 pocillos y añadir una muestra en blanco que no contiene células a un pocillo. </li><li> Medir los niveles de fluorescencia azul y amarillo de inmediato a una longitud de onda de excitación de 430/435 nm y de emisión de longitudes de onda de 465/435 nm (azul) y 520/510 nm (amarillo). </li><li> Calcular los ratios de FRET (relación de fluorescencia amarilla detectado más de fluorescencia azul detectada). </li></ol> 5. Metabolito Ensayos <ol><li> Tomar 2 ml de las muestras diarias retirados en el paso 3.2 anterior y se centrifuga a 100 × g durante 5 min a 4 ° C. Eliminar el sobrenadante. </li><li> Resuspender el sedimento celular en 3 ml de PBS enfriado en hielo y se centrifuga de nuevo a 100 xg durante 5 min. </li><li> Eliminar el sobrenadante y resuspender el botón celular en 3 ml PBS helado que el anterior. Sonicar el 5x muestra durante 3 minutos cada uno en un pulso de 15 segundos encendido y 15 segundos apagado. En este punto, la celda adicionalcts pueden congelarse si se desea. </li><li> Utilice un kit de ensayo de glucosa adecuado, según las instrucciones del fabricante, con muestras de extractos de células para determinar la concentración intracelular de glucosa (ver tabla de materiales). Nota: Utilice las normas apropiadas para construir una curva estándar. En este trabajo, la concentración de las normas osciló entre 0-100 mM. </li><li> El uso de las lecturas de las normas, construir la curva patrón y encontrar la ecuación lineal de mejor ajuste (en este caso: y = 1310.51x - 723,43 donde y es la lectura de la absorbancia y x es la concentración de glucosa). </li><li> Cálculo de las concentraciones de glucosa utilizando la curva estándar y teniendo en cuenta la dilución. La concentración intracelular de la glucosa puede entonces ser calculado utilizando la siguiente ecuación: <img alt="" fo:content-width="4.5in" src="/files/ftp_upload/51200/book_bg.png" width="500" /> Para las células CHO, el volumen de una sola célula se calculó utilizando un diámetro de célula de 12 M 12 </sup&gt;. </li><li> Utilice un kit de ensayo de glutamina adecuada según las instrucciones del fabricante, con muestras de extractos de células para determinar la concentración de glutamina intracelular (ver tabla de materiales). Nota: Utilice las normas apropiadas para construir una curva estándar. En este trabajo, la concentración de las normas osciló entre 0-2 mM. </li><li> Como en 5.5 anteriormente, construir la curva de calibración (en este caso y = 203.1x + 17,1 donde y es la lectura de la absorbancia y x es la concentración de glucosa) </li><li> Utilizando la curva estándar, calcular la concentración de glutamina de las muestras, teniendo en cuenta la dilución. La concentración de glutamina intracelular se puede calcular utilizando la Ecuación 1 anterior. </li></ol>

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Los autores no tienen nada que revelar.

Los FIBS permiten in vivo y en la cuantificación in situ de moléculas clave, en este caso nutrientes limitantes del crecimiento, la eliminación de incertidumbres derivadas de temple y métodos de extracción. Los hallazgos sugieren que existe una buena correlación entre la señal FIBS y las concentraciones intracelulares en el rango de 1-5 mm para la glucosa y 0,3-2 mM de glutamina. En el lote de cultivo de células CHO, estas concentraciones se encuentran en las fases de declive exponenci...

Monitoreo metabolito tiene diversas aplicaciones en el procesamiento biológico, incluyendo el desarrollo del proceso, los medios de comunicación y el diseño de alimentación, y la ingeniería metabólica. Varios métodos están disponibles para las mediciones de concentración a través de la utilización de 1 enzimática, química 2, o ensayos de unión 3. Una opción interesante es el uso de lámparas fluorescentes, en sensores biológicos in vivo como reporteros de la concentración intracelular de metabolitos clave a base de proteínas. En los ensayos de volumen ultra bajo, la fluorescencia es una herramienta conveniente como la miniaturización realmente mejora la relación de 4,5-señal-ruido y sensores basados ​​en proteínas puede ser codificado genéticamente el sentido de que no hay reactivos exógenos son necesarios para el análisis de metabolitos. Transferencia de energía de resonancia de Förster (FRET) biosensores consisten en dos proteínas fluorescentes conectadas por un dominio de unión a ligando. FRET es una transferencia de energía no radiante de un donante fotoexcitado a un aceptor fluorescente molécula locat<html> ed en estrecha proximidad (&lt;100). La escisión ligando o unión provoca un cambio conformacional en el sensor, que, a su vez, induce un cambio en la proximidad de los fluoróforos, lo que lleva a un cambio en la eficiencia de FRET medido por el cambio en el espectro de emisión. Sensores biológicos FRET integrados (FIBS) se producen de forma constitutiva dentro de la línea celular y sus características espectrales cambian en función de la concentración del metabolito dentro de la célula. FIBS tienen tiempos de respuesta rápidos lo que es ideal para la toma de mediciones para los modelos dinámicos 6. Aplicaciones anteriores incluyen el monitoreo individuales 7-9 y múltiples 10 metabolitos y proporcionar datos sobre la distribución espacio-temporal 11. FIBS se pueden crear en dos configuraciones: Apo-Max, donde la unión del ligando altera la proximidad de los fluoróforos bajada de transferencia de energía y Apo-Min, donde la unión del ligando trae los dos fluoróforos en contacto más estrecho (Figura 1). _content &quot;&gt; En este trabajo, se presenta un protocolo para la construcción de ovario de hámster chino (CHO) líneas celulares que expresan constitutivamente una FIBS de un metabolito, glucosa o glutamina. Se establece una metodología para la calibración del sensor in vivo para permitir el futuro cuantitativa mediciones de la concentración de metabolito intracelular, tal como se presenta en la Figura 2. Sobre la base de esto, la concentración intracelular de glucosa o glutamina pueden determinarse en discontinuos alimentados cultivos de células CHO, al que se añadieron los dos nutrientes en altas concentraciones en-proceso. Los resultados demuestran que el uso de la señal fluorescente de los FIBS y la curva de calibración previamente construido, la predicción precisa de la concentración intracelular es posible, como se confirmó mediante un ensayo enzimático independiente. Este método ofrece ventajas sustanciales sobre tecnologías analíticas actuales, ya que no es invasiva y de bajo costo y rápido, dando una señal en tiempo real de los FIBS que pueden ser lunmonitoriza todo el cultivo.

<table border="0" cellpadding="0" cellspacing="0" width="936">
	<colgroup>
		<col />
		<col span="2" />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">CHO-S Cells</td>
			<td style="width:120px;">Life Tecnologies</td>
			<td style="width:120px;">11619-012</td>
			<td style="width:459px;">Cell line will vary depending on the goals of the study</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">pCDNA4/TO vector&#160;</td>
			<td style="width:120px;">Life Tecnologies</td>
			<td style="width:120px;"></td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">TransIT-PRO Transfection Reagent</td>
			<td style="width:120px;">Mirus Bio</td>
			<td style="width:120px;">MIR 5700</td>
			<td style="width:459px;">Transfections can be accomplished using any method suitable for the cell line under study</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Zeocin</td>
			<td style="width:120px;">Invivogen</td>
			<td style="width:120px;"></td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">CD-CHO medium</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">10743-011</td>
			<td style="width:459px;">Cell growth medium is dependent upon the cells under study</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">100X HT Supplement</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">11067-030</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">L-Glutamine 200 mM (100X), Liquid</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">25030032</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">InfinitePRO 200 plate reader</td>
			<td style="width:120px;">Tecan</td>
			<td style="width:120px;">FLx800TBI</td>
			<td style="width:459px;">Any 96-well fluorescence plate reader that can access the required wavelengths can be substituted</td>
		</tr>
		<tr height="21">
			<td height="190" rowspan="6" style="height:190px;width:239px;">Filters for plate reader</td>
			<td rowspan="6" style="width:120px;">Tecan</td>
			<td align="right" style="width:120px;">30000463</td>
			<td rowspan="6" style="width:459px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">(520NM BW 10NM)</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;width:120px;">30022786</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">(430NM BW 35NM)</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;width:120px;">30022787</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:120px;">(465NM BW 35NM)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Maxiprep Plasmid Purification Kit</td>
			<td style="width:120px;">Qiagen</td>
			<td style="width:120px;">12163</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Amplex Red glucose/glucose oxidase assay kit</td>
			<td style="width:120px;">Invitrogen</td>
			<td style="width:120px;">A22189</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">EnzyChrom&#160; Glutamine Assay Kit</td>
			<td style="width:120px;">BioAssay systems</td>
			<td style="width:120px;">EOAC-100</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Improved Neubauer haemocytometer</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:120px;">MNK-420-010N</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Incubator&#160;</td>
			<td style="width:120px;">Nuaire</td>
			<td style="width:120px;">NU-5510E</td>
			<td style="width:459px;"></td>
		</tr>
	</tbody>
</table>

fibs-enabled noninvasive metabolic profiling

En la era de la biología computacional, nuevos sistemas experimentales de alto rendimiento son necesarios con el fin de poblar y refinar los modelos para que puedan ser validados para fines predictivos. Idealmente estos sistemas serían bajo volumen, que se opone a los análisis de muestreo y destructivas cuando los datos de curso de tiempo se han de obtener. Lo que se necesita es una herramienta de supervisión in situ en la que puede reportar la información necesaria en tiempo real y de forma no invasiva. Una opción interesante es el uso de lámparas fluorescentes, en sensores biológicos in vivo como reporteros de concentraciones intracelulares a base de proteínas. Una clase particular de in vivo biosensores que ha encontrado aplicaciones en la cuantificación metabolito se basa en Förster de transferencia de energía de resonancia (FRET) entre dos proteínas fluorescentes conectadas por un dominio de unión a ligando. FRET sensores biológicos integrados (FIBS) se producen de forma constitutiva dentro de la línea celular, que tienen tiempos de respuesta rápidos y su cha espectralrísticas cambios de acuerdo a la concentración del metabolito dentro de la célula. En este documento, se describe el método para la construcción de ovario de hámster chino (CHO) líneas celulares que expresan constitutivamente una FIBS para la glucosa y la glutamina y la calibración de los FIBS in vivo en cultivo celular por lotes con el fin de permitir que el futuro cuantificación de la concentración de metabolito intracelular. Los datos de fed-batch cultivos de células CHO demuestra que los FIBS fue capaz en cada caso para detectar el cambio resultante en la concentración intracelular. Uso de la señal fluorescente de los FIBS y la curva de calibración previamente construido, la concentración intracelular se determinó con precisión como se confirmó mediante un ensayo enzimático independiente.

Una visión general de la metodología se presenta en la Figura 2. En el trabajo presentado en este documento, las células CHO fueron transfectadas con el vector de FIBS y líneas celulares estables se seleccionaron a una presión de antibiótico de 400 mg / ml de zeocina. Por lo tanto, se crearon dos líneas celulares estables que expresan constitutivamente separados los sensores de glucosa y glutamina. Figura 1 representa las configuraciones de los dos biosensores utilizados en este ...

Watch this Scientific Journal Video about FIBS-enabled Noninvasive Metabolic Profiling at JoVE.com

FIBS-enabled Noninvasive Metabolic Profiling

Se presenta una descripción de cómo calibrar Transferencia Förster Resonance Energy sensores biológicos integrados (FIBS) en el perfil metabólico situ. Los FIBS se pueden utilizar para medir los niveles intracelulares de metabolitos de forma no invasiva ayudando en el desarrollo de modelos metabólicos y cribado de alto rendimiento de las condiciones de bioprocesos.

FIBS habilitadas no invasiva perfiles metabólicos

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

fibs-enabled-noninvasive-metabolic-profiling

Research

JoVE Journal

Bioengineering

9.6K vistas.  Imperial College London.  Se presenta una descripción de cómo calibrar Transferencia Förster Resonance Energy sensores biológicos integrados (FIBS) en el perfil metabólico situ. Los FIBS se pueden utilizar para medir los niveles intracelulares de metabolitos de forma no invasiva ayudando en el desarrollo de modelos metabólicos y cribado de alto rendimiento de las condiciones de bioprocesos. 

Video: FIBS-enabled Noninvasive Metabolic Profiling

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

Perfiles de microARN de alto rendimiento: Multiplex Optimizado QRT-PCR en la escala de nanolitros de la IFC Fluidigm dinámica ArrayTM

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

Investigación

Bioingeniería

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

In this study, the use of an in situ loading device coupled with micro-X-ray computed tomography for fibrous joint biomechanics will be discussed. Experimental readouts identifiable with an overall change in joint biomechanics will include: 1) reactionary force vs. displacement, i.e. tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics, i.e.&#160;geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. concentric or eccentric loads.

In situ carga de compresión y de imágenes no invasiva correlativa del hueso-ligamento periodontal de dientes fibroso Conjunta

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.

Cuantificación y Tamaño-perfiles de extracelular vesículas Usando Tunable resistiva Pulso Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Fácil y Precisa mecanoterapia perfiles en micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing termini of oligo- and polysaccharides, as well as its unique chemical characteristics, sialic acid is involved in a multitude of different receptor-ligand interactions. By modifying the expression of sialic acid on the cell surface, sialic acid-dependent interactions will consequently be influenced. This can be helpful to investigate sialic acid-dependent interactions and has the potential to influence certain diseases in a beneficial way. Via metabolic glycoengineering (MGE), the expression of sialic acid on the cell surface can be modulated. Herein, cells, tissues, or even entire animals are treated with C2-modified derivatives of N-acetylmannosamine (ManNAc). These amino sugars act as sialic acid precursor molecules and therefore are metabolized to the corresponding sialic acid species and expressed on glycoconjugates. Applying this method produces intriguing effects on various biological processes. For example, it can drastically reduce&#160;the expression of polysialic acid (polySia) in treated neuronal cells and thus affects neuronal growth and differentiation. Here, we show the chemical synthesis of two of the most common C2-modified N-acylmannosamine derivatives, N-propionylmannosamine (ManNProp) as well as N-butanoylmannosamine (ManNBut), and further show how these non-natural amino sugars can be applied in cell culture experiments. The expression of modified sialic acid species is quantified by high performance liquid chromatography (HPLC) and further analyzed via mass spectrometry. The effects on polysialic acid expression are elucidated via Western blot using a commercially available polysialic acid antibody.

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid is a typical monosaccharide-unit found in glycoconjugates. It is involved in a plethora of molecular and cellular interactions. Here we present a method to modify cell surface sialic acid expression using metabolic glycoengineering with N-acetylmannosamine derivatives.

Glycoengineering metabólico del ácido siálico utilizando N-acil-modificado Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing ...

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic resonance (MR) microscopy data. While it is possible to perform MR collections on living, excised mammalian tissue, such experiments have traditionally been constrained by resolution limits and are thus incapable of visualizing tissue microstructure. Conversely, MR protocols that did achieve microscopic image resolution required the use of fixed samples to accommodate the need for static, unchanging conditions over lengthy scan times. The current protocol describes the first available MR technique that enables imaging of living, mammalian tissue samples at microscopic resolutions. Such data is of great importance to the understanding of how pathology-based contrast changes occurring at the microscopic level influence the content of macroscopic MR scans such as those used in the clinic. Once such an understanding is realized, diagnostic methods with greater sensitivity and accuracy can be developed, which will translate directly to earlier disease treatment, more accurate therapy monitoring and improved patient outcomes.
While the described methodology focuses on brain slice preparations, the protocol is adaptable to any excised tissue slice given that changes are made to the gas and perfusate preparations to accommodate the tissue&#39;s specific metabolic needs. Successful execution of the protocol should result in living, acute slice preparations that exhibit MR diffusion signal stability for periods up to 15.5 h. The primary advantages of the current system over other MR compatible perfusion apparatuses are its compatibility with the MR microscopy hardware required to attain higher resolution images and ability to provide constant, uninterrupted flow with carefully regulated perfusate conditions. Reduced sample throughput is a consideration with this design as only one tissue slice may be imaged at a time.

The current protocol describes a method by which users can maintain viability of acute hippocampal and cortical slice preparations during the collection of magnetic resonance microscopy data.

Apoyo metabólico de suprimidos, los tejidos de cerebro vivos durante la adquisición de la microscopía de resonancia magnética

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Calorespirometry: Es un enfoque potente y no invasivo para investigar el metabolismo energético celular

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Nanosensores para detectar actividad proteasa en Vivo para el diagnóstico no invasivo

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Medición de metaloproteinasa de la matriz celular Global y actividad metabólica en hidrogeles 3D

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Un modelo de cultivo de matriz-adipocitos extracelulares 3D humano para el estudio de la conversación cruzada metabólica Matrix-Cell

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...