Este estudio fue parte de la Agriculture and Agri-Food Canada A-Base del proyecto &quot;Desarrollo de técnicas de alto rendimiento para las muestras de carne para reducir la brecha phenomic para caracteres de calidad multivariados en la selección asistida por marcadores&quot;. La asistencia especializada de la Unidad de carne de bovino y el personal de procesamiento de carne en el Centro de Investigación Lacombe se aprecia sinceramente. Los autores también desean agradecer la asistencia técnica especializada de Christine Burbidge-Boyd, Fran Costello, Glynnis Croken y Rhona Thacker.

1. Steak Collection <ol><li> Tras la clasificación a las 24 horas - 48 horas después de la masacre, el músculo longissimus (ya sea el extremo torácica y / o lumbar) se elimina de canales de ganado bovino y se recogió para el análisis de la calidad. </li><li> El músculo se recorta de la grasa subcutánea y fuera cuadrado. El músculo se corta entonces en 2,5 cm (1 pulgada) de filetes tras la seguridad y los protocolos de manipulación de alimentos. Tras el corte de filetes individuales, las etiquetas apropiadas (número del animal, lado, la ubicación del músculo, o el tiempo) se colocan en los respectivos filete. </li><li> Filetes A continuación se preparan para el almacenamiento. Si quieren seguir siendo fresca, se colocan en una bandeja y se llevan a la parrilla. Si las carnes frescas no se pueden cocinar de inmediato, que necesitan ser cubiertos con plástico hasta que la cocción pueda comenzar, para prevenir la deshidratación. Si filetes deben ser envejecido en un refrigerador o congelado, el peso se registra con dos decimales antes de ser colocado en un oxígeno impermeable ba vacío paqueteg y sellado utilizando una máquina de envasado al vacío. Filetes se colocan en sus respectivos lugares de almacenamiento a largo plazo. </li></ol> 2. Procedimiento de cocción <ol><li> Si la muestra ha sido congelada, saque del congelador y coloque en un refrigerador de 4 ° C para descongelar durante la noche. </li><li> En el día de la cocción, precalentar una parrilla-hogar abierto a 210 ° C para la cocción de la muestra, y preparar el sistema de registro de temperatura y el ordenador mediante la inserción de los termopares en los puntos de venta del dispositivo de exploración. Engrase ligeramente la parrilla con manteca vegetal de aproximadamente 10 minutos antes de que comience la cocción. </li><li> Antes de comenzar a cocinar, saque los filetes de la nevera. Retire la muestra de su embalaje y secar el exceso de humedad de la carne con papel de cocina. Registre el peso bruto de la carne con dos decimales. </li><li> Insertar un termopar en el punto medio de la carne a lo largo de su eje longitudinal. Inicie el escaneado termopar para registrar los datos de cocción. </li><li> Coloque elcarnes a la parrilla, manteniendo el termopar paralela a la superficie de cocción plana de la carne y la parrilla. Si la sonda no se mantiene paralelo, que puede dar lugar a lecturas de temperatura inadecuadas. </li><li> Cocine los filetes a una temperatura interna de 35,5 ° C, y luego voltear la muestra a lo largo y continuar la cocción a una temperatura interna definitiva de 71 ° C. Una vez que la temperatura interna es de 71 ° C, retire la muestra de la parrilla y tire del termopar de la carne. </li><li> Si se desea un peso de cocción, deje que la carne se enfríe un poco (1-2 min), luego seque la muestra con una toalla de papel para eliminar el exceso de humedad y el peso de la muestra con dos decimales. </li><li> Si se requieren datos de cocción, guardar los datos escaneados termopar. </li></ol> 3. Ajuste del ángulo y de la preparación de muestras <ol><li> Si bien la muestra está todavía caliente de la parrilla, hacer un corte 1-2 cm desde el extremo lateral de la carne para crear un extremo cuadrado. </li><li> Una sección de 5 cm en la mujer en el desarrolloª de la carne se prepara mediante el uso de una &quot;caja de muestras de tamaño &#39;con una muesca cuchillo a 5 cm, en paralelo al corte original. </li><li> El ángulo de orientación de la fibra muscular se puede medir mediante la colocación de un transportador en la superficie del cuchillo de corte de la carne. </li><li> El cuadro de corte en ángulo variable se establece a continuación, el ángulo correspondiente de la muestra por deslizamiento de la placa inferior de metal personalizado con los agujeros inferiores para el guiado de la cuchilla de doble hoja. Ajuste las perillas para asegurar la placa metálica inferior en el ángulo correspondiente. </li><li> Coloque la sección de 5 cm de la carne en la zona de corte de la caja de corte en ángulo variable, de manera que la dirección de la fibra muscular es paralelo al ángulo de la cuchilla de doble hoja. </li><li> El cuchillo de doble hoja se utiliza para cortar una rodaja de 1 cm en el lugar anteriormente cortado 5 cm pieza, así que asegúrese de que el cuchillo pasará a través de la muestra en el ángulo correspondiente. Al colocar la carne, asegúrese de que está en contacto con el borde de la zona de corte más cercano alusuario, ya que el movimiento de corte se tire de la carne hacia el usuario. </li><li> Para cortar, insertar el cuchillo de doble hoja en la caja de corte y tire el cuchillo para cortar la carne en una tira de 1 cm. Para evitar el desgarro de la carne, use un movimiento ligero mientras se tira el cuchillo hacia el usuario. Mantener las hojas de cuchillo contra un borde de la placa de guía para mantener proporciones iguales de corte. </li><li> El resultado debe ser una cálida rebanada 1 cm, grueso que se encuentra a 5 cm de largo, con las fibras corriendo constantemente paralela a la división. Esta parte se utiliza entonces para el análisis de la fuerza de corte en rebanada de la textura de la carne. </li></ol> 4. Análisis de la fuerza de corte <ol><li> Para determinar el análisis de la fuerza de corte, el corte cizalla protocolo vigente (SSF) utiliza los 1 cm por 5 cm rebanar obtenidos con la caja de corte en ángulo variable. Usando una máquina de analizador de textura con una lámina de la rebanada de corte plana, el análisis se realiza en la rebanada caliente. </li><li> Prepare el analizador de textura con un kg celular y set 50 cargala distancia de corte de 48 mm, con una velocidad de la cruceta de 500 mm / min. </li><li> El cizallamiento se lleva a cabo una vez, perpendicular a las fibras musculares para proporcionar un análisis preciso de la fuerza máxima requerida para romper las fibras de la rebanada. </li><li> Una vez que la rebanada ha sido cortado por completo por el centro, guardar y registrar los datos. </li></ol>

<ol>
	<li>Jayasooriya, S. D., Torley, P. J., D'Arcy, B. R., Bhandari, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+high+power+ultrasound+and+ageing+on+the+physical+properties+of+bovine+Semitendinosus+and+Longissimus+muscles.">Effect of high power ultrasound and ageing on the physical properties of bovine Semitendinosus and Longissimus muscles.</a> Meat Science. 75, 628-639 (2007).</li><li>Koohmaraie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+proteinases+and+meat+aging.">Muscle proteinases and meat aging.</a> Meat Science. 36, 93-104 (1994).</li><li>Lepetit, J., Culioli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+meat.">Mechanical properties of meat.</a> Meat Science. 36, 203-237 (1994).</li><li>Honikel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+methods+supported+by+OECD+and+their+use+in+Mediterranean+meat+products.">Reference methods supported by OECD and their use in Mediterranean meat products.</a> Food Chemistry. 59, 573-582 (1997).</li><li>Monin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+methods+for+predicting+quality+of+whole+meat.">Recent methods for predicting quality of whole meat.</a> Meat Science. 49, S231-S243 (1998).</li><li>Shackelford, S., Wheeler, T., Koohmaraie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+slice+shear+force+as+an+objective+method+of+assessing+beef+longissimus+tenderness.">Evaluation of slice shear force as an objective method of assessing beef longissimus tenderness.</a> Journal of Animal Science. 77, 2693-2699 (1999).</li><li>Ju&#225;rez, M., Hui, Y. H., 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=Beef+Texture+and+Juiciness.">Beef Texture and Juiciness.</a> Handbook of meat and meat processing. , 177-206 (2012).</li><li>Kerth, C. R., Montgomery, J. L., Lansdell, J. L., Ramsey, C. B., Miller, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+gradient+in+longissimus+steaks.">Shear gradient in longissimus steaks.</a> Journal of Animal Science. 80, 2390-2395 (2002).</li><li>Janz, J. A. M., Aalhus, J. L., Dugan, M. E. R., Price, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mapping+method+for+the+description+of+Warner-Bratzler+shear+force+gradients+in+beef+Longissimus+thoracis+et+lumborum+and+Semitendinosus.">A mapping method for the description of Warner-Bratzler shear force gradients in beef Longissimus thoracis et lumborum and Semitendinosus.</a> Meat Science. 72, 79-90 (2006).</li><li>Derington, A. 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=Relationships+of+slice+shear+force+and+Warner-Bratzler+shear+force+of+beef+strip+loin+steaks+as+related+to+the+tenderness+gradient+of+the+strip+loin.">Relationships of slice shear force and Warner-Bratzler shear force of beef strip loin steaks as related to the tenderness gradient of the strip loin.</a> Meat Science. 88, 203-208 (2011).</li><li>Shackelford, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slice+Shear+Force.">Slice Shear Force.</a> Beef Facts. , (2009).</li><li>Henrickson, R. L., Mjoseth, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tenderness+variation+in+two+bovine+muscles.">Tenderness variation in two bovine muscles.</a> Journal of Animal Science. 23, 325-331 (1964).</li><li>Ju&#225;rez, M., 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=Quantifying+the+relative+contribution+of+ante-+and+post-mortem+factors+to+the+variability+in+beef+texture.">Quantifying the relative contribution of ante- and post-mortem factors to the variability in beef texture.</a> Animal. FirstView, 1-10 (2012).</li><li>Shanks, B. C., Wulf, D. M., Maddock, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+note:+The+effect+of+freezing+on+Warner-Bratzler+shear+force+values+of+beef+longissimus+steaks+across+several+postmortem+aging+periods.">Technical note: The effect of freezing on Warner-Bratzler shear force values of beef longissimus steaks across several postmortem aging periods.</a> Journal of Animal Science. 80, 2122-2125 (2002).</li><li>Lawrence, T. E., King, D. A., Obuz, E., Yancey, E. J., Dikeman, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+electric+belt+grill,+forced-air+convection+oven,+and+electric+broiler+cookery+methods+for+beef+tenderness+research.">Evaluation of electric belt grill, forced-air convection oven, and electric broiler cookery methods for beef tenderness research.</a> Meat Science. 58, 239-246 (2001).</li><li>Aalhus, J., Ju&#225;rez, M., Aldai, N., Uttaro, B., Dugan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meat+preparation+and+eating+quality.">Meat preparation and eating quality.</a> , 1058-1063 (2009).</li><li>Sa&#241;udo, C., 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=The+effects+of+slaughter+weight,+breed+type+and+ageing+time+on+beef+meat+quality+using+two+different+texture+devices.">The effects of slaughter weight, breed type and ageing time on beef meat quality using two different texture devices.</a> Meat Science. 66, 925-932 (2004).</li></ol>

No tenemos nada que revelar.

El cuadro de ángulo ajustable asegura la hoja SSF siempre tijeras fibras musculares en un verdadero ángulo perpendicular, en lugar de aproximadamente perpendicular que podría suceder con un corte de 45 ° de ángulo fijo 8. La aplicación de la caja de corte en ángulo variable permite para representaciones más precisas de la fuerza de corte máxima en una muestra, y es en esta base de la mejora de las técnicas de análisis de calidad objetiva de que el cuadro de corte en ángulo variable se desarrolló....

La ternura es uno de los atributos más importantes de la calidad de la carne 1. Inconsistencia en la suavidad de la carne se ha identificado como uno de los principales problemas que enfrenta la industria de la carne 2. La fuerza (WBSF) prueba de Warner-Bratzler cizalladura, caracterizado por un agujero triangular en una placa de corte mecanizado con precisión, es el método más extendido utilizado para indicar la carne sensibilidad sensorial 3,4, ya que es el método instrumental que podría decirse que ha mostrado el mejor correlación con los resultados del panel sensorial para dureza carne 5. Sin embargo, la rebanada de cizallamiento protocolo de fuerza (SSF) se ha convertido en una técnica importante para el análisis de la textura muscular y la ternura 6, como una alternativa al protocolo estándar WBSF 7. Es beneficioso en casos en los que necesitan un análisis rápido o un alto número de muestras a procesar. Para la SSF, un solo núcleo se toma de la carne cuando todavía está caliente, en comparación con los múltiples núcleos (3-6) tomada de la carne por lo general popaer 24 horas de refrigeración para el WBSF 6. A partir de esta una rebanada, la SSF analiza la textura media de todo el filete de 8, ya que se ha encontrado que la sensibilidad varía entre los laterales a los lados mediales del filete de 9, con el centro y medio de filetes que tienen la mejor representación para el promedio WBSF . La caída de la SSF es que, dentro de un bistec, desde el lateral a los lados mediales, hay una variación en los valores de fuerza de corte 9, sin embargo, mediante el uso consistente del cuadro de dimensionamiento para hacer secciones de 5 cm, se puede reducir la variabilidad que podría venir el uso de diferentes sectores de la carne. Sin embargo, como algunas carnes son de diferentes tamaños, un segmento 5 cm puede ocurrir en diferentes lugares de la carne, que a su vez podrían afectar los valores de fuerza de corte 10. Por otro lado, el cuadro de corte original diseñado para obtener la sección a ser analizada en el protocolo SSF permite sólo 2 ángulos fijos, 45 ° y 90 °, sin embargo, el músculocambios de orientación de fibra dentro de filetes, y dentro del músculo 11. Shackelford y Wheeler 12 declararon que el ángulo medio longissimus fue 43,8 °, que estaba cerca de 45 °, por lo que se considera apropiado cuando las muestras siempre se obtuvieron de la misma localidad. Sin embargo, Derington et al. 11 informó de un rango en el ángulo de las fibras a lo largo del músculo longissimus entre 33,1 ° y 53,9 °. Por lo tanto, cuando, para fines de investigación, varios filetes del mismo animal necesita ser analizado, la asignación de todos los ángulos de fibra ya sea a 45 ° o 90 ° potencialmente reduce la precisión. El uso de una caja de corte en ángulo variable puede proporcionar una representación más exacta de la fuerza de corte máxima, como tener la capacidad para medir y ajustar el ángulo de corte permite la rebanada para ejecutar constantemente en paralelo con las fibras musculares.

<table border="1">
	<tbody>
		<tr>
			<td>Name of equipment</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Vacuum Packager</td>
			<td>Koch Equipment, Kansas City, MO, USA</td>
			<td>Model UV2100</td>
		</tr>
		<tr>
			<td>Vacuum bags</td>
			<td>Winpak Ltd., Winnipeg, MB, Canada</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Thermocouples and Scanning Device</td>
			<td>Agilent / Hewlett Packard, Santa Clara, CA, USA</td>
			<td>34970A Data Acquisition Switch Unit</td>
		</tr>
		<tr>
			<td>Grill</td>
			<td>Garland Commercial Ranges Ltd., Mississauga, ON, Canada</td>
			<td>Model ED-30-B</td>
		</tr>
		<tr>
			<td>Crisco Vegetable Shortening</td>
			<td>The J.M. Smucker Company, Orrville, OH, USA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sample sizing box</td>
			<td>G-R Manufacturing Co., Manhattan, KS, USA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Angle adjustable box</td>
			<td>Innovation Centre, Red Deer College, Red Deer, AB, Canada</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Texture Analyzer Machine</td>
			<td>Texture Technologies, Hamilton, MA, USA</td>
			<td>Model TA-XT Plus</td>
		</tr>
		<tr>
			<td>Load Cell, 50 kg</td>
			<td>Texture Technologies, Hamilton, MA, USA</td>
			<td>TA-XT Plus</td>
		</tr>
		<tr>
			<td>USDA Warner-Bratzler knife w/guillotine block</td>
			<td>Texture Technologies, Hamilton, MA, USA</td>
			<td>TA-7</td>
		</tr>
		<tr>
			<td>Flat rectangle Blade for Slice Shear Force</td>
			<td>Texture Technologies, Hamilton, MA, USA</td>
			<td>TA-7C</td>
		</tr>
	</tbody>
</table>

introducing an angle adjustable cutting box for analyzing slice shear force in meat

La investigación indica el ángulo de la fibra del músculo longissimus puede variar, dependiendo de la ubicación dentro de un filete y en todo el músculo. En lugar de utilizar los originales fijos 45 ° o 90 ° de ángulo de corte para probar la fuerza de corte, una caja de corte en ángulo variable puede ser ajustada de modo que los ángulos de las cuchillas corresponden al ángulo de la fibra de cada muestra. Dentro de 2 minutos después de la cocción a una temperatura interna de 71 ° C en una parrilla de horno de reverbero fijó en 210 º C, de 1 cm por 5 cm núcleo se corta de la carne, paralelo a la dirección de las fibras musculares, con 2 cuchillas set 1 cm de distancia. Este núcleo caliente se somete a continuación a la rebanada protocolo de la fuerza de corte (SSF) para evaluar la textura de la carne. El uso de la caja de corte en ángulo variable y el protocolo SSF proporciona una representación precisa de la fuerza de corte máxima, como las fibras de rebanada y el músculo son consistentemente paralelo. Por lo tanto, el cuadro de corte de ángulo variable, en conjunción con el protocolo de SSF, se puede utilizar como un alto-ésimoroughput técnica para evaluar con precisión ternura de la carne en diferentes lugares del músculo longissimus y, potencialmente, en otros músculos.

Treinta y un acabado novillos de carne comercial fueron asesinados y sus cadáveres (528-601 kg) se dividieron. Con el fin de crear la variabilidad en la sensibilidad, lado derecho fueron inmediatamente almacenados a 2 ° C, mientras que el lado izquierdo se mantuvieron a 10 ° C durante 3 horas y luego a 2 ° C. Los músculos longissimus de los lados derecho e izquierdo fueron retirados 24 horas después de la masacre. Las muestras de los lados derecho (tratamiento &quot;duro&quot;) se analizaron ese día, mie...

Watch this Scientific Journal Video about Introducing an Angle Adjustable Cutting Box for Analyzing Slice Shear Force in Meat at JoVE.com

Introducing an Angle Adjustable Cutting Box for Analyzing Slice Shear Force in Meat

Fuerza de corte Slice es un método de referencia para el análisis de la textura de carne. El uso de un cuadro de corte ajustable ángulo podría aumentar su precisión para fines de investigación. Los resultados de diferentes lugares dentro de la<em&gt; Longissimus</em&gt; Muscular muestra una alta correlación con Warner-Bratzler metodología de fuerza de corte y alta adaptabilidad potencial de diferentes músculos.

La introducción de un Cuadro de corte Ángulo ajustable para analizar Slice fuerza de corte en la carne

Research indicates the fibre angle of the longissimus muscle can vary, depending upon location within a steak and throughout the muscle. Instead of using ...

introducing-an-angle-adjustable-cutting-box-for-analyzing-slice-shear

Research

JoVE Journal

Biology

12.8K vistas.  Agriculture and Agri-Food Canada. Fuerza de corte Slice es un método de referencia para el análisis de la textura de carne. El uso de un cuadro de corte ajustable ángulo podría aumentar su precisión para fines de investigación. Los resultados de diferentes lugares dentro de la Longissimus Muscular muestra una alta correlación con Warner-Bratzler metodología de fuerza de corte y alta adaptabilidad potencial de diferentes músculos.

Video: Introducing an Angle Adjustable Cutting Box for Analyzing Slice Shear Force in Meat

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

Cultura Cortar organotípicos de los cerebros de ratas E18

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Investigación

Biología

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

De células enteras grabaciones de la Luz evocados excitatorios de las corrientes sinápticas en la porción de retina

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

Objetivos la identificación de los microARN humanos con el sistema de ensayo con luciferasa LightSwitch reportero 3&#39;UTR-Construye y microRNA un imitador de las células adherentes

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Un método para la selección de aptámeros Estructura de conmutación Aplicado a una de nanopartículas de oro ensayo colorimétrico

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

Análisis qPCRTag - Un alto rendimiento, Tiempo real ensayo PCR para Sc2.0 Genotipado

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

Diseño y Desarrollo de aptámeros y oro nanopartículas basados ​​en ensayos colorimétricos para aplicaciones en-el-campo

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

La Asamblea y la Aplicación de &#39;Shear Anillos&#39;: A Novel endotelial Modelo de Orbital, unidireccional y periódica flujo de fluido y la tensión de cizallamiento

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Detección y caracterización de autoensamblaje de proteínas en Vivo por citometría de flujo

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

Automated Charting of the Visual Space of Housefly Compound Eyes

This paper describes the automatic measurement of the spatial organization of the visual axes of insect compound eyes, which consist of several thousands of visual units called ommatidia. Each ommatidium samples the optical information from a small solid angle, with an approximate Gaussian-distributed sensitivity (half-width on the order of 1&#730;) centered around a visual axis. Together, the ommatidia gather the visual information from a nearly panoramic field of view. The spatial distribution of the visual axes thus determines the eye's spatial resolution. Knowledge of the optical organization of a compound eye and its visual acuity is crucial for quantitative studies of neural processing of the visual information. Here we present an automated procedure for mapping a compound eye's visual axes, using an intrinsic, in vivo optical phenomenon, the pseudopupil, and the pupil mechanism of the photoreceptor cells. We outline the optomechanical setup for scanning insect eyes and use experimental results obtained from a housefly, Musca domestica, to illustrate the steps in the measurement procedure.

The protocol here describes the measurement of the spatial organization of the visual axes of housefly eyes, mapped by an automatic device, using the pseudopupil phenomenon and the pupil mechanism of the photoreceptor cells.

Cartografía automatizada del espacio visual de los ojos compuestos de la mosca doméstica

This paper describes the automatic measurement of the spatial organization of the visual axes of insect compound eyes, which consist of several thousands ...

Collection and Long-Term Maintenance of Leaf-Cutting Ants (Atta) in Laboratory Conditions

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled environments enables an enriched comprehension of their biology that can contribute to applied research. This practice is usually employed in population control studies of species that cause economic loss, such as Atta ants. To cultivate their mutualistic fungus, these leaf-cutting ants collect leaves and for this are considered agricultural pests widely distributed throughout the American continent. They are highly socially organized and inhabit elaborated underground nests composed of a variety of chambers. Their maintenance in a controlled environment depends on a daily routine of several procedures and frequent care that are described here. It starts with the collection of queens during the reproductive season (i.e., nuptial flight), which are then individually transferred to plastic containers. Due to the high mortality rate of queens, a second collection can be carried out about 6 months after the nuptial flight, when incipient nests with developed fungus wad are excavated, hand-picked, and placed in plastic containers. In the laboratory, leaves are daily provided to established colonies, and ant-produced waste is weekly removed along with remaining dry plant material. As the fungus garden keeps growing, colonies are transferred to different types of containers according to the experimental purpose. Leaf-cutting ant colonies are placed in interconnected containers, representing the organizational system with functional chambers built by those insects in nature. This setup is ideal to monitor factors such as waste amount, fungus garden health, and the behavior of workers and queen. Facilitated data collection and more detailed observations are considered the greatest advantage of keeping ant colonies in controlled conditions.

Collection and Long-Term Maintenance of Leaf-Cutting Ants Atta in Laboratory Conditions

Here, a protocol is described to successfully collect and maintain healthy Atta (Hymenoptera: Formicidae) ant colonies in laboratory conditions. Additionally, different nest types and configurations are detailed together with possible experimental procedures.

Recolección y mantenimiento a largo plazo de hormigas cortadoras de hojas (Atta) en condiciones de laboratorio

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled ...