El estudio fue apoyado por subvenciones de la Fondazione Bambino Gesù y Ricerca Corrente (Ministerio de Salud italiano) a C.C.  Nos gustaría agradecer al Dr. Enrico Bertini (Departamento de Neurociencia, Unidad de Enfermedades Neuromusculares y Neurodegenerativas, Laboratorio de Medicina Molecular, Hospital de Investigación Infantil Bambino Gesù), Dr. Stefania Petrini (Centro Central de Microscopía Confocal, Laboratorios de Investigación, Hospital de Investigación Infantil Bambino Gesù), Giulia Pericoli (Departamento de Oncohematología, Terapia Génica y Celular, Hospital de Investigación Infantil Bambino Gesù) y Roberta Ferretti (Departamento de Oncohematología, Terapia Génica y Celular, Hospital de Investigación Infantil Bambino Gesù) para discusiones científicas y ayuda técnica. Maria Vinci recibió una "beca para niños con cáncer del Reino Unido".

1. Recubrimiento de placas de 96 pozos
NOTA: Se probaron diferentes recubrimientos en la misma placa pero en pocillos separados (consulte el Archivo suplementario).
<ol><li>Diluir el Matrigel 1:100 en DMEM. Añadir 100 μL por pocillo a las placas de 96 pocillos e incubar durante 1 h a temperatura ambiente. Después de esto, retire la solución y lave los pocillos con 100 μL de DMEM dos veces.</li>
	<li>Laminina diluida (20 μg/mL, LN-521) en PBS (con calcio y magnesio). Añadir 100 μL al pozo e incubar a 4 °C durante la noche. Al día siguiente realizar dos lavados con DMEM antes de sembrar las células.</li>
	<li>Vitronectina diluida (10 μg/mL) en tampón de dilución. Agregue 100 μL por pocillo a la placa de 96 pocillos e incube durante 1 hora a temperatura ambiente. Lave los pocillos con PBS (sin calcio y magnesio) antes de colocar las células.</li>
	<li>Diluir HU-fibronectina (30 μg/mL) en ddH2O. Añadir 100 μL a los pocillos e incubar a temperatura ambiente durante 45 minutos. Después de esto, lave los pocillos con el medio antes de sembrar las células.</li>
</ol>2. Mantenimiento de iPSCs en cultivo
NOTA: Las iPSC se compraron comercialmente. Las iPSCs se derivaron de fibroblastos humanos sanos y se reprogramaron utilizando tecnología episomal.
<ol><li>Desde el congelador de -80 °C o nitrógeno líquido, descongelar las iPSC criopreservadas en un baño maría a 37 °C. Limpie el vial que contiene las células con etanol al 70% antes de moverlo al gabinete de seguridad biológica.</li>
	<li>Agregue la suspensión celular a 5 ml de medios de cultivo celular precalentados (p. ej., mTeSR1) gota a gota con una pipeta de 1000 μL en un tubo cónico estéril de 15 ml.</li>
	<li>Centrifugar las células a 304 x g durante 5 min a temperatura ambiente (RT).</li>
	<li>Retire el medio y vuelva a suspender el pellet celular en 4 ml de medio de cultivo celular.</li>
	<li>Colocar la suspensión celular en dos pocillos de las placas de 6 pocillos (105 iPSCs por placa de cultivo celular de 6 pocillos), donde los fibroblastos embrionarios de ratón (MEF) han sido chapados previamente. Semillas MEFs dos días antes de emplatar iPSCs a una densidad de 2.4 x 104/cm2 en DMEM (que contiene 10% de suero fetal bovino, 1% de L-glutamina y 1% de penicilina-estreptomicina).</li>
	<li>Después de la siembra, complemente los medios celulares con 10 μM del inhibidor de ROCK Y-27632.</li>
	<li>Cultive las iPSC en MEF durante las primeras 4-5 semanas y luego en condiciones libres de alimentador (condición libre de MEF), utilizando uno de los recubrimientos de interés (consulte el paso 1 y la Tabla 1) en mTeSR1.</li>
	<li>Cuando las iPSCs son 70-80% confluentes, paso 1:4 usando 0,5 mM de tratamiento EDTA durante 3-5 min a RT. Añadir 1 mL de 0,5 mM EDTA para una placa de 6 pocillos (o cantidades proporcionales para otros tipos de placas). Transferir a nuevos pozos en condiciones libres de alimentadores e incubar a 37 °C, 5% CO2, 20%O2.</li>
	<li>Cambie el medio con mTeSR1 nuevo todos los días y divida las celdas cada 2 días.</li>
</ol>3. Caracterización de la confluencia celular
<ol><li>Use 96 placas de pocillos para los experimentos.</li>
	<li>Sembrar 10,000 células por pocillo después de al menos 1 mes de cultivo en condiciones libres de alimentador para asegurarse de que los MEF no fueron pasados. Use portaobjetos de conteo desechables para contar las células con el microscopio óptico.</li>
	<li>Realizar los experimentos por triplicado. Por lo tanto, pruebe cada condición de siembra en tres pozos.</li>
	<li>Realice la adquisición automatizada de imágenes desde el día 1 después de la siembra utilizando un citómetro en modo de campo claro. Realice la adquisición automatizada de imágenes cada 24 h durante 5 días. Para obtener información detallada sobre los parámetros experimentales, consulte el Archivo complementario.</li>
	<li>Utilice el contraste automático y la exposición automática para visualizar mejor las células.</li>
	<li>Establezca la configuración de análisis (consulte Archivo suplementario) para el análisis de confluencia para aplicar una máscara de 60%, 80% y 100% por pozo, con el fin de evaluar los cambios de enfoque debidos a la refracción de la luz en el borde de los pozos. Utilice la configuración de análisis de máscara diferente mencionada anteriormente para analizar la confluencia celular en cada punto de tiempo.</li>
</ol>4. Análisis estadísticos
<ol><li>Reporte los resultados cuantitativos como medias ± error estándar de la media (SEM).</li>
	<li>Para comparar las diferencias generales de las diferentes condiciones de recubrimiento, obtenga datos utilizando las mismas muestras y realice la prueba t de muestra pareada de Student. Los valores de p inferiores a 0,05 se consideran estadísticamente significativos, y todos los valores de p informados son bilaterales.</li>
</ol>5. Caracterización de los microfilamentos citoesqueléticos
<ol><li>Fijar las células con paraformaldehído al 4% (PFA al 4%) en PBS durante 10 min en RT, seguido de dos lavados en PBS (10 min en total).</li>
	<li>Añadir 100 μL de solución de bloqueo (compuesta por 5% BSA, 0,1% Triton en PBS) a cada pocillo durante 1 h a RT.</li>
	<li>Retire la solución de bloqueo y lave las muestras dos veces con PBS durante 10 minutos.</li>
	<li>Añadir 100 μL de la solución de trabajo conjugada con faloidina por muestra e incubar durante 1 h a RT.</li>
	<li>Lave las células dos veces con PBS (10 min en RT).</li>
	<li>Núcleos de tinción con Hoechst 33342 diluidos 1:10000 en PBS durante 10 min a RT.</li>
	<li>Retire la solución de Hoechst y lave las células dos veces con PBS durante 10 minutos cada vez.</li>
	<li>Lave la muestra conH2Oy deje secar bajo una campana química.</li>
	<li>Añadir 100 μL de medios de montaje (es decir, PBS:glicerol, 1:1) para cubrir las células y preservar la fluorescencia de las muestras.</li>
	<li>Observe la celda a Ex/Em 493/517 nm en un microscopio confocal de barrido láser equipado con una fuente láser de luz blanca (WLL) y un láser de diodo de 405 nm. Adquiera imágenes confocales secuenciales utilizando un objetivo de inmersión en aceite HC PLAPO 40x. Utilice la misma potencia láser, divisores de haz, ajustes de filtro, diámetros de agujero de alfiler y modo de escaneo para todas las muestras examinadas.</li>
</ol>

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Los autores no tienen nada que revelar.

El uso de iPSCs para el modelado de enfermedades y el futuro cribado de fármacos junto con su posible aplicación en medicina de precisión lo convierte en una tecnología de gran relevancia y por esta razón creemos que es necesario entender claramente la condición de cultivo in vitro que mejor se asemeja a la situación fisiológica de las células madre embrionarias. En este contexto, probamos diferentes recubrimientos ECM utilizando iPSC de tipo salvaje para comprender las condiciones que permiten que las células ...

Las células madre pluripotentes inducidas (iPSC) se obtienen de células somáticas y se pueden diferenciar en diferentes tipos de células. A menudo se utilizan como un sistema para modelar la patogénesis de la enfermedad o realizar la detección de drogas, y también ofrecen el potencial de ser utilizados en el contexto de la medicina personalizada. Dado que las iPSC tienen un gran potencial, es importante caracterizarlas completamente para su uso como un sistema modelo confiable. Anteriormente mostramos la importancia de cultivar iPSCs en un ambiente hipóxico, ya que estas células dependen de la glucólisis y un ambiente aeróbico puede causar desequilibrio redox1. Las iPSC también son vulnerables a otras condiciones de cultivo, particularmente al entorno extracelular. La optimización de las condiciones de cultivo es un tema clave para mantenerlas sanas y proliferantes. Un cultivo saludable de iPSC conducirá a células sanas diferenciadas que generalmente son el punto final del modelo utilizado para comprender las características moleculares, celulares y funcionales de trastornos humanos específicos o procesos celulares.
En este estudio, se ha utilizado un protocolo simple para probar la confluencia de iPSCs utilizando diferentes condiciones de recubrimiento en pozos separados. Las iPSC requieren una capa alimentadora de fibroblastos embrionarios murinos (MEF) para adherirse correctamente, pero la coexistencia de iPSCs y MEF dificulta la realización de análisis como ARN o extracción de proteínas, ya que hay dos poblaciones de células. Para evitar la capa de alimentación, se han utilizado diferentes proteínas pertenecientes a la matriz extracelular (ECM) para recrear el nicho celular natural y tener un cultivo de iPSC libre de alimentador. En particular, Matrigel es una preparación solubilizada de membrana basal extraída del sarcoma de ratón Engelbreth-Holm-Swarm (EHS), que está enriquecida en proteínas de la matriz extracelular (es decir, laminina, colágeno IV, proteoglicanos de heparán sulfato, entactina/nidógeno y factores de crecimiento)2,3. Las otras condiciones de recubrimiento utilizadas son, en cambio, proteínas purificadas con relevancia conocida en la construcción de las ECM: se sabe que la laminina-521 es secretada por células madre pluripotentes humanas (hPSC) en la masa celular interna del embrión y es una de las lamininas más comunes en el cuerpo después del nacimiento 4,5,6,7,8,9, 10,11; la vitronectina es una matriz de cultivo celular libre de xeno-conocida por apoyar el crecimiento y la diferenciación de hPSC 12,13,14,15,16; La fibronectina es una proteína ECM importante para el desarrollo de vertebrados y la unión y mantenimiento de células madre embrionarias en estado pluripotente 17,18,19,20,21,22,23,24,25. Dado que existen diferentes condiciones de recubrimiento, las comparamos en términos de su efecto sobre la confluencia de las iPSC.

<table border="0" cellpadding="0" cellspacing="0" style="width:1124px;" width="1123">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:589px;">10 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile</td>
			<td style="width:281px;">Corning</td>
			<td style="width:77px;">4488</td>
			<td style="width:176px;">Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL high-clarity polypropylene (PP) conical centrifuge tubes</td>
			<td>Falcon</td>
			<td>352097</td>
			<td>Tool</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">1x PBS (With Ca2+; Mg2+)</td>
			<td>Thermofisher</td>
			<td>14040133</td>
			<td>Medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">1x PBS (without Ca2+; Mg2+)</td>
			<td>Euroclone</td>
			<td>ECB4004L</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile</td>
			<td>Corning</td>
			<td>4487</td>
			<td>Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture microplate, 96 WELL, PS, F-Bottom</td>
			<td>Greiner Bio One</td>
			<td>655090</td>
			<td>Support</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture plate, 6 well</td>
			<td>Costar</td>
			<td>3516</td>
			<td>Support</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM (Dulbecco&#39;s Modified Eagle&#39;s Medium- high glucose)</td>
			<td>Sigma</td>
			<td>D5671</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma</td>
			<td>ED4SS-500g</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epi Episomal iPSC Reprogramming Kit</td>
			<td>Invitrogen</td>
			<td>A15960</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FAST - READ 102</td>
			<td>Biosigma</td>
			<td>BVS100</td>
			<td>Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin</td>
			<td>Merck</td>
			<td>FC010</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td>Reagent</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">H2O</td>
			<td></td>
			<td>MILLIQ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst</td>
			<td>Thermofisher</td>
			<td>33342</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laminin 521</td>
			<td>Stem Cell Technologies</td>
			<td>77003</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>LS25030081</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel</td>
			<td>Corning Matrigel hESC-Qualified Matrix</td>
			<td>354277</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:589px;">Mouse embryonic fibroblasts (MEF)</td>
			<td>Life Technologies</td>
			<td>A24903</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MTESR1 Medium</td>
			<td>Stem Cell Technologies</td>
			<td>85851</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MTESR1 Supplement</td>
			<td>Stem Cell Technologies</td>
			<td>85852</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phalloidin</td>
			<td>Sigma</td>
			<td>P1951</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vitronectin</td>
			<td>Stem Cell Technologies</td>
			<td>7180</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y-27632</td>
			<td>Sigma</td>
			<td>Y0503</td>
			<td>Reagent</td>
		</tr>
	</tbody>
</table>

measuring the confluence of ipscs using an automated imaging system

Este estudio se centra en comprender cómo el cultivo de iPSC en diferentes sustratos de recubrimiento ECM puede afectar la confluencia celular. Se ha establecido un protocolo para evaluar la confluencia de iPSC en tiempo real sin necesidad de contar células en suspensión unicelular para evitar cualquier perturbación del crecimiento. Se utilizó un sistema de análisis de imágenes de alto contenido para evaluar la confluencia de iPCS en 4 ECM diferentes a lo largo del tiempo de manera automatizada. Se utilizaron diferentes entornos de análisis para evaluar la confluencia celular de las iPSCs adherentes y solo se observó una ligera diferencia (a las 24 y 48 horas con laminina) si se aplicó una máscara al 60, 80 o 100%. También mostramos que la laminina conduce a la mejor confluencia en comparación con Matrigel, vitronectina y fibronectina.

En este estudio, investigamos la confluencia de iPSCs cuando se cultivan en diferentes condiciones de recubrimiento. Usando un citómetro, pudimos obtener resultados fácilmente informativos en triplicados en 5 días. Dado que las iPSC apenas se adhieren a recipientes de plástico y es necesario un recubrimiento para apoyar su proliferación, decidimos monitorear la confluencia de iPSCs humanas, ya que es indicativo de la salud del cultivo celular y puede reflejar su potencial de diferenciación. Después de la expansió...

Watch this Scientific Journal Video about Measuring the Confluence of iPSCs Using an Automated Imaging System at JoVE.com

Measuring the Confluence of iPSCs Using an Automated Imaging System

El objetivo del protocolo es comparar diferentes condiciones de recubrimiento de matriz extracelular (ECM) para evaluar cómo el recubrimiento diferencial afecta la tasa de crecimiento de las células madre pluripotentes inducidas (iPSC). En particular, nuestro objetivo es establecer las condiciones para obtener un crecimiento óptimo de los cultivos iPSC.

Medición de la confluencia de iPSCs utilizando un sistema automatizado de imágenes.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

measuring-the-confluence-of-ipscs-using-an-automated-imaging-system

Research

JoVE Journal

Biology

1.9K vistas.  Ospedale Pediatrico Bambino Gesù, IRCCS. El objetivo del protocolo es comparar diferentes condiciones de recubrimiento de matriz extracelular (ECM) para evaluar cómo el recubrimiento diferencial afecta la tasa de crecimiento de las células madre pluripotentes inducidas (iPSC). En particular, nuestro objetivo es establecer las condiciones para obtener un crecimiento óptimo de los cultivos iPSC.

Video: Measuring the Confluence of iPSCs Using an Automated Imaging System

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform routine activities, but prosthetic suspensions using traditional socket technology can prove to be cumbersome and uncomfortable for a person with limb loss.  Moreover, for those with high proximal amputations, limited residual limb length may prevent exoprosthesis attachment all together. Osseointegrated implant technology is a novel operative procedure which allows firm skeletal attachment between the host bone and an implant. Preliminary results in European amputees with osseointegrated implants have shown improved clinical outcomes by allowing direct transfer of loads to the bone-implant interface.  Despite the apparent advantages of osseointegration over socket technology, the current rehabilitation procedures require long periods of restrictive load bearing prior which may be reduced with expedited skeletal attachment via electrical stimulation.  The goal of the osseointegrated intelligent implant design (OIID) system is to make the implant part of an electrical system to accelerate skeletal attachment and help prevent periprosthetic infection. To determine optimal electrode size and placement, we initiated proof of concept with computational modeling of the electric fields and current densities that arise during electrical stimulation of amputee residual limbs.  In order to provide insure patient safety, subjects with retrospective computed tomography scans were selected and three dimensional reconstructions were created using customized software programs to ensure anatomical accuracy (Seg3D and SCIRun) in an IRB and HIPAA approved study.  These software packages supported the development of patient specific models and allowed for interactive manipulation of electrode position and size. Preliminary results indicate that electric fields and current densities can be generated at the implant interface to achieve the homogenous electric field distributions   required to induce osteoblast migration, enhance skeletal fixation and may help prevent periprosthetic infections.  Based on the electrode configurations experimented with in the model, an external two band configuration will be advocated in the future.

There is a need to develop alternative prosthesis attachment due to limb loss attributed to vascular occlusive diseases and trauma. The goal of the work is to introduce an osseointegrated intelligent implant design system to increase skeletal fixation and reduce periprosthetic infection rates for patients needing osseointegrated technology.

Los análisis bioeléctricos de una osteointegrados Sistema Inteligente diseño de los implantes para Amputados

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

Investigación

Biología

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

La medición de la resistencia a la flexión de las células bacterianas usando una trampa óptica

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

Un método simple para imágenes Arabidopsis Uso de hojas perfluorodecalina como un medio de imágenes infiltrativas

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

El uso de un sistema de seguimiento 3D Automatizado de Registro Individual y bajíos de adultos de pez cebra

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

Medición de los efectos de las bacterias en<em&gt; C. Elegans</em&gt; Comportamiento El uso de un ensayo de retención de huevo

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

La formación de imágenes reorganización espacial de una vía de señalización MAPK Usando el sistema de expresión transitoria del Tabaco

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

Estudiar el Sistema de Control de Calidad de la Proteína<em&gt; D. discoideum</em&gt; Uso de imágenes de células vivas con temperatura controlada

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Un Protocolo Automatizado Rápido de Fibra Análisis Población en músculo de rata Secciones representativas Uso de la cadena pesada de miosina inmunohistoquímica

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

Medida flexible de reporteros bioluminiscentes con un automatizado Luciferase Longitudinal imagen optimizada de Gas y temperatura grabadora (cocodrilo)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Establecimiento de un pH extracelular ácido sistema de cultivo

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...