El trabajo en el laboratorio de los autores fue apoyado por la Deutsche Forschungsgemeinschaft (http://www.dfg.de; CO 1139/1-1), el Fonds der Chemischen Industrie (http://www.vci.de/fonds) y el Göttingen Centre for Molecular Biology (GZMB). Los autores desean agradecer a Jörg Stülke por sus útiles comentarios y su lectura crítica del manuscrito.

1. Preparación de placas de agar, medios de cultivo, criostocks y precultivos
<ol><li>Prepare los medios de crecimiento y los reactivos requeridos (ver tabla de materiales y reactivos).</li>
	<li>Raya las cepas de B. subtilis (por ejemplo. BP40 (rocG+ gudBCR amyE::PgudB-yfp) y BP52 (rocG+ gudB+ amyE::PgudB-cfp) expresando uno y dos GDHs activos, respectivamente)2 que se utilizarán en el experimento de competencia en placas de agar medio SP para obtener colonias individuales. Incubar las placas durante la noche a 37 °C.</li>
	<li>Tome colonias individuales e inoculte tubos de cultivo estériles que contengan 4 ml de medio líquido LB. Crecen las bacterias durante la noche a 28 °C y 220 rpm.</li>
	<li>Crecer cultivos nocturnos a 28 °C para evitar que las células lise o esporular.</li>
	<li>Mida la densidad óptica de los cultivos a una longitud de onda de 600 nm (OD600)utilizando un espectrofotómetro estándar.</li>
	<li>Si los cultivos han alcanzado un OD600 de 2,0, mezcle 0,75 ml de los cultivos con 0,75 ml de una solución estéril de glicerol al 50% en tubos de reacción de 1,5 ml. El ODfinal 600 debe ser 1.0 para obtener criostocks que contengan aproximadamente 108 células/ml.</li>
	<li>Guarde los tubos a -80 °C.</li>
	<li>Hacer tres precultivos de cada cepa etiquetados con los genes de fluoróforo de codificación cfp e yfp. Inocular los precultivos (tubos de cultivo estériles que contengan 4 ml de lb de medio líquido) con células de 1 μl de criostocks de -80 °C. Incubar los cultivos durante la noche a 28 °C y 220 rpm.</li>
</ol>2. Cocultivación de bacterias, recolección de muestras y almacenamiento de muestras
<ol><li>Preparar recién 100 ml de medio mínimo C-Glc y CE-Glc (ver tabla de reactivos y materiales), y transferir 20 ml de cada medio en matraces estériles de 100 ml.</li>
	<li>Tomar 0,1 ml de los precultivos que se cultivaron durante la noche, diluirlos con 0,9 ml lb medio en una cubeta de 1,5 ml, y determinar el OD600.</li>
	<li>Para el experimento de competición, tome aquellas preculturas de las diferentes cepas que tienen un OD600 similar entre 1.0-1.5.</li>
	<li>Para obtener poblaciones de células mixtas, diluir las células de los precultivos que tenían el OD600 adecuado a un OD600 de 0,05 en 20 ml C-Glc y CE-Glc medio mínimo complementado en matraces de agitación de 100 ml. Para el experimento de competición, las dos cepas deben mezclarse en una proporción de 1:1.</li>
	<li>Tome muestras de 10 ml de los frascos, transfiéralos a tubos de plástico de 15 ml, coseche las células por centrifugación durante 10 min a 4.000 x g en una centrífuga de mesa estándar y deseche el sobrenadante.</li>
	<li>Resuspend las células en 0.5 ml de medio lb fresco, y transferir las células a una taza de reacción estéril de 1.5 ml. Añadir 0,5 ml de glicerol estéril al 50%, mezclar la suspensión mediante un riguroso vórtice y almacenar las muestras en un congelador de -80 °C hasta su posterior tratamiento.</li>
	<li>Incubar las células que quedan en los frascos de agitación durante un máximo de 24 horas a 37 °C y 220 rpm. Mantenga los frascos de agitación en la oscuridad para evitar el blanqueamiento fotográfico de los fluoróforos.</li>
	<li>Tomar muestras adicionales de 0,1 ml después de 7 horas y 24 horas de crecimiento. Mida y observe el OD600 de diluciones 1:10 (en medio mínimo C-Glc o CE-Glc) de las muestras.</li>
	<li>Tome la cantidad apropiada de células de cada cultivo para hacer criostocks que tengan un OD600 de 1.0. Guarde las muestras a -80 °C hasta su posterior tratamiento.</li>
</ol>3. Tratamiento de muestras, revestimiento e incubación para análisis cuantitativos
<ol><li>Después de recoger todas las muestras, descongelar los criostocks, y diluir las células en una solución salina al 0,9% (ver tabla de reactivos y materiales) hasta 10-3.</li>
	<li>Placa 0.1 ml de las 10-3 diluciones en placas de agar medio SP y distribuir las células utilizando pipetas de vidrio estériles.</li>
	<li>Incubar las placas durante la noche a 37 °C en la oscuridad hasta que hayan aparecido colonias individuales.</li>
</ol>4. Contar a los sobrevivientes por microscopía de fluorescencia estéreo para análisis cuantitativo
<ol><li>Divida la parte inferior de la placa de agar con un bolígrafo negro en cuatro partes para una mejor orientación mientras se cuentan los sobrevivientes bajo el microscopio de fluorescencia estéreo.</li>
	<li>Coloque la placa boca abajo bajo el microscopio y enfoque las colonias usando la fuente de luz fría.</li>
	<li>Una vez que las colonias están en foco, cambie al conjunto de filtros apropiado para CFP para visualizar las células sobrevivientes de la cepa etiquetada como CFP. Cuente a los sobrevivientes de esta cepa etiquetando las colonias con un bolígrafo y anote el número.</li>
	<li>Retire las etiquetas con etanol y cambie al conjunto de filtros YFP para visualizar las células sobrevivientes de la cepa etiquetada con yfp. Cuente a los sobrevivientes nuevamente etiquetando las colonias con un bolígrafo y anote el número.</li>
</ol>5. Tratamiento de muestras y microscopía para análisis semicuantitativos
<ol><li>Para obtener cifras ilustrativas, spot 10 μl de la dilución10-4 (aproximadamente 100 células) del paso 3.1 en una placa de agar SP (ver Figura 3).</li>
	<li>Incubar las placas durante la noche a 37 °C en la oscuridad hasta que hayan aparecido colonias individuales.</li>
	<li>Coloque la placa de Petri sin tapa debajo del microscopio y enfoque el punto usando la fuente de luz fría.</li>
	<li>Tome fotos de las manchas para las figuras ilustrativas. Elija un tiempo de exposición apropiado para tomar las fotos de las colonias con la fuente de luz fría.</li>
	<li>Sin mover la placa, cambie al conjunto de filtros CFP, ajuste el tiempo de exposición y tome una foto. Haga lo mismo con el conjunto de filtros YFP y guarde las imágenes para análisis adicionales.</li>
</ol>6. Análisis de datos
<ol><li>Utilice software como Excel para los análisis de datos cuantitativos. Sobre la base de las colonias contadas, calcular el porcentaje de colonias amarillas y azules con respecto al número total de colonias, que se establecen en 100%.</li>
	<li>Utilice los números calculados para crear un diagrama de barras apiladas (consulte la figura 4 y la figura 5). Utilice un programa de procesamiento de imágenes como Adobe Photoshop para construir imágenes combinadas de las imágenes tomadas desde los diferentes puntos. Alternativamente, el software de libre acceso ImageJ, descargable desde <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a> se puede utilizar para el procesamiento de imágenes.</li>
	<li>Abra las imágenes desde un punto que se tomaron con el conjunto de filtros CFP y el conjunto de filtros YFP. Optimice el contraste y el brillo para reducir cualquier fluorescencia de fondo del medio.</li>
	<li>Vaya a una de las imágenes y seleccione todas. Copie la imagen y péguela en la otra imagen.</li>
	<li>Utilice la función "esquivar el color" para combinar las imágenes o superponer las imágenes fluorescentes utilizando la pestaña de canales. Los cuadros combinados de las colonias de diversos medios y de diversos puntos del tiempo representan el crecimiento de las diversas tensiones dentro de la cultura líquida.</li>
</ol>7. Consejos específicos: Experimento de interruptor de tinte y cocultivo de cepas isógenas etiquetadas con cfp y yfp
La expresión de cualquiera de los dos genes que codifican fluoróforos en B. subtilis podría influir en la aptitud y, por lo tanto, en la tasa de crecimiento de las bacterias. Por lo tanto, se recomienda realizar los siguientes experimentos para excluir que la eliminación de una cepa competidora de la población celular durante el cultivo se deba simplemente a un efecto negativo del fluoróforo:
<ol><li>Repita todo el experimento y cocultiva las cepas etiquetadas inversamente(por ejemplo, la cepa etiquetada anteriormente con cfpahora está etiquetada con el gen yfp y viceversa). Aunque inversamente, los resultados obtenidos deben ser comparables a la observación anterior de que una cepa tiene una ventaja de crecimiento selectivo sobre la otra cepa.</li>
	<li>Repita todo el experimento y coculte las cepas isógenas etiquetadas con yfp y cfp (por ejemplo, BP40 (rocG+ gudBCR amyE: :PgudB-yfp) y BP41 (rocG+ gudBCR amyE: :PgudB-cfp)). Estimar el efecto negativo de cualquiera de los dos fluoróforos sobre la aptitud de las bacterias siguiendo la composición de la población celular a lo largo del tiempo.</li>
</ol>

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Los autores declaran que no tienen intereses financieros contrapuestos.

Se han desarrollado varios métodos para analizar la aptitud competitiva de las bacterias16. En muchos casos las bacterias fueron etiquetadas con diferentes casetes de resistencia a los antibióticos17. Similar a nuestro enfoque, el etiquetado de las células con casetes de resistencia a antibióticos permite la evaluación de la aptitud competitiva de las bacterias durante el cocultivo en condiciones de crecimiento definidas. Por otra parte, este método se puede utilizar para determinar la aptitud...

Las bacterias del suelo suelen estar dotadas de redes reguladoras flexibles y amplias capacidades metabólicas. Ambas características permiten a las células ajustar sus vías catabólicas y anabólicas para competir con sus compañeros y otros microorganismos por los nutrientes, que están disponibles en un nicho ecológico dado1. Sin embargo, si las bacterias son incapaces de adaptarse a su entorno, otros mecanismos pueden explicar la supervivencia de una especie. De hecho, como muchas bacterias proliferan rápidamente y las poblaciones pueden alcanzar altas densidades celulares, las subpoblaciones pueden haber acumulado espontáneamente mutaciones beneficiosas que proporcionan a las células una ventaja de crecimiento selectivo y, por lo tanto, aumentan su aptitud. Además, los puntos críticos mutacionales y la mutagénesis adaptativa inducida por el estrés pueden facilitar la evolución de una bacteria inadaptada2,3. Así, la acumulación de mutaciones y el crecimiento bajo selección continua es el origen de la enorme diversidad microbiana, incluso dentro del mismo género4,5. Al igual que en la naturaleza, la conformación de genomas bacterianos también ocurre en el laboratorio debido al cultivo continuo bajo selección. Esto se ejemplifica en la domesticación de la bacteria Gram-positiva B. subtilis, que se utiliza en todo el mundo en la investigación básica y en la industria. En la década de 1940, B. subtilis fue tratado con rayos X perjudiciales para el ADN, seguidos de un cultivo bajo una condición de crecimiento específica6. Las mutaciones que se han acumulado en las bacterias durante su domesticación explican la pérdida de muchas características de crecimiento, es decir, la cepa de laboratorio B. subtilis 168 perdió la capacidad de formar colonias complejas7,8.
Hoy en día, para las bacterias modelo mejor estudiadas Escherichia coli y B. subtilis,una variedad de herramientas poderosas está disponible para manipular genéticamente sus genomas con el fin de abordar cuestiones científicas específicas. A veces la inactivación de un gen de interés causa un defecto de crecimiento severo, que luego es claramente visible en el medio de crecimiento estándar9. Por el contrario, las mutaciones que causan un defecto de crecimiento débil y, por lo tanto, solo afectan ligeramente la aptitud de la cepa a menudo se ignoran. Sin embargo, en ambos casos la incubación prolongada y el paso de las cepas mutantes durante varias generaciones suelen dar lugar a la acumulación de mutantes supresores que han restaurado el fenotipo de la cepa madre2,9. La caracterización de mutantes supresores y la identificación de las mutaciones que han restaurado el defecto de crecimiento de la cepa mutante madre es un enfoque muy útil que permite la elucidación de procesos celulares importantes y a menudonovedosos 10,11.
Estamos interesados en el control de la homeostasis del glutamato en B. subtilis12. Similar a E. coli, B. subtilis responde a la perturbación de la homeostasis del glutamato(es decir,bloqueo en la degradación del glutamato2)por la acumulación de mutantes supresores. Se demostró que las alteraciones genómicas en estos mutantes supresores que fueron adquiridos por mutación espontánea restauran rápidamente la homeostasis del glutamato9,13. Por lo tanto, no es sorprendente que la adaptación de B. subtilis a una condición de crecimiento específica durante la domesticación de la bacteria se refleje en la síntesis enzimática y en las actividades enzimáticas evolucionadas, que están involucradas en el metabolismo del glutamato12. Se ha sugerido que la falta de glutamato exógeno en el medio de crecimiento durante el proceso de domesticación fue la fuerza impulsora para la aparición y fijación de la críptica glutamato deshidrogenasa (GDH) del gen GUDBCR en la cepa de laboratorio 1682,14. Esta hipótesis es apoyada por nuestra observación de que la cantidad reducida de actividad de GDH en la cepa de laboratorio proporciona a las bacterias una ventaja de crecimiento selectivo cuando el glutamato exógeno es escaso2. Por otra parte, el cultivo de una cepa de B. subtilis, sintetizando el GDH GudB, en ausencia de glutamato exógeno da lugar a la acumulación de mutantes supresores que han inactivado el gen gudB 2. Obviamente, la presencia de un GDH catabólicamente activo es desventajosa para la célula porque el glutamato producido endógenamente que de otra manera podría ser utilizado para el anabolismo se degrada a amonio y 2-oxoglutarato(Figura 1). Por el contrario, cuando el glutamato es proporcionado por el medio, una cepa de B. subtilis equipada con actividad GDH de alto nivel tiene una ventaja de crecimiento selectivo sobre una cepa que sintetiza solo un GDH funcional. Es razonable suponer que la actividad de GDH de alto nivel permite a las bacterias utilizar el glutamato como segunda fuente de carbono además de otras fuentes de carbono proporcionadas por el medio2 (ver Figura 1). Por lo tanto, la actividad de GDH afecta fuertemente la aptitud de las bacterias, dependiendo de la disponibilidad de glutamato exógeno.
Aquí presentamos un método muy ilustrativo para monitorear y visualizar la competencia intraespecípecíta entre dos cepas de B. subtilis que difieren en un solo lugar geométrico en el cromosoma(Figura 2). Las dos cepas fueron etiquetadas con los genes yfp y cfp que codifican los fluoróforos YFP y CFP, y cocultivadas bajo diferentes condiciones nutricionales. Mediante el muestreo a lo largo del tiempo y el enchapado de diluciones apropiadas en placas de agar, los sobrevivientes en cada uno de los cultivos podrían ser fácilmente monitoreados utilizando un microscopio de fluorescencia estéreo común. El procedimiento descrito en este papel es fácil de realizar y conveniente visualizar la extensión y la eliminación clónicas rápidas de mutaciones beneficiosas y perjudiciales, respectivamente, en una población de la célula en un cierto plazo.

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	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">(NH4)2SO4&#160;</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">3746</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Agar</td>
			<td style="width:175px;">Difco, USA</td>
			<td style="width:111px;">214010</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="40">
			<td height="61" rowspan="2" style="height:61px;width:363px;">Ammonium ferric citrate (CAF)</td>
			<td rowspan="2" style="width:175px;">Sigma-Aldrich, Germany</td>
			<td rowspan="2" style="width:111px;">9714</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">CaCl2&#160;</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">5239</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Glucose</td>
			<td style="width:175px;">Applichem, Germany</td>
			<td style="width:111px;">A3617</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Glycerol</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">4043</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">K2HPO4 x 3 H2O</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">6878</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">KCl</td>
			<td style="width:175px;">Applichem, Germany</td>
			<td style="width:111px;">A3582</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">KH2PO4</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">3904</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">KOH</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">6751</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">MgSO4 x 7 H2O&#160;</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">P027</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">MnCl2&#160;</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">T881</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="40">
			<td height="61" rowspan="2" style="height:61px;width:363px;">MnSO4 x 4 H2O</td>
			<td rowspan="2" style="width:175px;">Merck Millipore, Germany</td>
			<td rowspan="2" style="width:111px;">102786</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">NaCl</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">9265</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Nutrient broth</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">X929</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Potassium glutamate</td>
			<td style="width:175px;">Applichem, Germany</td>
			<td style="width:111px;">A3712</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Tryptone</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">8952</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Tryptophan</td>
			<td style="width:175px;">Applichem, Germany</td>
			<td style="width:111px;">A3445</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Yeast extract</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">2363</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">1.5 ml Reaction tubes</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">72,690,001</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">2.0 ml Reaction tubes</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">72,691</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">15 ml Plastic tubes with screw cap</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">62,554,001</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Petri dishes</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">82.1473</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">1.5 ml Polystyrene cuvettes</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">67,742</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">15 ml Glass culture tubes&#160;</td>
			<td rowspan="2" style="width:175px;">Brand, Germany</td>
			<td rowspan="2" style="width:111px;">7790 22</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">with aluminium caps</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">100 ml Shake flasks with aluminium caps</td>
			<td style="width:175px;">Brand, Germany</td>
			<td style="width:111px;">928 24</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Sterile 10 ml glass pipettes</td>
			<td style="width:175px;">Brand, Germany</td>
			<td style="width:111px;">278 23</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Incubator (28 and 37 &#176;C)</td>
			<td style="width:175px;">New Brunswick</td>
			<td style="width:111px;">M1282-0012</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="42">
			<td height="85" rowspan="3" style="height:85px;width:363px;">Standard pipette set (2-20 &#956;l, 10-100 &#956;l, 100-1000 &#956;l)</td>
			<td rowspan="3" style="width:175px;">Eppendorf, Germany</td>
			<td style="width:111px;">4910&#160;000.034, 4910 000.042,&#160;&#160;</td>
			<td rowspan="3" style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:111px;">4910 000.042,</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:111px;">4910 000.069&#160;</td>
		</tr>
		<tr height="80">
			<td height="101" rowspan="2" style="height:101px;width:363px;">Table top centrifuge for 1.5 and 2 ml reaction tubes</td>
			<td rowspan="2" style="width:175px;">Thermo Scientific, Heraeus Fresco 21, Germany</td>
			<td rowspan="2" style="width:111px;">75002425</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="60">
			<td height="81" rowspan="2" style="height:81px;width:363px;">Table top centrifuge for 15 ml plastic tubes</td>
			<td rowspan="2" style="width:175px;">Heraeus Biofuge Primo R, Germany</td>
			<td rowspan="2" style="width:111px;">75005440</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="80">
			<td height="101" rowspan="2" style="height:101px;width:363px;">Standard spectrophotometer</td>
			<td rowspan="2" style="width:175px;">Amersham Biosciences Ultrospec 2100 pro, Germany</td>
			<td rowspan="2" style="width:111px;">80-2112-21&#160;</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">Stereofluorescence microscope&#160;</td>
			<td style="width:175px;">Zeiss SteREO Lumar V12, Germany</td>
			<td style="width:111px;">495008-0009-000</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Freezer (-20 and -80 &#176;C)</td>
			<td style="width:175px;">-</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Fridge (4 &#176;C)</td>
			<td style="width:175px;">-</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">Autoclave</td>
			<td style="width:175px;">Zirbus, LTA 2x3x4, Germany</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">pH meter</td>
			<td style="width:175px;">pH-meter 766, Calimatic, Knick, Germany</td>
			<td style="width:111px;">766</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="40">
			<td height="61" rowspan="2" style="height:61px;width:363px;">Vortex</td>
			<td rowspan="2" style="width:175px;">Vortex&#160; 3, IKA, Germany</td>
			<td rowspan="2" style="width:111px;">3340000</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="21">
			<td height="64" rowspan="3" style="height:64px;width:363px;">Balance</td>
			<td rowspan="3" style="width:175px;">CP2202S, Sartorius, Germany</td>
			<td style="width:111px;">replaced by</td>
			<td rowspan="3" style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:111px;">CPA2202S</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:111px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Black pen (permanent marker)</td>
			<td style="width:175px;">Staedler, Germany</td>
			<td style="width:111px;">317-9</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Powerpoint program</td>
			<td style="width:175px;">Microsoft, USA</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">Office Excel program</td>
			<td style="width:175px;">Microsoft, USA</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">Program for data processing</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">Adobe Photoshop CS5</td>
			<td style="width:175px;">Adobe, USA</td>
			<td style="width:111px;">replaced by CS6, download</td>
			<td style="width:167px;">Computer program for image processing</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Computer</td>
			<td style="width:175px;">PC or Mac</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">ZEN pro 2011 software for the stereofluorescence microscope</td>
			<td style="width:175px;">Zeiss, Germany</td>
			<td style="width:111px;">410135 1002 110</td>
			<td style="width:167px;">AxioCam MRc Rev. Obtained through Zeiss</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:815px;">Specific solution recipes</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">SP medium</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">8 g Nutrient broth</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">0.25 mg MgSO4 x 7 H2O</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">1 g KCl</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">if required, add 15 g agar for solid SP medium</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">1 ml CaCl2 (0.5 M), sterilized by filtration</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">1 ml MnCl2 (10 mM) sterilized by filtration</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:815px;">2 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">LB medium</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 g Tryptone</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">5 g Yeast extract</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 g NaCl</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">if required, add 15 g agar for solid LB medium</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">C-Glc minimal medium</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">200 ml 5 x C salts</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml L-Tryptophan (5 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml III&#8217; salts</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">25 ml Glucose (20%), autoclaved for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">CE-Glc minimal medium</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">200 ml 5 x C salts</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml L-Tryptophan (5 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml III&#8217; salts</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">20 ml Glutamate (40%)</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">25 ml Glucose (20%), autoclaved for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">5 x C salts&#160;</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">20 g KH2PO4</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">80 g K2HPO4 x 3 H2O</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">16.5 g (NH4)2SO4</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">III&#8217; salts</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">0.232 g MnSO4 x 4 H2O</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">12.3 g MgSO4 x 7 H2O</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">40% Glutamate solution</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">200 g L-Glutamic acid</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">adjust the pH to 7.0 by adding approximately 80 g KOH</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 0.5 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">0.9% Saline (NaCl) solution</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">50% Glycerol solution</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">295 ml Glycerol (87%)</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 0.5 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:815px;">Bacteria (All strains are based on the Bacillus subtilis strain 168)</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:648px;">Bacillus subtilis BP40 (rocG+ gudBCR amyE::PgudB-yfp)&#160;</td>
			<td rowspan="4" style="width:167px;">Laboratory strain collection</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:648px;">Bacillus subtilis BP41 (rocG+ gudBCR amyE::PgudB-cfp)&#160;</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:648px;">Bacillus subtilis BP52 (rocG+ gudB+ amyE::PgudB-cfp)</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:648px;">Bacillus subtilis BP156 (rocG+ gudB+ amyE::PgudB-yfp)</td>
		</tr>
	</tbody>
</table>

monitoring intraspecies competition in a bacterial cell population by cocultivation of fluorescently labelled strains

Muchos microorganismos como las bacterias proliferan extremadamente rápido y las poblaciones pueden alcanzar altas densidades celulares. Pequeñas fracciones de células en una población siempre tienen mutaciones acumuladas que son perjudiciales o beneficiosas para la célula. Si el efecto de aptitud de una mutación proporciona a la subpoblación una fuerte ventaja de crecimiento selectivo, los individuos de esta subpoblación pueden superar rápidamente e incluso eliminar por completo a sus compañeros inmediatos. Así, los pequeños cambios genéticos y la acumulación selección-impulsada de células que han adquirido mutaciones beneficiosas pueden llevar a un cambio completo del genotipo de una población de la célula. Aquí presentamos un procedimiento para supervisar la rápida expansión clonal y la eliminación de mutaciones beneficiosas y perjudiciales, respectivamente, en una población celular bacteriana a lo largo del tiempo por cocultivación de individuos marcados fluorescentemente de la bacteria modelo Gram-positiva Bacillus subtilis. El método es fácil de realizar y muy ilustrativo para mostrar la competencia intraespecies entre los individuos en una población celular bacteriana.

El método descrito aquí se aplicó con éxito para visualizar la competencia intraespecies en una población celular que consiste en cepas de B. subtilis que fueron etiquetados con los genes cfp e yfp que codifican los fluoróforos CFP y YFP, respectivamente. Como se muestra en la Figura 3,el método se puede utilizar para visualizar la competencia intraespecies de una manera muy ilustrativa. Al detectar las muestras en áreas pequeñas, la composición clonal de la població...

Watch this Scientific Journal Video about Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains at JoVE.com

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Las bacterias pueden acumular mutaciones perjudiciales o beneficiosas durante su vida. En una población de células, los individuos que han acumulado mutaciones beneficiosas pueden superar rápidamente a sus semejantes. Aquí presentamos un procedimiento simple para visualizar la competencia intraespecies en una población de células bacterianas a lo largo del tiempo utilizando individuos marcados fluorescentemente.

Monitoreo De La Competencia Intraespecies En Una Población De Células Bacterianas Por Cocultivación De Cepas Marcadas Fluorescentemente

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a ...

monitoring-intraspecies-competition-bacterial-cell-population

Research

JoVE Journal

Biology

8.4K vistas.  Georg-August University. Las bacterias pueden acumular mutaciones perjudiciales o beneficiosas durante su vida. En una población de células, los individuos que han acumulado mutaciones beneficiosas pueden superar rápidamente a sus semejantes. Aquí presentamos un procedimiento simple para visualizar la competencia intraespecies en una población de células bacterianas a lo largo del tiempo utilizando individuos marcados fluorescentemente.

Video: Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Title Cell Encapsulation by Droplets

Título encapsulación celular a través de gotitas

Investigación

Biología

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivación de células estaminales embrionarias humanas por Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Seguimiento de los cambios dinámicos en niveles de calcio mitocondrial de la apoptosis mediante un sensor de calcio genéticamente codificados

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

La activación de la apoptosis mediante microinyección citoplasmática del citocromo C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

Seguimiento de las actividades de quinasa y fosfatasa través del ciclo celular por Proporcional FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking1,2 but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases3.
Yet their existence is still a matter of controversy4,5. Indeed, lipid raft size has been estimated to be around 20 nm6, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized7,8. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells.
Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts9,10. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models11.
FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently12. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change12.

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS

A technique to probe the lipid raft partitioning of fluorescent proteins at the plasma membrane of living cells is described. It takes advantage of the disparity in diffusion times of proteins located inside or outside of lipid rafts. Acquisition can be performed dynamically in control conditions or after drug addition.

Determinación de la balsa de lípidos Partición de las sondas marcadas con fluorescencia en las células vivas mediante espectroscopia de correlación de fluorescencia (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A largo plazo Prueba de toxicidad letal con el Crustáceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Perfiles de Células Madre Embrionarias Humanas individual por RT-PCR cuantitativa

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Análisis de glucocorticoides fecales: Monitoreo suprarrenal no invasiva en los équidos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and product development. Stress-strain relationships are typically obtained from mechanical testing data to enable comparative assessment among samples and in some cases identification of constitutive mechanical properties. However, errors may be introduced through the use of average strain measures, as significant heterogeneity in the strain field may result from geometrical non-uniformity of the sample and stress concentrations induced by mounting/gripping of soft tissues within the test system. When strain field heterogeneity is significant, accurate assessment of the sample mechanical response requires measurement of local strains. This study demonstrates a novel biomechanical testing protocol for calculating local surface strains using a mechanical testing device coupled with a high resolution camera and a digital image correlation technique. A series of sample surface images are acquired and then analyzed to quantify the local surface strain of a vascular tissue specimen subjected to ramped uniaxial loading. This approach can improve accuracy in experimental vascular biomechanics and has potential for broader use among other native soft tissues, engineered soft tissues, and soft hydrogel/polymeric materials. In the video, we demonstrate how to set up the system components and perform a complete experiment on native vascular tissue.

We describe the use of digital image correlation to characterize the local surface strain field on vascular tissue samples subjected to uniaxial tensile testing. These measurements facilitate precise quantification of the sample mechanical response and the generation of constitutive stress-strain relations.

El uso de Correlación Digital de Imágenes para caracterizar cepas locales en muestras de tejido vascular

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and ...