Producido por Loredana Manfra del Instituto para la protección del medio ambiente y la investigación (ISPRA-Departamento de Monitoreo de la Calidad Ambiental) y Federica Savorelli de la Agencia Regional para la Protección del Medio Ambiente (Emilia-Romaña, Italia). Los miembros de referencia para el subgrupo de trabajo sobre el crustáceo Artemia en el Grupo de Métodos biológicos - Agua salada / agua salobre y de sedimentos de la Comisión UNICHIM de la Calidad del Agua (el instituto para el funcionamiento de las normas técnicas en el sector químico). Grabación y edición de Marco Pisapia de Ispra (Unidad de la web). Producción de Coordinación de Anna Maria Cicerón y Erika Magaletti del ISPRA (Departamento de Monitoreo de la Calidad Ambiental). Nos gustaría agradecer a la Agencia Regional para la Protección del Medio Ambiente de Emilia-Romaña, Ferrara Rama, el Grupo de Métodos biológicos - Agua salada / salobreEl agua y los sedimentos de la Comisión UNICHIM de Calidad del Agua y Migliore Luciana, la Universidad Tor Vergata (Roma) por su cooperación; Rossella Boscolo y Massimo Gabellini de Ispra (Departamento de Prevención y Mitigación de Impactos) por su apoyo financiero; Matassa Fabio y Londres, el Escuela de Idiomas en Roma por su apoyo lingüístico.

1. Método Estándar Nuestras pruebas consiste en exponer larvas de Artemia a un intervalo de concentraciones de la sustancia o de una muestra de ensayo con el fin de determinar los niveles de concentración o dilución que pudieran causar la muerte del 50% de los organismos (CL 50) expuestos durante 14 días y cumplir con las condiciones definidas por este método. Si es necesario y posible, la siguiente también se puede determinar: a) el nivel de concentración que provoca la muerte de un 20% de los organismos expuestos (LC 20), b) el más alto nivel de concentración analizada que no determina una tasa de mortalidad superior a la de el control negativo (NOEC); el menor nivel de concentración de ensayo que determina, después de 14 días, una tasa de mortalidad mayor que la del control negativo (LOEC). 2. Ensayo de Materiales 2.1 huevos de Artemia Para llevar a cabo las pruebas, Artemia franciscana </emLas larvas&gt; de menos de 48 horas de edad (nauplios) son obligatorios, en la etapa II o III, obtenido a partir de los huevos recién nacidos 5. Los huevos o quistes certificados utilizados para la realización de análisis están disponibles comercialmente, por ejemplo, de la División de Control de Calidad Investigación, EE.UU. Agencia de Protección Ambiental, Cincinnati, OH 45268, EE.UU. o desde el Laboratorio de Investigaciones Biológicas de la Contaminación Acuática de la Universidad de Gante, Bélgica. 2,2 agua de dilución Agua salada sintética se puede utilizar como agua de dilución, preparada por disolución de reconocidos reactivos de calidad analítica o una formulación disponible comercialmente en agua destilada o desionizada. Mezclas de sal para crear el agua salada ideal son comercialmente disponibles, por ejemplo, las mezclas del Océano instantáneos. Se recomienda un agua de dilución con una salinidad igual a 35 (± 2) PSU, así como una concentración de oxígeno disuelto de más de 80% del valor de saturación y una temperatura de 25 (± 2) y°, C. Después de aireación durante 48 horas y la filtración en filtros con una porosidad de al menos 0,45 m, la dilución se pueden almacenar durante un máximo de 30 días en la oscuridad a una temperatura de 0-4 ° C. 2.3 cultivo de algas Se recomienda que los organismos de ensayo son alimentan de microalgas Dunaliella tertiolecta, que crecen exponencialmente y alcanzar densidades de 1.3x10 6 - 2.0x10 6 células / ml. Las pruebas en muestras del medio ambiente requiere de cultivos con densidades superiores a 2.0x10 6 células / ml 6. Se sugiere que Dunaliella tertiolecta crece en un medio de cultivo con agua de mar (30 (± 2) fuente de alimentación y 20 (± 1) ° C) con la adición de nutrientes esenciales, minerales y vitaminas. Algunas indicaciones sobre la composición de las algas y la preparación de los medios de comunicación se presentan en el Apéndice. La densidad celular inicial se sugiere a ser aproximadamente 10 5 células / ml. El cultivo de algas se mantiene en untermostato de ambiente a 20 (± 1) ° C, sistema de iluminación con lámparas fluorescentes de 3000 (± 300) lx, con una relación fotoperíodo de 16 horas de luz y 8 horas de oscuridad. Otras especies de fitoplancton también podría ser utilizado para la alimentación, pero sería necesario realizar estudios comparativos con estas especies de fitoplancton para encontrar el &quot;equivalente&quot; las especies de Dunaliella tertiolecta, debido a las condiciones experimentales (por ejemplo, densidades celulares iniciales, iluminación de luz, temperatura, composición de medios de cultivo y tiempo de exposición) varía entre las especies de fitoplancton. Además de la tolerancia de las microalgas a diferentes contaminantes debe tenerse en cuenta debido a los efectos contaminantes sobre el crecimiento y desarrollo de impacto fitoplancton también a la Artemia. 2.4 Sustancia de referencia La sustancia de referencia en este ensayo es dodecil sulfato de sodio. 2,5 de vidrio para laboratorio <ol><li> Contenedores de prueba con 100 vasos de vidrio de borosilicato (18 ml para realizar las pruebas y 18 para la transferencia de soluciones). </li><li> Un matraz (por ejemplo 500 matraz ml) para preparar la solución estándar. </li><li> Seis frascos (por ejemplo 200 matraces ml) para preparar las soluciones de prueba. </li><li> Vidrio o poliestireno 5 cm de diámetro placas de Petri con desprendible cubre a ser utilizado para la activación de los quistes y transferir Artemia desde el plato de la eclosión de los recipientes de ensayo. </li><li> Pipetas Pasteur de vidrio (con las puntas redondeadas por la llama) para la transferencia de los nauplios. </li><li> De vidrio o de cánulas desechables Pasteur 3 ml pipetas de plástico (para cortar) para la transferencia de Artemia. </li><li> Micropipetas y pipetas graduadas para preparar las soluciones de pruebas. </li><li> Seis cilindros graduados (por ejemplo 50 cilindros ml) para la transferencia de soluciones de evaluación de los matraces a tél probando contenedores. </li></ol> 3. Quiste de activación 3,1. Placas de Petri que contienen quistes + verter el agua Para generar organismos de ensayo, 20 mg de quistes se colocan en una placa de Petri que contiene 12 ml de agua de dilución 48 horas antes del ensayo. 3,2. La incubación en la luz y en la oscuridad La placa de Petri se mantiene a 25 (± 2) ° C y en un 4000 (± 1000) lux (lúmenes por metro cuadrado) intensidad de la luz durante una hora. 3,3. Sustitución del medio utilizando el microscopio Después de 24 horas, las larvas de la eclosión se transfieren a una nueva placa de Petri llena de agua artificial. La transferencia se realiza utilizando el microscopio, es decir, utilizando una fuente de luz de modo que la eclosión, las larvas fototrópico migrar hacia el haz de luz. Una pipeta Pasteur de vidrio se utiliza para realizar la transferencia, asegurando que solamente el naciente larvas are transferido y no los quistes o las larvas todavía en las membranas. 3,4. La incubación en la oscuridad El plato que contiene las larvas se coloca en una cámara termostática oscuridad durante 24 horas a 25 (± 2) ° C. 4. Preparación de las soluciones de prueba 4,1. Un peso de la sustancia + Introducir en el matraz Se recomienda que la solución estándar para la sustancia de prueba se preparó disolviendo 0,5 g de la sustancia en un matraz de 500 ml. El matraz se llena con agua desionizada o destilada y la solución se agita hasta que la sustancia de pruebas se ha disuelto completamente. Las soluciones tienen que ser preparados en el momento de uso, a menos que no se sabe si la sustancia es estable en solución. En ese caso, la solución estándar se pueden preparar hasta dos días antes de la prueba. 4,2. Preparación del control negativo, añadiendo agua de dilución y wi algasª de la pipeta de 10 ml El control negativo se prepara en un matraz (por ejemplo, 200 ml de volumen) mediante la adición de una alícuota de la suspensión de algas en el agua de dilución de modo que una densidad de 10 5 células / ml se obtiene. 4,3. Preparación de las soluciones de pruebas: la adición de agua de dilución, las algas y la solución estándar Se sugiere que las soluciones de ensayo se preparan en cinco matraces de 200 ml mediante la adición de la solución estándar para el agua de dilución en las cantidades especificadas de manera que las concentraciones deseadas para las pruebas se obtiene. Para alimentar los organismos, partes alícuotas de una suspensión microalgas Dunaliella tertiolecta pueden utilizarse y se añadió a alcanzar una densidad de 10 5 células / ml cuando se preparan soluciones de prueba. La siguiente orden de adición, se recomienda: agua de dilución, la suspensión de algas y la solución estándar. Después de la preparación de soluciones de prueba, tienen que ser utilizados en el ensayo tan pronto como sea posible. 4,4. La transferencia de las soluciones de la mufla a los contenedores de prueba, y luego a las placas de Petri Las cantidades iguales (50 ml) de las soluciones de ensayo se introduce en los recipientes de ensayo utilizando la probeta graduada. Tres réplicas para cada concentración se realizan. Para cada serie de pruebas, un contenedor de control se prepara con una cantidad de agua de dilución igual al volumen de las soluciones de ensayo. Volúmenes iguales (aproximadamente 12 ml) de las soluciones de ensayo se introducen en las placas de Petri. Para cada serie de pruebas, un plato de Petri de control se utiliza. 5. Preparación para las pruebas 5,1. Nauplios en la fase II-III Cuarenta y ocho horas después de la activación de quistes, los nauplios llegar a la fase II-III de las larvas y luego son utilizables para la prueba. 5,2. Nauplios de transferencia en las placas de Petri utilizando el microscopio La placa de Petri utilizado para quiste<html> activación se saca de la cámara termostática. Una pequeña cantidad de larvas se transfieren a las placas de Petri que contenían el control y las soluciones de prueba. Esta tarea se va a realizar con el microscopio utilizando una pipeta de diámetro suficientemente grande para que los organismos no se dañan. En esta fase de la prueba, la transferencia se puede realizar con una pipeta Pasteur de vidrio (con punta redondeada por llama) y una fuente de luz situada lateralmente que fomenta la Artemia a agregarse. 5,3. Nauplios transferencia de las placas de Petri en los contenedores de prueba con el microscopio Diez larvas se transfieren desde los platos Petri soluciones de ensayo a los recipientes de ensayo. También esta operación tiene que ser realizado mediante la utilización de la pipeta Pasteur y microscopio. Se recomienda que un volumen que no exceda de 1 ml se transfiere durante el paso de las larvas de no afectar el volumen total del sistema de prueba. e_step &quot;&gt; 5.4. los envases llenos de cubierto con parafilm Los vasos de precipitados de ensayo están cubiertas con parafilm (dejando un hueco para el paso de aire), y se mantuvo a una temperatura de 25 (± 2) ° C durante toda la duración de la prueba y en una iluminación de 900 (± 100) lx con un fotoperíodo razón de 14 horas de luz de 10 horas de oscuridad. 6. Sustitución del Suplemento media y la Alimentación 6,1. Supervivencia de control de la frecuencia Dos días después de la prueba ha comenzado, y después de los días 5, 7, 9 y 12 de la Artemia se observa bajo el microscopio para verificar la tasa de supervivencia y para reemplazar el medio y el suplemento alimenticio. Número de organismos vivos se cuenta en cada recipiente de ensayo. Después de la observación bajo el microscopio y la estimulación ligera mecánica (por ejemplo, tocar las larvas con una pipeta Pasteur de vidrio) los organismos que no muestran un cierto movimiento en torno al 10 segundos debe considerarse muerto. 6,2. Preparación de una solución de ensayo que muestra muestreo algas con una pipeta de 10 ml y de muestreo de la sustancia de ensayo con una micropipeta. La transferencia de las soluciones de la mufla a los recipientes de prueba Aunque las pruebas, las soluciones de ensayo debe ser reemplazado periódicamente, y se preparó en el mismo día que se van a utilizar. Las soluciones de ensayo se hacen de la solución estándar previamente preparada. 6,3. Artemia transferencia, utilizando una pipeta Pasteur de corte, de los contenedores de prueba de edad para los tres nuevos contenedores La transferencia de Artemia se realiza mediante el uso de una pipeta con un diámetro lo suficientemente amplio como para no dañar los organismos. Durante esta fase, un plástico de 3 ml pipeta Pasteur (a cortar) puede ser utilizado. 6,4. Las larvas en un vaso que contiene ya sea en vivo o muerto Artemia Después de 1<html> 4 días de exposición al final de la prueba, el número de larvas supervivientes se contaron y se registran en la hoja de cálculo. LC 50 y / o LC 20, NOEC y LOEC de 14 días, se calculan y registran, así como los límites de confianza al 95% su caso, métodos y cálculo. 7. Los resultados representativos Al final de la prueba (14 días), se calcula el porcentaje de mortalidad para cada concentración, de acuerdo con la siguiente fórmula: (M s / N) * 100 Donde: M s es el número promedio de personas murieron en la muestra analizada N es el número total de personas expuestas El cuadro 1 muestra un ejemplo de los datos de prueba (14d) con Artemia expuestos a dodecil sulfato de sodio. <table border="1"><tbody><tr><td> Las concentraciones (mg / l) </td>&quot;&gt; Número de personas murieron en cada réplica </td><td> Mortalidad (%) </td></tr><tr><td></td><td> Yo </td><td> II </td><td> III </td><td></td></tr><tr><td> El control negativo </td><td> 0 </td><td> 2 </td><td> 1 </td><td> 10 </td></tr><tr><td> 1,56 </td><td> 1 </td><td> 2 </td><td> 0 </td><td> 10 </td></tr><tr><td> 3,12 </td><td> 2 </td><td> 0 </td><td> 2 </td><td> 13 </td></tr><tr><td> 6,25 </td><td> 3 </td><td> 2 </td><td> 3 </td><td> 27 </td></tr><tr><td> 12,5 </td><td> 8 </td><td> 9 </td><td> 10 </td><td> 90 </td></tr><tr><td> 25,0 </td><td> 10 </td><td> 10 </td><td> 10 </td><td> 100 </td></tr><tr><td> 50,0 </td><td> 10 </td><td> 10 </td><td> 10 </td><td> 100 </td></tr></tbody></table> Además, determinar elCL 50 por un valor de trama gráfica gaussiana logarítmica o el uso de métodos estadísticos apropiados (por ejemplo, de Spearman-Karber o método de prueba). El cálculo de LC 50 para los datos de la tabla 1 es 8,18 (7,15-9,37) mg / l. Si la concentración máxima probada causó una menor tasa de mortalidad del 50%, no debe proceder al cálculo del valor LC 50 que luego sería poco fiable o incluso no computable. En este caso, sin embargo, sería apropiado para repetir la prueba mediante la ampliación de la gama de concentraciones ensayadas. Por otra parte, el valor LC 50 más correctamente expresado como mayor que la concentración más alta probada, lo que posiblemente indica el porcentaje de mortalidad en la concentración más alta probada y la mayor concentración corresponde a un efecto que no. Los resultados se consideran válidas si al final de la prueba de las siguientes condiciones se han cumplido: <ol class="lalpha"><li> el control de la tasa media de mortalidades ≤ 20%; </li><li> donde utilizando dodecilsulfato de sodio, la LC 50 a los 14 días está incluido en el 8,0 (± 5) mg / l de intervalo. </li></ol> Si las condiciones anteriores no se han cumplido, todos los datos obtenidos con el mismo lote de organismos debe ser considerada no válida y se repite la prueba.

<ol>
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No hay conflictos de interés declarado.

Artemia es uno de los organismos de prueba más valiosas disponibles para los ensayos de ecotoxicidad y la investigación realizada hasta el momento nos permite afirmar que es posible sostener varias opciones relacionadas con el uso de Artemia en toxicología y ecotoxicología. Las características que hacen de este organismo en una especie adecuada para los bioensayos son: una amplia distribución geográfica, alta adaptabilidad a condiciones ambientales adversas y nutrientes variados, la cultura de l...

Producido por Loredana Manfra del Instituto para la protección del medio ambiente y la investigación (ISPRA-Departamento de Monitoreo de la Calidad Ambiental) y Federica Savorelli de la Agencia Regional para la Protección del Medio Ambiente (Emilia-Romaña, Italia). Los miembros de referencia para el subgrupo de trabajo sobre el crustáceo Artemia en el Grupo de Métodos biológicos - Agua salada / agua salobre y de sedimentos de la Comisión UNICHIM de la Calidad del Agua (el instituto para el funcionamiento de las normas técnicas en el sector químico). Grabación y edición de Marco Pisapia de Ispra (Asesor Científico y Servicios de Documentación). Producción de Coordinación de Anna Maria Cicerón y Erika Magaletti del ISPRA (Departamento de Monitoreo de la Calidad Ambiental). Nos gustaría agradecer a la Agencia Regional para la Protección del Medio Ambiente de Emilia-Romaña, Ferrara y Poder de la BiologGrupo de Metodología en iCal - Agua salada / agua salobre y de sedimentos de la Comisión UNICHIM de la Calidad del Agua por su cooperación; Rossella Boscolo y Massimo Gabellini de Ispra (Departamento de Prevención y Mitigación de Impactos) por su apoyo financiero, y la London School of Languages ​​en Roma por su apoyo lingüístico. <table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número del producto </td></tr><tr><td> Dodecilsulfato sódico </td><td> Sigma-Aldrich </td><td> L4390 </td></tr></tbody></table> Apéndice El alga los medios de comunicación tiene que estar preparado con reactivos analíticos de calidad. Un litro de solución nutriente se preparó disolviendo 300 g de NaNO 3, 30 g de KH 2 g PO 4 y 20 de NH 4 Cl en agua destilada. Cuatro soluciones iniciales (A, B, C, D) De minerales se preparan como sigue: <ul><li> Un litro de solución &quot;A&quot; que contiene 30 g de ZnSO 4 H 2 O, 25 g de CuSO 4 5 H 2 O, 30 g de CoSO 4 7 H 2 O y 20 g de MnSO 4 H 2 O en agua destilada; </li><li> Un litro de solución &quot;B&quot; que contiene 50 g de FeCl 3 • 6 H 2 O en agua destilada; </li><li> Un litro de solución &quot;C&quot; que contiene 25 g de Na 2 MoO 4 • 2 H 2 O en agua destilada; </li><li> Un litro de solución &quot;D&quot; que contiene 50 g de Na 2 EDTA di • 2 H 2 O en agua destilada. </li></ul> Un litro de una solución final de los minerales (solución &quot;E&quot;) se obtiene añadiendo partes alícuotas de las soluciones arriba mencionadas a agua destilada. La siguiente orden de adición se recomienda: 100 ml de solución de &quot;D&quot;, 10 ml de solución de &quot;C&quot;, 10 ml de solución &quot;B&quot; y 10 ml de solución &quot;A&quot;. Tres iniciativassoluciones l (F, G, H) de las vitaminas se preparó como sigue: <ul><li> Un litro de solución &quot;F&quot;, que contiene 100 mg de biotina en agua destilada; </li><li> Un litro de solución &quot;T&quot; que contiene 10 g de tiamina en agua destilada; </li><li> Un litro de solución &quot;H&quot; que contiene 100 mg de cobalamina en agua destilada. </li></ul> Un litro de una solución final de vitaminas (solución &quot;I&quot;) se obtiene añadiendo 10 ml de las tres soluciones iniciales a agua destilada. Un litro de la algas medios se prepara disolviendo 1 ml de la solución nutritiva, 1 ml de la solución mineral final (solución &quot;E&quot;) y 1 ml de la solución de vitaminas final (solución &quot;I&quot;) en agua de mar. Este último puede ser obtenida disolviendo reconocidos reactivos de calidad analítica o una formulación disponible comercialmente en agua destilada o desionizada. Mezclas de sal para crear el agua salada ideal son comercialmente disponibles, por ejemplo, las mezclas del Océano instantáneos. Se trata de recomendacionesDed la utilización de agua de mar con una salinidad de 30 (± 2) fuente de alimentación y temperatura de 20 (± 1) ° C.

long-term lethal toxicity test with the crustacean artemia franciscana

Nuestras actividades de investigación como objetivo el uso de métodos biológicos para la evaluación de la calidad ambiental, con especial referencia al agua salada / agua salobre y de sedimentos. La elección de los indicadores biológicos debe estar basada en el conocimiento científico confiable y, posiblemente, de la disponibilidad de procedimientos normalizados. En este artículo, presentamos un protocolo estandarizado que se utiliza el medio marino crustáceo Artemia para evaluar la toxicidad de productos químicos y / o de matrices ambientales marinas. Los científicos proponen que la artemia (Artemia) es un candidato adecuado para el desarrollo de un bioensayo estándar para la utilización en todo el mundo. Una serie de documentos han sido publicados sobre los efectos tóxicos de diversos productos químicos y sustancias tóxicas en artemia (Artemia). La principal ventaja de este crustáceo de los estudios de toxicidad es la disponibilidad total de los quistes secos, los cuales pueden ser inmediatamente utilizado en las pruebas y difícil cultivo no se exigió <sup&gt; 1,2. Quiste basados ​​en ensayos de toxicidad son baratos, disponible de forma continua, sencilla y fiable y, por tanto una respuesta importante a las necesidades rutinarias de detección de toxicidad, por los requisitos de control industrial o con fines de regulación 3. El método propuesto consiste en la mortalidad como criterio de valoración. El número de supervivientes se contaron y el porcentaje de muertes se calcularon. Las larvas se considera muerto si no realiza movimientos internos o externos durante varios segundos de observación 4. Este procedimiento fue las pruebas estandarizadas de una sustancia de referencia (dodecil sulfato de sodio), y algunos resultados se presentan en este trabajo. En este artículo se acompaña de un vídeo que describe la realización de las pruebas de toxicidad de procedimiento, que muestra todos los pasos relacionados con el protocolo.

Watch this Scientific Journal Video about Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana at JoVE.com

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Este estudio se refiere al desarrollo y la estandarización de un protocolo metodológico para determinar el valor a largo plazo (14 días) toxicidad letal ejercida por las sustancias químicas, aguas residuales industriales o de aguas residuales y líquidos de muestras ambientales en el crustáceo de agua salada,<em&gt; Artemia franciscana</em&gt;.

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 ...

long-term-lethal-toxicity-test-with-the-crustacean-artemia-franciscana

Research

JoVE Journal

Biology

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

19.5K vistas.  Institute for Environmental Protection and Research. Este estudio se refiere al desarrollo y la estandarización de un protocolo metodológico para determinar el valor a largo plazo (14 días) toxicidad letal ejercida por las sustancias químicas, aguas residuales industriales o de aguas residuales y líquidos de muestras ambientales en el crustáceo de agua salada, Artemia franciscana.

Video: Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

A largo plazo de imágenes usando las células de mamíferos Wide-Field Microscopía

Investigación

Biología

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account.
We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies.
We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. 
Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.

We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.

A largo plazo la cultura de muestras humanas del cáncer de pecho y su análisis mediante tomografía óptica de proyección

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

A largo plazo, de alta resolución de imagen confocal Time Lapse de<em&gt; Cotiledones Arabidopsis</em&gt; Epidermis durante la germinación

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a promising model organism for laboratory studies of development, providing a useful outgroup comparison to the well studied arthropod model organism Drosophila melanogaster. In contrast to the syncytial cleavages of Drosophila, the early cleavages of Parhyale are holoblastic. Fate mapping using tracer dyes injected into early blastomeres have shown that all three germ layers and the germ line are established by the eight-cell stage. At this stage, three blastomeres are fated to give rise to the ectoderm, three are fated to give rise to the mesoderm, and the remaining two blastomeres are the precursors of the endoderm and germ line respectively. However, blastomere ablation experiments have shown that Parhyale embryos also possess significant regulatory capabilities, such that the fates of blastomeres ablated at the eight-cell stage can be taken over by the descendants of some of the remaining blastomeres. Blastomere ablation has previously been described by one of two methods: injection and subsequent activation of phototoxic dyes or manual ablation. However, photoablation kills blastomeres but does not remove the dead cell body from the embryo. Complete physical removal of specific blastomeres may therefore be a preferred method of ablation for some applications. Here we present a protocol for manual removal of single blastomeres from the eight-cell stage of Parhyale embryos, illustrating the instruments and manual procedures necessary for complete removal of the cell body while keeping the remaining blastomeres alive and intact. This protocol can be applied to any Parhyale cell at the eight-cell stage, or to blastomeres of other early cleavage stages. In addition, in principle this protocol could be applicable to early cleavage stage embryos of other holoblastically cleaving marine invertebrates.

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a promising model organism for studies of crustacean embryology and comparative arthropod development and evolution. This protocol describes a method for manual removal of single blastomeres from early cleavage stage embryos of Parhyale.

Ablación de una sola célula de ocho células de embriones de los crustáceos anfípodos<em&gt; Parhyale hawaiensis</em

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

Multicapa de montaje para microscopía de luz Hoja de largo plazo del pez cebra

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because the larval body wall and most internal organs are transparent. However, continuous live imaging of intact Drosophila larvae for longer than 30 min has been challenging because it is difficult to noninvasively immobilizeimmobilizing larvae for a long time. Here we present a larval mounting method called LarvaSPA that allows for continuous imaging of live Drosophila larvae with high temporal and spatial resolution for longer than 10 hours. This method involves partially attaching larvae to the coverslip using a UV-reactive glue and additionally restraining larval movement using a polydimethylsiloxane (PDMS) block. This method is compatible with larvae at developmental stages from second instar to wandering third instar. We demonstrate applications of this method in studying dynamic processes of Drosophila somatosensory neurons, including dendrite growth and injury-induced dendrite degeneration. This method can also be applied to study many other cellular processes that happen near the larval body wall.

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

This protocol describes a method for mounting Drosophila larvae to achieve longer than 10 h of uninterrupted time-lapse imaging in intact live animals. This method can be used to image many biological processes close to the larval body wall.

LarvaSPA, un método para montar larvas de Drosophila para imágenes de lapso de tiempo a largo plazo

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

Aislamiento, Transfección y Cultura a Largo Plazo de Cardiomiocitos adultos de ratón y rata

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

Aplicación del movimiento pasivo de la cabeza para generar aceleraciones definidas en las cabezas de roedores

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

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

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

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

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

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

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

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

Cultivo a largo plazo de Caenorhabditis elegans individuales en medios sólidos para la monitorización longitudinal de fluorescencia e intervenciones aversivas

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...