Plantas de Nicotiana benthamiana se cultivan en una cámara de crecimiento con temperatura controlada a 25 ° C. Totalmente expandido hojas sanas de plantas de 3-6 semanas de edad se utilizan. Consejo: Se obtienen mejores resultados mediante el uso de hojas de reciente aparición 1. Agrobacterium protocolo de infiltración transitoria: Día 1 <ol><li> LB racha / rifampicina (25 mg / ml) / kanamicina (100 mg / ml) placas de agar con las acciones de glicerol de Agrobacterium tumefaciens (cepa LBA4404) que contiene los vectores apropiados con el gen de la (s) a ensayar para la muerte celular y el vector que contiene el casete de GUS bajo un promotor constitutivo. Siempre incluyen un control de vector vacío como control negativo. </li><li> Se incuba a 28 ° C durante 2 días. </li></ol> Día 3 <ol><li> Inocular 2 ml de LB con rifampicina (25 mg / ml) y kanamicina (100 mg / ml) con una sola colonia de cada LB / Rifampicina / kanamicina placa previamente rayada. </li><li> Incubar cada cultura por agitación a 28 ° C durante 24 horas a 200 rpm hasta que la densidad máxima de crecimiento se alcanza. </li></ol> Sugerencia: Aglutinación veces se observa, en el que las culturas caso de necesidad de estar bien resuspendido (es decir, pipeteando arriba y abajo) antes de proceder. Día 4 <ol><li> Después de la incubación, aumentar el volumen total de 10 ml con LB fresco (con los antibióticos adecuados) y acetosiringona (concentración final 25 mM). </li><li> Incubar cada cultura por agitación a 28 º C durante otras 16 horas. </li></ol> Día 5 <ol><li> Al día siguiente, lavar dos veces cada cultura mediante la adición de 10 ml (no añadir acetosiringona) medio de la infiltración (10 mM MgSO 4 .7 H 2 O, 9 mM MES, pH 5,6) y se centrifuga a 4000 xg durante 10 min a temperatura ambiente. </li><li> Después de la centrifugación final, resuspender cada cultura, en medio de la infiltración de 5 ml contiene acetosiringona (100 M). </li><li> Medir el OD 600 nm, las mediciones de entre 0,1 y 0,9 son aceptables. Sugerencia: OD 600nm de tan bajas como 0.1 se han utilizado sin ningún problema, sin embargo, el aumento de las EP pueden producir resultados más consistentes. </li><li> Incubar los cultivos durante más tres horas para preparar los cultivos de Agrobacterium para la infección. </li><li> Culturas mezcla que contiene el gen de la (s) a analizar y el control negativo (Agrobacterium que contiene el vector vacío) en una proporción de 1:1 con la cultura que contiene el casete de GUS. </li><li> Infiltrarse en la abaxial (bajo) parte de las nuevas hojas que emergen con un 1 ml de jeringa sin aguja (Fig. 1). Sugerencia: Infiltrarse en tanto la mezcla de control negativo (vector vacío + casete GUS) y la mezcla que contiene el gen que se vaya a analizar (gen x + casete GUS) de hojas opuestas de la misma planta. Utilice plantas por triplicado para cada tratamiento. </li><li> 3 días después de la infiltración de los impuestos especiales se infiltraron hojas y ensayo para la expresión de la proteína GUS. </li></ol> 2. Histoquímico GUS ensayo Día 8 <ol><li> Vacío infiltrarse en el medio de sustrato X-gluc en las hojas eliminado. </li><li> Incubar en la oscuridad a temperatura ambiente durante la noche o hasta que la tinción de azul bien marcada. </li></ol> Día 9 <ol><li> Enjuague con agua destilada. </li><li> Incubar en el 70% de etanol hasta que la clorofila es eliminado, entonces la transferencia de agua destilada. Visualmente evaluar los niveles de expresión de GUS. </li></ol> 3. Ensayo fluorométrico MUG: Día 8 <ol><li> Aislar la proteína total de molienda se infiltraron discos de hoja en un mortero con nitrógeno líquido. Añadir 100 uL de tampón de extracción GUS (50 mM NaPi pH 7,0, 10 mM EDTA, 0,1% Triton X-100, el 0,1% de N-lauroylsarcosine sal de sodio, β-mercaptoetanol (0,7 l / ml), manteniendo la muestra en hielo. </li><li> Centrifugar la suspensión a 14.000 g por 5 min a 4 ° C y tomar el sobrenadante como la proteína soluble total. </li><li> Medir la concentración mediante el uso de un nanodrop de acuerdo a las instrucciones del fabricante. </li><li> Ajustar la concentración de proteínas y 100 mg de proteína soluble total para cada muestra usando un tampón de extracción de GUS. </li><li> Añadir el sustrato MUG (4-metilumbeliferil-β-D-glucurónido trihidrato), preparado en tampón de extracción GUS a una concentración final de 2 mM. Un volumen total de 100 l por muestra (proteína + sustrato MUG) debería ser suficiente. </li><li> Incubar las reacciones de 1 hora a 37 ° C. </li><li> Detener las reacciones añadiendo 800 l de GUS dejar de tampón (0,2 M Na 2 CO 3). </li><li> Medida de fluorescencia utilizando un lector de placas a una longitud de onda de excitación de 365 nm y la longitud de onda de emisión de 455 nm y comparar los valores de una curva estándar de MU. Los niveles de informe de la actividad GUS como nmol MU / mg de proteína soluble total / min. </li></ol> 4. Acciones y soluciones: <ol><li> Acetosiringona (1 M) de acciones (FW = 196.2 g) </sub&gt; - 0.981 g en 5 ml de DMSO </li><li> Kanamicina (100 mg / ml) de archivo - preparar en dH 2 0 - filtro de esterilización </li><li> Rifampicina (25 mg / ml) de valores - preparar en DMSO </li><li> Luria-Bertani (LB) medio líquido de crecimiento: (1 litro) 1% (w / v) Bacto-triptona, 10 g 0,5% (w / v) Bacto-extracto de levadura, 5 g 170 mM de cloruro de sodio 10 g pH - 7 </li><li> Acetosiringona (25 M) = 25 l de una acción M 1 en un volumen final de 1 ml </li><li> Media infiltración: (1 litro) 10 mM MgSO 4 .7 dH 2 0 (FW = 246,48 g), 2,4648 g 9 mM MES (FW = 213,25 g), 1,91925 g pH - 5,6 </li><li> Acetosiringona (100 M) = 100 l de una acción 1M en un volumen final de 1 ml </li><li> GUS tampón de extracción 50 mM NaPi pH 7,0, 10 mM EDTA, 0,1% Triton X-100, el 0,1% de N-lauroylsarcosine sal de sodio, β-mercaptoetanol (0,7 l / ml) </li><li> 2x tampón fosfato 0,2 M NaH 2 PO 4 y 0,2 M Na 2 HPO 4 pH 7 </li><li> X-Gluc sustrato solución Disolver 1 mg de 5-bromo-4-cloro-3-indolil bD-glucurónido (X-gluc) en metanol de 0,1 ml. Añadir 1 ml de tampón fosfato 2x, 20 l de potasio 0,1 M ferricianuro, 10μl Triton X-100 el 10% y 850 l de agua destilada. </li></ol> 5. Los resultados representativos: Siguiendo este protocolo, la pérdida de expresión de GUS se espera cuando se produce la muerte celular. Tenemos al mismo tiempo expresaron el gen reportero β-glucuronidasa (GUS) con un miembro de la Arabidopsis cito-protector de Bcl-2 asociada athanoGene (BAG) de la familia. Colaboramos infiltrado N. benthamiana deja con un cassette de 35S conducido expresión bolsa y el GUS vector pCambia 2301 utilizando LBA4404 cepa de Agrobacterium. Como se muestra en la figura 2, un aumento visible en la tinción de GUS se observó después de esta infiltración. Por el contrario, cuando el conocido pro-apoptóticos de la familia de Bcl-2 BAX se utilizó, una marcada reducción de la tinción de GUS se observó (Fig. 2). En ambos casos, la expresión de GUS estaba visiblemente diferente en comparación con el control. Sin embargo, cuando la diferencia en la expresión es menos evidente, ensayos fluorométricos MUG se puede realizar para quanitate expresión de GUS. <img src="/files/ftp_upload/2680/2680fig1.jpg" alt="Figura 1"/> Figura 1. Ejemplo de la infiltración de la mezcla de Agrobacterium lado abaxial de N. benthamiana hojas con un 1 ml jeringa sin aguja. <img src="/files/ftp_upload/2680/2680fig2.jpg" alt="Figura 2"/> Figura 2. Un vector GUS fue co-expresados ​​en N. benthamiana hojas con el gen anti-muerte, y un conocido inductor de muerte celular. Los niveles de GUS se compararon con un control de vector vacío (GUS control).

<ol>
	<li>Jefferson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GUS+fusions:+,+&#946;-glucuronidase+as+a+sensitive+and+versatile+gene+fusion+marker+in+higher+plants.">GUS fusions: , &#946;-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.</a> EMBO J. 6, 3901-3901 (1987).</li><li>Nishihara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+&#946;-Glucuronidase+Gene+in+Pollen+of+Lily+(Lilium+longiflorum),+Tobacco+(Nicotiana+tabacum),+Nicotiana+rustica,+and+Peony+(Paeonia+lactiflora)+by+Particle+Bombardment.">Expression of the &#946;-Glucuronidase Gene in Pollen of Lily (Lilium longiflorum), Tobacco (Nicotiana tabacum), Nicotiana rustica, and Peony (Paeonia lactiflora) by Particle Bombardment.</a> Plant Physiol. 102, 357-357 (1993).</li><li>Hodal, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Science. 87, 115-115 (1992).</li><li>Kabbage, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BAG+proteins:+a+ubiquitous+family+of+chaperone+regulators.">The BAG proteins: a ubiquitous family of chaperone regulators.</a> Cell Mol. Life Sci. . 65, 1390-1390 (2008).</li><li>Kang, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AtBAG6,+a+novel+calmodulin-binding+protein,+induces+programmed+cell+death+in+yeast+and+plants.">AtBAG6, a novel calmodulin-binding protein, induces programmed cell death in yeast and plants.</a> Cell Death Differ. 13 (1), 84-84 (2006).</li><li>Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Science. 165 (1), 1-1 (2006).</li><li>Takayama, S., Reed, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperone+targeting+and+regulation+by+BAG+family+proteins.">Molecular chaperone targeting and regulation by BAG family proteins.</a> Nat Cell Biol. 3 (10), 237-237 (2001).</li><li>Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+functional+analysis+of+BAG-1:+a+novel+Bcl-2-binding+protein+with+anti-cell+death+activity.">Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity.</a> Cell. 80 (2), 279-279 (1995).</li><li>Vitha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+&#946;-glucuronidase+assay+in+transgenic+plants.">Quantitative &#946;-glucuronidase assay in transgenic plants.</a> Biol. Plant. 35, 151-151 (1993).</li><li>Otha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+Expression+in+Tobacco+of+a+&#946;-Glucuronidase+(GUS)+Reporter+Gene+Containing+an+Intron+Within+the+Coding+Sequence.">Construction and Expression in Tobacco of a &#946;-Glucuronidase (GUS) Reporter Gene Containing an Intron Within the Coding Sequence.</a> Plant Cell Physiol. 31, 805-805 (1990).</li></ol>

<p class="jove_content"&gt; A menudo es difícil de usar las técnicas de detección de células de muerte en las plantas que son comunes en sistemas de mamíferos. En combinación con un sistema reportero GUS, se presenta un método a base de plantas, sensibles para la detección y análisis de los jugadores de la muerte celular. Este método aprovecha el simple hecho de que las células vivas son necesarios para la expresión de GUS a ocurrir. Para asegurar resultados significativos y repetibilidad, es fundamental que las culturas que alberga...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> Rifampicina </td><td> VWR </td><td> IC19549001 </td><td> 25mg/mL en DMSO </td></tr><tr><td> 4&#39;-hidroxi-3 &#39;, 5&#39;-dimethoxyacetophenone (acetosiringona) </td><td> VWR </td><td> TCD2666 </td><td> 0,981 g / ml en DMSO (1M stock) </td></tr><tr><td> 2 - (4-morfolino) etanosulfónico monohidrato de ácido (MES) </td><td> VWR </td><td> EM-6110 </td><td> 1,92 g / L en el agua para hacer 1 litro de los medios de infiltración </td></tr><tr><td> Bacto-triptona </td><td> Pescador </td><td> BP1421-2 </td><td> 10 g / L en el agua para hacer una L de medio LB </td></tr><tr><td> Bacto-extracto de levadura </td><td> VWR </td><td> EM1.03753.0500 </td><td> 5 g / L en el agua para hacer una L de medio LB </td></tr><tr><td> Cloruro de sodio </td><td> VWR </td><td> EM-7710 </td><td> 10 g / L en el agua para hacer una L de medio LB </td></tr><tr><td> Fosfato de sodio monobásico monohidrato </td><td> VWR </td><td> MK-7868 a 12 </td><td> 2,5 g / L en el agua para la toma de 50 mM de tampón NaPi </td></tr><tr><td> Fosfato de sodio dibásico heptahidratado </td><td> VWR </td><td> EMD-SX0715-1 </td><td> 5 g / L en el agua para la toma de 50 mM de tampón NaPi </td></tr><tr><td> Magnesio heptahidrato de sulfato </td><td> VWR </td><td> EM-MX0070-1 </td><td> 2,5 g / L en el agua para hacer 1 litro de los medios de infiltración </td></tr><tr><td> N-Lauroylsarcosine </td><td> VWR </td><td> TCL0151-500G </td><td> 0,1% v / v en buffer GUS </td></tr><tr><td> 5-bromo-4-cloro-3-indoxyl-beta-D-glucurónido cyclohexylammonium sal (X-gluc) </td><td> Oro Biotecnología </td><td> G1281C </td><td> 1mg/100uL en metanol 100% </td></tr><tr><td> 4-metilumbeliferil-μ-D-glucurónido hidrato (MUG) </td><td> Sigma </td><td> M5664 </td><td> 2 mM en 100 uL de tampón de extracción GUS </td></tr><tr><td> Trihidrato de ferrocianuro potásico </td><td> VWR </td><td> EM-PX1460-1 </td><td> De valores de 100 mm para la solución de sustrato X-gluc </td></tr><tr><td> β-metilumbeliferona (MU) </td><td> Sigma-Aldrich </td><td> M1381 </td><td> Por MU tampón estándar utilizar la curva de extracción de GUS </td></tr><tr><td> De carbonato de sodio </td><td> VWR </td><td> EM-SX0395-11 </td><td> 0,2 M en agua para la toma de GUS tampón de detención </td></tr></tbody></table>

a &#946;-glucuronidase (gus) based cell death assay

Hemos desarrollado un novedoso sistema de expresión transitoria de plantas que al mismo tiempo expresa el gen reportero β-glucuronidasa (GUS), con la supuesta reguladores positivos o negativos de la muerte celular. En este sistema, N. benthamiana hojas son co-infiltrado con un casete de expresión 35S impulsado que contiene el gen para ser analizados, y el vector GUS pCambia 2301 utilizando LBA4404 cepa de Agrobacterium como un vehículo. Dado que las células vivas son necesarios para la expresión de GUS que se produzca, la pérdida de la actividad GUS se espera que cuando este gen marcador es co-expresada con reguladores positivos de la muerte celular. Igualmente, el aumento de la actividad GUS se observa cuando los genes anti-apoptóticos se utilizan en comparación con el control de vectores. Como se muestra a continuación, hemos utilizado con éxito este sistema en nuestro laboratorio para analizar los dos jugadores a favor y en contra la muerte. Estos incluyen la planta de anti-apoptótica Bcl-2 asociada athanoGene (BAG) de la familia, así como, los mamíferos conocidos inductores de la muerte celular, tales como BAX. Además, hemos utilizado este sistema para analizar la función de la muerte de truncamientos específicos dentro de las proteínas, lo cual podría proporcionar pistas sobre la posible modificación post-traduccional / activación de estas proteínas. A continuación, presentamos un método rápido y sensible de plantas basado, como un paso inicial en la investigación de la función de la muerte de genes específicos.

Watch this Scientific Journal Video about A Î²-glucuronidase GUS Based Cell Death Assay at JoVE.com

A &amp;#946;-glucuronidase GUS Based Cell Death Assay

Ensayos muerte celular programada de uso común en los sistemas de mamíferos, tales como escalamiento de ADN o los ensayos de TUNEL, a menudo son difíciles de reproducir en las plantas. En combinación con un sistema reportero GUS, se propone una vía rápida, a base de plantas de ensayo transitorio para analizar las propiedades de la muerte potencial de genes específicos.

A β-glucuronidasa (GUS), basada en células de ensayo Muerte

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

a-glucuronidase-gus-based-cell-death-assay

Research

JoVE Journal

Biology

A &#946;-glucuronidase (GUS) Based Cell Death Assay

27.9K vistas.  Texas A&M University. Ensayos muerte celular programada de uso común en los sistemas de mamíferos, tales como escalamiento de ADN o los ensayos de TUNEL, a menudo son difíciles de reproducir en las plantas. En combinación con un sistema reportero GUS, se propone una vía rápida, a base de plantas de ensayo transitorio para analizar las propiedades de la muerte potencial de genes específicos.

Video: A &#946;-glucuronidase GUS Based Cell Death Assay

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

El estudio de biogénesis de la membrana con un ensayo de luciferasa basado en genes

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Investigación

Biología

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios &lt;0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (&lt; 5% events with cytoplasmic PI staining).

An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.

Modificado anexina V / yoduro de propidio ensayo Apoptosis una evaluación precisa de la muerte celular

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

Modificado madre embrionarias de ratón basado en células para la cuantificación de la eficiencia de inducción cardiogénico

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Análisis de RNA Global Síntesis en el nivel de células individuales siguiente Hipoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ultimately kill. Such A/B toxins generally consist of an enzymatically active Asubunit (e.g., ricin toxin A (RTA)) and one or more cell binding Bsubunit(s), which are responsible for toxin binding to specific cell surface receptors. Our current knowledge of how A/B toxins are capable of efficiently intoxicating cells helped scientists to understand fundamental cellular mechanisms, like endocytosis and intracellular protein sorting in higher eukaryotic cells. From a medical point of view, it is likewise important to identify the major toxin trafficking routes to find adequate treatment solutions for patients or to eventually develop therapeutic toxin-based applications for cancer therapy.
Since genome-wide analyses of A/B toxin trafficking in mammalian cells is complex, time-consuming, and expensive, several studies on A/B toxin transport have been performed in the yeast model organism Saccharomyces cerevisiae. Despite being less complex, fundamental cellular processes in yeast and higher eukaryotic cells are similar and very often results obtained in yeast can be transferred to the mammalian situation.
Here, we describe a fast and easy to use reporter assay to analyze the intracellular trafficking of RTA in yeast. An essential advantage of the new assay is the opportunity to investigate not only RTA retro-translocation from the endoplasmic reticulum (ER) into the cytosol, but rather endocytosis and retrograde toxin transport from the plasma membrane into the ER. The assay makes use of a reporter plasmid that allows indirect measurement of RTA toxicity through fluorescence emission of the green fluorescent protein (GFP) after in vivo translation. Since RTA efficiently prevents the initiation of protein biosynthesis by 28S rRNA depurination, this assay allows the identification of host cell proteins involved in intracellular RTA transport through the detection of changes in fluorescence emission.

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

In the manuscript, we describe the use of a yeast-based fluorescence reporter assay to identify cellular components involved in the trafficking and killing processes of the cytotoxic A subunit of the plant toxin ricin (RTA).

Un análisis Simple reportero basado en fluorescencia para identificar los componentes celulares necesarios para la toxina de ricina una cadena (RTA) trata de levadura

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in the biogenesis of melanosomes, a lysosome-related organelle produced by melanocytes. REs deliver the melanocyte-specific cargo to premature melanosomes during their formation. Blockage in the generation of REs, observed in several mutants of Hermansky-Pudlak syndrome, results in hypopigmentation of skin, hair, and eye. Therefore, studying the dynamics (refer to number and length) of REs is useful to understand the function of these organelles in normal and disease conditions. In this study, we aim to measure the RE dynamics using a resident SNARE STX13.

Recycling endosomes are part of the endosomal tubular network. Here we present a method to quantify the dynamics of recycling endosomes using GFP-STX13 as an organelle marker.

El ensayo basado en microscopía para estudiar y analizar los endosomas de reciclaje utilizando el tráfico de SNARE

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in ...

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

Un método paso a paso para detectar anticuerpos neutralizantes contra AAV utilizando un ensayo colorimétrico basado en células

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...

Histology Basics and Cell Death Detection in Honeybee Tissue

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather changes, various pesticides, pathogens, and malnutrition, mainly entering through the mouth and primarily affecting the digestive tracts of adult bees. To understand and prevent the effects of such external and internal stressors on honeybees, one useful research method is the immunohistochemical method. A basic protocol is described to prepare the midgut (ventriculus) and hypopharyngeal glands (HPGs) of adult bees for histological analysis. A detailed methodology is described to assess the level of cell damage and distinguish necrosis from programmed cell death (apoptosis) as a natural process of tissue regeneration. The results of adult honeybee treatment with oxalic acid and pesticides (insecticide and acaricide) and the determination of cell death in the ventriculus and HPGs are presented. The pros and cons of the methodology are also discussed.

Immunohistochemical methods are useful in honeybee research to detect and assess the level of apoptosis and necrosis in the midgut and hypopharyngeal glands of adult bees.

Conceptos básicos de histología y detección de muerte celular en tejido de abeja melífera

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather ...

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous research indicated that multiple pathways could mediate the spontaneous death of neutrophils. A cocktail was developed by simultaneously targeting these pathways, caspases-lysosomal membrane permeabilization-oxidant-necroptosis inhibition plus granulocyte colony-stimulating factor (CLON-G), which prolonged the neutrophil lifespan to greater than 5 days without significantly compromising the neutrophil function. Concurrently, a reliable and stable protocol for assessing and evaluating neutrophil death was also developed. In this work, we show that CLON-G can prolong the neutrophil lifespan in vitro to more than 5 days, and we exhibit the lengthening of the neutrophil lifespan with FACS and confocal fluorescence microscopy. This report introduces procedures for the preparation of CLON-G and showcases an in vitro spontaneous death assay of neutrophils, which can be used for the study of neutrophils and for subsequently interrogating neutrophil death, thus providing a reliable resource for the neutrophil community.

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

This protocol details the preparation of CLON-G to extend the neutrophil lifespan to greater than 5 days and provides a reliable procedure for evaluating neutrophil death with flow cytometry and confocal fluorescence microscopy.

Extensión de la vida útil de los neutrófilos con CLON-G y un ensayo de muerte espontánea in vitro

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous ...

Cuantificación del inicio de la muerte celular a través de imágenes continuas

Studying Cell Death Initiation Using a Digital Microscope

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as the formation of cell death zones on inoculated leaves. Here, a protocol for studying the rate of cell death initiation by imaging inoculated leaves in the time between the cell death initiation and the cell death appearance using a digital microscope is presented. The digital microscope enables a continuous imaging process in desired intervals, which allows an accurate determination of cell death initiation rate up to minutes exactly, as opposed to hours in traditional methods. Imaging with the digital microscope is also independent of light and can therefore be used during day and night without disturbing the circadian rhythm of the plant. Different pathosystems resulting in programmed cell death development could be studied using this protocol with minor modifications. Overall, the protocol thus allows simple, accurate, and inexpensive identification of cell death initiation rate.

Autor destacado: Un enfoque simplificado para estudiar el inicio de la muerte celular en la respuesta hipersensible 

Here we present a protocol for studying the rate of programmed cell death initiation by continuously imaging inoculated leaves following cell death induction.

Estudio de la iniciación de la muerte celular mediante un microscopio digital

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as ...