Este trabajo fue financiado por los Institutos Nacionales de Salud Subvenciones AR055686 y AR060836 y beca postdoctoral de AD por la Asociación Francesa de Distrofia Muscular (AFM). La instalación de imagen celular utilizado en este estudio es apoyado por los Institutos Nacionales de Salud Subvenciones HD040677.

1. Imágenes de células de reparación de membrana Usando granel (grano de cristal) Herir Este protocolo permite el marcado por separado las células dañadas y las que no cicatrizan. La cuantificación de estas poblaciones de células requiere el uso de tres condiciones: 1. Prueba (C1) - Las células se dejan reparar en presencia de Ca 2 +, 2. Control de 1 (sin lesión C2) - Las células se incubaron en presencia de Ca 2 +, pero no heridos, y 3. Control 2 (sin reparación C3) - las células se les permite reparar en ausencia de Ca 2 +. <ol><li> Se cultivan las células a&gt; 50% de confluencia en tres cubreobjetos estériles y lavar C1 y C2 dos veces con CIM a 37 ° C y C3 con PBS a 37 ° C y cubreobjetos de transferencia de juntas tóricas de silicona. </li><li> Añadir 100 l de solución de dextrano FITC precalentado en CIM en C1 y C2 o en PBS en C3. </li><li> Herir membrana plasmática en C1 y C3 por suaves ondulaciones 40 mg de perlas de vidrio sobre el portaobjetos a temperatura ambiente por cubreobjetos inclinación manualmente hacia atrás ysucesivamente 6-8 veces en un ángulo de 30 °. Nota: Para las lesiones leves, asegúrese de que las cuentas de vidrio se distribuyen de manera uniforme, minimizando las lesiones repetidas. Para mejorar la reproducibilidad de lesiones entre las muestras, realización simultánea de la lesiones de perlas de vidrio de las diferentes muestras que se van a comparar. </li><li> Evitar más de rodadura de cuentas, transferir todos los cubreobjetos a una incubadora a 37 ° C en ambiente de CO 2 y permitir la reparación de proceder durante 5 min. </li><li> Sin dejar que las bolas rueden, retire las cuentas de vidrio y FITC dextrano por lavado con CIM (C1 y C2) o PBS (C3) a 37 ° C. </li><li> Lugar cubreobjetos de vuelta en el ring y añadir 100 l de solución precalentada lisina fixable dextrano TRITC en CIM en C1 y C2 o en PBS en C3. </li><li> Incubar a 37 ° C durante 5 min a temperatura ambiente de CO 2. </li><li> Lavar dos veces con cubreobjetos precalentado CIM y fijar con 4% PFA durante 10 minutos a temperatura ambiente. </li><li> Lavar dos veces con PBS y se incuba durante 2 min a temperatura ambiente en el reactivo de Hoechst. </li><li> Lave samples dos veces con PBS, montar en una diapositiva utilizando medio de montaje y células de imagen usando un microscopio de epifluorescencia. </li><li> Usar células de C2 para determinar la intensidad de la tinción de fondo rojo y verde, y utilizar este valor para el umbral de los canales rojo y verde para todas las muestras (C1-C3). </li><li> Puntuación número total de células que son a) verde (lesionado y reparado) yb) de color rojo o rojo y verde (lesionado, pero no pudieron reparar) en muestras C1 y C3. </li><li> Conde&gt; 100 células verdes para cada condición y expresar la fracción de células que han fallado para reparar como un porcentaje de todas las células lesionadas (verde y rojo). </li></ol> 2. Imágenes en vivo de la cinética de la reparación de la membrana de la célula después de la lesión con láser <ol><li> Lavar las células con precalentado CIM y luego poner el cubreobjetos en CIM con el tinte FM. </li><li> Coloque el cubreobjetos en un soporte en la incubadora de etapa superior se mantiene a 37 ° C. </li><li> Seleccione una región de 1-2 mm 2 de la membrana celular, y para irradiar &lt;; 10 mseg con el láser pulsado. Atenuar la potencia del láser a través del software para el 40-50% de la potencia de pico. Potencia óptima permite lesión consistente, pero no letal y esto debe ser determinado por ensayo y error para cada instrumento individual y célula de la línea que se utiliza. </li><li> Para supervisar la reparación, la imagen cada 10 segundos en epifluorescencia y campo claro, comenzando antes de la lesión y continuar durante 3-5 min después de una lesión. Nota: Para control de reparación, repita los pasos 2.1 a 2.4 con las células dañadas en PBS que contenía colorante FM. </li><li> Para cuantificar la cinética de reparación, medir FM celular colorante fluorescente y trazar el cambio en la intensidad (ΔF/F0) durante el curso de formación de imágenes. Estos datos deben promediarse durante más de 10 células de cada condición y se representa como promedio o el valor de la celda individual, según sea necesario. </li></ol> 3. Imaging granel (grano de cristal) lesión inducida Lysosomal Exocitosis Las muestras incluyen siguientes células cultivadas hasta&gt; 50%confluencia: 1. Test (C1) - Las células se permiten reparar en la presencia de Ca 2 +, 2. Control de 1 (C2; Ninguna lesión) - Las células no lesionadas ni incubadas con el anticuerpo primario, y 3. Control 2 (C3; Ninguna reparación) - Las células se permiten reparar en ausencia de Ca 2 +. <ol><li> Lave cubreobjetos C1 y C2 dos veces con CIM a 37 ° C y C3 con PBS a 37 º C y transferirlos en silicona las juntas tóricas. </li><li> Añadir 100 l de precalentado lisina fixable dextrano TRITC en CIM en C1 y C2 y C3 en PBS. </li><li> Herir membrana plasmática en C1 y C3 como en el paso 1.3. </li><li> Evitar más de rodadura de cuentas, transferir todos los cubreobjetos a una incubadora a 37 ° C en ambiente de CO 2 y permitir la reparación de proceder durante 5 min. </li><li> Retire las perlas de vidrio y TRITC dextrano mediante el lavado de los cubreobjetos en medio de crecimiento frío, asegurando de nuevo sin rodadura de las perlas de vidrio en las células. </li><li> Enjuagar los portaobjetos dos veces con medio de crecimiento frío y la transferencia a las juntas tóricas. </li><li> Para C1 y C3, Añadir 100 ml de rata anticuerpo anti ratón LAMP1 en medio de crecimiento completo de frío y añadir el, medio de crecimiento completo fría para C2. </li><li> Para permitir la unión del anticuerpo cubreobjetos se incuba durante 30 min a 4 ° C. </li><li> Lavar los cubreobjetos tres veces con CIM frío y corregir todos los cubreobjetos con PFA al 4% durante 10 min a TA y luego enjuague 3x con la CIM. </li><li> Incubar todos los cubreobjetos en 100 l de solución de bloqueo durante 15 min a TA </li><li> Incubar todos los cubreobjetos en 100 l Alexa Fluor 488 anticuerpo anti rata durante 15 min a 4 ° C. </li><li> Lavar dos veces con PBS y se incuba durante 2 min a RT en Hoechst. </li><li> Lavar las células dos veces con PBS, montar en una diapositiva utilizando medio de montaje y la imagen usando un microscopio de epifluorescencia. </li><li> El uso de imágenes de células C2, determinar la tinción inespecífica de fondo en el rojo (dextrano TRITC) y verde (Alexa Fluor 488 anticuerpos) canales y utilizar estos valores de tinción de fondo con el umbral de los canales rojo y verde para todas las muestras (C1-C3). &lt;/ Li&gt; <li> Utilice los&gt; 100 marcados con rojo células de C1 y C3 para medir la intensidad de la tinción LÁMPARA1 (verde) en células lesionadas. Para un experimento exitoso la tinción LÁMPARA1 en las células de C3 será significativamente menor que en las células de C1. </li></ol> 4. Imágenes en vivo del daño celular membrana Se activa el Tráfico subcelular <ol><li> Fluorescently etiquetar el compartimento de interés transfectando reportero apropiado (por ejemplo CD63-GFP para lisosomas 8) o mediante el uso de colorantes fluorescentes 9. </li><li> Para lisosomas etiquetado con colorante fluorescente, incubar las células cultivadas a 50% de confluencia en medio de cultivo conteniendo FITC-dextrano </li><li> Permitir dextrano a ser endocitosis durante 2 horas (o más largo, según sea necesario para el etiquetado suficiente endosomal con dextrano por la línea celular de interés) en la incubadora de CO 2. </li><li> Lavar las células con medio de cultivo y se incuban precalentadas en ella durante 2 horas en una incubadora de CO2 para permitir a todosendocitosis dextrano a acumularse en el lisosoma. </li><li> Antes de imágenes, enjuague el cubreobjetos precalentado CIM y montar en un soporte de portaobjetos en la incubadora de etapa superior a 37 ° C. </li><li> Realizar campo amplio (para el movimiento a lo largo de la célula) o TIRF (movimiento en la superficie celular y la exocitosis) de imágenes - las células que no tienen abarrotadas FITC dextrano lisosomas marcados son ideales para obtener imágenes de los movimientos y la exocitosis de los lisosomas individuales. </li><li> Alineación de los láseres TIRF para microscopio de imagen: Use 60X o 100X objetivo con&gt; 1,45 NA. Configure el ángulo de incidencia del rayo láser TIRF utilizando el enfoque del fabricante. </li><li> Lesionar la membrana celular mediante la irradiación de una región pequeña (1-2 mm 2) de &lt;100 ms, con el láser pulsado a 40-50% de atenuación. </li><li> Para monitorear la respuesta del lisosoma a la celda de lesiones, celdas de imagen a los 4-6 cuadros / seg durante al menos 2 minutos o más dependiendo de la dinámica de la exocitosis en las células de interés. </li></ol>

<ol>
	<li>Bi, G. Q., Alderton, J. M., Steinhardt, 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=Calcium-regulated+exocytosis+is+required+for+cell+membrane+resealing.">Calcium-regulated exocytosis is required for cell membrane resealing.</a> Cell Biol. 131, 1747-1758 (1995).</li><li>Togo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+plasma+membrane+stimulates+rearrangement+of+microtubules+and+lipid+traffic+toward+the+wound+site.">Disruption of the plasma membrane stimulates rearrangement of microtubules and lipid traffic toward the wound site.</a> Cell Sci. 119, 2780-2786 (2006).</li><li>Miyake, K., McNeil, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle+accumulation+and+exocytosis+at+sites+of+plasma+membrane+disruption.">Vesicle accumulation and exocytosis at sites of plasma membrane disruption.</a> Cell Biol. 131, 1737-1745 (1995).</li><li>Reddy, A., Caler, E. V., Andrews, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+repair+is+mediated+by+Ca2+-regulated+exocytosis+of+lysosomes.">Plasma membrane repair is mediated by Ca2+-regulated exocytosis of lysosomes.</a> Cell. 106, 157-169 (2001).</li><li>Babiychuk, E. B., Monastyrskaya, K., Potez, S., Draeger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blebbing+confers+resistance+against+cell+lysis.">Blebbing confers resistance against cell lysis.</a> Death Differ. 18, 80-89 .</li><li>Bansal, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+membrane+repair+in+dysferlin-deficient+muscular+dystrophy.">Defective membrane repair in dysferlin-deficient muscular dystrophy.</a> Nature. 423, 168-172 (2003).</li><li>Abreu-Blanco, M. T., Verboon, J. M., Parkhurst, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+wound+repair+in+Drosophila+occurs+through+three+distinct+phases+of+membrane+and+cytoskeletal+remodeling.">Cell wound repair in Drosophila occurs through three distinct phases of membrane and cytoskeletal remodeling.</a> Cell Biol. 193, 455-464 (2011).</li><li>Jaiswal, J. K., Chakrabarti, S., Andrews, N. W., Simon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptotagmin+VII+restricts+fusion+pore+expansion+during+lysosomal+exocytosis.">Synaptotagmin VII restricts fusion pore expansion during lysosomal exocytosis.</a> PLoS Biol. 2, 1224-1232 (2004).</li><li>McNeil, P. L., Clarke, M. F., Miyake, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+wound+assays.">Cell wound assays.</a> Protoc. Cell Biol. Chapter 12, (2001).</li><li>McNeil, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+introduction+of+molecules+into+cells.">Direct introduction of molecules into cells.</a> Protoc. Cell Biol. Chapter 20, (2001).</li><li>Knight, M. M., Roberts, S. R., Lee, D. A., Bader, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+imaging+using+confocal+microscopy+induces+intracellular+calcium+transients+and+cell+death.">Live cell imaging using confocal microscopy induces intracellular calcium transients and cell death.</a> J. Physiol. Cell Physiol. 284, C1083-C1089 (2003).</li><li>McNeil, P. L., Miyake, K., Vogel, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endomembrane+requirement+for+cell+surface+repair.">The endomembrane requirement for cell surface repair.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 4592-4597 (2003).</li><li>Benink, H. A., Bement, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentric+zones+of+active+RhoA+and+Cdc42+around+single+cell+wounds.">Concentric zones of active RhoA and Cdc42 around single cell wounds.</a> Cell Biol. 168, 429-439 (2005).</li><li>Schmoranzer, J., Goulian, M., Axelrod, D., Simon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+constitutive+exocytosis+with+total+internal+reflection+fluorescence+microscopy.">Imaging constitutive exocytosis with total internal reflection fluorescence microscopy.</a> Cell Biol. 149, 23-32 (2000).</li><li>Steyer, J. A., Horstmann, H., Transport Almers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=docking+and+exocytosis+of+single+secretory+granules+in+live+chromaffin+cells.">docking and exocytosis of single secretory granules in live chromaffin cells.</a> 388, 474-478 (1997).</li><li>Jaiswal, J. K., Andrews, N. W., Simon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+proximal+lysosomes+are+the+major+vesicles+responsible+for+calcium-dependent+exocytosis+in+nonsecretory+cells.">Membrane proximal lysosomes are the major vesicles responsible for calcium-dependent exocytosis in nonsecretory cells.</a> Cell Biol. 159, 625-635 (2002).</li><li>Jaiswal, J. K., Simon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+4.12.">Ch. 4.12.</a> Current Protocols in Cell Biology. , 4.12.1-4.12.15 (2003).</li><li>Johnson, D. S., Jaiswal, J. K., Simon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+internal+reflection+fluorescence+(TIRF)+microscopy+illuminator+for+improved+imaging+of+cell+surface+events.">Total internal reflection fluorescence (TIRF) microscopy illuminator for improved imaging of cell surface events.</a> Protoc. Cytom. Chapter 12, (2012).</li><li>Jaiswal, J. K., Simon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+single+events+at+the+cell+membrane.">Imaging single events at the cell membrane.</a> Chem. Biol. 3, 92-98 (2007).</li></ol>

Los autores no tienen nada que revelar.

Lesión de la membrana celular in vivo se produce debido a una variedad de factores de estrés fisiológicos y se han desarrollado varios enfoques experimentales para imitar éstos. Estos incluyen hiriendo membrana celular de las células adherentes al quitarlas de la antena o haciendo pasar a través de un estrecho orificio 9,10 jeringa. Después de este tipo de lesiones se curan las células en suspensión y no adherido a la matriz extracelular como lo hacen normalmente en el tejido. Y otros, tales...

La membrana de la célula mantiene la integridad de las células, proporcionando una barrera entre la célula y el medio ambiente extracelular. Un estímulo químico, eléctrico o mecánico que excede el umbral fisiológico normal, así como la presencia de los patógenos invasores puede cada resultado de lesiones a la membrana celular y desencadenar una respuesta celular posterior para reparar esta lesión. Para sobrevivir a estas lesiones a la membrana celular, las células poseen un mecanismo eficiente para la reparación. Este mecanismo es dependiente de calcio e implica el tráfico intracelular de proteínas como anexinas y MG53, entre otros, así como los compartimentos subcelulares tales como endosomas, lisosomas, vesículas de Golgi y mitocondrias derivadas de la membrana de la célula lesionada 1-7. Sin embargo, los detalles de la secuencia de eventos moleculares y subcelulares involucrados en la reparación de la membrana celular dañada sigue siendo poco conocida. Respuesta de reparación de la célula se puede segregar en el oídomente y las respuestas tardías. Las primeras respuestas, que se producen dentro de segundos a minutos escala de tiempo, son tremendamente importante en la determinación de la naturaleza de las respuestas tardías que conducen a la reparación celular éxito o muerte celular. Ensayos de punto final basado en el análisis bioquímico y celular a granel han ayudado a establecer la participación de los procesos moleculares y celulares de la reparación. Pero, debido a la heterogeneidad y la rapidez de respuesta de reparación celular, ensayos de punto final no proporcionan los detalles cinéticos y espaciales de la secuencia de eventos que conducen a reparar. Los enfoques que permitan lesión controlada de la membrana celular y permiten el seguimiento de la reparación de la membrana celular y subcelular respuestas asociadas a una alta resolución espacial y temporal son ideales para este tipo de estudios. En este caso, se han presentado este tipo de enfoques. Dos de los protocolos describen enfoques para controlar la cinética en tiempo real de reparación de la membrana celular y las respuestas subcelulares asociados con el proceso de reparación en células vivas siguientes inj láserury. Como complemento a estos ensayos basados ​​en imágenes de células vivas, los ensayos de punto final también se han descrito que proporcionan una medida basada en la población para la reparación de la vigilancia de las células individuales y las respuestas asociadas subcelulares. Para demostrar su utilidad estos enfoques se han utilizado para vigilar el tráfico y exocitosis de lisosomas en respuesta a la lesión de la membrana celular.

<table border="0" cellpadding="0" cellspacing="0" width="814" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>HBSS</td>
			<td>Sigma</td>
			<td>H1387</td>
			<td>Used to prepare CIM (HBSS/10 mM Hepes/2 mM Ca2+ pH 7.4)</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>BP410</td>
			<td>Used to prepare CIM (HBSS/10 mM Hepes/2 mM Ca2+ pH 7.4)</td>
		</tr>
		<tr>
			<td>FITC dextran (Lysine fixable)</td>
			<td>Invitrogen</td>
			<td>D1820</td>
			<td>2 mg/ml in CIM or PBS</td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>Sigma</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITC dextran (Lysine fixable)</td>
			<td>Invitrogen</td>
			<td>D1818</td>
			<td>2 mg/ml in CIM or PBS</td>
		</tr>
		<tr>
			<td>PFA 16%</td>
			<td>EMS</td>
			<td>15710</td>
			<td>4% in PBS</td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>1/10 000 in PBS</td>
		</tr>
		<tr>
			<td>FM dye</td>
			<td>Invitrogen</td>
			<td>T3163</td>
			<td>1 &#181;g/&#181;l in CIM or PBS</td>
		</tr>
		<tr>
			<td>FITC dextran</td>
			<td>Sigma</td>
			<td>FD40S</td>
			<td>2 mg/ml in growth medium</td>
		</tr>
		<tr>
			<td>LAMP1&#160;</td>
			<td>Santa Cruz</td>
			<td>sc-19992</td>
			<td>1/300 in growth medium</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 chicken anti-rat IgG</td>
			<td>Invitrogen</td>
			<td>A21470</td>
			<td>1/500 in blocking solution</td>
		</tr>
		<tr>
			<td>Mounting media</td>
			<td>Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Fisher</td>
			<td>12-545-86</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom petridish</td>
			<td>MatTek corporation</td>
			<td>P35G-1.0-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone O ring</td>
			<td>Bellco</td>
			<td>1943-33315</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip holder</td>
			<td>Bellco</td>
			<td>1943-11111</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen</td>
			<td>A-7816</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>VWR</td>
			<td>12001-566</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>VWR</td>
			<td>MP1400-500H</td>
			<td>Used for growth medium: 10% FBS, 1% P/S in DMEM</td>
		</tr>
		<tr>
			<td>Penicillin/Sreptomycin</td>
			<td>VWR</td>
			<td>12001-350</td>
			<td>Used for growth medium: 10% FBS, 1% P/S in DMEM</td>
		</tr>
		<tr>
			<td>Chicken serum</td>
			<td>Sigma</td>
			<td>C5405</td>
			<td>5% chicken serum in CIM is used for blocking solution during immunostaining</td>
		</tr>
		<tr>
			<td>PBS (Ca2+ and Mg2+ free)</td>
			<td>VWR</td>
			<td>12001-664</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence microscope</td>
			<td>Olympus America, PA</td>
			<td>IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>TIRF illuminator&#160;</td>
			<td>Olympus America, PA</td>
			<td>Cell^TIRF (IEC60825-1:2007)</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence illuminator</td>
			<td>Sutter Instruments, Novato CA</td>
			<td>Lambda DG-4 (DG-4 30)</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD Camera</td>
			<td>Photometrics, Tucson, AZ</td>
			<td>Evolve 512 EMCCD&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Image acquisition and anaysis software</td>
			<td>Intelligent Imaging Innovations, Inc. Denver, CO</td>
			<td>Slidebook 5</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulsed laser</td>
			<td>Intelligent Imaging Innovations, Inc. Denver, CO</td>
			<td>Ablate (3iL13)</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage top incubator</td>
			<td>Tokaihit, Japan</td>
			<td>INUBG2ASFP-ZILCS</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging cell membrane injury and subcellular processes involved in repair

La capacidad de las células para curar heridas es un proceso celular fundamental, pero los mecanismos celulares y moleculares implicados en las células curativas heridos son poco conocidos. Aquí se describen ensayos para controlar la capacidad y la cinética de la curación de las células cultivadas después de una lesión localizada. El primer protocolo describe un método de punto final a base de evaluar simultáneamente la capacidad de reparación de la membrana celular de cientos de células. El segundo protocolo describe un enfoque de formación de imágenes en tiempo real para controlar la cinética de la reparación de la membrana celular en las células individuales después de una lesión localizada con un láser pulsado. Como las células curativas heridos implica el tráfico de proteínas específicas y compartimentos subcelulares a la zona de la lesión, el tercer protocolo describe el uso de la aproximación al punto final más arriba basado para evaluar el tráfico de uno de esos eventos (exocitosis lisosomales) en cientos de células dañadas de forma simultánea y el último protocolo describe el uso de lesión láser pulsado junto con micros TIRFcopia para monitorear la dinámica de los compartimentos subcelulares individuales en las células dañadas en alta resolución espacial y temporal. Si bien los protocolos siguientes describen la utilización de estos enfoques para estudiar la relación entre la reparación de la membrana celular y exocitosis lisosomal en las células musculares cultivadas, que se pueden aplicar como tal para cualquier otra célula en cultivo adherente y compartimento subcelular de elección.

Protocolos que se describen aquí para obtener imágenes de células individuales son para controlar la capacidad y la cinética de la reparación de la membrana celular (Protocolos 1 y 2) y el tráfico subcelular y fusión de lisosomas durante la reparación (Protocolos 3 y 4). Protocolo 1 muestra un ensayo mayor que permite el marcado todas las células dañadas y la identificación de las células dañadas que no pudieron reparar. Los resultados en la Figura 1 muestran qu...

Watch this Scientific Journal Video about Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair at JoVE.com

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

El proceso de las células de curación heridas implica el tráfico de proteínas específicas y los compartimentos subcelulares al sitio de la lesión de la membrana celular. Este protocolo describe ensayos para controlar estos procesos.

Imágenes de células lesión de la membrana y procesos subcelulares implicadas en la reparación

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

imaging-cell-membrane-injury-subcellular-processes-involved

Research

JoVE Journal

Biology

14.9K vistas.  Children's National Medical Center.  El proceso de las células de curación heridas implica el tráfico de proteínas específicas y los compartimentos subcelulares al sitio de la lesión de la membrana celular. Este protocolo describe ensayos para controlar estos procesos. 

Video: Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

Visualización de MG53 mediada por reparación de la membrana celular mediante In vivo Y In vitro Sistemas de

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

Investigación

Biología

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Rata Exteriorización Mesenterio: un modelo para investigar la dinámica celulares implicados en la angiogénesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca2+-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca2+ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca2+-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca2+.

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

Viva Imaging Ensayo para la evaluación de los Roles de Ca<sup&gt; 2 +</sup&gt; Y esfingomielinasa en la reparación de los poros de la formación de toxina Heridas

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Microscopia intravital para formar imágenes de estructuras subcelulares en ratones vivos que expresan proteínas fluorescentes

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

La proyección de imagen de resolución súper Estado de agotamiento de la tierra en células de mamíferos

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

Super-Resolución De Imágenes De Células Vivas De Estructuras Subcelulares

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

Imágenes ópticas basadas en fotodiodos para registrar dinámicas de red con resolución de una sola neurona en invertebrados no transgénicos

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, propulsive contractions. Regular RP contractions rely on pacemaker activity, which originates from the most proximal region of the RP at the pelvis-kidney junction (PKJ). Due to the difficulty in accessing and preserving intact preparations of the PKJ, most investigations on RP pacemaking have focused on single-cell electrophysiology and Ca2+ imaging experiments. Although important revelations on RP pacemaking have emerged from such work, these experiments have several intrinsic limitations, including the inability to accurately determine cellular identity in mixed suspensions and the lack of in situ imaging of RP pacemaker activity. These factors have resulted in a limited understanding of the mechanisms that underlie normal rhythmic RP contractions. In this paper, a protocol is described to prepare intact segments of mouse PKJ using a vibratome sectioning technique. By combining this approach with mice expressing cell-specific reporters and genetically encoded Ca2+ indicators, investigators may be able to more accurately study the specific cell types and mechanisms responsible for peristaltic RP contractions that are vital for normal urine transport.

The goal of this protocol is to isolate intact pacemaker regions of the mouse renal pelvis using vibratome sectioning. These sections can then be used for in situ Ca2+ imaging to elucidate Ca2+ transient properties of pacemaker cells and other interstitial cells in vibratome slices.

Aislamiento e imágenes de regiones de marcapasos intactas y vivas de la pelvis renal del ratón mediante seccionamiento del vibratomo

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

Aislamiento de células musculares lisas cardíacas y vasculares de peces cebra adultos, juveniles, larvales y embrionarios para estudios electrofisiológicos

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...

Imaging Neutrophil Migration in the Mouse Skin to Investigate Subcellular Membrane Remodeling Under Physiological Conditions

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first immune cells to reach the site of inflammation and to participate in the innate immune response during infection or tissue damage. So far, neutrophil migration has been successfully visualized using various in vitro experimental systems based on uniform stimulation, or confined migration under agarose, or micro-fluidic channels. However, these models do not recapitulate the complex microenvironment that neutrophils encounter in vivo. The development of multiphoton microscopy (MPM)-based techniques, such as intravital subcellular microscopy (ISMic), offer a unique tool to visualize and investigate neutrophil dynamics at subcellular resolutions under physiological conditions. In particular, the ear of a live anesthetized mouse provides an experimental advantage to follow neutrophil interstitial migration in real-time due to its ease of accessibility and lack of surgical exposure. ISMic provides the optical resolution, speed, and depth of acquisition necessary to track both cellular and, more importantly, subcellular processes in 3D over time (4D). Moreover, multi-modal imaging of the interstitial microenvironment (i.e., blood vessels, resident cells, extracellular matrix) can be readily accomplished using a combination of transgenic mice expressing select fluorescent markers, exogenous labeling via fluorescent probes, tissue intrinsic fluorescence, and second/third harmonic generated signals. This protocol describes 1) the preparation of neutrophils for adoptive transfer into the mouse ear, 2) different settings for optimal sub-cellular imaging, 3) strategies to minimize motion artifacts while maintaining a physiological response, 4) examples of membrane remodeling observed in neutrophils using ISMic, and 5) a workflow for the quantitative analysis of membrane remodeling in migrating neutrophils in vivo.

Neutrophil migration relies on the rapid and continuous remodeling of the plasma membrane in response to chemoattractant and its interactions with the extracellular microenvironment. Described herein is a procedure based on Intravital Subcellular Microscopy to investigate the dynamic of membrane remodeling in neutrophils injected in the ear of anesthetized mice.

Obtención de imágenes de la migración de neutrófilos en la piel del ratón para investigar la remodelación de la membrana subcelular en condiciones fisiológicas

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first ...