Reconocemos el Dr. Harold Dvorak útil para los debates, incluyendo la sugerencia de probar la fijación en 37 ° C PFA. Este trabajo fue apoyado por el NIH subvención CA92644 P01 y por un contrato de la Fundación Nacional para la Investigación del Cáncer.

1. Cultivo de células en el disco de vidrio recubierto de colágeno <ol><li> Lugar discos de vidrio (12 mm de diámetro) en un frasco de vidrio (4 oz) y autoclave para esterilizar los discos. </li><li> Ponga etanol 25 ml de 70% en un tubo Falcon de 50 ml y colocarlo en una campana de cultivo celular. Esta solución puede ser reutilizado varias veces hasta que el nivel de la solución se reduce a 20 ml. </li><li> Coloque unas afiladas pinzas con un punto extra fino en el etanol al 70% durante 5 minutos antes de usarla para manejar el disco de vidrio. </li><li> Retire con cuidado la pinza del tubo y cerrar la tapa, a continuación, coloque suavemente la pinza de etanol tratada en la parte superior del tubo se seque al aire durante 2 min. No permita que la punta del fórceps tocar nada en la campana. </li><li> Utilice las pinzas estériles para agarrar uno de los discos de vidrio estéril del frasco de vidrio y lo coloca en un pozo de la placa de cultivo celular de 24 pocillos. Repita hasta que el número deseado de pozos se ha rellenado con los discos. </li><li> Ponga 500 l de solución de colágeno bovino (50 ng / ml) en cada pocillo que contiene un disco de vidrio y colocarlo en un 37 ° C, 5% de CO 2 de células cultura incubadora durante al menos 30 min. No hay ningún daño en una incubación más largo. </li><li> Harvest HUVEC (células endoteliales de vena umbilical humana) o PC3 (células de tumor de próstata) que ya se cultivan en 150 mm placa de cultivo celular mediante tripsinización y bloquean la actividad de la tripsina utilizando su medio de cultivo completo. Recoger las células en un tubo Falcon de 15 ml. </li><li> Contar el número de células y asegúrese de que la viabilidad es superior al 90%. </li><li> Sedimentar las células por centrifugación a 200 xg durante 10 min a 4 ° C. Eliminar el sobrenadante. </li><li> Utilice el medio de cultivo para diluir las células a 10 5 células / ml. Coloque las células en hielo. </li><li> Aspirar el colágeno en la campana de cultivo de células y colocar suavemente 500 l de suspensión celular a cada pocillo en el que los discos de vidrio se recubrieron previamente con colágeno. 5 x 10 4 células / pocillo en la placa de 24 pocillos darán 60% confluency para HUVEC y 30% de confluencia para células PC3. Nota: añadir más suspensión de células de más alta densidad celular. </li><li> Cultura de las células en un 37 ° C, 5% de CO 2 incubadora durante 1 hora, 2 horas, 4 horas, 6 horas, o de 24 horas para realizar un seguimiento de las actividades nanopodia y pase celular. Normalmente, las células se adherirán a la disco de vidrio en los primeros 30 minutos, y luego comienzan a polarizarse y migrar en la primera hora, y llevar a cabo las interacciones intercelulares y la división celular en las horas siguientes. </li></ol> 2. La fijación de la célula <ol><li> Cada pozo tendrá 500 l 4% PFA (paraformaldehído) para fijar las células. De cualquier fabricado comercialmente o recién hecho PFA al 4% puede ser utilizado. Calcular la cantidad de PFA necesario basado en el número de pozos preparados, y colocar PFA en un tubo Falcon de 15 ml. PRECAUCIÓN: paraformaldehído es un posible carcinógeno. </li><li> Coloque el tubo de halcón en un soporte de espuma de poliestireno que ya estaba en su lugar en un 37 ° C incubadora. Deje el tubo en la incubadora durante 30 min a la guerram hasta la PFA a 37 ° C. </li><li> Coloque una almohadilla térmica en la campana de cultivo y vuelva a encenderlo. </li><li> Saque la placa de cultivo celular de 24 pocillos y la PFA precalentado de la incubadora y colóquelo en la parte superior de la almohadilla. </li><li> Utilice una mano para aspirar el medio de un pozo, e inmediatamente después de usar la otra mano para deslizar suavemente 500 l PFA en el pozo a través del borde también. Para la aspiración más fácil, la inclinación de la placa de cultivo a aproximadamente 45 º, que se inclina contra el soporte de espuma de poliestireno de modo que es estable en ese ángulo. Esto facilitará la aspiración y la adición de solución de PFA al pozo sin molestar a las células en cultivo. Este es el paso más importante para preservar la estructura celular original de actividades celulares. </li><li> Repita el proceso hasta que todos los pozos con un disco de vidrio han recibido PFA. Nota: todos los pasos que hay que cambiar la solución pre-existente del pozo se aplicarán mismos procedimientos de aspiración. </li><li> Colocar la placa en 37 ° C durante 5 min. </li&gt; <li> Tome la placa posterior de la campana de cultivo y la inclinación en contra de espuma de poliestireno, como se describe en el paso 2.5. Utilice una mano para transferir suavemente la PFA desde el pozo en un tubo de recogida; utilizar la otra mano para colocar inmediatamente después de al menos 500 l temperatura de PBS habitación en el pozo. Después de todos los pozos se han lavado, volver y repetir el lavado PBS una vez más en todos los pozos. Vacíe la PFA recogido en un contenedor de residuos peligrosos. </li><li> Prepare el tampón de bloqueo ICC mediante la adición de 2% de suero bovino fetal al PBS. Azida de sodio 0,04% (diluir de 20% solución de reserva) se añade como conservante. ATENCIÓN: La azida de sodio es un compuesto tóxico. El tampón puede ser almacenado en 4 ° C durante un largo período de tiempo sin la contaminación bacteriana. </li><li> Con la placa de cultivo todavía inclinado en contra de un stand, una vez más, pasar por cada pozo de aspiración para eliminar PBS e inmediatamente después de añadir 500 l de tampón de bloqueo de la CPI. Las células están listas para la tinción de inmunofluorescencia. Si es necesario, lacélulas fijadas se pueden almacenar en un refrigerador a 4 º C durante una semana sin pérdida significativa de proteína TM4SF1. </li></ol> 3. TM4SF1 Inmunofluorescencia La tinción de Nanopodia <ol><li> En cada pocillo, reemplace el amortiguador de bloqueo 500 l ICC con un nuevo tampón de bloqueo que contenía 0,01% de Triton X-100 y se deja reposar a temperatura ambiente durante 1 hora. No utilice más alta que 0,03% de Triton X-100 ya que eliminará TM4SF1 de las membranas celulares. PBS lavó las células de la etapa 2.10 puede ser movido directamente al tampón de bloqueo que contenía Triton si la tinción está listo para ser empezar de inmediato. </li><li> Diluir el anticuerpo primario anti-TM4SF1 (o anti-CD9) a 0,5 g / ml en tampón de bloqueo CPI. </li><li> En cada pozo, eliminar el tampón Triton ICC/0.01% y reemplazarlo con 300 l de la solución anti-TM4SF1-anticuerpo. Incubar 2 horas a temperatura ambiente o dejar toda la noche a 4 ° C. </li><li> Lavar cada pocillo con no menos de 500 l de PBS. Repita 3 veces; cada vez que dan en least 5 min de tiempo de incubación. </li><li> Preparar una solución de anticuerpo y faloidina secundaria en CPI tampón de bloqueo, utilizando una dilución de 1000 veces de Alexa 488 anticuerpo secundario de burro anti-ratón marcado (/ ml de concentración final 2 g) y 1000 x dilución de faloidina (50 ng / ml de concentración final). </li><li> Retire PBS y añadir 300 l de la solución de anticuerpo secundario y phalloidin a cada pocillo y se incuba durante 2 horas o deja toda la noche a 4 ° C. </li><li> Repita los pasos de lavado PBS con al menos una incubación de 5 min por lavado. En el último lavado, deje cada pocillo en PBS durante 1 hora. </li><li> Caída de una sola gota (~ 10 l) de montaje anti-fade medios en un portaobjetos de vidrio. </li><li> Doble la punta de un 19 G 1 ½ en jeringa de aguja 90 ° presionando suavemente sobre una superficie dura. Utilice una mano para sostener la aguja doblada y suavemente levante un disco de vidrio del pozo, y con la otra mano para agarrar el disco con las pinzas cortantes. </li><li> Gire el disco y vidrio# 160; boca abajo dejando que el contacto con las células del medio de montaje en la placa de vidrio. Coloque con cuidado el disco de vidrio y deje que se seque durante la noche en un lugar oscuro a temperatura ambiente. </li><li> Proceder a la imagen de la inmunofluorescencia manchado nanopodia o almacenar las diapositivas de una caja de portaobjetos a -20 ° C. Las diapositivas permanecerán visibles durante unos meses en -20 ° C. </li></ol>

<ol>
	<li>Chhabra, E. S., Higgs, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+faces+of+actin:+matching+assembly+factors+with+cellular+structures.">The many faces of actin: matching assembly factors with cellular structures.</a> Nat. Cell Biol. 9, 1110-1121 (2007).</li><li>Shih, S. C., 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=The+L6+protein+TM4SF1+is+critical+for+endothelial+cell+function+and+tumor+angiogenesis.">The L6 protein TM4SF1 is critical for endothelial cell function and tumor angiogenesis.</a> Cancer Res. 69, 3272-3277 (2009).</li><li>Zukauskas, A., 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=TM4SF1:+a+tetraspanin-like+protein+necessary+for+nanopodia+formation+and+endothelial+cell+migration.">TM4SF1: a tetraspanin-like protein necessary for nanopodia formation and endothelial cell migration.</a> Angiogenesis. 14, 345-354 (2011).</li><li>Hellstrom, I., Beaumier, P. L., Hellstrom, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+effects+of+L6,+an+IgG2a+antibody+that+reacts+with+most+human+carcinomas.">Antitumor effects of L6, an IgG2a antibody that reacts with most human carcinomas.</a> Proc. Natl. Acad. Sci. U.S.A. 83, 7059-7063 (1986).</li><li>Jang, Y. Y., Ye, Z., Cheng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+and+stem+cell+research.">Molecular imaging and stem cell research.</a> Mol. Imag. 10, 111-122 (2011).</li><li>Hemler, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetraspanin+functions+and+associated+microdomains.">Tetraspanin functions and associated microdomains.</a> Nat. Rev. Mol. 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No hay conflictos de interés declarado.

Nanopodia son canales de la membrana celular delgadas que se adhieren firmemente a la matriz a través de TMED y pueden extenderse a más de 100 m de una célula móvil polarizada para detectar el medio ambiente y mediar en las interacciones intercelulares 2,3. Nanopodia adherirse a la matriz tan firmemente que los residuos se quedan atrás ya que los celulares se aleja (Figuras 1 y 2). Así Nanopodia permite el estudio de cómo las células perciben su entorno y determinar l...

Durante la polarización para el movimiento, las células animales se extienden una variedad de salientes, de membrana dinámicos de sus superficies, incluyendo filopodios, lamelipodios, fibras de retracción, y volantes 1. Recientemente añadido a esta lista fueron nanopodia, un tipo recién reconocido de fina (100-300 m de diámetro), alargadas (hasta 50-100 m de largo) de proyección de la membrana que proporcionan los canales de membrana para la extensión de las estructuras de actina F, como filopodios y retracción de la fibra, y que la mancha positivamente con TM4SF1 (transmembrana-4-L-seis-familia-1) en endoteliales y tumorales células cultivadas 2,3. TM4SF1 es una proteína con topología de tetraspanin como que se conocía originalmente como un antígeno de célula tumoral 4 antes de que el descubrimiento de que la molécula es un biomarcador de células endoteliales que desempeña un papel esencial en la proliferación celular endotelial y la migración 2,3. La tinción de inmunofluorescencia reveló que TM4SF1 se localiza en perinucLear y vesículas a la membrana plasmática, y se enriquece en TM 4SF1 e nriched omains d micro (TMED). TMED nanopodia anclaje a la matriz y el presente en un patrón de bandas espaciadas regularmente de 1-3 longitud TMED / m de nanopodia. Nanopodia típicamente se extienden desde delante líder de una célula y de salida trasera durante la polarización de las células para el movimiento. Debido a la naturaleza adherente firme de TMED, nanopodia son incapaces de retraer de nuevo en la celda de medida que se aleja; por tanto, los residuos abandonados nanopodia trazan la trayectoria de movimiento celular. Nanopodia proporcionar canales de membrana para la extensión F-actina y la retracción, y son los sitios de las interacciones intercelulares y comunicaciones 2,3. Estas características hacen que nanopodia proporcionan una oportunidad única para estudiar los mecanismos subyacentes conjunto de F-actina durante la polarización celular, la detección celular del medio ambiente, la determinación de la ruta de acceso y la dirección de movimiento de la célula, y las interacciones intercelulares unND comunicaciones. Debido a la naturaleza altamente hidrofóbica de TMED y la naturaleza membranosa delgada y frágil de nanopodia, especial cuidado debe tomarse con el fin de preservar la TMED y nanopodia. La destrucción de nanopodia y remoción de TMED por métodos comunes de laboratorio es una posible razón por la que sólo sesenta y tres publicaciones han aparecido en TM4SF1 desde su primer descubrimiento en 1986 1 y por la falta total de conocimiento de enriquecimiento TM4SF1 en 100-300 micras microdominios en la superficie de la célula hasta que el informe de TM4SF1 en las células endoteliales en 2009 2. Métodos de inmunotinción convencionales suelen utilizar disolventes orgánicos como etanol, metanol, o acetona para fijar las células y el uso de 0,1% o mayor concentración de Triton X-100 para permeabilizar las células 5. Los estudios aquí descritos implementaron tres grandes cambios con el método convencional para revelar TMED y nanopodia: (i) utilizan 37 ° C 4% PFA y fijar las células en un 3776; incubadora, (ii) aplicar el lavado de PBS temperatura ambiente suave, y (iii) el uso de menos de 0,03% de Triton X-100 sólo brevemente para permeabilizar las células antes de la adición de anticuerpo primario, como X-100 mayor que 0,03% extraerá Tritón TM4SF1. Todos los tetraspanins forman microdominios en la membrana de la célula 6 y algunos colocalize con TMED en endoteliales y células tumorales 2,3. Como tetraspanins como CD9, CD81, CD151 y se expresan de manera ubicua, el protocolo de tinción descrito aquí se puede extender a muchos tipos diferentes de células que carecen de TM4SF1 para los estudios de la función nanopodia.

<table border="0" cellpadding="0" cellspacing="0" width="1597">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">glass discs&#160;</td>
			<td style="width:160px;">Fisher Scientific</td>
			<td style="width:133px;">12-545-82&#160;</td>
			<td style="width:983px;">12-mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">glass jar</td>
			<td>Fisher Scientific</td>
			<td>02-912-310</td>
			<td>Certified Clean Clear Glass Straight-Sided Jars, 4 oz.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">glass slide</td>
			<td>Fisher Scientific</td>
			<td style="width:133px;">12-544-1</td>
			<td>Fisherfinest Premium Plain Glass Microscope Slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">50-ml falcon tube&#160;</td>
			<td style="width:160px;">BD Falcon</td>
			<td style="width:133px;">352070</td>
			<td>50-ml high-clarity polypropylene conical centrifuge tube, 16,000 rcf rating. Sterile.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">15-ml falcon tube with Styrofoam rack</td>
			<td style="width:160px;">Corning</td>
			<td style="width:133px;">430790</td>
			<td>Corning 15-ml PP Centrifuge Tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sharp forceps&#160;</td>
			<td>Fisher Scientific</td>
			<td style="width:133px;">13-812-42</td>
			<td>Dissecting Extra-Fine-Pointed Splinter Forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">24-well cell culture plate</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">353047</td>
			<td>24-well Cell Culture Plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">150-mm cell culture plate&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">353025</td>
			<td>150-mm cell culture plate&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">19G 1 &#189; syringe needle&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">309635</td>
			<td>19G 1 &#189;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Reagent</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">ethanol</td>
			<td>Decon Laboratories, Inc.</td>
			<td>2701</td>
			<td>Decon&#39;s Pure Ethanol 200 Proof</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">collagen solution&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">354249</td>
			<td>Collagen I, High Concentration, rat tail, 100 mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">trypsin/EDTA</td>
			<td style="width:160px;">Cellgro</td>
			<td style="width:133px;">25-053-CI</td>
			<td><a name="RANGE!D15">0.25% Trypsin-EDTA 1X</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">DMEM</td>
			<td>Life Technologies</td>
			<td>11965-092</td>
			<td>DMEM high glucose (1X), liquid, with L-glutamine, without sodium pyruvate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">10X PBS</td>
			<td style="width:160px;">Affymetrix</td>
			<td style="width:133px;">75889 5 LT</td>
			<td style="width:983px;">PBS, 10X Solution, pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">PFA (paraformaldehyde)</td>
			<td style="width:160px;">Affymetrix</td>
			<td style="width:133px;">19943 1 LT</td>
			<td>4% in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F4135-500ML</td>
			<td><a name="RANGE!D19">Fetal Bovine Serum</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">EGM2-MV</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td>EGM-2 BulletKit, EBM-2 Basal Medium 500 ml and EGM-2 SingleQuot Kit Suppl. &#38; Growth Factors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td style="width:133px;">T8787</td>
			<td>Triton X-100</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaN3</td>
			<td>Sigma-Aldrich</td>
			<td style="width:133px;">S2002-25G</td>
			<td>solubilized in water. 4% stock solution and working concentration is 0.4%.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">mouse anti-human TM4SF1 antibody&#160;</td>
			<td style="width:160px;">Millipore</td>
			<td style="width:133px;">MAB3127</td>
			<td>Epitope is located in extracellular domain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">mouse anti-human CD9 antibody&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">555370</td>
			<td>Epitope is located in extracellular domain</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Alexa Fluor 488 Donkey anti-mouse 2nd antibody</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:133px;">A-21202</td>
			<td>Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Phalloidin</td>
			<td style="width:160px;">Chemicon</td>
			<td style="width:133px;">90324</td>
			<td>Rhodamine-conjugated Phalloidin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">anti-fade mounting media&#160;</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:133px;">P-36931</td>
			<td>ProLong Gold Antifade Reagent with DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Buffers and solutions</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">70% ethanol&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td>17.5 ml of ethanol (200 proof) 
			7.5 ml of double deionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">10X PBS (make 200 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">20 ml of 10X PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">180 ml of double deionized water</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">20% Triton X-100 (make 20 ml)&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">4 ml</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">16 ml double deionized water</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">4% NaN3 (make 25 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">1 g of NaN3</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">25 ml of double deionized water</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">50 ng/ml bovine collagen solution in PBS (make 5 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Make stock soln. of 50 &#956;g/ml (50 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">830 &#956;l of Collage I (3 mg)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">50 ml PBS&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">ICC Blocking Buffer (50 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">49 ml PBS</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td style="width:983px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">2% FBS (add 1 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td style="width:983px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">0.04% NaN3 (add 100 &#956;l of 4% NaN3)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">ICC Blocking Buffer/0.01% Triton X-100 (make 50 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">50 ml of ICC Blocking Buffer&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">25 &#956;l of 20% Triton X-100</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Cell</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">HUVEC&#160;</td>
			<td style="width:160px;">Lonza&#160;</td>
			<td>C2517A</td>
			<td><a href="http://www.lonza.com/products-services/bio-research/primary-and-stem-cells/human-cells-and-media/endothelial-cells-and-media/huvec-human-umbilical-vein-endothelial-cells.aspx">http://www.lonza.com/products-services/bio-research/primary-and-stem-cells/human-cells-and-media/endothelial-cells-and-media/huvec-human-umbilical-vein-endothelial-cells.aspx</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">PC3</td>
			<td style="width:160px;">ATCC</td>
			<td style="width:133px;">CRL-1435</td>
			<td><a href="http://www.atcc.org/products/all/CRL-1435.aspx#85786B46AA23451B94BC5D45200673F7">http://www.atcc.org/products/all/CRL-1435.aspx#85786B46AA23451B94BC5D45200673F7</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Equipment</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">cell culture hood</td>
			<td style="width:160px;">NuAIRE</td>
			<td style="width:133px;">Nu-425-600</td>
			<td>NU-425 (Series 60) BIOLOGICAL SAFETY CABINET&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">37 &#176;C, 5% CO2 cell culture incubator&#160;</td>
			<td style="width:160px;">CellStar</td>
			<td style="width:133px;">QWJ300DABA&#160;</td>
			<td>Cellstar CO2 Water Jacketed Incubator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">heating pad&#160;</td>
			<td>K&#38;H Manufacturing</td>
			<td style="width:133px;">1020</td>
			<td style="width:983px;">K&#38;H Lectro Kennel Heated Pad with Free Fleece Cover (http://www.amazon.com/Lectro-Kennel-Heat-Pad-22-5x28-5-In/dp/B000PSRN20/ref=pd_bxgy_petsupplies_img_y)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">centrifuge</td>
			<td style="width:160px;">Sorvall&#160;</td>
			<td style="width:133px;">T6000B&#160;</td>
			<td>Sorvall T6000 (B) Benchtop Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemacytometer</td>
			<td>Sigma-Aldrich</td>
			<td style="width:133px;">Z359629</td>
			<td>Bright-Line Hemacytometer</td>
		</tr>
	</tbody>
</table>

nanopodia - thin, fragile membrane projections with roles in cell movement and intercellular interactions

Las células adherentes en cultivo mantienen un estado polarizado para apoyar el movimiento y las interacciones intercelulares. Nanopodia son delgadas, alargadas, en gran parte proyecciones de membrana F-actina negativos en las células endoteliales y de cáncer que se pueden visualizar a través TM4SF1 (transmembrana-4-L-seis-familia-1) la tinción de inmunofluorescencia. Racimos TM4SF1 en 100-300 micras de diámetro TMED (TM 4SF1 e nriched micro omains d) que contiene 3 a un máximo de 14 TM4SF1 moléculas individuales. TMED están dispuestas intermitentemente a lo largo nanopodia con una separación regular de 1 a 3 TMED por μ m y anclar firmemente nanopodia a la matriz. Esto permite nanopodia extender más de 100 μ m de la parte delantera o trasera que conduce trasera del endoteliales tumorales o células polarizadas, y hace que los residuos de la membrana a quedar atrás en matrIX cuando la célula se aleja. TMED y nanopodia han pasado por alto debido a su extrema fragilidad y sensibilidad a la temperatura. Lavado de rutina y fijación interrumpen la estructura. Nanopodia se conservan por fijación directa en paraformaldehído (PFA) a 37 ° C, seguido por una breve exposición a 0,01% de Triton X-100 antes de la tinción. Nanopodia abrir nuevas perspectivas en la biología celular: prometen a remodelar nuestra comprensión de cómo las células perciben su entorno, detectar e identificar otras células a distancia, iniciar interacciones intercelulares en estrecho contacto, y de los mecanismos de señalización que participan en el movimiento, la proliferación celular y la comunicaciones en células. Los métodos que se desarrollan para el estudio nanopodia derivados de TM4SF1 pueden ser útiles para los estudios de nanopodia que se forman en otros tipos de células a través de la agencia de tetraspanins clásicos, especialmente el CD9 expresión ubicua, CD81, CD151 y.

Para el Paso 1: Si las células (como HUVEC y PC3 utilizado en este estudio) son capaces de crecer normalmente, las células se unen al disco revestido con colágeno dentro de 30 minutos después de que se siembran, se polarizan y se convierten en móvil poco después, y se extienden nanopodia por delante de su camino de movimiento. Figuras 1A y 1B, respectivamente, muestran un polarizada y la proliferación de HUVEC. Figura 2A muestra células...

Watch this Scientific Journal Video about Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions at JoVE.com

Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

Nanopodia son los canales de membrana delgada pero frágiles que se extienden hasta 100 micras de los principales delantero o trasero trasero de una célula y perciben el entorno celular. La fijación directa a 37 ° C, lavado suave, y la evitación de disolventes orgánicos como etanol, metanol, o acetona y de mayores Triton X-100 concentraciones están obligados a observar estas estructuras celulares.

Nanopodia - Proyecciones membrana delgada y frágil con papeles en Movimiento Celular y intercelulares Interacciones

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely ...

nanopodia-thin-fragile-membrane-projections-with-roles-cell-movement

Research

JoVE Journal

Biology

10.2K vistas.  Harvard Medical School.  Nanopodia son los canales de membrana delgada pero frágiles que se extienden hasta 100 micras de los principales delantero o trasero trasero de una célula y perciben el entorno celular. La fijación directa a 37 ° C, lavado suave, y la evitación de disolventes orgánicos como etanol, metanol, o acetona y de mayores Triton X-100 concentraciones están obligados a observar estas estructuras celulares. 

Video: Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

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

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

El Protein Complex Plataforma de Producción MultiBac en el EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

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 ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Brecha ocasiones intercelular de comunicación: un biomarcador funcional para evaluar los efectos adversos de las sustancias tóxicas y toxinas, y de Salud Beneficios de Productos Naturales

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

Análisis de una sola partícula de código abierto para el Super-resolución Microscopía con VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, &#34;flash-and-freeze,&#34; that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution.

We developed a novel technique in electron microscopy, &#34;flash-and-freeze,&#34; that enables the visualization of membrane dynamics with ms temporal resolution. This technique combines the optogenetic stimulation of neurons with high-pressure freezing. Here, we demonstrate the procedures and describe the protocols in detail.

Flash-y-Freeze: una técnica novedosa para capturar la dinámica de la membrana con Microscopía Electrónica

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and ...

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

Tirando de nanotubos de membrana de las vesículas unilaminar gigante

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.

F&#246;rster Resonance Energy Transfer (FRET) between two fluorophore molecules can be used for studying protein interactions in the living cell. Here, a protocol is provided as to how to measure FRET in live cells by detecting sensitized emission of the acceptor and quenching of the donor molecule using confocal laser scanning microscopy.

Evaluación de las interacciones de proteínas en células vivas con emisión sensibilizada por FRET

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the ...

Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membrane&#160;interactions, and viral budding, among other vital cellular processes. For efficient biological activity, cell membranes should be rigid enough to maintain the integrity of the cell and its compartments yet fluid enough to allow membrane components, such as proteins and functional domains, to diffuse and interact. This delicate balance of elastic and fluid membrane properties, and their impact on biological function, necessitate a better understanding of collective membrane dynamics over mesoscopic length and time scales of key biological processes, e.g., membrane deformations and protein binding events. Among the techniques that can effectively probe this dynamic range is neutron spin echo (NSE) spectroscopy. Combined with deuterium labeling, NSE can be used to directly access bending and thickness fluctuations as well as mesoscopic dynamics of select membrane features. This paper provides a brief description of the NSE technique and outlines the procedures for performing NSE experiments on liposomal membranes, including details of sample preparation and deuteration schemes, along with instructions for data collection and reduction. The paper also introduces data analysis methods used to extract key membrane parameters, such as the bending rigidity modulus, area compressibility modulus, and in-plane viscosity. To illustrate the biological importance of NSE studies, select examples of membrane phenomena probed by NSE are discussed, namely, the effect of additives on membrane bending rigidity, the impact of domain formation on membrane fluctuations, and the dynamic signature of membrane-protein interactions.

This paper describes the protocols for sample preparation, data reduction, and data analysis in neutron spin echo (NSE) studies of lipid membranes. Judicious deuterium labeling of lipids enables access to different membrane dynamics on mesoscopic length and time scales, over which vital biological processes occur.

Espectroscopía de eco de espín de neutrones como una sonda única para la dinámica de la membrana lipídica y las interacciones membrana-proteína

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membrane&#160;interactions, and viral ...

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 ...