We thank the Pathology Research Core for assistance with electron microscope analysis studies at Cincinnati Children's Hospital Medical Center. The work was supported by NIH (American Society of Hematology Bridge award to-MDF; R01 DK102890 to MDF).

Todos los procedimientos experimentales fueron aprobados por el comité institucional de los animales en la Fundación de Investigación Infantil de Cincinnati. Para este estudio, se aislaron células madre hematopoyéticas de la médula ósea de ratones C57BL / 6 ratones endogámicos de entre 2 - 4 meses. Clasificación de células mediante FACS después de la tinción de BM con diferentes fabricantes de superficie incluyendo Lineage, c-Kit, Sca-1, Flt3 y CD34 se utilizó para la purificación de HSC basado en Flt3 Lin- Sca-1 + c-Kit + - estrategia gating - CD34 como protocolo estándar descrito antes de las 8. PRECAUCIÓN: Varios productos químicos altamente tóxicos se utilizan durante este procedimiento. Estos son tóxicos por inhalación y contacto con la piel. Por favor, use guantes y ropa de protección. Trabajar en campana de humos mientras se trabaja con estos productos químicos. 1. células, la fijación, tinción y Pre-incrustación <ol><li> Ordenar 10.000 células madre hematopoyéticas (utilizando Lin - Sca-1 <sup&gt; + c-Kit + Flt3 - CD34 - gating estrategia; población LT-HSC) en un tubo de microcentrífuga de 1,5 ml que contiene 600 l de modificación de Dulbecco de Iscove Medio (IMDM) con suero bovino fetal al 10% usando FACS. </li><li> Centrifugar las CMH ordenados a 1.000 xg durante 10 min en un tubo de microcentrífuga de 1,5 ml utilizando una centrífuga con cubos que oscila hacia afuera. Retire medio por aspiración suave y dejar 200 l de medio en los tubos en cada paso. En esta etapa, el sedimento celular permanece invisible en el tubo. </li><li> Fijar las células mediante la adición de 0,2 ml de solución de fijación 2x a temperatura ambiente (RT) durante 1 hora 1,9. <ol><li> Hacer la solución de fijación fresca antes de su uso. En 1x, la solución se compone de 3% de paraformaldehído y 2,5% de glutaraldehído en tampón de cacodilato 0,1 M. </li></ol></li><li> Centrifugar las células a 1000 xg durante 10 minutos a 30 ° C utilizando una centrífuga con cubos basculante. </li><li> Lavar las células con 600 l de tampón de 0,1 M de cacodilatousando centrifugación y dejar tampón de 200 l en el tubo después de la centrifugación. </li><li> Tinción de las células fijadas mediante la adición de 200 l de solución / ml 2 mg de azul de Evans en tampón cacodilato y se incuba durante 20 min a TA. Las células serán de color azul. </li><li> Lavar las células 3 veces con 600 l de tampón de cacodilato 0,1 M usando centrifugación y dejar tampón de 200 l en el tubo cada vez. </li><li> Vuelva a suspender las células en tampón de 200 l y transferir la suspensión celular a un tubo de 0,5 ml. Centrifugar a 1000 xg durante 10 min utilizando una centrífuga de mesa. Eliminar el tampón suavemente sin alterar el sedimento celular pequeña ahora visible, y dejar tampón de 50 l en el tubo. </li><li> Añadir 200 mg bajo punto de fusión de agarosa para 5 ml de tampón de cacodilato 0,1 M en el tubo de 15 ml de una solución de agarosa al 4%. Fundir la agarosa mediante la transferencia del tubo de agua hirviendo en una botella de vidrio de 100 ml y mantener tibia hasta que se usaron. </li><li> Añadir 200 l de 4% de agarosa de baja fusión disolvieron en 0,1 M cacodylatampón TE a la suspensión celular en un tubo de 0,5 ml e inmediatamente se centrifuga a 1000 xg durante 10 min a 30 ° C usando una centrífuga de mesa. </li><li> Después de confirmar que las células sedimentaron en el fondo del tubo de 0,5 ml en la agarosa semisólida después de la centrifugación, transferir inmediatamente el tubo a 4 ° C o de hielo durante 20 min para solidificar la agarosa. Este paso es muy crítico, ya que un pequeño sedimento celular visible en este paso resulta en un buen grupo de células bajo el microscopio electrónico. </li><li> Transferir con cuidado la agarosa solidificada que contiene el sedimento celular del tubo de plástico a un plato de 35 mm de Petri que contenía tampón utilizando un G 3,10 27 aguja. </li><li> Recortar los agarosa solidificada que contiene el sedimento celular en una sola pieza de aproximadamente 1 - 2 mm con un bisturí. La transferencia de la pieza de agarosa que contiene el sedimento celular a un nuevo tubo de 1,5 ml. </li><li> Lavar 2 - 3 veces con 600 l de tampón cacodilato 0,1 M mediante la adición y la eliminación de buffer usando la pipeta sin centrifugación. Utilice esta pieza de agarosa que contiene el sedimento celular para el siguiente paso. En este paso, la muestra se puede almacenar a 4 ° C durante la noche antes de pasar al siguiente paso. </li></ol> 2. Osmification, deshidratación, incrustación <ol><li> Eliminar el tampón de cacodilato desde el tubo de 1,5 ml con el sedimento celular que contiene agarosa usando una pipeta. Añadir 1,0 ml de 1% tetraóxido de osmio (OsO4) de solución en una campana de humos, y se incuba durante 1 hora a 4 ° C. </li><li> Lavar tres veces con 600 l de tampón de cacodilato 0,1 M y transferir la muestra a un vial de centelleo de vidrio de 20 ml con una tapa. </li><li> Procesar la muestra para la deshidratación, la infiltración y la incrustación en resina (por ejemplo, LX-112) con los cambios de serie de las siguientes soluciones en una placa de 6 pocillos. Mover la pieza de agarosa por células de pellets con unas pinzas. <ol><li> Sumergir la muestra en 25% de etanol a temperatura ambiente durante 15 min. </li><li> Sumergir la muestra en 50% de etanol a temperatura ambiente durante 15 min. </li><li> Sumergir la muestra en el 75%etanol a TA durante 15 min. </li><li> Sumergir la muestra en 95% de etanol a temperatura ambiente durante 15 min. </li><li> Sumergir la muestra en 100% de etanol a temperatura ambiente durante 15 min, dos veces. </li><li> Sumergir la muestra en etanol: resina (3: 1) a TA durante 30 min. </li><li> Sumergir la muestra en etanol: resina (1: 1) a TA durante 30 min. </li><li> Sumergir la muestra en etanol: resina (1: 3) a TA durante 30 min. </li><li> Sumergir la muestra en resina pura a temperatura ambiente durante 60 min, dos veces. </li></ol></li><li> Transferir la muestra a la parte inferior de la punta de la pirámide molde de goma en forma de uso de fórceps con cuidado y añadir la resina más pura en la parte superior de la muestra para llenar el molde. Incubar la muestra a 60 ° C durante 72 hr para la polimerización de la resina. </li><li> Retire la pirámide resina polimerizada de los moldes girando el molde de goma. Un pequeño grupo de células negro debe ser visible en la pirámide de resina polimerizada, como se ha descrito anteriormente 7. Coloque la pirámide de resina polimerizada en el cilindro de montaje con cianoacrilato. </li></ol><ol><li> Recorte el bloque de pirámide con una hoja de afeitar y un cuchillo de diamante después de montar el bloque en un ultra-micrótomo. Hacer una forma trapezoidal ancha corta con la parte superior y la parte inferior del bloque en paralelo entre sí, y con los lados de manera uniforme en ángulo. Esta forma de la pirámide ayuda de cortes seriados. </li><li> Además, recortar el bloque de todo el sedimento celular con el bisturí de diamante y quitar las secciones de plástico alrededor de la pieza de agarosa al sedimento celular. </li><li> Cortar 1 micras secciones usando un ultra-micrótomo. Mover las secciones 1 m desde el barco del agua a un portaobjetos de vidrio utilizando una herramienta de pestañas y una herramienta de lazo de metal como se informó anteriormente 7. secciones de transferencia de diapositivas y diapositivas de transferencia a la placa caliente (37 ° C) para el secado. </li><li> Añadir una gota de solución de azul de toluidina a las secciones utilizando una jeringa equipada con 0,22 micras filtro y se incuba durante 3 min. Enjuague los portaobjetos con agua destilada y ly las secciones se sequen. Consulte con un microscopio de luz para identificar la posición de las células. </li><li> Los cortes de secciones ultrafinas (70 - 100 nm) utilizando un cuchillo de diamante. Mueve 2 - 3 secciones en las redes de malla 200 de la embarcación del agua a un plato de Petri de vidrio usando una herramienta de pestañas. </li><li> Transferir el cristal placa de Petri que contiene cuadrículas con secciones de una placa caliente a 30 ° C para secar las secciones durante 30 minutos. </li><li> Tinción de las rejillas con gotas de acetato de uranilo al 1% a temperatura ambiente durante 10 min y luego enjuague en agua destilada 6 - 8 veces. Mancha con citrato de plomo de Reynolds, a TA durante 5 min y enjuague en agua destilada 6 - 8 veces. Transferir el cristal placa de Petri que contiene las cuadrículas en una placa caliente a 37 ° C para secar las secciones. </li><li> Analizar las secciones con un microscopio electrónico, a 80 kV 7. </li></ol>

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	<li>Bozzola, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conventional+specimen+preparation+techniques+for+transmission+electron+microscopy+of+cultured+cells.">Conventional specimen preparation techniques for transmission electron microscopy of cultured cells.</a> Methods Mol Biol. 369, 1-18 (2007).</li><li>Leapman, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+techniques+in+electron+microscopy.">Novel techniques in electron microscopy.</a> Curr Opin Neurobiol. 14, 591-598 (2004).</li><li>Anderson, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+preparing+peripheral+leucocytes+for+electron+microscopy.">A method of preparing peripheral leucocytes for electron microscopy.</a> J Ulstract Res. 13, 263-268 (1965).</li><li>Taupin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+scarce+biological+samples+for+light+and+transmission+electron+microscopy.">Processing scarce biological samples for light and transmission electron microscopy.</a> Eur J Histochem. 52, 135-139 (2008).</li><li>Mather, J., Stanbridge, C. M., Butler, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+removal+of+selected+cells+from+cytological+smear+preparations+for+transmission+electron+microscopy.">Method for the removal of selected cells from cytological smear preparations for transmission electron microscopy.</a> J Clin Pathol. 34, 1355-1357 (1981).</li><li>Oorschot, V., de Wit, H., Annaert, W. G., Klumperman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+flat-embedding+method+to+prepare+ultrathin+cryosections+from+cultured+cells+in+their+in+situ+orientation.">A novel flat-embedding method to prepare ultrathin cryosections from cultured cells in their in situ orientation.</a> J Histochem Cytochem. 50, 1067-1080 (2002).</li><li>Kumar, S., Ciraolo, G., Hinge, A., Filippi, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+and+reproducible+process+for+transmission+electron+microscopy+(TEM)+of+rare+cell+populations.">An efficient and reproducible process for transmission electron microscopy (TEM) of rare cell populations.</a> J Immunol Methods. 404, 87-90 (2014).</li><li>Chen, J., 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=Enrichment+of+hematopoietic+stem+cells+with+SLAM+and+LSK+markers+for+the+detection+of+hematopoietic+stem+cell+function+in+normal+and+Trp53+null+mice.">Enrichment of hematopoietic stem cells with SLAM and LSK markers for the detection of hematopoietic stem cell function in normal and Trp53 null mice.</a> Exp Hematol. 36, 1236-1243 (2008).</li><li>Hopwood, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reactions+between+formaldehyde,+glutaraldehyde+and+osmium+tetroxide,+and+their+fixation+effects+o+bovine+serum+albumin+and+on+tissue+blocks.">The reactions between formaldehyde, glutaraldehyde and osmium tetroxide, and their fixation effects o bovine serum albumin and on tissue blocks.</a> Histochemistry. 24, 50-64 (1970).</li><li>Saini, R., 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=Nitric+oxide+synthase+localization+in+the+rat+neutrophils:+immunocytochemical,+molecular,+and+biochemical+studies.">Nitric oxide synthase localization in the rat neutrophils: immunocytochemical, molecular, and biochemical studies.</a> J Leukoc Biol. 79, 519-528 (2006).</li><li>Kumar, S., 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+small+GTPase+Rap1b+negatively+regulates+neutrophil+chemotaxis+and+transcellular+diapedesis+by+inhibiting+Akt+activation.">The small GTPase Rap1b negatively regulates neutrophil chemotaxis and transcellular diapedesis by inhibiting Akt activation.</a> J Exp Med. 211, 1741-1758 (2014).</li></ol>

Los autores no tienen nada que revelar.

Este método permite el análisis TEM de bajo número de células, mediante el uso de la tinción de azul de Evans y la incrustación de agarosa para localizar un pequeño sedimento celular durante la deshidratación, la incrustación de resina y los procesos de corte. Es importante destacar, que mantiene juntos los grupos de células y preserva la ultra-estructura de la célula, que es deseable para examinar células múltiples para la cuantificación de datos. En estudios TEM, a menudo se ...

Detalles-Ultra estructurales y sub-orgánulos de células se han puesto de manifiesto principalmente por microscopía electrónica de transmisión (TEM) estudios de tejidos o sedimentos celulares 1,2. piezas sólidas de tejidos se pueden utilizar fácilmente para estudios de microscopía electrónica. Sin embargo, los análisis de TEM 1,3 en las células en suspensión siguen siendo difíciles y requieren altos números de células, es decir, millones de células. Debido a esto, los análisis ultra-estructurales de las poblaciones de células raras en suspensión, por ejemplo, células madre hematopoyéticas (HSC), no son evaluados fácilmente. Múltiples intentos para el análisis de TEM de las muestras de los escasos utilizando cytospin o monocapa enfoques no pueden conseguir datos cuantitativos significativa del número limitado de células. Por lo tanto, el requisito de un número alto de células limita el uso de esta potente herramienta para entender los detalles ultraestructurales subcelulares de las poblaciones de células raras. Una limitación clave para los estudios TEM con un número limitado de célulass en suspensión es la localización de las células para el procesamiento y, por tanto, los estudios de TEM de las células limitadas con particular tamaños pequeños siguen siendo difíciles. Varios enfoques alternativos se han adoptado para hacer frente a esta limitación: la BSA / bisacrilamida (BSA-BA) polimerización mediada por la suspensión celular, la tinción de las células con un tinte para que sean visibles en un soporte de película delgada incluyendo cubreobjetos, y análisis TEM de las preparaciones de citospina 4-6. Sin embargo, se logró un éxito muy limitado, como se encontraron muy pocas células en secciones después de ultra-seccionamiento. El desafío clave de identificación de células dispersas persiste debido a la monocapa de células se mantiene prácticamente invisible en el gel solidificado para seccionar. Además, la preparación de citospina de células puede alterar su organización celular debido a la propagación de células y la fragilidad de la estructura celular. Por lo tanto, estos inconvenientes inherentes justifican un enfoque novedoso para llevar a cabo estudios de TEM de las poblaciones de células raras con másconsistencia. Para superar este problema, hemos descrito un método nuevo y preparación de la muestra alternativa para los estudios de TEM de las poblaciones de células raras 7. Aquí, se presenta un protocolo de preparación de muestras eficiente para llevar a cabo TEM de las muestras biológicas con escasos resultados cualitativos y cuantitativos consistentes. Evans tinción con azul se llevó a cabo después de la fijación para localizar un pequeño sedimento celular a partir de células de bajo número, es decir, 10.000 médula ósea madre hematopoyéticas y células progenitoras que de otro modo habrían permanecido invisible, y el sedimento se incrusta en agarosa antes de procesos de deshidratación y de la resina de inclusión. Este método agrupa las células juntos y permite el análisis eficaz de la ultraestructura y organización subcelular de las células madre hematopoyéticas (identificado como Lin - Sca-1 + c-Kit + Flt3 - CD34 -; HSC), una rara 0,2 - población de células 0,5% en la médula ósea. Este pro experimentaltocol puede ser útil para llevar a cabo estudios de ultra-estructurales y obtener resultados cuantitativos en muchas poblaciones raras pero altamente importantes.

<table border="0" cellpadding="0" cellspacing="0" width="1096">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:327px;">Paraformaldehyde 20% Aqueous Soln</td>
			<td style="width:216px;">Electron microscopy Sciences</td>
			<td style="width:113px;">15713</td>
			<td style="width:440px;">CAUTION Toxic via inhalation, skin contact. Wear gloves, use fume hood.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:327px;">Gluteraldehyde 70% Aqueous Soln</td>
			<td style="width:216px;">Electron microscopy Sciences</td>
			<td>16350</td>
			<td>CAUTION Toxic via inhalation, skin contact. Wear gloves, use fume hood.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Cacodyladate buffer&#160;</td>
			<td style="width:216px;">Electron microscopy Sciences</td>
			<td style="width:113px;">12300</td>
			<td>Make 0.1 M Cacodyladate buffer (pH 7.4) in distilled water, store at 4&#176;C. CAUTION Irritant by inhalation or skin contact, wear gloves and work in fume hood.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Evans Blue&#160;</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:113px;">E-2129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low melting agarose&#160;</td>
			<td style="width:216px;">Sigma Aldrich</td>
			<td style="width:113px;">A-5030</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Osmium tetraoxide&#160;</td>
			<td style="width:216px;">Electron microscopy Sciences</td>
			<td style="width:113px;">19130</td>
			<td>1 gm in 50 ml of 0.1M Cacodyladate buffer to make 2% OsO4 stock, CAUTION Very toxic by inhalation or skin contact, strong oxidizer, wear protective clothing, eye protection, use fume hood.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LX-112 Embedding Kits (LADD&#174;)</td>
			<td style="width:216px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">DDSA (Dodecenylsuccinic anhydride)</td>
			<td style="width:216px;">LADD</td>
			<td style="width:113px;">21340</td>
			<td>Mix 16% DDSA; 30% NMA; 54% LX-112 together and then add 1.4% DMP-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">NMA (Nadic methyl anhydride)</td>
			<td style="width:216px;">LADD</td>
			<td style="width:113px;">21350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Epoxy Resins Epon 812 (LX-112)</td>
			<td style="width:216px;">LADD</td>
			<td style="width:113px;">21310</td>
			<td>CAUTION Toxic by inhalation, skin contact. Wear gloves, protective clothing, use fume hood.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:327px;">DMP-30 (2,4,6 -Tri(dimethylaminomethyl)phenol)</td>
			<td style="width:216px;">LADD</td>
			<td style="width:113px;">21370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reynolds lead citrate (EM Stain II)</td>
			<td style="width:216px;">Leica</td>
			<td style="width:113px;"></td>
			<td>CAUTION Toxic by inhalation, avoid contact with skin or eyes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Uranyl acetate (EM Stain I)</td>
			<td style="width:216px;">Leica</td>
			<td style="width:113px;">8072820</td>
			<td>CAUTION Toxic by inhalation, avoid contact with skin or eyes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)&#160;</td>
			<td style="width:216px;">ATCC</td>
			<td style="width:113px;">30-2005</td>
			<td>composition: NaCl, NaHCO3, HEPES, Glucose, Ca, Mg, sod pyruvate etc. For for details https://www.atcc.org/~/media/Attachments/E/9/7/E/4893.ashx</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pyramid tip mold&#160;</td>
			<td>Ted Pella</td>
			<td style="width:113px;">10585</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mounting cylinders</td>
			<td style="width:216px;">Ted Pella</td>
			<td style="width:113px;">10580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra-microtome&#160;</td>
			<td style="width:216px;">(Leica EM UC7)</td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">200 mesh grids (nikil)</td>
			<td style="width:216px;">Electron microscopy Sciences</td>
			<td style="width:113px;">G-200 Ni</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Electron Microscope</td>
			<td>Hitachi model H-7650&#160;</td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Image capture engine software</td>
			<td>AMT-600&#160;</td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm plastic petri dishes</td>
			<td>Fisher scientific&#160;</td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Quick Bond Super glue Cyanoacrylate&#160;</td>
			<td style="width:216px;">Electron microscopy Sciences</td>
			<td style="width:113px;">72588</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor blades</td>
			<td style="width:216px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass scintillation vials&#160;</td>
			<td>Fisher scientific&#160;</td>
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an alternative approach for sample preparation with low cell number for tem analysis

microscopía electrónica de transmisión (TEM) proporciona detalles de la organización celular y ultraestructura. Sin embargo, el análisis de TEM de las poblaciones de células raras, especialmente células en suspensión, tales como células madre hematopoyéticas (HSC), sigue siendo limitada debido a la exigencia de un número de células de alta durante la preparación de la muestra. Hay algunos enfoques Cytospin o monocapa para el análisis TEM de las muestras de los escasos, pero estos enfoques no pueden conseguir datos cuantitativos significativos a partir del número limitado de células. Aquí, se describe un enfoque alternativo y novedoso para la preparación de muestras en los estudios TEM para poblaciones de células raras que permite el análisis cuantitativo. Un número relativamente bajo de células, es decir, 10.000 HSC, se utilizó con éxito para el análisis de TEM en comparación con los millones de células utilizadas normalmente para estudios de TEM. En particular, la tinción de azul de Evans se realizó después de paraformaldehído-glutaraldehído fijación (PFA-GA) para visualizar la pequeña pel celulardejar, lo que facilitó la incorporación de agarosa. No se observaron agrupaciones de numerosas células en cortes ultrafinos. Las células tenían una morfología bien conservada, y los detalles ultra-estructurales de la mitocondria complejos y varios de Golgi eran visibles. Este protocolo eficiente, fácil y reproducible permite la preparación de muestras de un número bajo de células y se puede utilizar para el análisis TEM cualitativa y cuantitativa sobre las poblaciones de células raras de muestras biológicas limitados.

Un protocolo eficiente y consistente para la preparación de muestras para realizar análisis de TEM se describe de bajo número de células. Tinción posterior a la fijación con azul de Evans y la transferencia de las células a un tubo de microcentrífuga de 0,5 ml ayudó a visualizar el pequeño sedimento celular 7. Osmification con tetraóxido de osmio en agarosa condujo a una fácil detección del sedimento celular oscuro durante la deshidratación y la incrustación. </...

Watch this Scientific Journal Video about An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis at JoVE.com

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

Un enfoque alternativo para la preparación de muestras con bajo número de celular para el análisis TEM

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

an-alternative-approach-for-sample-preparation-with-low-cell-number

Research

JoVE Journal

Biology

13.6K vistas.  Cincinnati Children’s Research Foundation. Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis. 

Video: An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applications. It yields beautiful images1 and allows for techniques such as fluorochrome assessment and histomorphometry2. Fluorochrome analysis is a technique where fluorescent dyes that bind to calcium are injected at a particular time point, which allows for quantification of the amount of mineralisation at that given time. Histomorphometry is a process of bone quantification at the microscopic level. 
Performing undecalcified bone histology is technically challenging, particularly with large size specimens. It requires variations in technique from those used in standard paraffin embedded histology. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them. Specimen preparation, fixation and processing are achieved with a manner similar to other soft tissues, however due to the density and lower permeability of bone considerably longer fixation and processing times are required, often taking several weeks. Embedding is achieved using a supporting medium with similar or equal hardness and density to the bone such as methacrylate- based resins, but unlike paraffin infiltration and embedding, this is an irreversible step. Sectioning can be achieved by grinding which produces a thicker section, which is optimal for studies such as fluorochrome analysis. This is best achieved using a diamond blade on a macrotome. Alternatively, thinner sections can be produced for light microscopy and this is achieved using a sledge microtome with a very sharp blade. The sledge microtome provides the additional strength and stability required for large, hard blocks. Resin embedded sections can be stained with a variety of stains, which are demonstrated.

Undecalcified bone histology provides important information for a variety of clinical and research applications. It is technically challenging, particularly with large size specimens. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them.

Preparación ósea descalcificados para histología, histomorfometría y Análisis Fluorocromo

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides ...

Investigación

Biología

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

Preparación de muestras para microscopía de fuerza atómica de virión único e imágenes de fluorescencia de súper resolución

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Culturas alternativas para Pluripotentes Stem Cell Production, mantenimiento y análisis genético

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

Preparación de muestras para análisis de masas Citometría

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

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 ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Preparación de la muestra y proyección de imagen de exosomas por microscopía electrónica de transmisión

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

Preparación de muestras miniatura para microscopia electrónica de transmisión

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is often difficult to isolate and thus studying them often provides a challenge. 
This study establishes a stomatal guard cell enrichment method. The protocol utilizes Scotch tape to isolate guard cells and extract proteins from the cells. This method improves the integrity and yield of guard cell during isolation and provides a reliable sample for the study of cell signaling processes and how they correlate to stomatal movement phenotypes. In this method, the plant leaves were partitioned between two portions of tape and peeled apart to remove the abaxial side. This protocol was applied to Arabidopsis leaf tissue to isolate guard cells. The cells were treated with fluorescein diacetate (FDA) to determine viability and with abscisic acid (ABA) to assess stomata movement in response to ABA. In conclusion, this protocol proves useful for preparing enriched stomatal guard cells and isolating proteins. The quality of the cells and proteins obtained here enable physiological and other biological studies.

This protocol describes a method of preparing enriched stomatal guard cells that is useful for physiological and other biological studies.

Cinta-cáscara de estomas: Un método mejorado para la preparación de la muestra de protector de la célula

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

Preparación de muestras para análisis de proteómica basado en la espectrometría de masa de microvasos Ocular

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

Microaspiración de células únicas como estrategia alternativa a la clasificación celular activada por fluorescencia para la separación de mezclas de virus gigantes

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...