El trabajo fue apoyado por la Fundación Sueca para la Investigación Estratégica Ambiental (MISTRA) a través del programa de DOM y Grant 211684 (BACSIN) del Programa Marco de la Unión Europea Comunidad 7PM. Damos las gracias a J. Engstrand de asistencia en la filmación del análisis de rayos X y L. Tang por su ayuda en la narrativa conceptual.

1. El cultivo y la cosecha de P. putida <ol><li> Preparar un cultivo iniciador de Pseudomonas putida mediante la inoculación de 100 ml de caldo de soja tríptico (TSB) con una colonia de P. células putida cultivadas en agar suplementado con caldo de soja tríptico (TSA). Mantener el cultivo a 30 ° C en un tablero de agitación fijado en 130 rpm. </li><li> Después de 7 h transferir una alícuota del cultivo iniciador equivalente a 1/10 del volumen final del cultivo principal a la TSB-medio fresco. Mantener las células a 30 ° C en un tablero de agitación a 130 rpm durante 16 horas. </li><li> Después de 16 h de crecimiento de las células se recogen por centrifugación del cultivo celular a 1500 xg durante 20 min a TA. Decantar el sobrenadante y lavar las células mediante re-suspensión del sedimento celular se encuentra en una solución de NaCl que es isotónica con el medio de crecimiento. En este caso se utilizó una solución de NaCl 150 mM. Las células se centrifugan una vez más y se decanta el sobrenadante antes de resuspender las células en el medio de formulación,véase el paso 3.2. </li></ol> 2. El cultivo de otras especies <ol><li> Para adaptar el protocolo a otras especies bacterianas los procedimientos de cultivo y la cosecha deben ser cambiados de acuerdo a los requerimientos de esa especie. Para evitar el choque osmótico al manipular las células durante la cosecha y la etapa de lavado (s), la osmolalidad de las soluciones debe coincidir con la del medio de crecimiento en la cosecha. </li></ol> 3. Formulación de las células <ol><li> Preparar las soluciones de formulación pesando los componentes de la matriz respectivas y disolverlos en agua. Para lograr condiciones isotónicas, o bien ajustar la cantidad de excipiente o añadir NaCl o algún otro soluto compatible celular para llegar a la osmolalidad deseada. Para las sustancias de bajo peso molecular, tales como disacáridos las cantidades se pueden ajustar fácilmente para llegar a condiciones isotónicas. En sustancias como polímeros que son de alto peso molecular, la tonicidad tiene que ser ajustado con células compatibles por lolaúdes, por ejemplo, NaCl o disacáridos. Tenga en cuenta que cualquier adición de una sustancia influirá en las propiedades físicas de la formulación, que a su vez puede influir en el comportamiento de la liofilización y la supervivencia celular. </li><li> Decantar la solución de lavado (en este caso NaCl) y volver a suspender las células en los respectivos medios de formulación. </li><li> Asegúrese de que las células se dispersan homogéneamente. Las formulaciones de baja viscosidad se agitaron mientras que las formulaciones de viscosidad más altos se mezclan usando añadiendo, por ejemplo., Una barra de agitación y agitar el recipiente hasta que se logra una mezcla homogénea. </li><li> Divida las formulaciones en viales de congelación de pelo. Los viales se deben pesar vacío, con la muestra antes y después de secado por congelación para calcular la cantidad de agua eliminada durante la liofilización. </li><li> Añadir tapones de goma de los viales si los viales deben ser sellados en el interior del liofilizador, consulte el paso 4.3. </li><li> Enumerar las células en cada formulación antes de la liofilización, consulte el paso 6. </li> &lt;/ Ol&gt; 4. La liofilización <ol><li> Las condiciones de secado por congelación se tienen que ajustar a las propiedades físicas de la formulación. El parámetro más importante es en este caso la temperatura de transición vítrea, Tg, de la formulación, denota Tg &#39;para la muestra de congelación y concentrada para indicar que el agua está todavía presente. La Tg &#39;de la formulación de congelación-concentrada se mide fácilmente utilizando calorimetría de barrido diferencial (DSC), véase el paso 7. </li><li> Ajustar los parámetros del proceso de liofilización de manera que la temperatura de la muestra es siempre por debajo de la Tg de la muestra y que la presión de la cámara permite una rápida sublimación del hielo, es decir, el secado primario, y el agua residual en la formulación, es decir, secundaria secado. Si la temperatura de la muestra está por encima de Tg &#39;durante el proceso de secado hay un gran riesgo de colapso estructural de la muestra lo que puede disminuir significativamente la supervivencia celular. </li><li> Para proteger los productos secos después de freeze-secado de la atmósfera de la muestra tiene que ser controlado. Si es posible, sellar las muestras en el secador por congelación antes de que el vacío se libera al final del ciclo de secado por congelación. Alternativamente, los frascos se pueden llenar con una atmósfera preferida. Es importante evitar la exposición de las muestras a las condiciones ambientales como la humedad o el oxígeno presente en la atmósfera de almacenamiento se influye en la supervivencia de las células. </li><li> Pesar los viales. </li></ol> 5. Rehidratación <ol><li> Rehidratar las muestras con agua desionizada y estéril. La cantidad de agua que debe añadirse debe ser la misma que la cantidad eliminado durante la liofilización y se calcula a partir del peso del vial vacío, el mismo vial con la muestra antes y después de la liofilización. Vortex las muestras una y otra vez hasta que las soluciones a continuación, aparece homogénea. </li></ol> 6. Enumeración <ol><li> Dibuje 100 l de cada muestra y hacer diluciones en serie de 10 veces. A partir de las diluciones de integración100 l resto se siembra en placas de TSA. Las placas se incubaron a la temperatura y el tiempo requerido. </li></ol> 7. Caracterización de la formulación de comportamiento de la congelación <ol><li> Tomar una pequeña cantidad, 5 a 10 mg, de la muestra y encerrarlo en una sartén DSC-aluminio con tapa. Prepare un recipiente vacío como referencia. </li><li> Establecer el programa de análisis de temperatura DSC. El programa de enfriamiento está configurado para imitar la etapa de congelación en el programa de secado por congelación. Esto se hace como la cinética de enfriamiento pueden influir en la Tg &#39;de las formulaciones hidratadas. Antes de la exploración de calefacción comienza a reducir la temperatura por debajo de la Tg esperado &quot;de las formulaciones investigadas, normalmente -100 ° C. </li><li> Elija un tipo adecuado de calentamiento, normalmente una velocidad de calentamiento de 5 - 40 º C / min se utiliza. La señal de flujo de calor registrada se expresa en vatios y se verá influido por la velocidad de calentamiento. A mayor velocidad de calentamiento dará una señal más grande de Tg. </li><li> Ajuste el rango de temperatura de manera que it cubre tanto Tg &#39;y el derretimiento de la formulación. </li></ol> 8. Las mediciones de tensión superficial de las formulaciones hidratadas <ol><li> El montaje experimental usado en este experimento es de acuerdo con el método du Noüy para medir la superficie. </li><li> Limpie el anillo de platino y el recipiente de medición con cuidado. El recipiente se enjuagó con acetona, se secó con un paño libre de pelusa, y luego quemada en el interior con una llama incolora. El anillo se ilumina en rojo en una llama incolora. </li><li> Llene el recipiente con la solución y deje que la superficie resolver antes de medir. Para las soluciones que se alcanza el estado de equilibrio sólo después de un largo tiempo, por ejemplo, polímeros, la sencilla configuración utilizada aquí proporcionarán datos cualitativos. </li><li> Para las formulaciones con altas viscosidades, por ejemplo soluciones de 2% (w / w) de HEC o de HPMC, la tensión superficial tiene que ser medido en soluciones de concentraciones más bajas y luego extrapolada. Esto se puede hacer tomando ventaja del hecho de quela caída en los niveles de tensión superficial frente a concentraciones por debajo de 0,5% (w / w). </li></ol> 9. Análisis de rayos X de las formulaciones secas <ol><li> Tomar una pequeña cantidad de matriz / bacterias y cargarlo en un soporte de muestras. </li><li> Monte el soporte de la muestra en el X-ray-difractómetro. </li><li> Para analizar cualquier fase cristalina presente en la muestra el paquete de programas de Bruker EVA se puede utilizar. </li></ol> 10. Microscopía Electrónica <ol><li> Tomar una pequeña cantidad de matriz / bacterias de los viales y fijarlo en un talón SEM. </li><li> Cargar el titular de SEM en el dispositivo de recubrimiento por pulverización catódica (Thermo Polaron VGScientific SC7640 utilizado en el presente trabajo) y la capa de la muestra con unos pocos nm de Au / Pd o Pt. En estos experimentos, los parámetros de pulverización fueron: 1900 V, 20 mA y 20 segundos, para un ~ 5-10 nm recubrimiento Au / Pd. El recubrimiento reduce la carga eléctrica durante la adquisición de imagen. Se recomienda comenzar con una capa fina y, si persiste la carga y hace que la imagenartefactos, la muestra deben estar recubiertos de nuevo. </li><li> Cargar el titular de SEM en la cámara de SEM. Seleccione los parámetros de imagen correspondientes. Para el instrumento utilizado en la Figura 2 (FEI Strata DB235), se utilizó 5 kV de voltaje de aceleración, tamaño de spot 3, una distancia de trabajo de 5 mm y el detector de electrones secundarios. </li><li> Opcionalmente, es posible utilizar el haz de iones enfocado para cortar secciones transversales de las bacterias incrustadas en el polímero para revelar más detalles. </li></ol>

<ol>
	<li>Wessman, P., Mahlin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+matrix+properties+on+the+survival+of+freeze-dried+bacteria.">Impact of matrix properties on the survival of freeze-dried bacteria.</a> J. Sci. Food Agric. 91 (14), 2518-2528 (2011).</li><li>Ablett, S., Izzard, M. 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=Differential+Scanning+Calorimetric+Study+of+Frozen+Sucrose+and+Glycerol+Solutions.">Differential Scanning Calorimetric Study of Frozen Sucrose and Glycerol Solutions.</a> Journal of the Chemical Society-Faraday Transactions. 88 (6), 789-794 (1992).</li><li>Knopp, S. A., Chongprasert, 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+relationship+between+type+TMDSC+curve+of+frozen+sucrose+solutions+and+collapse+during+freeze-drying.">The relationship between type TMDSC curve of frozen sucrose solutions and collapse during freeze-drying.</a> Journal of Thermal Analysis and Calorimetry. 54 (2), 659-672 (1998).</li><li>Crowe, J. H., Leslie, S. B., 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=Is+Vitrification+Sufficient+to+Preserve+Liposomes+during+Freeze-Drying.">Is Vitrification Sufficient to Preserve Liposomes during Freeze-Drying.</a> Cryobiology. 31 (4), 355-366 (1994).</li><li>Jain, P., Sen, 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=Effect+of+glass-forming+biopreservatives+on+head+group+rotational+dynamics+in+freeze-dried+phospholipid+bilayers:+A+P-31+NMR+study.">Effect of glass-forming biopreservatives on head group rotational dynamics in freeze-dried phospholipid bilayers: A P-31 NMR study.</a> Journal of Chemical Physics. 131 (2), (2009).</li><li>Stock, J. B., Rauch, B., 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=Periplasmic+Space+in+Salmonella-Typhimurium+and+Escherichia-Coli.">Periplasmic Space in Salmonella-Typhimurium and Escherichia-Coli.</a> Journal of Biological Chemistry. 252 (21), 7850-7861 (1977).</li><li>Crowe, J. H., Hoekstra, F. 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=Anhydrobiosis.">Anhydrobiosis.</a> Annual Review of Physiology. 54, 579-599 (1992).</li><li>Leslie, S. B., Israeli, E., 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=Trehalose+and+Sucrose+Protect+Both+Membranes+and+Proteins+in+Intact+Bacteria+during+Drying.">Trehalose and Sucrose Protect Both Membranes and Proteins in Intact Bacteria during Drying.</a> Applied and Environmental Microbiology. 61 (10), 3592-3597 (1995).</li><li>Nesarikar, V. V., Nassar, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cations+and+anions+on+glass+transition+temperatures+in+excipient+solutions.">Effect of cations and anions on glass transition temperatures in excipient solutions.</a> Pharmaceutical Development and Technology. 12 (3), 259-264 (2007).</li><li>You, Y., Ludescher, 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=The+effect+of+sodium+chloride+on+molecular+mobility+in+amorphous+sucrose+detected+by+phosphorescence+from+the+triplet+probe+erythrosin+B.">The effect of sodium chloride on molecular mobility in amorphous sucrose detected by phosphorescence from the triplet probe erythrosin B.</a> Carbohydr. Res. 343 (2), 350-363 (2008).</li><li>Crowe, J. H., Hoekstra, F. A., Nguyen, K. H. N., Crowe, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+vitrification+involved+in+depression+of+the+phase+transition+temperature+in+dry+phospholipids.">Is vitrification involved in depression of the phase transition temperature in dry phospholipids.</a> Biochim. Biophys. Acta Biomembr. 1280, 187-196 (1996).</li></ol>

No hay conflictos de interés declarado.

El motivo de este estudio fue investigar algunas propiedades de formulación que puedan ser de importancia para la supervivencia celular durante la liofilización. Aunque la tolerancia intrínseca secado varía entre las diferentes especies, como se ilustra en la Figura 1A, la tendencia de qué tan bien la diferente supervivencia de las células de soporte formulaciones es similar. Es informativo para comenzar con una comparación de la sacarosa y Ficoll. Se cree que un factor clave para una formulació...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Ficoll PM 400</td>
			<td>GE-healthcare</td>
			<td>17-0300-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEC (Natrosol-M Pharm Grade)</td>
			<td>Ashland</td>
			<td>&nbsp;</td>
			<td>Gift from Ashland</td>
		</tr>
		<tr>
			<td>HPMC (Methocel F4M)</td>
			<td>Dow</td>
			<td>&nbsp;</td>
			<td>Gift from the Department of Pharmacy, Uppsala University</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S2395</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71376</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tryptic Soy broth</td>
			<td>Merck</td>
			<td>105459</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tryptic Soy Agar</td>
			<td>Merck</td>
			<td>105458</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lyostar II</td>
			<td>FTS Kinetics</td>
			<td>N.A.</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pyris Diamond DSC</td>
			<td>Perkin-Elmer</td>
			<td>N.A.</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bruker AXS SMART CCD 1k Diffractometer</td>
			<td>Bruker</td>
			<td>N.A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dual-beam FEI Strata DB235 FIB/SEM</td>
			<td>FEI</td>
			<td>N.A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Kr&uuml;ss Educational Tensiometer</td>
			<td>Kr&uuml;ss</td>
			<td>N.A.</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

formulations for freeze-drying of bacteria and their influence on cell survival

Agua celular se puede quitar para inactivar microorganismos reversible para facilitar el almacenamiento. Uno de tales métodos de eliminación es la liofilización, que se considera un método de deshidratación suave. Para facilitar la supervivencia celular durante el secado, las células se formulan a menudo de antemano. La formulación forma una matriz que incorpora las células y los protege de diversas tensiones perjudiciales impuestas a las células durante la congelación y el secado. Se presenta aquí un método general para evaluar la tasa de supervivencia de las células después de la liofilización y que se ilustra mediante la comparación de los resultados obtenidos con cuatro formulaciones diferentes: la sacarosa, el disacárido sacarosa polímero derivado de Ficoll PM400, y los respectivos polisacáridos hidroxietil celulosa (HEC ) e hidroxipropil metil celulosa (HPMC), en dos cepas de bacterias, P. putida KT2440 y A. A6 chlorophenolicus. En este trabajo se ilustra cómo preparar formulaciones para la liofilización y cómo investigar la mecánicaismos de la supervivencia celular después de la rehidratación mediante la caracterización de la formulación utilizando de calorimetría diferencial de barrido (DSC), mediciones de tensión superficial, análisis de rayos X, y la microscopía electrónica y en relación a los datos de las tasas de supervivencia. Los polímeros se eligen para obtener una estructura monomérica del respectivo polisacárido sacarosa se asemeja a un mayor o menor grado. El uso de este método de instalación que mostramos que los polímeros pueden apoyar la supervivencia de las células tan eficazmente como disacáridos si ciertas propiedades físicas de la formulación son controlados 1.

La Tabla 1 muestra los datos sobre la composición de la formulación, eventos térmicos registrados por DSC durante el calentamiento de las formulaciones congeladas, la estructura de las muestras secas y la tensión superficial de las soluciones de formulación. La Tg &#39;de sacarosa se ​​ha determinado a -40 ° C 2, 3 y puede ser difícil de detectar para concentraciones de sacarosa inferior al 20% w / w. El evento térmico a -35 ° C está probablemente relacionado...

Watch this Scientific Journal Video about Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival at JoVE.com

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

La liofilización es a menudo una manera fácil y conveniente de obtener productos secos de células bacterianas viables. Un problema del proceso es la supervivencia celular. Nos detalle aquí un procedimiento para investigar cómo la supervivencia celular durante la liofilización está influenciada por las propiedades de la formulación utilizada.

Las formulaciones para la liofilización de las bacterias y su influencia en la supervivencia celular

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

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Biology

23.2K vistas.  Swedish University of Agricultural Sciences. La liofilización es a menudo una manera fácil y conveniente de obtener productos secos de células bacterianas viables. Un problema del proceso es la supervivencia celular. Nos detalle aquí un procedimiento para investigar cómo la supervivencia celular durante la liofilización está influenciada por las propiedades de la formulación utilizada.

Video: Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made significant contributions to our understanding of the co-evolution of eukaryotes with their microbiota. The symbiotic association between the Hawaiian bobtail squid, Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri has been utilized as a model system for understanding the effects of beneficial bacteria on animal development. Recent studies have shown that macrophage-like hemocytes, the sole cellular component of the squid host's innate immune system, likely play an important role in mediating the establishment and maintenance of this association. This protocol will demonstrate how to obtain hemocytes from E. scolopes and then use these cells in bacterial binding assays. Adult squid are first anesthetized before hemolymph is collected by syringe from the main cephalic blood vessel. The host hemocytes, contained in the extracted hemolymph, are adhered to chambered glass coverslips and then exposed to green fluorescent protein-labeled symbiotic Vibrio fischeri and non-symbiotic Vibrio harveyi. The hemocytes are counterstained with a fluorescent dye (Cell Tracker Orange, Invitrogen) and then visualized using fluorescent microscopy.

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

This video will demonstrate how to obtain hemocytes (blood cells) from the Hawaiian bobtail squid, Euprymna scolopes for use in cell biological and bacterial adhesion assays. Hemocytes will be stained with a fluorescent dye and exposed to GFP-labeled bacteria.

La obtención de hemocitos de la hawaiana Squid Bobtail Euprymna scolopes Y la observación de su adhesión a las bacterias simbióticas y no simbióticas-

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made ...

Investigación

Biología

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

La detección de modificaciones post-traduccionales de los nativos intactos nucleosomas por ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

El análisis de la función de las pequeñas GTPasas de la microinyección de plásmidos en células epiteliales polarizadas

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Perfiles de transcripción de los organismos unicelulares de los cardiomiocitos de ratón adulto

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Aislamiento y cultivo de fibras individuales y sus células satélite del músculo esquelético adulto

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

Estrategias para Anastasis Seguimiento, una célula de supervivencia Fenómeno que Invierte Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

Un método estandarizado para el análisis de las células del hígado sinusoidal endoteliales y sus Fenestraciones por Microscopía Electrónica de Barrido

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

Determinación de la supervivencia en el tiempo<em&gt; Brachionus</em&gt; Los rotíferos: Sincronización de Condiciones maternas

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Investigar los efectos perjudiciales de baja presión Plasma esterilización sobre la supervivencia de vivir el Bacillus subtilis esporas usando el celular microscopia

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Cuantificación de proteína específica lisina acetilación y Succinylation estequiometría usando Spectrometry total de la adquisición de datos-independiente

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...