Este trabajo fue apoyado por becas fromthe Fondo Belga para la Investigación Científica (FNRS), Les Amis de l&#39;Institut Bordet, FNRS-Opération Télévie, Cáncer del Plan de Bélgica, Fonds Lambeau-Marteaux, Fonds JC Heuson y Fonds Barsy.

NOTA: Todos los especímenes fueron adquiridos utilizando un protocolo aprobado por el Comité de Ética Médica del Instituto Jules Bordet con el consentimiento informado por escrito obtenido de cada paciente. 1. Preparación del homogeneizado de tejido <ol><li> Diseccionar tejidos resecados (tejido maligno y normal resecado de la sala de operaciones) están en el laboratorio de patología por personal capacitado para su recogida inmediata. Tumor, NANT (tomada la distancia más alejada del tumor como sea posible) y fragmentos de tejido normal se procesan de forma rutinaria plazo de 1 - 3 horas de la escisión quirúrgica en un laboratorio BSL2 usando procedimientos de bioseguridad estándar para los tejidos humanos. Un diagrama de flujo del protocolo se ilustra en la Figura 1. </li><li> Pesar todos los fragmentos de tejido (normal, NANT y tumorales) y medir la longitud, anchura y altura (largo x ancho x alto). Este es un paso importante para la posterior normalización de las subpoblaciones de células, el ARN extraído, etc. NOTE: El rango de tamaño de la muestra es de 100 a 10.000 mm 3 con nada de grasa si es posible. </li><li> Pie de imprenta el fragmento del tumor en un portaobjetos de vidrio para tinción H &amp; E para verificar que el tejido es en realidad parte del tumor. <ol><li> Para ello, pulsando un microscopio portaobjetos de vidrio en el fragmento del tumor y aplicar una suave presión con los dedos durante unos segundos. </li><li> Fijar el portaobjetos con isopropanol durante 2 min seguido de una etapa de lavado en agua. Contratinción el tejido durante 30 segundos con hematoxilina de Mayer. </li><li> Lave la diapositiva en seis baños de agua. Teñir en Floxina B 2% durante 15 s. </li><li> Lave en un baño de agua, seguido de cuatro baños de isopropanol y terminar con un baño de agua. </li><li> Incubar en isopropanol durante 1 min y de drenaje. Claro en dos baños de xileno. </li><li> Montar con medio de montaje basado xileno. Examine para celularidad tumoral (Figura 2). NOTA: Principalmente, las células tumorales se adhieren a la diapositiva improntado - el estroma, linfoide o adipose células raramente permanecen, dejando espacios entre las células tumorales (Figura 2). </li></ol></li><li> Coloque el fragmento de tejido en un pequeño plato de cultivo que contiene 1 ml de la, medio de células hematopoyéticas libre de suero definido químicamente (en lo sucesivo denominado medio) a temperatura ambiente y trocea en pedazos pequeños (~ 1 - 2 mm 2) utilizando un estéril bisturí. </li><li> Transferir todo (fragmentos de tejido + medio) a un tubo disociador C mecánica. </li><li> Enjuague la placa de Petri y el bisturí con 2 ml de medio con una pipeta Pasteur y añadir esta al tubo C (volumen de medio para la disociación = 3 ml). </li><li> Utilice la A.01 programa disociador mecánico para tubos C (el programa más suave) para homogeneizar los fragmentos de tejido en una suspensión de células individuales. Coloque el tubo de C en el aparato y ejecutar el programa dos veces seguidas (un ciclo = 25 segundos). NOTA: Este procedimiento de homogeneización se ha establecido y validado por el tejido mamario humano, otra tumo tejido o tipos pueden necesitar usar un programa diferente y debe ser probado primero. </li><li> Retire el tubo C del aparato y decantar el homogeneizado directamente en un filtro de células de 40 micras sentado en un tubo de 50 ml. Usando la misma pipeta Pasteur como en el paso 1.6, transferir cualquier líquido restante en el tubo C para el filtro de células. </li><li> Transferir el líquido filtrado en un tubo de 15 ml con una punta de micropipeta 1 ml. Temporalmente mantener el filtro de células y su tubo 50 ml. </li><li> Enjuague el tubo C con un adicional de 3 ml de medio y transferir este, de nuevo utilizando la misma pipeta Pasteur como en el paso 1.6, para el filtro de células todavía sentado en el tubo de 50 ml. Exprima una cantidad máxima del líquido residual atrapado en el tejido no homogeneizada en el tubo de 50 ml moviendo suavemente alrededor del filtro con una pipeta Pasteur limpia o 1 ml punta que posteriormente desechado para evitar contaminar el eluido. </li><li> Coloque el colador celular al revés de tél tubo C original y enjuague con 3 ml de medio para que el tejido no homogeneizada cae de nuevo en el tubo C. </li><li> Vuelva a homogeneizar como en el paso 1.7 para dos ciclos del programa A.01. </li><li> Verter esta segunda homogeneizado a través del filtro de células sentado en el tubo de 50 ml, lavar el tubo C de nuevo con medio de 3 ml (como en el paso 1,10) y transferir con la pipeta Pasteur para el filtro de células sentado en el tubo de 50 ml de nuevo apretando un máximo cantidad de líquido desde el tejido conectivo residual atrapado en el filtro de células. </li><li> En este punto, un volumen de ~ 2,5 ml es en el tubo de 15 ml y ~ 9 ml en el tubo de 50 ml. </li></ol> 2. La separación del sobrenadante de tejidos y células <ol><li> Centrifugar los homogeneizados en los 15 ml y 50 ml tubos durante 15 min a 600 xg a temperatura ambiente. </li><li> Decantar el SN desde el tubo de 15 ml en un tubo limpio y almacenar temporalmente a 4 ° C. Este sobrenadante = tumor primario, NANT o Ti normalesDICIÓN SN (volumen final de 2,5 ml) se precisó y alicuotó antes de su almacenamiento a -80 ° C para análisis futuros (ver más abajo). </li><li> Desechar el sobrenadante del tubo de 50 ml. </li><li> Resuspender suavemente ambos sedimentos celulares en un volumen final de 1 ml de medio. Brevemente, primero romper suavemente el sedimento celular en ambos tubos (tocando el tubo sobre una superficie dura). Resuspender el sedimento celular suelta en los 50 ml con 500 l de medio y transferir la suspensión celular al tubo de 15 ml para resuspender el pellet segundos. Repita este paso una vez con los segundos 500 l de medio para la recuperación máxima de las células. </li><li> Transferencia de 10 l de la suspensión celular a un tubo pequeño, mezcle con 10 l de azul de tripano (dilución 1: 1) y contar el número de células viables utilizando un hemocitómetro. NOTA: En este punto una fracción de la suspensión celular puede también ser analizada por citometría de flujo para evaluar el tamaño celular, granularidad y si se desea un número limitado de marcadores subpoblación para más precise evaluación de la distribución relativa de célula en el homogeneizado antes de extenso análisis o experimentación. Todos los análisis por citometría de flujo incorporan etiquetado CD45 para la normalización de las subpoblaciones. </li><li> Sedimentar las células por centrifugación a 300 xg durante 10 min a temperatura ambiente. Las células del tumor, NANT, o tejido normal están ahora listos para la purificación adicional o análisis. Estos pasos adicionales son mejores cuando se realizan en el mismo día de la cirugía. NOTA: Para el análisis de citometría de flujo pero no de clasificación de células de las células rojas de la sangre residual debe ser lisadas después del marcaje de anticuerpos mediante la adición de 0,4 ml de sangre roja tampón de lisis celular al sedimento celular, inmediatamente agitación durante 1 seg y la incubación de un mínimo de 10 min en la sala de temperatura (protegido de la luz) antes del análisis. </li></ol> 3. Aclaración del sobrenadante de Tejidos <ol><li> Centrifugar los tubos de 1,5 ml con SN tejido a 15.000 xg durante 15 min a 4 ° C. </li><li> Retire con cuidado tél sobrenadante sin tocar o alterar el sedimento. Transferir a un tubo limpio (o tubos) dependiendo del número y volumen de alícuotas deseados. </li><li> Almacenar el sobrenadante a -80 ° C para uso futuro. </li></ol> 4. sangre del paciente <ol><li> Se recoge una muestra de sangre de cada paciente como un control por punción venosa en tubos heparinizados el día antes de la cirugía. Centrifugar la sangre a 400 xg durante 10 min a 20 ºC con el freno para obtener el plasma y la capa leucocitaria. </li><li> Retire el plasma y clarificar por centrifugación a 10.000 xg durante 15 min a 20 ºC. Alícuota y almacenar plasma a -80 ° C para uso futuro. Se diluye la capa leucocitaria en medio </li><li> Separar las células mononucleares utilizando estándar de centrifugación en gradiente de Ficoll-Hypaque antes del análisis inmediato, el aislamiento subpoblación, ADN / ARN / extracción de la proteína o la criopreservación. </li></ol> 5. Citometría de Flujo <ol><li> Las células de la etiqueta según fabricacióninstrucciones del r a 4 ° C, protegido de la luz. Lisar las células rojas de la sangre en las suspensiones de células a partir de fragmentos de tejido después del marcaje de anticuerpos mediante la adición de 0,4 ml de tampón de lisis celular al sedimento celular. </li><li> Vortex inmediatamente durante 1 segundo e incubar un mínimo de 10 minutos a temperatura ambiente, protegido de la luz. Pasar las células marcadas en un citómetro de flujo para la adquisición de datos sin lavar. </li></ol>

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The authors declare that no conflict of interest exists.

Este estudio describe un método optimizado para la rápida preparación de los homogeneizados de tejidos normales y malignos de mama sin digestión enzimática para la clasificación posterior de células, la extracción, la criopreservación y / o análisis fenotípico de CD45 + subpoblaciones. El objetivo de este enfoque experimental es para producir imágenes de la TIL que refleja de cerca su estado in vivo y compararlos con los tejidos normales con mínima manipulación de los tejidos frescos de...

The tumor microenvironment is composed of various cell types with numerous studies showing they each play distinct and important roles in tumorigenesis1,2. These include, but are not limited to, infiltrating immune cells, stromal cells, endothelial cells and tumor cells3. Ex vivo studies of tumor infiltrating lymphocytes (TIL; CD45+ cells or leukocytes, which are predominantly lymphocytes in breast tumors) from fresh human tissue samples is made difficult by their low frequency, the small sample sizes often available for research and the potential for loss of viability during extraction. Because immune cells infiltrating tumors are usually present as passengers rather than permanent residents in general they are easier to release from the tissue matrix.
Dissociating tumor tissue while maintaining cellular integrity is technically challenging and has traditionally been performed using a combination of mechanical and enzymatic steps to prepare single cell suspensions4-8. This approach involves lengthy incubation periods and is associated with a significant reduction in cell viability as well as the loss of cell surface receptors by enzymatic cleavage. High quality flow cytometric studies characterizing TIL in the tumor microenvironment as well as clean purifications of CD45+ subpopulations by flow cytometry or antibody-coated beads are more difficult to achieve from enzyme-digested tumor tissue. In addition, the supernatant (SN) from the resulting tumor homogenate is not amenable to further analysis including quantification of secreted proteins (cytokines, chemokines, immunoglobulins or tumor antigens) or experimental treatment of normal cells, because of the potential for protein degradation in the enzymatic digests.
In our search for a method to prepare single cell homogenates from breast tissues [including tumor, non-adjacent non-tumor (NANT) and normal (from mammary reductions) breast tissues] without enzymatic digestion, we tested a variety of mechanical homogenization techniques. Homogenates prepared using a mechanical dissociator had increased cell viability (2-fold) and total cell recovery (2-fold) while preserving surface receptor expression. Enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells suggesting they were all released in the initial homogenate. Thus, this rapid and simple approach allows both qualitative and quantitative assessment of the CD45+ subpopulations present in various normal and malignant human tissues. An added advantage of this approach is that the SN from the initial homogenate (primary tissue SN) can be collected and stored for further analysis or experimentation.

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			<td height="26" style="height:27px;width:272px;">Equipment</td>
			<td style="width:163px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:409px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">GentleMacs Dissociator&#160;</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-093-235</td>
			<td>BD Medimachine is somewhat equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Centrifuge 5810 R</td>
			<td style="width:163px;">Eppendorf</td>
			<td style="width:137px;">N/A</td>
			<td>or other standard table top centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Centrifuge 5417 R</td>
			<td style="width:163px;">Eppendorf</td>
			<td style="width:137px;">N/A</td>
			<td>or other standard microcentrifuge</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Esco Class II A2 Biosafety Cabinet</td>
			<td>ESCO global</td>
			<td style="width:137px;">N/A</td>
			<td>or other standard BSL2 hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Inverted Microscope</td>
			<td style="width:163px;">Nikon eclipse TS100</td>
			<td style="width:137px;">N/A</td>
			<td>or other microscope compatible for a hemacytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">B&#252;rker Chamber</td>
			<td style="width:163px;">Marienfield&#160;</td>
			<td style="width:137px;">640210</td>
			<td>or other standard hemacytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Navios Flow Cytometer</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:137px;">N/A</td>
			<td>or other flow cytometer (8-10 color recommended)</td>
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		<tr height="26">
			<td height="26" style="height:27px;width:272px;"></td>
			<td style="width:163px;"></td>
			<td style="width:137px;"></td>
			<td></td>
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		<tr height="26">
			<td height="26" style="height:27px;width:272px;">Materials</td>
			<td style="width:163px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">GentleMacs C-Tube</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-096-344</td>
			<td>BD Medimachine uses Filcon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Cell Culture Dish</td>
			<td style="width:163px;">Sarstedt</td>
			<td style="width:137px;">72,710</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Disposable Scalpel</td>
			<td style="width:163px;">Swann-Morton</td>
			<td style="width:137px;">0510</td>
			<td>or standard single use sterile scalpel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD Cell Strainer 40&#181;m</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">734-0002</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD Falcon Tube 50mL</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">352070</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD Falcon Tube 15mL</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">352097</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD FACS Tube 5mL</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">352008</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Sterile Pasteur Pipette 5 mL&#160;</td>
			<td style="width:163px;">VWR</td>
			<td style="width:137px;">612-1685</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Microfuge Tube 1.5 mL</td>
			<td style="width:163px;">Eppendorf</td>
			<td style="width:137px;">7805-00</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;"></td>
			<td style="width:163px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;">Reagents</td>
			<td style="width:163px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">X-Vivo 20</td>
			<td style="width:163px;">Lonza</td>
			<td style="width:137px;">BE04-448Q</td>
			<td>serum-free medium recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Phosphate buffered saline</td>
			<td style="width:163px;">Lonza</td>
			<td style="width:137px;">BE17-516F</td>
			<td>standard physiological PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Trypan blue&#160;</td>
			<td style="width:163px;">VWR</td>
			<td style="width:137px;">17942E</td>
			<td>or other vital stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">VersaLyse</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:137px;">A09777</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Fixable viability Dye eFluor 780&#160;</td>
			<td style="width:163px;">eBioscience</td>
			<td style="width:137px;">65-0865-14</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD3 FITC</td>
			<td style="width:163px;">BD Biosciences</td>
			<td style="width:137px;">345763</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD3 Vio Blue</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-094-363</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD4 PE</td>
			<td style="width:163px;">BD Biosciences</td>
			<td style="width:137px;">345769</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD4 APC</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-091-232</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD8 ECD</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:137px;">737659</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD8 PerCP</td>
			<td style="width:163px;">BD Biosciences</td>
			<td style="width:137px;">345774</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD19 APC-Vio770</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-096-643</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD45 VioGreen</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-096-906</td>
			<td>for flow cytometry experiments</td>
		</tr>
	</tbody>
</table>

a simple and rapid protocol to non-enzymatically dissociate fresh human tissues for the analysis of infiltrating lymphocytes

La capacidad de las células malignas para evadir el sistema inmune, caracterizado por el escape del tumor de ambos respuestas inmunitarias innatas y adaptativas, se acepta ahora como un sello importante de cáncer. Nuestra investigación sobre el cáncer de mama se centra en el papel activo que desempeñan linfocitos infiltrantes de tumores en la progresión tumoral y la evolución del paciente. Hacia este objetivo, hemos desarrollado una metodología para el aislamiento rápido de células linfoides intactos de tejidos normales y anormales en un esfuerzo para evaluar los próximo a su estado nativo. Los homogeneizados preparados usando un espectáculo disociador mecánica tanto una mayor recuperación de la viabilidad celular y preservando al mismo tiempo la expresión receptor de superficie en comparación tejidos digeridos con enzima para. Por otra parte, la digestión enzimática del material insoluble restante no se recuperó células CD45 + adicionales que indican que las mediciones cuantitativas y cualitativas en el homogeneizado primaria probablemente reflejen realmente la infiltración de las subpoblaciones en la Fragm tejidoent. Las células linfoides en estos homogeneizados pueden ser fácilmente caracterizado usando inmunológica (fenotipo, la proliferación, etc.) o molecular (ADN, ARN y / o proteína) enfoques. Células CD45 + también pueden ser utilizados para la purificación subpoblación, la expansión in vitro o crioconservación. Un beneficio adicional de este enfoque es que el sobrenadante tejido primario a partir de los homogeneizados se puede utilizar para caracterizar y comparar citocinas, quimiocinas, inmunoglobulinas y antígenos presentes en tejidos normales y malignos. Este funciones de protocolo extremadamente bien para los tejidos de mama humanos y deberían ser aplicables a una amplia variedad de tejidos normales y anormales.

La digestión enzimática de fragmentos de tejido, ya sea con soluciones disponibles en el mercado de disociación de tejido o diversas mezclas de laboratorio de colagenasa, DNasa y / o inhibidores de hialuronidasa, escinden una amplia variedad de receptores en la superficie de las células. Nuestros estudios, se centró inicialmente en las células T CD4 + que infiltran los tumores de mama, se presentaron rápidamente con un problema técnico importante debido a la escisión de los receptores de CD4 de la su...

Watch this Scientific Journal Video about A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes at JoVE.com

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

Un protocolo simple y rápida para no enzimáticamente disociarse Tejidos Humanos frescas para el Análisis de los linfocitos infiltrantes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

a-simple-rapid-protocol-to-non-enzymatically-dissociate-fresh-human

Research

JoVE Journal

Immunology and Infection

19.9K vistas.  Université Libre de Bruxelles. This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

Video: A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Combinación de adhesivo basado en cinta de muestreo y de fluorescencia In situ La hibridación para la detección rápida de Salmonella En los productos frescos

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Investigación

Inmunología e Infección

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

Un protocolo Chelex simple para la extracción de ADN a partir de<em&gt; Anopheles</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

Un protocolo simple para el agrupamiento de plaquetas mediada por<em&gt; Plasmodium falciparum</emEritrocitos&gt; por el VIH en un recurso pobre ajuste

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Análisis rápido de aberraciones cromosómicas en linfocitos B de ratón por PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Protocolo Aislamiento eficiente para B y linfocitos T a partir Humanos Palatine amígdalas

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

Un enfoque semi-automatizado de preparación de cócteles de anticuerpos para el Análisis Inmunofenotípicos de la sangre periférica humana

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Un método rápido para el procesamiento de sangre para aumentar el rendimiento de los niveles plasmáticos de péptido en la sangre humana

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Un enfoque simple y eficiente de construcción de mutantes de virus vaccinia Vectores

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

Un protocolo optimizado para analizar la glucólisis y respiración mitocondrial en los linfocitos

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

La cuantificación rápida de Blastogénesis inducida por mitógeno de linfocitos T para identificar fármacos inmunomoduladores

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...