Los autores desean agradecer a Nancy Mora por su asistencia técnica. Este trabajo fue financiado por becas de investigación 48573-M y 168098 de Consejo Nacional de Ciencia y Tecnología, México, y por subvenciones IN212308 y IN205311-2 de Dirección General de Asuntos del Personal Académico de la Universidad Nacional Autónoma de México, México.

1. Aislamiento de neutrófilos (PMN) de la sangre humana <ol><li> Use aproximadamente 20 ml de sangre humana con heparina (10 U / ml) como anticoagulante. Se recogió sangre de voluntarios adultos sanos por venopunción. Todos los experimentos se realizaron bajo la aprobación del Comité de Bioética de la Instituto de Investigaciones Biomédicas - UNAM. </li><li> Dejar 2 ml de dextrano al 6% T500 en PBS en un tubo de 15 ml de centrífuga cónico y añadir 10 ml de sangre. Mezclar invirtiendo el tubo dos o tres veces y se deja reposar durante 45 minutos para permitir la sedimentación de eritrocitos. </li><li> En un nuevo máximo de 15 ml tubo de centrífuga cónico poner 5 ml de Ficoll-Paque. </li><li> Tome el plasma rico en leucocitos que se formó encima de los eritrocitos sedimentados, y cuidadosamente la pipeta en la parte superior de la Ficoll-Paque formar una segunda capa. Asegúrese de que hay dos fases. </li><li> Centrifugar a 516 xg durante 20 min a 4 ° C. Después de la centrifugación, en la interfase de plasma y Ficoll Paque-existe una capa de células mononucleares. Nordesteutrophils están en la parte inferior del tubo. </li><li> Eliminar el sobrenadante, el sedimento celular romper tocando el tubo contra un bastidor, y resuspender las células mediante la adición de 10 ml de frío (4 ° C) PBS Nota:. Resuspender las células pipeteando arriba y abajo no es recomendable, ya que esto es demasiado dañar a las células. </li><li> Transferir la suspensión celular a un tubo de 50 ml cónicos de centrífuga y centrifugar a 290 xg durante 5 min a 4 ° C. </li><li> Romper el pellet de células como antes y añadir 10 ml de frío (4 ° C) con una solución hipotónica (0,2% de NaCl, 1% de BSA, 20 mM Hepes, pH = 7,4) para lisar los eritrocitos. Mezclar por el tubo suavemente con la mano durante exactamente un minuto. </li><li> Añadir 10 ml de frío (4 ° C) solución hipertónica (1,6% de NaCl, 1% de BSA, 20 mM de HEPES, pH = 7,4), se mezcla y se cuentan las células usando un hemocitómetro (generalmente&gt; 95% son PMN). Nota: Mantener el tubo con la suspensión de células en hielo mientras contando las células. </li><li> Se centrifuga como antes y se resuspende en 10 PMN7 células / ml en frío (4 ° C) PBS. Mantenga en hielo. Nota: Los neutrófilos siendo viable y funcional a 4 ° C durante aproximadamente 6 a 8 horas. </li></ol> 2. La activación de PMN <ol><li> Poner 100 l de suspensión de PMN (1 x 10 7 células / ml) en un tubo Eppendorf de 1,5 ml. Nota: Los tubos de muestra se debe hacer por lo menos por duplicado. Habituales controles negativos deben incluir sólo primer anticuerpo, y el anticuerpo secundario solamente. </li><li> Añadir el anticuerpo del receptor anti-integrina correspondiente o anti-Fc monoclonal (mAb) a 10 ug / ml y se incuba en hielo durante 15 min. </li><li> Lavar PMN por la adición de 1 ml de frío (4 ° C) PBS, centrifugar a 1.743 xg (4.500 rpm) en una microcentrífuga, y aspirar el sobrenadante. </li><li> Romper el sedimento celular tocando el fondo del tubo contra un bastidor, añadir 1 ml de PBS frío, y se lava dos veces más como en el paso anterior. Nota: Estos lavados eliminar el anticuerpo no unido. </li><li> Resuspender PMN en el mismo (enitial) volumen de tibia (37 ° C) PBS que contenía 60 mg / ml de F (ab &#39;) 2 de cabra anti-IgG de ratón. Nota: El anticuerpo secundario anti-ratón de anticuerpos IgG se une a la primera anticuerpo anti-receptor y causa la reticulación del receptor. </li><li> Incubar a 37 ° C durante 1 a 20 minutos, dependiendo del factor nuclear de interés. Para NF-kB por lo general 15 min, y para Elk-1 generalmente 5 min. Nota: incubar las células a 37 ° C entrecruzamiento del receptor induce, que no puede tener lugar a 4 ° C. </li><li> Añadir 1 ml de PBS frío y centrifugar 3 min a 1.743 xg en microcentrífuga. </li><li> Remover el sobrenadante </li><li> Congelar PMN inmediatamente en un baño de dry-ice/ethanol y mantenerlos allí durante 10 minutos. </li></ol> 3. El aislamiento y la fijación de los núcleos <ol><li> Resuspender pellet congelado PMN en 100 l frío (4 ° C) tampón hipotónico (HEPES 10 mM, 10 mM KCl, 1,5 mM de MgCl 2, y 1 mM recién añadido DL-ditiotreitol [DTT]; PH = 7,9). Se recomienda tomar los tubos del baño de dry-ice/ethanol uno por uno, y para limpiar la parte inferior del tubo antes de añadir el tampón hipotónico. </li><li> Colocar sobre hielo, y comprobar la integridad de los núcleos mediante la tinción de una parte alícuota de la suspensión de núcleos con azul de tripano. Núcleos mirar a su alrededor y azul. PMN sin avería no mancharse, y los restos celulares aparece como partículas azules de forma irregular. Nota: Si suspensión de núcleos contiene muchas células intactas o desechos, es mejor desechar la preparación y repetir el procedimiento con otra muestra. </li><li> Centrifugar los núcleos a 775 xg (3.000 rpm) en microcentrífuga durante 10 min en el interior de una habitación fría. En este punto los núcleos están muy frágil y cuidado adicional debe ser tomado en mantener las muestras frías y no centrifugar a fuerzas excesivas. </li><li> Eliminar el sobrenadante por pipeteo muy cuidadoso. </li><li> Añadir 100 l de paraformaldehído al 4% frío en PBS para fijar los núcleos. Nota: El sedimento es muy aflojar unad adición del tampón es suficiente para volver a suspender los núcleos. Pipeteando arriba y abajo debe ser evitado. </li><li> Incubar en hielo durante 20 min. </li><li> Inmunomarcador núcleos para citometría de flujo o mantenerlos durante un máximo de 24 horas a 4 ° C. </li></ol> 4. Núcleos inmunomarcación para el análisis de citometría de flujo <ol><li> Centrifugar los núcleos a 1.743 xg (4.500 rpm) en microcentrífuga durante 1 min, y eliminar el sobrenadante mediante pipeteo muy suave. </li><li> Añadir 100 l de frío (4 º C) 0,1% de Triton X-100, 4% de paraformaldehído en PBS para permeabilizar núcleos. Incubar en hielo durante 10 min. </li><li> Centrífuga permeabilized núcleos a 1.743 xg en microcentrífuga y retire con cuidado el sobrenadante. En este punto gránulos de núcleos no se adhieren bien en la parte inferior del tubo. Se recomienda centrifugar de nuevo si el sedimento se suelta. </li><li> Resuspender los núcleos en 500 l de frío 4% de suero fetal bovino (FBS) en PBS para bloquear sitios de unión inespecíficos, e incubar en hielo durante 20 min. Centrífugonúcleos a 1.743 xg durante 3 minutos en microcentrífuga y retire con cuidado el sobrenadante. </li><li> Resuspender los núcleos en 100 l de PBS frío que contenía 4% de FBS y 2,5 g / ml del mAb contra el factor nuclear de interés. Incubar en hielo durante 20 min. Nota: habituales controles negativos deben incluir anticuerpos antinucleares único factor, y sólo marcado con FITC anticuerpo secundario. </li><li> Lavar dos veces con 500 l de PBS frío con FBS al 4% y centrifugando a 1743 xg durante 3 min. </li><li> Eliminar el sobrenadante cuidadosamente, los núcleos se resuspende en 100 l de PBS frío que contenía 4% de FBS y 10 ug / ml de la correspondiente marcado con FITC anticuerpo secundario, y se incuba en hielo durante 20 min. </li><li> Lavar dos veces con 500 núcleos l de PBS frío con FBS al 4%, como en el paso anterior. </li><li> Resuspender los núcleos en 400 l de paraformaldehído al 4% frío en PBS. Nota: Los núcleos se colocan en paraformaldehído al permitir que la muestra se almacena por varios días sin contaminaciónproblemas que pueden ocurrir en presencia de suero. </li><li> Analizar mediante citometría de flujo inmediatamente o almacenar en la oscuridad a 4 ° C durante un máximo de tres días. </li></ol> 5. Análisis de citometría de flujo <ol><li> Analizar núcleos inmunomarcadas en un citómetro de flujo tal FACScan (Becton Dickinson; Franklin Lakes, NJ) o aparato similar. </li><li> Ajustar la configuración de adquisición para: FSC en escala logarítmica con 10-1, SSC en escala logarítmica en el 196, y los núcleos puerta en un punto de parcelas. </li><li> Adquirir diez mil núcleos por muestra. </li><li> Analizar la fluorescencia de FITC núcleos teñidos por el canal FL-1 (506 nm), fijado en escala logarítmica en 400. </li></ol>

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Los autores declaran que no tienen intereses en conflicto financieros.

El método de purificación descrito aquí permite el aislamiento de neutrófilos no estimulados (PMN) con una pureza mayor que 95% (evaluado por observación microscópica), en un corto tiempo. A veces, los neutrófilos pueden estar contaminados por los eritrocitos, si éste no se lisan completamente. Esto no suele afectar a la técnica, ya que los eritrocitos y los PMN se pueden distinguir fácilmente como poblaciones celulares distintas por citometría de flujo. Aislado PMN entonces puede ser estimulado por los recep...

Los neutrófilos son las más abundantes en leucocitos de sangre periférica 1. Durante los neutrófilos inflamación y la infección son las primeras células que aparecen en el sitio afectado cuando actúan como la primera línea de defensa 2. Los neutrófilos poseen varios mecanismos antimicrobianos 3 incluyendo la fagocitosis, la producción de especies reactivas de oxígeno, liberación de enzimas líticas por la desgranulación y la producción de citocinas proinflamatorias 4,5. Los neutrófilos son las células de vida corta que se activa rápidamente a través de la señalización de los distintos receptores de la superficie celular. Aunque los neutrófilos se han considerado las células terminales debido a su corta vida y porque se someten a apoptosis menos que sea activado durante el proceso inflamatorio 6, ahora está claro que también pueden modificar su fenotipo cambiando el nivel de la transcripción de determinados genes. La producción de citocinas 5 y la inhibición de la apoptosis 7,8 son dos importantes de activación dependientes de funciones de la célula regulada en el nivel de transcripción en los neutrófilos. El factor nuclear kB (NF-kB) participa en el control transcripcional de la producción de citoquinas y 4 en la regulación de la supervivencia celular y la apoptosis 9-11 en varios tipos de células. Las vías de señalización que conducen a la activación del factor nuclear generalmente se estudian por ensayos de gen reportero o por ensayos de cambio de movilidad electroforética (EMSA). Sin embargo, debido a los neutrófilos son las células de vida corta, el estudio de respuestas regulados transcripcionalmente en estas células no se puede realizar con ensayos de gen reportero, ya que no hay técnicas eficaces para la transfección de neutrófilos. Ensayos de EMSA se han utilizado en los neutrófilos para explorar la activación del factor nuclear 12,13; sin embargo, esta metodología es complicada y cara, ya que implica el uso de materiales radiactivos. Nucleofection es otra técnica que se ha utilizado successfully para transfectar monocitos 14. Por lo tanto, al menos en teoría, la activación del factor nuclear puede ser detectado en los neutrófilos por transfección (a pesar de la baja eficiencia). Sin embargo, esta técnica sería más costoso, consume tiempo y, probablemente, menos cuantitativo. El análisis microscópico de células inmuno también podría ser utilizado para detectar los factores nucleares en el núcleo. En efecto, hemos detectado NF-kappa B translocación en el núcleo de esta manera 15. Desafortunadamente, esta técnica es también consume mucho tiempo, menos cuantitativo, y sujeto al sesgo del observador. Aquí, se presenta un método simple y eficiente que permite la detección y cuantificación de los factores nucleares en núcleos aislados y immunolabeled por citometría de flujo. Se describen técnicas para aislar los neutrófilos de sangre periférica humana, estimular estas células a través de integrinas o receptores de Fc con anticuerpos anti-receptor, aislar y núcleos inmunomarcador, y analizar núcleos por citometría de flujo (Figura 1). El método ha sido utilizado con éxito para detectar NF-kB 15 y Elk-1 16 factores nucleares en los núcleos de neutrófilos. La sensibilidad de este método permite la detección de pequeños cambios en los niveles de factor nuclear en el núcleo. Este método también se puede utilizar para analizar el nivel de factores de transcripción en los núcleos de otros tipos de células.

<table border="1">
<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td >Heparin </td>
 <td >PiSA (Mexico)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td >Dextran T500</td>
 <td >Pharmacosmos A/S (Holbaek, Denmark) </td>
 <td>T1-Dextran T500</td>
 <td></td>
 </tr>
 <tr>
 <td >Ficoll-Paque</td>
 <td >Pharmacia</td>
 <td>17-0320-01 </td>
 <td></td>
 </tr>
 <tr>
 <td >Sodium chloride </td>
 <td >Sigma</td>
 <td>S7653 </td>
 <td></td>
 </tr>
 <tr>
 <td >Sodium phosphate monobasic</td>
 <td >Sigma</td>
 <td>S9638</td>
 <td></td>
 </tr>
 <tr>
 <td >Sodium phosphate dibasic</td>
 <td >Sigma</td>
 <td>S9390</td>
 <td></td>
 </tr>
 <tr>
 <td >Bovine serum albumin (BSA)</td>
 <td >Sigma</td>
 <td>A2153</td>
 <td>Cohn Fraction V</td>
 </tr>
 <tr>
 <td >HEPES</td>
 <td >Sigma</td>
 <td>H3375 </td>
 <td></td>
 </tr>
 <tr>
 <td >Potassium chloride</td>
 <td >Sigma</td>
 <td>P9541 </td>
 <td></td>
 </tr>
 <tr>
 <td >Magnesium chloride anhydrous </td>
 <td >Sigma</td>
 <td>M8266 </td>
 <td></td>
 </tr>
 <tr>
 <td >DL-dithiothreitol (DTT) </td>
 <td >Sigma</td>
 <td>D9163</td>
 <td></td>
 </tr>
 <tr>
 <td >Trypan Blue (0.4 % solution)</td>
 <td >Sigma</td>
 <td>T8154</td>
 <td></td>
 </tr>
 <tr>
 <td >Paraformaldehyde</td>
 <td >Sigma</td>
 <td>P6148 </td>
 <td></td>
 </tr>
 <tr>
 <td >Triton X-100 </td>
 <td >Sigma</td>
 <td>X100</td>
 <td></td>
 </tr>
 <tr>
 <td >Fetal bovine serum (FBS)</td>
 <td >GIBCO</td>
 <td>10437-028</td>
 <td></td>
 </tr>
 <tr>
 <td >Monoclonal antibody IV.3</td>
 <td >Medarex (Annandale, NJ) </td>
 <td>025-1 </td>
 <td>Human-specific anti-FcRII (CD32) </td>
 </tr>
 <tr>
 <td >Monoclonal antibody 3G8</td>
 <td >Medarex (Annandale, NJ) </td>
 <td>028-2 </td>
 <td>Human-specific anti-FcRIII (CD16) </td>
 </tr>
 <tr>
 <td >Monoclonal antibody TS2/16</td>
 <td >Dana Farber Cancer Research Institute (Boston, MA) </td>
 <td>Donated by Dr. Martin Hemler </td>
 <td>Human-specific anti-&beta;1 integrin (CD29)</td>
 </tr>
 <tr>
 <td >Monoclonal antibody IB4</td>
 <td >University of California, San Francisco </td>
 <td>Donated by Dr. Eric J. Brown </td>
 <td>Human-specific anti-&beta;2 integrin (CD18)</td>
 </tr>
 <tr>
 <td >F(ab')2 goat anti-mouse IgG </td>
 <td >Cappel (Aurora, OH)</td>
 <td>55468</td>
 <td></td>
 </tr>
 <tr>
 <td>FITC-conjugated F(ab')2 goat anti-mouse IgG</td>
 <td >Cappel (Aurora, OH)</td>
 <td>55522</td>
 <td></td>
 </tr>
 <tr>
 <td>FITC-conjugated F(ab')2 goat anti-rabbit IgG</td>
 <td >Cappel (Aurora, OH)</td>
 <td>55665</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-NF-&kappa;B p50</td>
 <td >Santa Cruz Biotechnology (Santa Cruz, CA)</td>
 <td>sc-114</td>
 <td>Rabbit polyclonal antibody</td>
 </tr>
 <tr>
 <td>Anti-NF-&kappa;B p65</td>
 <td >Santa Cruz Biotechnology (Santa Cruz, CA)</td>
 <td>sc-109</td>
 <td>Rabbit polyclonal antibody</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>15-ml centrifuge tube</td>
 <td >Corning</td>
 <td>430791</td>
 <td></td>
 </tr>
 <tr>
 <td>50-ml centrifuge tube</td>
 <td >Corning</td>
 <td>430291</td>
 <td></td>
 </tr>
 <tr>
 <td>Centrifuge, Sorvall Tabletop</td>
 <td >Dupont Instruments</td>
 <td>RT 6000D</td>
 <td></td>
 </tr>
 <tr>
 <td>pH-meter</td>
 <td >Corning</td>
 <td>340</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipetman pipette P-20</td>
 <td >Gilson </td>
 <td>F123600</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipetman pipette P-200</td>
 <td >Gilson </td>
 <td>F123601</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipetman pipette P-1000</td>
 <td >Gilson </td>
 <td>F123602</td>
 <td></td>
 </tr>
 <tr>
 <td>Hemocytometer</td>
 <td >Fisher Scientific</td>
 <td>0267110</td>
 <td></td>
 </tr>
 <tr>
 <td>Microscope</td>
 <td >Nikon </td>
 <td>Eclipse E600</td>
 <td></td>
 </tr>
 <tr>
 <td>Inverted microscope</td>
 <td >Nikon </td>
 <td>TMS</td>
 <td></td>
 </tr>
 <tr>
 <td>Water Bath Incubator</td>
 <td >Fisher Scientific </td>
 <td>2IS-M</td>
 <td></td>
 </tr>
 <tr>
 <td>Microcentrifuge</td>
 <td >Eppendorf</td>
 <td>5414C</td>
 <td></td>
 </tr>
 <tr>
 <td>Microcentrifuge</td>
 <td >Eppendorf</td>
 <td>5418</td>
 <td></td>
 </tr>
 <tr>
 <td>Flow Cytometer</td>
 <td >Becton Dickinson (Franklin Lakes, NJ) </td>
 <td>FACScalibur </td>
 <td></td>
 </tr>
 </tbody>
</table>

a simple and efficient method to detect nuclear factor activation in human neutrophils by flow cytometry

Los neutrófilos son las más abundantes en leucocitos de sangre periférica. Estas células son el primero en aparecer en sitios de inflamación e infección, convirtiéndose así en la primera línea de defensa contra los microorganismos invasores. Los neutrófilos poseen importantes funciones antimicrobianos, tales como fagocitosis, la liberación de enzimas líticas, y la producción de especies reactivas del oxígeno. Además de estas importantes funciones de defensa, neutrófilos realizar otras tareas en respuesta a la infección, tales como la producción de citocinas proinflamatorias y la inhibición de la apoptosis. Las citoquinas reclutar leucocitos otros que ayudan a eliminar la infección, y la inhibición de la apoptosis permite el neutrófilo a vivir más tiempo en el sitio de la infección. Estas funciones se regulan a nivel de la transcripción. Sin embargo, debido a los neutrófilos son las células de vida corta, el estudio de respuestas regulados transcripcionalmente en estas células no se puede realizar con los métodos convencionales de genes informadores ya que no hay eficienciatécnicas eficientes para la transfección de neutrófilos. Aquí, se presenta un método simple y eficiente que permite la detección y cuantificación de los factores nucleares en núcleos aislados y immunolabeled por citometría de flujo. Se describen técnicas para aislar los neutrófilos puros a partir de sangre periférica humana, estimular estas células con anticuerpos anti-receptor, aislar y núcleos inmunomarcador, y analizar núcleos por citometría de flujo. El método ha sido utilizado con éxito para detectar NF-kB y Elk-1 factores nucleares en los núcleos de los neutrófilos y otros tipos celulares. Por lo tanto, este método representa una opción para el análisis de la activación de factores de transcripción en los núcleos aislados a partir de una variedad de tipos de células.

El método de purificación descrito aquí proporciona generalmente los neutrófilos no estimulados (PMN) con una pureza mayor que 95% (Figura 1A). Aislado PMN entonces puede ser estimulado por los receptores de reticulación particular con anticuerpos monoclonales específicos. Hemos PMN estimulados a través de los receptores Fc y las integrinas (Figura 1B). Una vez estimulado, PMN son lisadas y los núcleos se aislaron con rendimientos altos. Los núcleos son entonces immunolabeled p...

Watch this Scientific Journal Video about A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry at JoVE.com

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Los neutrófilos son las más abundantes en leucocitos de sangre. Los neutrófilos poseen funciones regulados transcripcionalmente tales como la producción de citocinas proinflamatorias y la inhibición de la apoptosis. Estas funciones pueden ser estudiadas con el método presentado aquí, que permite la detección y cuantificación de los factores nucleares por citometría de flujo en los núcleos aislados

Un método simple y eficiente para detectar la activación del factor nuclear en neutrófilos humanos por citometría de flujo

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

a-simple-efficient-method-to-detect-nuclear-factor-activation-human

Research

JoVE Journal

Immunology and Infection

17.7K vistas.  University of Alberta. Los neutrófilos son las más abundantes en leucocitos de sangre. Los neutrófilos poseen funciones regulados transcripcionalmente tales como la producción de citocinas proinflamatorias y la inhibición de la apoptosis. Estas funciones pueden ser estudiadas con el método presentado aquí, que permite la detección y cuantificación de los factores nucleares por citometría de flujo en los núcleos aislados

Video: A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

Evaluación cuantitativa de las células inmunes en el tejido de la médula espinal lesionada por citometría de flujo: un nuevo uso de un método de purificación celular

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Investigación

Inmunología e Infección

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Un método simple y eficaz para aislar los macrófagos de la Mezcla de cultivos primarios de células de hígado en adultos

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Humano In Vitro Supresión como herramienta de cribado para el reconocimiento de un estado inicial de desequilibrio inmunológico

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

El examen de selección tímica positivo y negativo por citometría de flujo

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

El estudio de las interacciones de<em&gt; Staphylococcus aureus</em&gt; Con neutrófilos mediante citometría de flujo y microscopía Tiempo Lapso

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

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

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

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 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 ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Imágenes en vivo de la actividad antifúngica por los neutrófilos humanos primarios y los monocitos en respuesta a las<em&gt; A. fumigatus</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Aislamiento y cultivo In Vitro de los macrófagos alveolares murinos y humanos

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Caracterización de subconjuntos de monocitos humanos de sangre análisis de citometría de flujo

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...