Les auteurs tiennent à remercier Nancy Mora pour son assistance technique. Ce travail a été financé par des subventions de recherche 48573-M et 168098 du Consejo Nacional de Ciencia y Tecnologia, au Mexique, et par des subventions IN212308 et IN205311-2 à partir de Dirección General de Asuntos del Personal Academico, Universidad Nacional Autonoma de Mexico, au Mexique.

1. Isolement des neutrophiles (PMN) à partir de sang humain <ol><li> Utilisez environ 20 ml de sang humain avec de l&#39;héparine (10 U / ml) comme anticoagulant. Le sang a été recueilli à partir adultes volontaires en bonne santé par ponction veineuse. Toutes les expériences ont été réalisées en cours d&#39;approbation du Comité de bioéthique à l&#39;Instituto de Investigaciones Biomédicas - UNAM. </li><li> Mettre 2 ml de 6% de dextrane T500 dans du PBS dans un tube à centrifuger conique de 15 ml et ajouter 10 ml de sang. Mélanger en inversant le tube deux ou trois fois et laisser reposer pendant 45 min pour permettre de sédimentation. </li><li> Dans un tube à centrifuger frais 15 ml conique mettre 5 ml de Ficoll-Paque. </li><li> Prenez le plasma riche en leucocytes qui s&#39;est formé au-dessus érythrocytes sédimentés, et soigneusement pipette au-dessus du Ficoll-Paque former une deuxième couche. Assurez-vous qu&#39;il ya deux phases. </li><li> Centrifuger à 516 xg pendant 20 min à 4 ° C. Après centrifugation, à l&#39;interphase de plasma et de Ficoll-Paque se trouve une couche de cellules mononucléées. Neutrophils sont au fond du tube. </li><li> Eliminer le surnageant, briser le culot cellulaire en appuyant sur ​​le tube contre une grille, et remettre les cellules en ajoutant 10 ml d&#39;eau froide (4 ° C) PBS. Remarque: La remise en suspension des cellules par pipetage de haut en bas n&#39;est pas recommandée car elle est trop dommageables pour les cellules. </li><li> Transférer la suspension cellulaire dans un tube à centrifugation de 50 ml coniques et centrifuger à 290 xg pendant 5 min à 4 ° C. </li><li> Briser le culot cellulaire comme avant et ajouter 10 ml d&#39;eau froide (4 ° C) une solution hypotonique (0,2% de NaCl, 1% de BSA, 20 mM de HEPES, pH = 7,4) pour lyser les érythrocytes. Mélanger en agitant doucement le tube à la main pendant exactement une minute. </li><li> Ajouter 10 ml d&#39;eau froide (4 ° C) solution hypertonique (1,6% de NaCl, 1% de BSA, 20 mM d&#39;HEPES, pH = 7,4), mélanger et compter les cellules en utilisant un hémocytomètre (généralement&gt; 95% sont des PMN). Remarque: Conservez le tube avec la suspension cellulaire sur la glace tout en comptant les cellules. </li><li> Centrifuger comme avant et remettre en suspension PMN à 107 cellules / ml dans le froid (4 ° C) PBS. Garder sur la glace. Remarque: Les neutrophiles demeurer viable et fonctionnelle à 4 ° C pendant environ 6 à 8 heures. </li></ol> 2. Activation PMN <ol><li> Mettez 100 pl de suspension de PMN (1 x 10 7 cellules / ml) dans un tube Eppendorf de 1,5 ml. Remarque: Les tubes échantillons doivent se faire au moins en double. Habituelles du contrôle négatif doit inclure premier anticorps seul, et l&#39;anticorps secondaire. </li><li> Ajouter l&#39;anticorps récepteur correspondant anti-intégrine ou anti-Fc monoclonal (Acm) à 10 pg / ml et incuber sur de la glace pendant 15 min. </li><li> Laver PMN en ajoutant 1 ml d&#39;eau froide (4 ° C) PBS, centrifugation à 1743 xg (4.500 rpm) dans une microcentrifugeuse, et l&#39;aspiration du surnageant. </li><li> Briser le culot cellulaire en appuyant sur la partie inférieure du tube contre une grille, ajouter 1 ml de PBS froid, et laver deux fois plus que lors de l&#39;étape précédente. Remarque: Ce lave éliminer les anticorps non liés. </li><li> PMN remettre en suspension dans la même (enitial) le volume d&#39;eau tiède (37 ° C) PBS contenant 60 ug / ml de F (ab &#39;) 2 de chèvre anti-IgG de souris. Remarque: Le secondaire anti-IgG de souris se lie à la première anticorps anti-récepteur et provoquer la réticulation du récepteur. </li><li> Incuber à 37 ° C pendant 1 à 20 min, en fonction du facteur nucléaire de l&#39;intérêt. Pour le NF-kB généralement de 15 min, et pour Elk-1 habituellement de 5 min. Remarque: L&#39;incubation des cellules à 37 ° C réticulation récepteur induit, ce qui ne peut avoir lieu à 4 ° C. </li><li> Ajouter 1 ml de PBS froid et centrifuger 3 min à 1743 xg dans microcentrifugeuse. </li><li> Retirer le surnageant </li><li> Congelez PMN immédiatement dans le bain dry-ice/ethanol et les y maintenir pendant 10 min. </li></ol> 3. L&#39;isolement et la fixation des noyaux <ol><li> Remettre en suspension congelée PMN concentré dans 100 ul froid (4 ° C) tampon hypotonique (10 mM HEPES, 10 mM KCl, 1,5 mM MgCl 2, et 1 mM fraîchement ajoutée DL-dithiothreitol [DTT], PH = 7,9). Il est recommandé de prendre les tubes du bain une dry-ice/ethanol par un, et pour essuyer le fond du tube avant d&#39;ajouter le tampon hypotonique. </li><li> Placer sur la glace, et de vérifier l&#39;intégrité des noyaux par coloration d&#39;une aliquote de la suspension noyaux avec du bleu trypan. Noyaux regarder autour et bleu. PMN Intact ne se tacher, et les débris cellulaires apparaît sous forme de particules bleues de forme irrégulière. Remarque: Si la suspension contient de nombreux noyaux des cellules intactes ou des débris, il est préférable de jeter la préparation et répéter la procédure avec un autre échantillon. </li><li> Centrifuger à 775 xg noyaux (3.000 rpm) microcentrifugeuse pendant 10 min dans une chambre froide. A ce point de noyaux sont très fragiles et des soins supplémentaires doivent être prises pour maintenir les échantillons à froid et non centrifugation à des forces excessives. </li><li> Retirer le surnageant par pipetage très prudent. </li><li> Ajouter 100 ul de paraformaldéhyde 4% dans du PBS froid pour fixer les noyaux. Remarque: Le culot est très facilement perdre uned ajoutant le tampon est suffisant pour remettre en suspension les noyaux. Aspiration et refoulement doit être évitée. </li><li> Incuber dans la glace pendant 20 min. </li><li> Noyaux Immunolabel la cytométrie de flux ou de les conserver jusqu&#39;à 24 heures à 4 ° C. </li></ol> 4. L&#39;immunomarquage des noyaux d&#39;analyse de cytométrie en flux <ol><li> Centrifuger à 1743 xg noyaux (4.500 rpm) microcentrifugeuse pendant 1 min, puis retirez surnageant par pipetage très doux. </li><li> Ajouter 100 ul de froid (4 ° C) 0,1% de Triton X-100, le paraformaldéhyde 4% dans du PBS à perméabiliser noyaux. Incuber dans la glace pendant 10 min. </li><li> Centrifugeuse perméabilisées noyaux à 1743 xg dans microcentrifugeuse et retirer délicatement le surnageant. A ce stade, pellets noyaux n&#39;attachent pas bien au fond du tube. Il est recommandé de centrifuger à nouveau si le culot se détache. </li><li> Noyaux remettre en suspension dans 500 ul de sérum froid 4% de veau fœtal (FBS) dans du PBS pour bloquer les sites de liaison non spécifiques, et incuber sur de la glace pendant 20 min. Centrifugernoyaux à 1743 xg pendant 3 min dans microcentrifugeuse et retirer délicatement le surnageant. </li><li> Resuspendre noyaux dans 100 ul de PBS froid contenant 4% de FBS et 2,5 ug / ml de l&#39;anticorps monoclonal contre le facteur nucléaire des intérêts. Incuber dans la glace pendant 20 min. Remarque: Usual contrôles négatifs doivent inclure anticorps anti-facteur nucléaire seulement, et marqué au FITC anticorps secondaire seul. </li><li> Laver deux fois avec 500 ul de PBS froid avec 4% de FBS et centrifugation à 1743 xg pendant 3 min. </li><li> Retirer le surnageant avec beaucoup de soin, les noyaux remettre en suspension dans 100 ul de PBS froid contenant 4% de FBS et 10 pg / ml de la correspondante marqué au FITC anticorps secondaire et incuber sur de la glace pendant 20 min. </li><li> Laver deux fois les noyaux avec 500 ul de PBS froid avec du FBS 4% comme dans l&#39;étape précédente. </li><li> Remettre en suspension dans 400 ul noyaux de paraformaldéhyde 4% dans du PBS froid. Remarque: Les noyaux sont placés dans du paraformaldéhyde à permettre à l&#39;échantillon doit être stocké pendant plusieurs jours sans contaminationproblèmes qui peuvent se produire en présence de sérum. </li><li> Analyser immédiatement par cytométrie en flux ou conserver à l&#39;obscurité à 4 ° C pendant un maximum de trois jours. </li></ol> 5. Analyse par cytométrie en flux <ol><li> Analyser noyaux immunomarquées dans un cytomètre de flux tels un FACScan (Becton Dickinson, Franklin Lakes, NJ) appareil ou similaire. </li><li> Réglez les paramètres d&#39;acquisition d&#39;: FSC à échelle logarithmique à 10-1, SSC à échelle logarithmique à 196, et les noyaux de grille dans un point-parcelle. </li><li> Acquérir dix mille noyaux par échantillon. </li><li> Analyser la fluorescence du FITC-noyaux colorés par l&#39;intermédiaire du canal FL-1 (506 nm) fixé à échelle logarithmique à 400. </li></ol>

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Les auteurs déclarent n&#39;avoir aucun conflit d&#39;intérêts financiers.

Le procédé de purification décrit ici permet d&#39;isoler les neutrophiles non stimulés (PMN) de pureté supérieure à 95% (évaluée par l&#39;observation microscopique), dans un court laps de temps. Parfois, les neutrophiles peuvent être contaminés par les érythrocytes si celui-ci ne sont pas lysées complètement. Cela ne devrait pas nuire à la technique, les érythrocytes et les PMN peuvent facilement être distingués comme des populations cellulaires distinctes par cytométrie en flux. PMN isolé peut ens...

Les neutrophiles sont les leucocytes les plus abondants dans le sang périphérique 1. Au cours de neutrophiles inflammation et l&#39;infection sont les premières cellules à apparaître sur le site touchée où ils agissent comme première ligne de défense 2. Les neutrophiles possèdent plusieurs mécanismes antimicrobiens 3, notamment la phagocytose, production d&#39;espèces réactives de l&#39;oxygène, la libération d&#39;enzymes lytiques par la dégranulation et la production de cytokines pro-inflammatoires 4,5. Les neutrophiles sont des cellules de courte durée qui se rapidement activés par la signalisation de divers récepteurs de surface cellulaire. Bien que les neutrophiles ont été considérés cellules terminales en raison de leur courte durée de vie et parce qu&#39;ils subissent une apoptose à moins activées pendant le processus inflammatoire 6, il est maintenant clair qu&#39;ils peuvent également modifier leur phénotype en changeant le niveau de la transcription de gènes particuliers. La production de cytokines 5 et l&#39;inhibition de l&#39;apoptose 7,8 sont deux important fonctions cellulaires dépendant de l&#39;activation régulée au niveau de la transcription dans les cellules neutrophiles. Le facteur nucléaire kB (NF-kB) participe à la régulation transcriptionnelle de 4 la production de cytokines et dans la régulation de la survie cellulaire et l&#39;apoptose 9-11 dans différents types cellulaires. Les voies de signalisation qui conduisent à l&#39;activation du facteur nucléaire sont habituellement étudiées par des essais de gène rapporteur ou par électrophorèse des essais de décalage de mobilité (EMSA). Cependant, parce que les neutrophiles sont des cellules de courte durée, l&#39;étude des réponses transcription régulés dans ces cellules ne peuvent pas être réalisées avec des dosages de gènes rapporteurs, car il n&#39;existe pas de techniques efficaces pour la transfection des neutrophiles. Analyses EMSA ont été utilisés dans les neutrophiles d&#39;explorer l&#39;activation du facteur nucléaire 12,13; toutefois, cette méthode est compliquée et coûteuse car elle implique l&#39;utilisation de matières radioactives. Nucléofection est une autre technique qui a été utilisée successfully pour transfecter monocytes 14. Ainsi, au moins en théorie, l&#39;activation du facteur nucléaire a pu être détectée dans les neutrophiles par transfection (malgré la faible efficacité). Cependant, cette technique serait plus coûteux, fastidieux et probablement moins quantitative. L&#39;analyse microscopique des cellules immunocolorées pourrait également être utilisé pour détecter les facteurs nucléaires dans le noyau. En effet, nous avons détecté une translocation de NF-kB dans le noyau de cette façon 15. Malheureusement, cette technique est aussi beaucoup de temps, moins quantitative, et sujettes à des biais de l&#39;observateur. Ici, nous présentons une méthode simple et efficace qui permet la détection et la quantification des facteurs nucléaires dans des noyaux isolés et immunomarquées par cytométrie en flux. On décrit des techniques pour isoler des neutrophiles du sang périphérique humain, stimuler ces cellules par l&#39;intermédiaire des intégrines ou des récepteurs Fc avec des anticorps anti-récepteurs, d&#39;isoler et de noyaux immunolabel, et analyser les noyaux par cytométrie de flux (Figure 1). La méthode a été utilisée avec succès pour détecter NF-kB 15 et Elk-1 16 facteurs nucléaires dans les noyaux des neutrophiles. La sensibilité de cette méthode permet la détection de petits changements dans les niveaux de facteurs nucléaires dans le noyau. Cette méthode peut également être utilisée pour analyser le niveau des facteurs de transcription dans le noyau à partir d&#39;autres types cellulaires.

<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

Les neutrophiles sont les leucocytes les plus abondants dans le sang périphérique. Ces cellules sont les premiers à apparaître sur les sites de l&#39;inflammation et de l&#39;infection, devenant ainsi la première ligne de défense contre les micro-organismes envahisseurs. Les neutrophiles possèdent d&#39;importantes fonctions antimicrobiennes tels que la phagocytose, la libération d&#39;enzymes lytiques, et la production d&#39;espèces réactives de l&#39;oxygène. En plus de ces fonctions de défense importants, les neutrophiles effectuer d&#39;autres tâches en réponse à l&#39;infection, comme la production de cytokines pro-inflammatoires et l&#39;inhibition de l&#39;apoptose. Cytokines recruter d&#39;autres leucocytes qui aident à éliminer l&#39;infection, et l&#39;inhibition de l&#39;apoptose des neutrophiles permet de vivre plus longtemps sur le site de l&#39;infection. Ces fonctions sont réglementés au niveau de la transcription. Cependant, parce que les neutrophiles sont des cellules de courte durée, l&#39;étude des réponses transcriptionnellement réglementées dans ces cellules ne peuvent pas être effectuées avec les méthodes classiques de gènes rapporteurs puisqu&#39;il n&#39;y a pas effitechniques santes pour la transfection des neutrophiles. Ici, nous présentons une méthode simple et efficace qui permet la détection et la quantification des facteurs nucléaires dans des noyaux isolés et immunomarquées par cytométrie en flux. Nous décrivons les techniques pour isoler les neutrophiles purs à partir de sang périphérique humain, de stimuler ces cellules avec des anticorps anti-récepteurs, d&#39;isoler et de noyaux immunolabel, et d&#39;analyser les noyaux par cytométrie en flux. La méthode a été utilisée avec succès pour détecter NF-kB et Elk-1 facteurs nucléaires dans les noyaux des neutrophiles et d&#39;autres types cellulaires. Ainsi, cette méthode représente une option pour analyser l&#39;activation des facteurs de transcription dans des noyaux isolés à partir d&#39;une variété de types de cellules.

Le procédé de purification décrit ici fournit habituellement neutrophiles non stimulés (PMN) de pureté supérieure à 95% (figure 1A). PMN isolé peut ensuite être stimulée par les récepteurs spécifiques de réticulation avec des anticorps monoclonaux spécifiques. Nous avons stimulé PMN par le biais des récepteurs Fc et les intégrines (figure 1B). Une fois stimulés, PMN sont lysées et les noyaux sont isolés avec des rendements élevés. Les noyaux sont ensuite immunomarq...

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

Les neutrophiles sont les leucocytes les plus abondants dans le sang. Les neutrophiles possèdent des fonctions transcription régulés tels que la production de cytokines pro-inflammatoires et l&#39;inhibition de l&#39;apoptose. Ces fonctions peuvent être étudiés avec la méthode présentée ici, qui permet la détection et la quantification des facteurs nucléaires par cytométrie en flux dans les noyaux isolés

Une méthode simple et efficace pour détecter l&#39;activation du facteur nucléaire dans les neutrophiles humains par cytométrie en flux

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 vues.  University of Alberta. Les neutrophiles sont les leucocytes les plus abondants dans le sang. Les neutrophiles possèdent des fonctions transcription régulés tels que la production de cytokines pro-inflammatoires et l'inhibition de l'apoptose. Ces fonctions peuvent être étudiés avec la méthode présentée ici, qui permet la détection et la quantification des facteurs nucléaires par cytométrie en flux dans les noyaux isolés

Vidéo: 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.

Évaluation quantitative des cellules immunitaires dans le tissu blessés médullaires par cytométrie en flux: une nouvelle utilisation pour un procédé de purification cellulaire

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

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Immunologie et infection

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.

Une méthode simple et efficace pour isoler les macrophages de mixte cultures primaires de cellules hépatiques adultes

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.

Homme In Vitro</emRépression> comme outil de dépistage pour la reconnaissance d&#39;un Etat précoce de déséquilibre immunitaire

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.

L&#39;examen de sélection positive et négative thymique par cytométrie en flux

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.

L&#39;étude des interactions de<em&gt; Staphylococcus aureus</em&gt; Avec des neutrophiles par cytométrie en flux et microscopie Time Lapse

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 protocole simple et rapide aux non enzymatique dissocier frais tissus humains pour l&#39;analyse des lymphocytes infiltrant

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.

Une approche simple et efficace de construire Mutant Vaccinia vecteurs de virus

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.

Imagerie en direct de l&#39;activité par Human Antifongique primaire neutrophiles et des monocytes en réponse à<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.

Isolement et la Culture In Vitro de Macrophages alvéolaires murines et humaines

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).

Caractérisation des sous-ensembles de Monocyte humain de sang Flow Cytometry analyse

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