Le projet a été soutenu par le NIH NIAID Marché n HHSN2662000500021C. Nous remercions Ming Chen pour son assistance technique.

1. Programme du 96 Amaxa ainsi Nucleofector navettes <ol><li> Ouvrez un nouveau fichier de paramètres. </li><li> Sélectionnez le nombre de puits que vous allez utiliser pour la transfection standards en faisant glisser le curseur sur le diagramme de plaque de 96 puits. Utiliser un minimum de trois puits à la piscine pour chaque échantillon expérimental. </li><li> Entrez le code du programme: en part1 sélectionnez &#39;FF&#39; et choisissez part2 &#39;168 &#39;à partir des menus déroulants </li><li> De la boîte de solutions, sélectionnez &#39;monocytes, humaine » </li><li> Selon l&#39;option de configuration, sélectionnez «standard». </li><li> Cliquez sur Appliquer. </li><li> Pour inclure un contrôle sans transfection, sélectionnez puits supplémentaires à partir du schéma tel que requis, puis choisissez «No Control Program &#39;de l&#39;option de configuration et cliquez sur Appliquer. </li><li> Sélectionnez l&#39;un des puits inutilisés sur le diagramme et cliquez sur la plaque undefine. </li></ol> 2. Préparer les PED pour la transfection <ol><li> Tout travail doit être effectué dans des conditions stériles sous une hotte de culture cellulaire lorsque cela est possible. Préparer la solution en ajoutant nucléofection de 96 puits complément de monocytes humains de 96 puits Solution Nucleofector dans le rapport de 450 à 2025. Mélanger et laisser réchauffer à température ambiante. Vous aurez besoin de 20 pi de solution nucléofection par puits. Faire un excès de 10% pour permettre de pipetage erreur. </li><li> Placez le nombre de modules nucleocuvette vous avez besoin dans la plaque nucleocuvette dans la bonne orientation à savoir insérer le premier pour les lignes 1 et 2. </li><li> Déterminer le nombre de PED nécessaire pour le calcul de votre expérience sur les 500 000 cellules par puits et de culot par centrifugation à 400g pendant 10min. Retirer délicatement le surnageant. </li><li> Resuspendre les cellules dans la solution par un léger pipetage nucléofection haut et en bas à quelques reprises. </li><li> Divisez le volume correct de cellules en suspension dans eppendorfs étiquetés pour le particulier par exemple le traitement GLO et RIG-I. </li><li> Ajouter siARN 0.25μg par 500.000 cellules et mélanger par pipetage. Non-utilisation de siRNA ciblant dans votre échantillon de contrôle sans-transfection. </li><li> Pipeter 20 pi du mélange ci-dessus dans les modules nucleocuvette, en fonction de votre configuration expérimentale, assurant que le liquide est livré au fond du puits. </li><li> Recouvrir la plaque nucleocuvette avec le couvercle et appuyez sur le plateau sur une surface dure une couple de fois pour aider à assurer l&#39;élimination des bulles d&#39;air. </li></ol> 3. Transfecter les PED <ol><li> Insérer la plaque dans le bac de navette de 96 puits Nucleofector, cliquez sur Télécharger et puis démarrer. </li><li> Suivez les progrès des processus de transfection sur l&#39;écran du lap top, une croix noire sur un fond vert signifie une transfection réussie dans ce puits, tandis qu&#39;une barre noire sur un fond rouge signifie qu&#39;il a échoué. </li><li> A l&#39;issue du processus de transfection, enlever la plaque et ajouter 80μl de milieu de croissance DC à chaque puits en utilisant une pipette multicanaux. </li><li> Incuber la plaque pendant 10 min à 37 ° C et 5% de CO 2. </li><li> Transférer le volume 100 ul de la nucleocuvettes dans des tubes contenant 100 pi de la matrice pré-chauffé milieu de croissance DC, le maintien de l&#39;orientation correcte. </li><li> Retirez et jetez les tubes où la transfection n&#39;a pas eu lieu. </li><li> Incuber à 37 ° C et 5% de CO 2 pour l&#39;intervalle de temps ou d&#39;autres 24h désiré. </li></ol> 4. Infecter les cellules avec NDV <ol><li> Retirer les tubes de matrice de l&#39;incubateur de la hotte de culture cellulaire et piscine tubes pour chaque échantillon expérimental dans eppendorfs </li><li> Pellet les cellules doucement par rotation dans une centrifugeuse de bureau pendant 5 min et retirez surnageant. </li><li> Resuspendre les cellules dans un milieu de croissance sans sérum contenant NDV à une MOI de 1 et incuber à 37 ° C et 5% de CO 2 pendant 45 min, avec eppendorfs vaguement recouverte d&#39;une manière stérile. </li><li> Ajouter 900μl de milieu de croissance DC, et re-incuber pendant 8-10 h. </li></ol> 5. Récolte des cellules <ol><li> Cellules Pellet en faisant tourner eppendorfs dans une centrifugeuse de bureau et retirer le surnageant. </li><li> Cellules de récolte pour l&#39;ARN ou de l&#39;extraction des protéines en fonction de votre protocole. </li></ol> 6. Les résultats représentatifs: En utilisant notre protocole optimisé, nous transfectées avec les PED RIGI ciblage siRNA pour 24 h et ensuite infecté les cellules avec NDV (un paramyxovirus détecté par RIG-I) pour stimuler la voie de réponse interféron. Par QRT-PCR, nous avons démontré renverser du gène de 75% au niveau de la transcription. Nous avons également observé une réduction similaire de l&#39;expression de IFNβ, qui est un effecteur en aval de RIG-I dans la cascade de signalisation IFN. Par ailleurs, nous avons observé que l&#39;expression de IFNβ chez les non-infectés, le contrôle de cellules transfectées n&#39;était pas détectable alors que celle de MxA, un gène de réponse IFNβ aval, a été minime (figure 1A). Une seconde transfection des PED avec RIGI ciblage siRNA a été réalisée en utilisant suffisamment de cellules pour y inclure une analyse Western blot. Lorsque leRT-PCR résultats étaient semblables à ce que nous avons vu précédemment (62% et 66% d&#39;abattre RIG-I et d&#39;expression IFNβ respectivement) (figure 1B), le Western blot sondé pour la RIG-I a révélé que l&#39;expression de ce gène avait été complètement bloqué (figure 1C). <img src="/files/ftp_upload/2766/2766fig1.jpg" alt="Figure 1A"/> Figure 1. (A) ont été transfectées avec MoDCs soit siRNA ciblant RIG-I ou siRNA GLO non spécifique et l&#39;effet sur ​​l&#39;expression de la RIG-I, IFNβ et MxA déterminée par qRT-PCR. Le protocole de transfection utilisé un tampon de monocytes spécifiques et FF168 programme nucleoporation (Lonza Walkersville Inc) Après incubation pendant 24 h, les cellules transfectées ont été soit infectés par le NDV (+) ou vers la gauche non infectées (-). Après une incubation de 10 h, les cellules ont été récoltées et les niveaux d&#39;ARN extrait Transcription représentent les résultats de deux expériences répétées. Tiré de Bowles et al 16 (B). MoDCs obtenu à partir d&#39;un pelage différent de Buffy que celles utilisées pour la figure 1A ont de nouveau été transfectées avec RIG-I-ciblage siRNA ou siARN GLO non spécifiques en utilisant le protocole de transfection même que ci-dessus. Un contrôle supplémentaire à l&#39;aide MoDCs non transfectées a été incorporé dans l&#39;expérience. Après une incubation de 24 h toutes les cellules ont été infectées par le NDV et incubé pendant encore 10 h avant d&#39;être récolté à la fois pour l&#39;ARN et l&#39;extraction des protéines. Niveaux Transcription de RIG-I, IFNβ et MxA tel que déterminé par qRT PCR représentent les résultats de deux expériences répétées. Tiré de Bowles et al 16. (C) des lysats de cellules décrit dans la figure 1B ci-dessus ont été analysés par Western blot. Les pistes 1 et 2, des lysats de cellules non transfectées; pistes 3 et 4, des lysats de cellules transfectées siARN GLO; voies 5 et 6, des lysats de cellules transfectées avec RIG-I-ciblage siRNA. Les échantillons ont été sondées pour RIG-I et aussi GAPDH (comme un contrôle du chargement) et ont été analysés en double. Tiré de Bowles et al 16.

<ol>
	<li>Reis e Sousa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+dendritic+cells:+translating+innate+into+adaptive+immunity.">Activation of dendritic cells: translating innate into adaptive immunity.</a> Curr. Opin. Immunol. 16, 21-25 (2004).</li><li>Bancherau, J., Steinman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+and+the+control+of+immunity.">Dendritic cells and the control of immunity.</a> Nature. 392, 245-252 (1998).</li><li>Clark, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+dendritic+cells+in+the+innate+immune+system.">The role of dendritic cells in the innate immune system.</a> Microbes Infect. 2, 257-272 (2000).</li><li>Hamm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transfection+method+for+primary+cells.">Efficient transfection method for primary cells.</a> Tissue Eng. 8, 235-245 (2002).</li><li>Henderson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dendritic+cells+genetically+engineered+to+express+high+levels+of+the+human+epithelial+tumor+antigen+mucin+(MUC-1).">Human dendritic cells genetically engineered to express high levels of the human epithelial tumor antigen mucin (MUC-1).</a> Cancer Res. 56, 3763-3770 (1996).</li><li>Reeves, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+transduction+of+human+dendritic+cells+with+a+tumor-associated+antigen+gene.">Retroviral transduction of human dendritic cells with a tumor-associated antigen gene.</a> Cancer Res. 56, 5672-5677 (1996).</li><li>Aicher, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+retroviral+mediated+transduction+of+a+reporter+gene+in+human+dendritic+cells:+feasibility+of+therapy+with+gene-modified+antigen+presenting+cells.">Successful retroviral mediated transduction of a reporter gene in human dendritic cells: feasibility of therapy with gene-modified antigen presenting cells.</a> Exp. Hematol. 25, 39-44 (1997).</li><li>Thomas, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+problems+with+the+use+of+viral+vectors+for+gene+therapy.">Progress and problems with the use of viral vectors for gene therapy.</a> Nat Rev Genet. 4, 346-358 (2003).</li><li>Jooss, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transduction+of+dendritic+cells+by+DNA+viral+vectors+directs+the+immune+response+to+transgene+products+in+muscle+fibers.">Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers.</a> J. Virol. 72, 4212-4223 (1998).</li><li>Mitchell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-transfected+dendritic+cells+in+cancer+immunotherapy.">RNA-transfected dendritic cells in cancer immunotherapy.</a> J. Clin. Invest. 106, 1065-1069 (2000).</li><li>Mincheff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+transfection+and/or+cross-priming+of+dendritic+cells+following+DNA+and+adenoviral+immunizations+for+immunotherapy+of+cancer-changes+in+peripheral+mononuclear+subsets+and+intracellular+IL-4+and+IFN-gamma+lymphokine+profile.">In vivo transfection and/or cross-priming of dendritic cells following DNA and adenoviral immunizations for immunotherapy of cancer-changes in peripheral mononuclear subsets and intracellular IL-4 and IFN-gamma lymphokine profile.</a> Crit. Rev. Oncol. Hematol. 39, 125-132 (2001).</li><li>Roth, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helper-dependent+adenoviral+vectors+efficiently+express+transgenes+in+human+dendritic+cells+but+still+stimulate+antiviral+immune+responses.">Helper-dependent adenoviral vectors efficiently express transgenes in human dendritic cells but still stimulate antiviral immune responses.</a> J. Immunol. 169, 4651-4656 (2002).</li><li>Tendeloo, V. F. V. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+gene+delivery+by+mRNA+electroporation+in+human+hematopoietic+cells:+superiority+to+lipofection+and+passive+pulsing+of+mRNA+and+to+electroporation+of+plasmid+cDNA+for+tumor+antigen+loading+of+dendritic+cells.">Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells.</a> Blood. 98, 49-56 (2001).</li><li>Lenz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleoporation+of+dendritic+cells:+efficient+gene+transfer+by+electroporation+into+human+monocyte-derived+dendritic+cells.">Nucleoporation of dendritic cells: efficient gene transfer by electroporation into human monocyte-derived dendritic cells.</a> FEBS Lett. 538, 149-154 (2003).</li><li>Prechtel, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+interfering+RNA+(siRNA)+delivery+into+monocyte-derived+dendritic+cells+by+electroporation.">Small interfering RNA (siRNA) delivery into monocyte-derived dendritic cells by electroporation.</a> J. Immunol. Methods. 311, 139-152 (2006).</li><li>Bowles, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+efficient+high-throughput+plasmid+and+siRNA+transfection+of+human+monocyte-derived+dendritic+cells+without+cell+maturation.">Validation of efficient high-throughput plasmid and siRNA transfection of human monocyte-derived dendritic cells without cell maturation.</a> J. Immunol. Methods. , .</li><li>Kato, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+involvement+of+RIG-I+in+antiviral+response.">Cell type-specific involvement of RIG-I in antiviral response.</a> Immunity. 23, 19-28 (2005).</li><li>Kato, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+roles+of+MDA5+and+RIG-I+helicases+in+the+recognition+of+RNA+viruses.">Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses.</a> Nature. 441, 101-105 (2006).</li><li>Haller, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mx+GTPase+family+of+interferon-induced+antiviral+proteins.">The Mx GTPase family of interferon-induced antiviral proteins.</a> Microbes Infect. 9, 1636-1643 (2007).</li></ol>

Transfection efficace des cellules dendritiques naïves primaire est importante pour l&#39;analyse à haut débit et l&#39;ingénierie inverse des voies cellulaires inflammatoires dans cette cellule clé de la médiation de la transition innée-immune adaptative. Cependant, la plupart des chercheurs estiment que ces cellules sont difficiles à transfecter fois efficace et sans la maturation de transfection de cellules induisant la procédure lors de l&#39;utilisation des techniques de transfection standard. Nous avons r...

<table border="1"><tbody><tr><th> Equipement / Réactif </th><th> Société </th><th> Catalogue # </th><th> Commentaires </th></tr><tr><td> Amaxa Nucleofector de 96 puits navette </td><td> Lonza </td><td> 108S0109 </td><td> Numéro de série </td></tr><tr><td> Amaxa monocytes humains de 96 puits Kit Nucleofector </td><td> Lonza </td><td> VHPA-2007 </td><td> Contient les monocytes humains de 96 puits Solution Nucleofector, le Supplément de 96 puits et l&#39;Nucleocuvettes et plaques </td></tr><tr><td> RIG-I siARN </td><td> Dharmacon </td><td> L-012511-00 </td><td></td></tr><tr><td> SiARN GLO </td><td> Dharmacon </td><td> D-001600-01-20 </td><td></td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 11875 </td><td> Complété avec 10% de SVF, 2 mM de L-glutamine, 100 U / ml de pénicilline et 100 pg / ml de streptomycine pour rendre le milieu de croissance DC </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11965 </td><td></td></tr><tr><td> L-glutamine </td><td> Invitrogen </td><td> 25030081 </td><td></td></tr><tr><td> Pénicilline / streptomycine </td><td> Invitrogen </td><td> 15070063 </td><td></td></tr><tr><td> Sérum de veau foetal </td><td> HyClone </td><td> 3070.03 </td><td></td></tr><tr><td> Cellules dendritiques </td><td> New York Blood Centre </td><td></td><td> PED sont purifiés à partir de couches leuco-en utilisant une procédure standard </td></tr></tbody></table>

optimized protocol for efficient transfection of dendritic cells without cell maturation

Cellules dendritiques (CD) peuvent être considérés comme des sentinelles du système immunitaire qui jouent un rôle critique dans son initiation et une réponse à l&#39;infection. Détection de l&#39;antigène pathogènes par les CD naïve est à travers des récepteurs de reconnaissance des motifs (PRR) qui sont capables de reconnaître des structures spécifiques conservé appelé pathogène motifs moléculaires associés (PAMPS). Détection des PAMPs par les CD déclenche une cascade de signalisation intracellulaire entraîne leur activation et de transformation pour les PED matures. Ce processus est généralement caractérisé par une production d&#39;interféron de type 1 avec d&#39;autres cytokines pro-inflammatoires, la régulation positive des marqueurs de surface cellulaire, comme CMHII et CD86 et la migration des DC matures dans les ganglions lymphatiques drainant, où l&#39;interaction avec les cellules T initie la réponse immunitaire adaptative 2, 3. Ainsi, les PED relier les systèmes immunitaire innée et adaptative. La capacité à disséquer les réseaux moléculaires qui sous-tendent la réponse à divers pathogènes DC est cruciale pour une meilleure compréhension de la régulation de ces voies de signalisation et de leurs gènes induits. Il devrait également aider à faciliter le développement de vaccins à base de DC contre les maladies infectieuses et les tumeurs. Cependant, cette ligne de recherche a été gravement entravée par la difficulté de transfecter des primaires PED 4. Méthodes de transduction virus, tels que le système lentiviral, sont généralement utilisés, mais portent de nombreuses limitations telles que la complexité et de la bio-dangereux risque (avec les coûts associés) 5,6,7,8. En outre, la livraison de produits de gènes viraux augmente l&#39;immunogénicité de ces transduites PED 9,10,11,12. L&#39;électroporation a été utilisée avec des résultats mitigés 13,14,15, mais nous sommes les premiers à signaler l&#39;utilisation d&#39;un protocole de transfection à haut débit et de manière concluante démontrer son utilité. Dans ce rapport, nous résumons un protocole optimisé commerciale pour la transfection à haut débit des humains PED primaire, avec une toxicité cellulaire limitée et une absence de maturation des DC 16. Efficacité de la transfection (de plasmide GFP) et la viabilité des cellules ont été plus de 50% et 70% respectivement. FACS analyse a établi l&#39;absence d&#39;augmentation de l&#39;expression des marqueurs de la maturation du CD86 et CMH II dans des cellules transfectées, tandis qRT-PCR n&#39;a démontré aucune régulation à la hausse des IFNβ. En utilisant ce protocole d&#39;électroporation, nous fournir des preuves pour la transfection réussie des PED avec le siRNA et efficace abattre des gènes ciblés RIG-I, un récepteur de reconnaissance clés virale 16,17, à la fois l&#39;ARNm et de protéine.

Watch this Scientific Journal Video about Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation at JoVE.com

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Nous présentons notre protocole optimisé nucléofection à haut débit comme un moyen efficace de transfection humaines primaires dérivées de monocytes des cellules dendritiques avec soit l&#39;ADN plasmidique ou siRNA sans causer la maturation des cellules. En outre, nous fournir des preuves pour faire taire les siARN réussie de gène ciblé RIG-I à la fois au niveau ARNm et des protéines.

Protocole optimisé pour une transfection efficace des cellules dendritiques, sans maturation cellulaire

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

optimized-protocol-for-efficient-transfection-dendritic-cells-without

Research

JoVE Journal

Immunology and Infection

20.2K vues.  Mount Sinai School of Medicine. Nous présentons notre protocole optimisé nucléofection à haut débit comme un moyen efficace de transfection humaines primaires dérivées de monocytes des cellules dendritiques avec soit l'ADN plasmidique ou siRNA sans causer la maturation des cellules. En outre, nous fournir des preuves pour faire taire les siARN réussie de gène ciblé RIG-I à la fois au niveau ARNm et des protéines.

Vidéo: Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Génération et d&#39;étiquetage des murins de la moelle osseuse des cellules dendritiques dérivées des nanocristaux Qdot pour les études de suivi

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Recherche

Immunologie et infection

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

L&#39;analyse de la maturation des cellules dendritiques pulmonaire et de la migration au cours de l&#39;inflammation allergique des voies respiratoires

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Tueur antigène artificielle cellules présentatrices (KaAPC) d&#39;utilisation efficace<em&gt; In Vitro</em&gt; L&#39;épuisement des lymphocytes T humains spécifiques de l&#39;antigène

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

La transfection très efficace des droits de THP-1 par les macrophages nucléofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Une méthode de rendement efficace et haute pour l&#39;isolement des souris dendritiques sous-ensembles cellulaires

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Génération de Immature, d&#39;âge mûr et tolérogène cellules dendritiques avec Metabolic Différentes phénotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

Développement et Validation d’une seule molécule ultrasensible de tableau numérique Enzyme-linked Immunosorbent Assay for Human interféron-α

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

Nouveau protocole pour la génération de cellules dendritiques immunogéniques physiologiques

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

Méthode de dépistage à cellule unique pour la sélection et la récupération d'anticorps ayant les spécificités souhaitées des populations enrichies de cellules de la mémoire humaine B

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

Transfection très efficace des macrophages primaires avec ARNm transcrit in vitro

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...