Il progetto è stato sostenuto da NIH Contratto NIAID No. HHSN2662000500021C. Ringraziamo Ming Chen per la sua assistenza tecnica.

1. Programmare il Amaxa 96 pozzetti navetta Nucleofector <ol><li> Aprire un nuovo file di parametri. </li><li> Selezionare il numero di pozzi che verrà utilizzato per la trasfezione di serie trascinando il cursore sul diagramma 96 pozzetti. Usare un minimo di 3 pozzi di piscina per ogni campione sperimentale. </li><li> Inserire il codice del programma: in part1 selezionare &#39;FF&#39; e in part2 selezionare &#39;168 &#39;dal menu a discesa </li><li> Dalla soluzione casella di selezione &#39;Monocita, umano&#39; </li><li> Secondo l&#39;opzione di controllo, selezionare &#39;standard&#39;. </li><li> Fare clic su Applica. </li><li> Per includere una no-transfezione di controllo, selezionare ulteriori pozzi dal diagramma come richiesto e poi scegliere &#39;No Control Program&#39; Opzione di controllo e fare clic su Applica. </li><li> Selezionare qualsiasi pozzi rimasti inutilizzati sul diagramma piatto e cliccare su undefine. </li></ol> 2. Preparare DC per la trasfezione <ol><li> Tutto il lavoro dovrebbe essere fatto in condizioni di sterilità in una cappa di coltura cellulare, se possibile. Preparare la soluzione nucleofection con l&#39;aggiunta di ben 96-supplemento al monociti umani a 96 pozzetti Soluzione Nucleofector nel rapporto di 450 al 2025. Mescolare e lasciare a temperatura ambiente. Avrete bisogno di 20μl di soluzione nucleofection per bene. Fai un eccesso del 10% per consentire pipettaggio errore. </li><li> Collocare il numero di moduli nucleocuvette è necessario nella piastra nucleocuvette nel corretto orientamento cioè inserendo la prima in a righe 1 e 2. </li><li> Determinare il numero di controller necessari per l&#39;esperimento calcolo su 500.000 cellule per bene e pellet per centrifugazione a 400g per 10 minuti. Rimuovere con attenzione il sopranatante. </li><li> Risospendere le cellule nella soluzione nucleofection pipettando gentilmente su e giù un paio di volte. </li><li> Dividere la giusta quantità di cellule risospese in eppendorfs l&#39;etichetta per il particolare trattamento ad esempio GLO e RIG-I. </li><li> Aggiungi siRNA 0.25μg per 500.000 cellule e mescolare pipettando. Uso non-targeting siRNA nel no-trasfezione campione di controllo. </li><li> Pipettare 20μl delle miscele sopra nel nucleocuvette moduli, in base al layout sperimentale, assicurando il liquido viene consegnato al fondo del pozzo. </li><li> Coprire la piastra nucleocuvette con il coperchio e toccare la piastra su una superficie dura un paio di volte per contribuire a garantire la rimozione di bolle d&#39;aria. </li></ol> 3. Trasfezione DC <ol><li> Inserire la piastra nel cassetto Nucleofector navetta a 96 pozzetti, clicca su Carica e quindi avviare. </li><li> Seguire i progressi del processo di trasfezione sul display del computer portatile, una croce nera su sfondo verde significa una trasfezione di successo in quel pozzo, mentre una barra nera su sfondo rosso, significa che non ha avuto successo. </li><li> Al termine del processo di trasfezione, rimuovere la piastra e aggiungere 80μl di terreno di crescita continua in ciascun pozzetto utilizzando una pipetta multicanale. </li><li> Incubare piastra per 10 minuti a 37 ° C e 5% di CO 2. </li><li> Trasferire il volume di 100μl dal nucleocuvettes in provette contenenti 100μl di matrice pre-riscaldato medio DC crescita, mantenendo l&#39;orientamento corretto. </li><li> Rimuovere e scartare quei tubi in cui la transfezione non si è verificato. </li><li> Incubare a 37 ° C e 5% di CO 2 per 24 ore o altri intervallo di tempo desiderato. </li></ol> 4. Infettare le cellule con NDV <ol><li> Rimuovere i tubi matrice dal termostato alla cappa di coltura cellulare e tubi in piscina per ogni campione sperimentale in eppendorfs </li><li> Agglomerare le cellule dolcemente facendo girare in una centrifuga da tavolo per 5 minuti e rimuovere il surnatante. </li><li> Risospendere le cellule nel siero senza mezzo di crescita contenente NDV ad una MOI di 1 e incubare a 37 ° C e 5% di CO 2 per 45 min, con eppendorfs vagamente coperto in maniera sterile. </li><li> Aggiungere 900μl di mezzo di crescita continua, e ri-incubare per 8-10 h. </li></ol> 5. Raccogliere cellule <ol><li> Celle a pellet dal giro eppendorfs in una centrifuga da tavolo e rimuovere il surnatante. </li><li> Celle di raccolta per l&#39;RNA o l&#39;estrazione di proteine ​​secondo il protocollo. </li></ol> 6. Rappresentante dei risultati: Usando il nostro protocollo ottimizzato abbiamo DC transfettate con RIGI-targeting siRNA per 24 ore e poi le cellule infettate con NDV (un paramyxovirus rilevato da RIG-I) per stimolare il percorso di risposta all&#39;interferone. Da qRT-PCR abbiamo dimostrato abbattere del gene del 75% a livello di trascrizione. Abbiamo anche osservato una riduzione simile nell&#39;espressione di IFNβ, che è un effettore a valle di RIG-I nella cascata IFN segnalazione. Inoltre, abbiamo osservato che l&#39;espressione di IFNβ a non infette, controllo transfettate cellule non era rilevabile mentre quello di MxA, un gene IFNβ risposta a valle, è stato minimo (figura 1A). Una seconda trasfezione DC con RIGI-targeting siRNA è stata effettuata utilizzando cellule sufficiente per comprendere l&#39;analisi Western Blot. Qualora ilRT-PCR risultati sono stati simili a quanto visto in precedenza (62% e il 66% di abbattere RIG-I ed espressione IFNβ rispettivamente) (Figura 1B), il Western Blot sondato per RIG-I ha rivelato che l&#39;espressione di questo gene era stato completamente bloccato (Figura 1C). <img src="/files/ftp_upload/2766/2766fig1.jpg" alt="Figura 1A"/> Figura 1. (A) MoDCs sono state trasfettate con siRNA o targeting RIG-I o siRNA GLO aspecifici e l&#39;effetto sulla espressione di RIG-I, IFNβ e MxA determinato dalla qRT-PCR. Il protocollo di trasfezione usato un monociti-specifico buffer e programma nucleoporation FF168 (Lonza Walkersville Inc.) Dopo incubazione per 24 ore, le cellule transfettate erano o infettati con NDV (+) o sinistra non infetti (-). Dopo un ulteriore incubazione per 10 ore, le cellule sono state raccolte e dei livelli di trascrizione RNA estratto rappresentano i risultati di due esperimenti di replicare. Tratto da Bowles et al 16. (B) MoDCs ottenuti da una mano diversa buffy come usato per la figura 1A sono stati nuovamente trasfettate sia con RIG-I-targeting siRNA o siRNA GLO aspecifico utilizzando lo stesso protocollo di trasfezione come sopra. Un ulteriore controllo utilizzando MoDCs untransfected è stato incorporato l&#39;esperimento. A seguito di una 24 ore di incubazione tutte le cellule sono state infettate con NDV e incubate per 10 h prima di essere raccolte sia per estrazione di RNA e proteine. I livelli di trascrizione di RIG-I, IFNβ e MxA come determinato dal qRT-PCR rappresentano i risultati di due esperimenti di replicare. Tratto da Bowles et al 16. (C) lisati da cellule descritto nella Figura 1B sopra sono stati analizzati mediante Western Blot. Corsie 1 e 2, lisati da cellule untransfected; corsie 3 e 4, lisati da cellule transfettate siRNA GLO, corsie 5 e 6, lisati da cellule transfettate con RIG-I-targeting siRNA. I campioni sono stati sondati per RIG-I e anche GAPDH (come controllo di carico) e sono stati eseguiti in duplicato. Tratto da Bowles et al 16.

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Nessun conflitto di interessi dichiarati.

Trasfezione efficiente ingenuo cellule dendritiche primario è importante per l&#39;analisi di un elevato throughput e reverse engineering di cellulari vie infiammatorie in questa cellula fondamentale della mediazione innata-adaptive transizione del sistema immunitario. Tuttavia, la maggior investigatori scoprire che queste cellule sono di difficile trasfezione in modo efficiente e senza la maturazione di transfezione delle cellule procedura di induzione quando si utilizzano tecniche di trasfezione standard. Abbiamo stu...

<table border="1"><tbody><tr><th> Attrezzature / Reagente </th><th> Azienda </th><th> Catalogo # </th><th> Commenti </th></tr><tr><td> Amaxa Nucleofector 96-ben Shuttle </td><td> Lonza </td><td> 108S0109 </td><td> Numero di serie </td></tr><tr><td> Amaxa monociti umani a 96 pozzetti Kit Nucleofector </td><td> Lonza </td><td> VHPA-2007 </td><td> Contiene i monociti umani a 96 pozzetti Soluzione Nucleofector, il supplemento a 96 pozzetti e la Nucleocuvettes e piastre </td></tr><tr><td> RIG-I siRNA </td><td> Dharmacon </td><td> L-012511-00 </td><td></td></tr><tr><td> GLO siRNA </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> Supplementato con 10% FCS, 2 mM L-glutammina, 100 U / ml penicillina e 100 mg / ml di streptomicina per rendere terreno di crescita continua </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11965 </td><td></td></tr><tr><td> L-glutammina </td><td> Invitrogen </td><td> 25030081 </td><td></td></tr><tr><td> Penicillina / streptomicina </td><td> Invitrogen </td><td> 15070063 </td><td></td></tr><tr><td> Siero di vitello fetale </td><td> Hyclone </td><td> 3.070,03 </td><td></td></tr><tr><td> Le cellule dendritiche </td><td> New York Sangue centro </td><td></td><td> DC sono purificati da buffy coats utilizzando una procedura standard </td></tr></tbody></table>

optimized protocol for efficient transfection of dendritic cells without cell maturation

Le cellule dendritiche (DC) può essere considerato sentinelle del sistema immunitario che svolgono un ruolo critico nella sua iniziazione e la risposta alle infezioni 1. Rilevazione dell&#39;antigene patogeni da DC ingenuo è attraverso i recettori pattern recognition (PRR) che sono in grado di riconoscere specifiche strutture conservate denominate modelli molecolari patogeni associati (PAMPs). Rilevazione di PAMPs da DC innesca una cascata di segnalazione intracellulare con conseguente loro attivazione e la trasformazione di DC mature. Questo processo è tipicamente caratterizzata da produzione di interferone di tipo 1 insieme ad altre citochine proinfiammatorie, sovraregolazione di marcatori cellulari di superficie come MHCII e CD86 e la migrazione delle DC mature ai linfonodi drenanti, dove l&#39;interazione con le cellule T avvia la risposta immunitaria adattativa 2, 3. Così, DC collegare i sistemi immunitario innato e adattativo. La capacità di sezionare le reti molecolari alla base della risposta DC a vari agenti patogeni è fondamentale per una migliore comprensione del regolamento di queste vie di segnalazione e le loro geni indotti. Dovrebbe anche contribuire a facilitare lo sviluppo di DC-based vaccini contro le malattie infettive e tumori. Tuttavia, questa linea di ricerca è stato gravemente ostacolato dalla difficoltà di trasfezione primario DC 4. Metodi di trasduzione del virus, come il sistema lentivirale, sono tipicamente utilizzati, ma portano molti limiti come la complessità e bio-pericolosi rischio (con i costi associati) 5,6,7,8. Inoltre, la consegna dei prodotti gene virale aumenta l&#39;immunogenicità di coloro trasdotte DC 9,10,11,12. Elettroporazione è stata utilizzata con risultati alterni 13,14,15, ma siamo i primi a segnalare l&#39;utilizzo di un high-throughput protocollo di trasfezione e definitivamente dimostrare la sua utilità. In questa relazione si riassumono un protocollo ottimizzato commerciale high-throughput per la transfezione di DC umane primarie, con limitata tossicità delle cellule e l&#39;assenza di maturazione DC 16. L&#39;efficienza di trasfezione (di plasmide GFP) e la vitalità delle cellule erano più del 50% e 70% rispettivamente. FACS analisi stabilito l&#39;assenza di aumento di espressione della maturazione e MHCII marker CD86 in cellule trasfettate, mentre qRT-PCR ha dimostrato di non sovraregolazione IFNβ. Usando questo protocollo di elettroporazione, forniamo le prove per la trasfezione di successo di DC con siRNA ed efficace di abbattere genica mirata RIG-I, un recettore chiave riconoscimento virale 16,17, sia a livelli di mRNA e proteine.

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

Vi presentiamo il nostro sito ottimizzato high-throughput protocollo nucleofection come un modo efficace di trasfezione delle cellule dendritiche umane primarie derivate da monociti sia con DNA plasmidico o siRNA senza provocare la maturazione delle cellule. Abbiamo inoltre fornire le prove per il silenziamento del gene siRNA successo mirati RIG-I, sia a livelli di mRNA e proteine.

Protocollo ottimizzato per Transfection efficiente delle cellule dendritiche, senza la maturazione delle cellule

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 Views.  Mount Sinai School of Medicine. Vi presentiamo il nostro sito ottimizzato high-throughput protocollo nucleofection come un modo efficace di trasfezione delle cellule dendritiche umane primarie derivate da monociti sia con DNA plasmidico o siRNA senza provocare la maturazione delle cellule. Abbiamo inoltre fornire le prove per il silenziamento del gene siRNA successo mirati RIG-I, sia a livelli di mRNA e proteine.

Video: 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.

Generazione e l&#39;etichettatura di Murine osseo cellule dendritiche derivate dal midollo con nanocristalli Qdot per gli Studi inseguimento

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

Ricerca

Immunologia e infezioni

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.

Analisi della maturazione polmonare delle cellule dendritiche e migrazione durante l&#39;infiammazione allergica delle vie aeree

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.

Killer artificiale cellule presentanti l&#39;antigene (KaAPC) per una efficiente<em&gt; In Vitro</em&gt; L&#39;esaurimento delle Umana antigene-specifiche cellule T

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.

Trasfezione altamente efficiente di umani THP-1 da macrofagi Nucleofection

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.

Un metodo del rendimento efficiente e di alta per l&#39;isolamento di mouse dendritiche sottopopolazioni cellulari

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.

Generazione di Immaturo, matura e tollerogenico dendritiche cellule con differenti fenotipi metabolica

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.

Sviluppo e validazione di una singola molecola ultrasensibile di matrice digitale analisi enzima-collegata dell'immunosorbente per umana dell'interferone-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.

Nuovo protocollo per la generazione di cellule dendritiche fisiologiche immunogene

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.

Metodo di screening a singola cellula per la selezione e il recupero di anticorpi con specifiche desiderate da popolazioni di cellule di memoria umana b arricchite

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.

Trasfezione altamente efficiente dei macrofagi primari con mRNA trascritto 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 ...