Questo lavoro sostenuto in parte dal NIH sotto Grant CA137499 R15-01 (PC) e un fondo di avvio da Ohio State University (FB).

1. Dissezione di femori e tibie del mouse e della cultura di cellule del midollo osseo <ol><li> Sacrificio 2 topi da CO 2 asfissia e con attenzione sezionare tibie e femori senza tagliare le estremità delle ossa. </li><li> Pulire le ossa da tutti i tessuti attaccati utilizzando carta velina. Fare attenzione a non rompere le ossa. </li><li> Sterilizzare le ossa per immersione in etanolo al 70% per 10 minuti in una capsula di Petri 35 millimetri. Da questo momento, il lavoro all&#39;interno di una cappa di biosicurezza per evitare la contaminazione delle colture cellulari. </li><li> Recuperare le ossa dal etanolo e lasciarli asciugare per 5 minuti in una capsula di Petri all&#39;interno del cabinet biosicurezza. </li><li> Tagliare i femori a metà, e la tibia con la sua punta più sottile. Infondere la parte interna dell&#39;osso con 1 ml di terreno RPMI (senza siero ma con antibiotici) utilizzando una siringa sterile in una piastra di Petri sterile. </li><li> La sospensione cellulare viene raccolto e lavato 2X in RPMI medio di 10 min centrifugazione in una provetta da 15 ml per centrifuga a 1.100 RPM in una centrifuga refrigerata (4 ° C) con un rotore oscillante. </li><li> Dopo l&#39;ultimo lavaggio, risospendere le cellule in 2 ml di tampone di lisi ACK e incubare per 5 minuti a temperatura ambiente per eliminare i globuli rossi. </li><li> Aggiungere 13 ml di RPMI con 10% FBS, risospendere e lavare 2X in questo mezzo con le stesse impostazioni, come descritto in 1.6. </li><li> Contare le celle, regolare a 2 x 10 5 cellule / ml con RPMI 10% FBS, e aggiungere rm-GM-CSF (20 ng / ml concentrazione finale) 5. </li><li> Aggiungere 10 ml di questa sospensione ad una sterile, qualità microbiologica, 10 centimetri piatto di Petri, e la cultura in un incubatore CO 2 (37 ° C, 5% CO 2). </li><li> Tre giorni dopo, aggiungere altri 10 ml di RPMI 10% FBS con 20 ng / l di rmGM-CSF a ciascuno dei piatti preparati. </li><li> Tre giorni dopo, 10 ml di sospensione cellulare vengono recuperati da ciascuna piastra di Petri, centrifugato come in 1.6, risospeso nello stesso volume di RPMI 10% FBS con rmGM-CSF e restituito alla piastra Petri. Le cellule sono coltivate per 2 giorni aggiuntivi in CO 2 incubatore. </li></ol> 2. La raccolta e l&#39;etichettatura dei PVS <ol><li> Dopo 8 giorni di coltura le cellule debolmente aderenti sono recuperati dal lavare i piatti di Petri con terreno fresco. Questo protocollo rende circa 2-4 x10 8 DC nelle nostre mani. Le celle possono essere analizzati per fenotipo DC mediante citometria di flusso. </li><li> Cellule raccolte vengono poi etichettati con il kit 655 celle Qtracker Etichettatura rigorosamente seguendo le istruzioni del produttore. </li><li> Abbiamo ottenuto ottimi risultati etichettatura derivate dal midollo osseo murino DC con Qdots ad una concentrazione 10 nM. Per fare questo, aliquote di componenti Etichettatura Qtracker cellulare Kit A e B sono mescolati in volumi uguali, incubate per 5 minuti a temperatura ambiente, e subito diluito 1 / 100 in un mezzo fresco con vortex per 30 s. </li><li> A macchia 5 x 10 6 DC, mescolare 5 ml di ciascuno i componenti del kit A e B in una provetta Eppendorf e incubare per 5 minuti a temperatura ambiente. Quindi, aggiungere 1 ml di incubare RPMI 10% FBS, e vortex per 30 secondi. </li><li> Aggiungere 0,5 ml di sospensione cellulare contenente 5 x10 6 DC, e incubare a 37 ° C per 60 min. </li><li> Poi, lavare le cellule 2X in RPMI 10% FBS (1.100 rpm). Le celle possono essere risospese in RPMI per la cultura più o in PBS per l&#39;iniezione nei topi sperimentali. </li></ol> 3. Valutazione della DC etichettati <ol><li> L&#39;etichettatura delle cellule possono essere valutati mediante microscopia a fluorescenza. Per fare questo, una goccia di sospensione cellulare viene aggiunto a un vetrino da microscopio, coperta con un coprioggetto di vetro, e valutata con un microscopio a fluorescenza tenendo conto che questi Qdots hanno spettri di emissione e di eccitazione di 565 nm e 405-525nm, rispettivamente (Fig. 3 ) </li><li> A questo punto, le cellule etichettate può essere usata per iniettare gli animali, o la maturazione può essere indotta da incubando queste cellule per 48 ore (37 ° C, 5 CO 2) in presenza di LPS (100 ng / ml) e TNF-( 20 ng / ml). Fluorescente etichettatura non è influenzato dalla maturazione DC (Fig. 4). </li></ol> 4. Rappresentante dei risultati: Con la presente abbiamo descritto come preparare derivate dal midollo osseo murino DC e come li etichettati con Qdots fluorescenti. Fig. 1 riassume la procedura per ottenere le cellule, mentre la fig. 2 mostra la procedura di etichettarli con Qdots. Come mostrato in fig. 3, quasi tutte le cellule sono etichettati da questa procedura, e questo non è influenzato dalla maturazione DC con fattori infiammatori (Fig. 4). Fig. 5 mostra che Qdot macchiato di DC (Fig.5a) si comportano simili a cellule non colorate quando vengono trattati con un cocktail infiammatorio. Entrambe le preparazioni cellulari similmente upregulate espressione delle molecole co-stimolatorie (Fig. 5b), e producono quantità simili di IL-6 (fig. 5c) e l&#39;ossido nitrico (Fig. 5d) sugli stimoli maturazione. Questo concorda con preceOU dati pubblicati indicano che Qdot colorazione non influisce sulla capacità di DC di trasformarsi in cellule mature capaci di suscitare risposte immunitarie [6]. Queste cellule possono quindi essere utilizzati per l&#39;iniezione animali da esperimento. Come mostrato in fig. 6, 2 giorni dopo l&#39;iniezione endovenosa di DC etichettati in topi, siamo stati in grado di rilevare Qdot macchiato di cellule nella milza. Questo è anche in accordo con precedenti dati pubblicati mostrano l&#39;utilizzo di nanocristalli Qdot per valutare la migrazione delle DC in vivo [6] di imaging non invasiva e di citometria a flusso. <img src="/files/ftp_upload/2785/2785fig1.jpg" alt="Figura 1"/> Figura 1. Diagramma di flusso della generazione di corrente continua dal midollo osseo. Con la presente si raffigurano il processo per ottenere dal midollo osseo DC. Entrambe le tibie e femori sono sezionati e puliti dal tessuto circostante. Suggerimenti ossa sono conservate e l&#39;interno delle ossa sono lavata con media. Dopo aver eliminato i globuli rossi, le cellule del midollo osseo sono coltivate per 8 giorni in presenza di GM-CSF per differenziarle in PVS. <img src="/files/ftp_upload/2785/2785fig2.jpg" alt="Figura 2"/> Figura 2. Diagramma di flusso della procedura di etichettatura. Aliquote uguali di componenti Etichettatura Qtracker cellulare Kit A e B sono mescolati in un tubo eppendorf. Le cellule dendritiche sono raccolti da culture osso di 8 giorni midollo con GM-CSF e miscelato con la tintura di etichettatura per 60 minuti a 37 ° C. Quindi le cellule vengono lavate in media per eliminare le particelle di etichettatura in eccesso. <img src="/files/ftp_upload/2785/2785fig3.jpg" alt="Figura 3"/> Figura 3. Microfotografia fluorescente di DC subito dopo l&#39;etichettatura. Una goccia di cellule marcate è stato depositato su un vetrino e coperta da un coprioggetto. Poi, i campioni sono stati valutati in un microscopio a fluorescenza e le immagini sono state acquisite attraverso un 5,0 Colore Micropublisher fotocamera digitale CCD (Qimaging, Surrey, BC Canada). <img src="/files/ftp_upload/2785/2785fig4.jpg" alt="Figura 4"/> Figura 4. Microfotografia fluorescente di DC dopo 48 ore di maturazione in vitro. DC etichettati sono state coltivate per 48 ore con LPS (100 ng / ml) e TNF-α (20 ng / ml) su vetrini su un incubatore CO 2 (37 ° C, 5% CO 2). Poi, i campioni sono stati lavati con PBS (5 min, 2X), fissate con acetone (15, min 4 ° C) e di contrasto con DAPI. Le cellule sono state valutate in un microscopio a fluorescenza come sopra. <img src="/files/ftp_upload/2785/2785fig5.jpg" alt="Figura 5"/> Figura 5. L&#39;attività biologica del Qdot DC macchiato maturo. DC etichettati maturato in vitro di cui sopra sono stati analizzati mediante citometria di flusso (A) e (B). (A) Qdot macchiato di cellule dare un segnale forte nella (PerCP) canale FL3. Ombreggiato istogramma: controllo senza macchia. (B) DC Qdot macchiato e senza macchia sono stati ulteriormente colorate con anticorpi specifici contro la maturazione marker CD86 e CD40, e controlli isotipo. Le cellule sono state poi analizzate in un citofluorimetro FACSort (Becton Dickinson, San Jose, CA). Inoltre, IL-6 (C) e l&#39;ossido nitrico (NO) sono stati determinati in surnatanti di maturità Qdot macchiati di DC e controlli. I livelli di IL-6 e l&#39;ossido di azoto sono stati determinati per mezzo di analisi e test ELISA Griess come abbiamo descritto precedentemente 7, 8. Tutti i dati hanno mostrato in questa figura è rappresentativa di due o tre esperimenti indipendenti che mostrano risultati simili. I dati sono stati analizzati mediante ANOVA utilizzando il software GraphPad. <img src="/files/ftp_upload/2785/2785fig6.jpg" alt="Figura 6"/> Figura 6. Rilevamento delle DC marcati nei tessuti sezionati. Etichettati DC mature (1x10 6 celle) sono state iniettate endovena in topi C57BL / 6. (A) Due giorni dopo i topi sono stati sacrificati e milza raccolti, congelati a scatto, integrati in ottobre, e 8 sezioni mM preparati utilizzando un criostato. Poi, i campioni sono stati fissati con acetone (15, min 4 ° C) e di contrasto con DAPI. Le cellule sono state valutate in un microscopio a fluorescenza come sopra Fig. 6a:. Ingrandimento 40X, Fig. 6b: ingrandimento 100X, fig. 6C, ingrandimento 400X.

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Nessun conflitto di interessi dichiarati.

Murine mieloidi) DC sono stati ampiamente utilizzati per determinare l&#39;efficacia e il miglioramento della DC a base di vaccini; indagare DC: le interazioni delle cellule T o di sviluppo corrente continua, e determinare il loro ruolo nelle varie patologie 9-11. Con la presente si mostra come generare DC da precursori recuperato zucchine osso di tibie e femori. Noi recuperare le ossa senza tagliare le punte, che ci permette di sterilizzare loro da immersione in etanolo al 70%, riducendo così la probabilità di contaminazione. Per differenziare i controller di dominio a partire da cellule del midollo osseo usiamo solo GM-CSF come descritto in precedenza 5. Anche se alcuni protocolli di utilizzare anche IL-4, è stato riferito che questa citochina non è necessaria quando si lavora con alti livelli di GM-CSF 12. Infatti, abbiamo già dimostrato che questi controller di dominio sono in grado di indurre risposte immunitarie 13. Inoltre, la cura deve essere presa per recuperare le cellule solo vagamente aderente da 8 giorni di culture lavando i piatti di Petri con il mezzo da quando le cellule allegato mostrano un fenotipo più monociti-like. Qui si mostra l&#39;etichettatura dei PVS con particelle fluorescenti Qdot. Tale etichettatura ha alcuni vantaggi rispetto ad altri metodi. In primo luogo, le particelle Qdots sono facilmente incorporato nelle cellule. In secondo luogo, il segnale fluorescente è molto alta e non è influenzata dal maturazione DC. In terzo luogo, la fluorescenza non è persa quando le cellule o tessuti sono fissati con solventi come l&#39;acetone, contrariamente a quanto succede se GFP è usato per codificare DC 14, dando maggiore flessibilità al momento di scegliere protocolli di colorazione. Infine, il segnale di fluorescenza ad alta dato da queste particelle permette di visualizzare le cellule dei tessuti, nonostante l&#39;auto-fluorescenza. Come descritto in precedenza 6, colorazione Qdot non ha influenzato la capacità di maturazione di queste cellule. Con la presente ci dimostrano che Qdot macchiati DC si comportano in modo simile a come non macchiato DC, upregulating molecole co-stimolatorie, e la produzione di IL-6 e l&#39;ossido nitrico in risposta a stimoli infiammatori. Sebbene DC sono cellule specializzate nello scatenare la risposta immunitaria, hanno dimostrato di partecipare in condizioni patologiche come il cancro e l&#39;aterosclerosi 4, 15, 16. Sono stati inoltre affermato di partecipare al processo di angiogenesi 17, 18, ​​anche suggerito come strutturalmente partecipare alla sviluppo di nuovi vasi 19, 20. Così, i metodi che permettono di DC monitoraggio in vivo, e determinare la loro localizzazione geografica in diversi tessuti 4, 21, 22 sono molto preziosi.

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> C57BL / 6 topi </td><td> Jackson laboratori </td><td></td><td> Le femmine, 6-8 settimane di età </td></tr><tr><td> RPMI </td><td> Invitrogen </td><td> 11875-119 </td><td></td></tr><tr><td> Siero fetale bovino, qualificato </td><td> Invitrogen </td><td> 10437-028 </td><td></td></tr><tr><td> Antibiotico-antimicotico </td><td> Invitrogen </td><td> 15240-096 </td><td></td></tr><tr><td> PBS </td><td> Invitrogen </td><td> 10010-049 </td><td></td></tr><tr><td> Buffer di lisi ACK </td><td> Lonza Walkersville, Inc </td><td> 10-548E </td><td></td></tr><tr><td> Ricombinante murino GM-CSF </td><td> Peprotech Inc </td><td> 315-03 </td><td></td></tr><tr><td> Qtracker 655 celle Kit Etichettatura </td><td> Invitrogen </td><td> Q25021MP </td><td></td></tr><tr><td> Lipopolisaccaride </td><td> Invivogen </td><td> tlrl-eblps </td><td></td></tr><tr><td> Ricombinante murino TNF alfa </td><td> Peprotech </td><td> 315-01A </td><td></td></tr><tr><td> Anticorpi CD86 </td><td> BD Biosciences </td><td> 553691 </td><td></td></tr><tr><td> Anticorpi CD40 </td><td> BD Biosciences </td><td> 553791 </td><td></td></tr><tr><td> Sistema reagente Griess </td><td> Promega </td><td> G2930 </td><td></td></tr><tr><td> IL-6 la cattura anticorpale </td><td> eBioscience </td><td> 13-7061-81 </td><td></td></tr><tr><td> IL-6 rilevazione degli anticorpi </td><td> eBioscience </td><td> 13-7062-81 </td><td></td></tr></tbody></table>

generation and labeling of murine bone marrow-derived dendritic cells with qdot nanocrystals for tracking studies

Le cellule dendritiche (DC) sono cellule che presentano l&#39;antigene professionali (APC) ha trovato nei tessuti periferici e negli organi immunologiche come timo, midollo osseo, milza, linfonodi e placche di Peyer 1-3. DC presenti nel campione dei tessuti periferici dell&#39;organismo per la presenza di antigeni, che occupano, di processo e presente nella loro superficie, nel contesto di molecole di istocompatibilità (MHC). Poi, antigene-caricato DC migrare verso gli organi immunologici quando queste presentano l&#39;antigene ai linfociti T trattati innescare risposte immunitarie specifiche. Un modo per valutare le capacità migratorie di DCS è di etichettarli con coloranti fluorescenti 4. Con la presente ci dimostrano l&#39;utilizzo di nanocristalli Qdot fluorescenti di etichettare derivate dal midollo osseo murino DC. Il vantaggio di questa etichettatura è che Qdot nanocristalli in possesso di stabile e di lunga durata fluorescenza che li rendono ideali per la rilevazione di cellule marcate nei tessuti recuperati. Per fare questo, le prime cellule saranno recuperati dal zucchine osso murini e coltivate per 8 giorni in presenza di granulociti e macrofagi fattore stimolante le colonie al fine di indurre la differenziazione DC. Queste cellule verranno poi etichettati con Qdots fluorescente da breve incubazione in vitro. Cellule colorate possono essere visualizzati con un microscopio a fluorescenza. Le celle possono essere iniettati in animali da laboratorio, a questo punto o può essere in cellule mature al momento in incubazione in vitro con stimoli infiammatori. Nelle nostre mani, la maturazione DC non ha determinato la perdita di segnale fluorescente né colorazione Qdot influire sulle proprietà biologiche dei PVS. Una volta iniettato, queste cellule possono essere identificate in organi immunitario con la microscopia a fluorescenza seguenti dissezione tipici e le procedure di fissaggio.

Watch this Scientific Journal Video about Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies at JoVE.com

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

Antigeni assorbimento delle cellule dendritiche e migrare verso gli organi immunitario di presentare antigeni alle cellule T trattati. Qdot nanocristalli etichettatura fornisce un segnale di lunga durata e stabile fluorescenti. Questo permette di tenere traccia delle cellule dendritiche a diversi organi mediante microscopia a fluorescenza.

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 ...

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Research

JoVE Journal

Immunology and Infection

24.7K Views.  Ohio University. Antigeni assorbimento delle cellule dendritiche e migrare verso gli organi immunitario di presentare antigeni alle cellule T trattati. Qdot nanocristalli etichettatura fornisce un segnale di lunga durata e stabile fluorescenti. Questo permette di tenere traccia delle cellule dendritiche a diversi organi mediante microscopia a fluorescenza.

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

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

Indagine di polarizzazione dei macrofagi Utilizzando Bone Marrow macrofagi derivati

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Ricerca

Immunologia e infezioni

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Derivate dal midollo osseo macrofagi Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Isolamento e iniezione endovenosa di Murine derivate dal midollo osseo monociti

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

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. ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Generazione e identificazione di GM-CSF Derivato alveolo-come macrofagi e dendritiche cellule da midollo osseo di topo

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Generazione di derivate da monociti cellule dendritiche umane da sangue intero

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Generazione di un gran numero di progenitori mieloidi e precursori delle cellule dendritiche dal midollo osseo murino utilizzando una romanzo cella ordinamento strategia

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Rilevamento di attivazione Inflammasome e morte delle cellule Pyroptotic in macrofagi murini derivate da midollo osseo

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

An &lt;em&gt;In Vivo&lt;/em&gt; Mouse Model to Measure Na&amp;#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Un modello di topo In Vivo di attivazione delle cellule T Naïve CD4 di misura, la proliferazione e la differenziazione Th1 indotta dalle cellule dendritiche derivate da midollo osseo

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

Protocollo economico ed efficiente per l'isolamento e la coltura di cellule dendritiche derivate dal midollo osseo da topi

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...