National Institutes of comunione Salute Kirchstein Fellowship predoctoral AI-64128 (MPM), GM-41514 (MDC), GM-48071 (PI)

1) Rimuovere entrambe le ossa di femore da un topo <ol><li> Usare le forbici per tagliare via la dissezione dei muscoli ed esporre l&#39;osso del femore sopra e sotto le articolazioni (ginocchio e anca). Afferrare il centro del femore con una pinzetta dissezione e taglio sopra e sotto le articolazioni di lasciare il più delle epifisi intatto possibile. </li><li> Pulire i muscoli il più possibile con le forbici la dissezione di piccole dimensioni. Trasferire il femore in un piatto di RPMI. </li><li> Tutte le procedure devono essere effettuate nel cappuccio da questo punto in poi, utilizzando solo mezzi sterili, strumentazione, pipette e piatti della cultura. </li></ol> 2) Sterilizzare le ossa del femore: <ol><li> Nel cofano, il trasferimento delle ossa dal RPMI a un piatto pieno di cultura piccolo etanolo al 70% su ghiaccio. </li><li> Lasciare a bagno le ossa in etanolo per 5 minuti in ghiaccio. </li></ol> 3) Raccogliere midollo osseo in condizioni sterili: <ol><li> Trasferire le ossa di un piatto cultura piccolo contenente sterile filtrata primaria terreni di coltura DC. </li><li> Con una pinzetta sterile, tenere l&#39;osso su un piatto scartare o un tubo, e con le forbici sterili, tagliare ogni epiphesis (le estremità delle ossa) al largo. Il taglio dovrebbe esporre il midollo osseo, che è rosso brillante al centro dell&#39;osso. </li><li> Con una siringa sterile (26-28 gauge), aspirare circa 100-200 ml di media sterile. </li><li> Tenere l&#39;osso con la pinzetta sterile su un piatto fresco di media sterile e inserire l&#39;ago in un lato del tessuto osseo. Posizionare la punta dell&#39;ago vicino alla parte superiore delle ossa e lentamente lavare l&#39;osso con i media sterile. Se si è al centro delle ossa, l&#39;ago deve scorrere con facilità. </li><li> Il midollo osseo sbiadisce, sia in piccoli pezzi o come un pezzo unico. Va lavata delle ossa e nel piatto dei media sterile. </li><li> Ripetere questo passaggio come necessario per lavare completamente il midollo delle ossa. </li><li> Quando l&#39;osso è pulito, sarà bianca e trasparente. </li><li> Ripetete questa procedura con l&#39;osso del femore altri. Eliminare ogni osso del femore, una volta che sia pulito. </li></ol> 4) Re-Spin sospendere e le cellule di midollo osseo: <ol><li> Trasferire le cellule in una provetta sterile falco. </li><li> Se il midollo è ancora intatto, molto delicatamente pipetta multimediali su e giù nel piatto, per rompere il midollo in una sospensione di cellule singole. Questa operazione potrebbe richiedere alcuni minuti e dovrebbe essere fatto lentamente per evitare di uccidere le cellule con la forza pura. </li><li> Cellule centrifuga come qualsiasi altre cellule di mammifero. </li></ol> 5) cellule Lysis: <ol><li> Rimuovere il tubo dalla centrifuga dopo lo spin è fatto e versare mezzi di comunicazione off (decantare) in un piccoli rifiuti (50 ml di tubo di falco) nel cappuccio. </li><li> Risospendere il pellet con delicatezza sfogliando il fondo della provetta. </li><li> Usando acqua sterile filtrata e DPBS 10x o 10x PBS, effettuare una lisi acqua per rimuovere i globuli rossi (RBC), utilizzando i seguenti volumi: <ul><li> 900 ml di acqua sterile filtrata </li><li> 100 ml DPBS 10x o 10x PBS </li></ul></li><li> Aggiungere 100 ml di PBS 10x secondo 50-10 dopo aver aggiunto l&#39;acqua. E &#39;molto importante fare questo immediatamente in modo che solo i globuli rossi vengono lisati. E &#39;una buona idea avere entrambe le pipette pieno e pronto. </li><li> Aggiungere 5 ml - 10 ml di sterili DC supporti di base. </li></ol> 6) Conteggio di celle: <ol><li> Nel cofano, mescolare 6 al 11 mL di cellule del midollo osseo rimestando delicatamente il tubo con una leggera inversione, una inclinazione di 45 gradi (non così che i media tocca la parte superiore del tubo) </li><li> Rimuovere 10 ml di media in una provetta sterile per il conteggio. </li><li> Richiudere le cellule e centrifugare di nuovo. </li></ol> 7) Placcatura cellule dendritiche: <ol><li> Le cellule devono essere placcato ad una densità di 1 x 106/ml. </li><li> Rimuovere le cellule da centrifuga, media decantare e risospendere le cellule in quantità appropriata di primaria media DC per la placcatura. </li><li> Luogo nel tessuto della cultura incubatore. </li></ol> 8) Cura Cultura e Maturazione: Controllare sempre le culture prima di aggiungere altri mezzi di comunicazione o utilizzando in qualsiasi esperimenti. Controllare la salute generale delle cellule e cercare potenziale contaminazione. GIORNO 0: le cellule dendritiche placcato. GIORNO 3: Rimuovere il 75% dei media e non adherant celle e aggiungere di nuovo primario dei media DC. 6 ° GIORNO: Dopo Placcatura cella iniziale: Re-plate le cellule con la seguente procedura: <ol><li> Rimuovere i supporti, che dovrebbe contenere alcuni non-adherant DC a questo punto, e mettere in un tubo da 50 ml sterile falco, lasciando le piastre leggermente umido (non volete le cellule aderenti ad asciugare) </li><li> Aggiungere 3 mM EDTA in soluzione salina in ogni piatto e lasciar riposare per 5 minuti. </li><li> Tenere il piatto in un angolo di 45 gradi e delicatamente pipetta dei media su e giù contro il fondo del piatto per rimuovere delicatamente non adherant cellule. </li><li> EDTA / PBS deve diventare più spessa cellule che indicano vengono raccolti dal fondo delpiatto. </li><li> Dopo alcuni minuti di questo, miscela di cellule possono essere trasferiti al tubo 50 ml falco. </li><li> Spin tutte le cellule dopo aver eseguito questa procedura per ogni piatto. </li><li> Conte e la piastra le cellule ad una densità di 1 x 10 ^ 6 per ogni piastra in media secondaria DC in nuovi piatti sterile 10 centimetri cultura. Ritorno alle cellule di tessuto cultura incubatrice. </li></ol> DAY 10-11 DC maturo pronto per la stimolazione / antigene di carico. 9) La stimolazione delle cellule dendritiche: <ol><li> Usa LPS (lipopolyscaccharide) in combinazione con il peptide desiderato per l&#39;attivazione e il caricamento del peptide. A seconda della fonte di LPS, diverse condizioni di stimolazione può dare risultati migliori. Inoltre, la quantità desiderata di peptidi per la presentazione DC devono essere ottimizzate. Condizioni di stimolo in questo protocollo sono stati 100 ng / ml LPS e 100 ug / mL OVA (ovoalbumina). </li><li> Antigene di carico / trattare con LPS per 18-24 ore prima del raccolto / uso. </li></ol> Media dendritiche Cell: filtro sterile dopo tutto è stata aggiunta Si prega di consultare la sezione di discussione per una revisione delle condizioni delle cellule dendritiche diverse culture e per il fenotipo risultante delle cellule dendritiche. Queste condizioni di coltura sono stati progettati per produrre mature cellule dendritiche che a casa rapidamente ai linfonodi drenanti, 18-24 ore dopo l&#39;iniezione sottocutanea e l&#39;antigene presente in modo efficiente. Primaria DC Media: filtro sterile dopo tutto è stata aggiunta <ul><li> 500 ml IMDM (togliere 55-60 ml per 500mL volume finale) </li><li> 50 ml di calore FBS inattivato </li><li> 5 ml di 200 mM di L-Gln, concentrazione finale di 2 mM </li><li> 100 UI / ml di penicillina e 100 mg / mL di streptomicina (5 ml di 10.000 UI / ml Pen e 10.000 mg / mL magazzino Strep) </li><li> 50 uM B-Me (14,3 M B-Me magazzino, diluire 1:100 in cappa chimica, utilizzare 35 ul per 100 ml per 50 mezzi Dosi micromolari) </li><li> 20-30 ng / mL GM-CSF </li><li> 100-400 UI / mL di IL-4 di circa 10-40 ng / mL </li></ul> Mezzi di comunicazione per SecondaryDC * maturazione 100 ng / mL TNF-alfa (aggiungere alla primaria DC media) * Nota: GM-CSF + IL-4 dovrebbe produrre cellule dendritiche immature. Utilizzando GM-CSF + IL4 (400 UI / ul) + TNF-alfa (100 ug / ml) creerà le cellule che assomigliano mature monociti cellule dendritiche derivate. Mature cellule dendritiche sono noti per non prendere l&#39;antigene nel modo più efficiente le cellule dendritiche immature e le culture possono essere endogeni TNF-alfa rilasciata dalle cellule stromali 4.

<ol>
	<li>Miller, M. J., Safrina, O., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+single+cell+dynamics+of+CD4++T+cell+activation+by+dendritic+cells+in+lymph+nodes.">Imaging the single cell dynamics of CD4+ T cell activation by dendritic cells in lymph nodes.</a> J Exp Med. 200, 847-856 (2004).</li><li>Henrickson, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+sensing+of+antigen+dose+governs+interactive+behavior+with+dendritic+cells+and+sets+a+threshold+for+T+cell+activation.">T cell sensing of antigen dose governs interactive behavior with dendritic cells and sets a threshold for T cell activation.</a> Nat Immunol. 9, 282-291 (2008).</li><li>Jefford, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+comparison+of+DCs+generated+in+vivo+with+Flt3+ligand+or+in+vitro+from+blood+monocytes:+differential+regulation+of+function+by+specific+classes+of+physiologic+stimuli.">Functional comparison of DCs generated in vivo with Flt3 ligand or in vitro from blood monocytes: differential regulation of function by specific classes of physiologic stimuli.</a> Blood. 102, 1753-1763 (2003).</li><li>Inaba, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+large+numbers+of+dendritic+cells+from+mouse+bone+marrow+cultures+supplemented+with+granulocyte/macrophage+colony-stimulating+factor.">Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.</a> J Exp Med. 176, 1693-1702 (1992).</li><li>Lutz, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+functions+of+IL-4+receptor+types+I+and+II+for+dendritic+cell+maturation+and+IL-12+production+and+their+dependency+on+GM-CSF.">Differential functions of IL-4 receptor types I and II for dendritic cell maturation and IL-12 production and their dependency on GM-CSF.</a> J Immunol. 169, 3574-3580 (2002).</li><li>Xu, Y., Zhan, Y., Lew, A. M., Naik, S. H., Kershaw, M. 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+development+of+murine+dendritic+cells+by+GM-CSF+versus+Flt3+ligand+has+implications+for+inflammation+and+trafficking.">Differential development of murine dendritic cells by GM-CSF versus Flt3 ligand has implications for inflammation and trafficking.</a> J Immunol. 179, 7577-7584 (2007).</li><li>Inaba, K., Swiggard, W. J., Steinman, R. M., Romani, N., Schuler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+dendritic+cells.">Isolation of dendritic cells.</a> Curr Protoc Immunol. 3, (2001).</li><li>Winzler, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+stages+of+mouse+dendritic+cells+in+growth+factor-dependent+long-term+cultures.">Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures.</a> J Exp Med. 185, 317-328 (1997).</li><li>Koch, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis+factor+alpha+maintains+the+viability+of+murine+epidermal+Langerhans+cells+in+culture,+but+in+contrast+to+granulocyte/macrophage+colony-stimulating+factor,+without+inducing+their+functional+maturation.">Tumor necrosis factor alpha maintains the viability of murine epidermal Langerhans cells in culture, but in contrast to granulocyte/macrophage colony-stimulating factor, without inducing their functional maturation.</a> J Exp Med. 171, 159-171 (1990).</li><li>Miller, M. J., Hejazi, A. S., Wei, S. H., Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+repertoire+scanning+is+promoted+by+dynamic+dendritic+cell+behavior+and+random+T+cell+motility+in+the+lymph+node.">T cell repertoire scanning is promoted by dynamic dendritic cell behavior and random T cell motility in the lymph node.</a> Proc Natl Acad Sci U S A. 101, 998-1003 (2004).</li><li>Wei, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++signals+in+CD4++T+cells+during+early+contacts+with+antigen-bearing+dendritic+cells+in+lymph+node.">Ca2+ signals in CD4+ T cells during early contacts with antigen-bearing dendritic cells in lymph node.</a> J Immunol. 179, 1586-1594 (2007).</li><li>Castellino, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+enhance+immunity+by+guiding+naive+CD8++T+cells+to+sites+of+CD4++T+cell-dendritic+cell+interaction.">Chemokines enhance immunity by guiding naive CD8+ T cells to sites of CD4+ T cell-dendritic cell interaction.</a> Nature. 440, 890-895 (2006).</li></ol>

Le cellule dendritiche sono mediatori chiave della risposta immunitaria e la cellula che presenta l&#39;antigene più efficiente caratterizzato fino ad oggi. Metodi per l&#39;uomo e topo di cellule dendritiche cultura in letteratura si differenziano per il tipo di citochine usato per influenzare lo sviluppo di diversi tipi di cellule dendritiche. In particolare, GM-CSF, flt3L, IL4, IL13, TNF-alfa e IFN-gamma sono utilizzati in diverse combinazioni per produrre maturo, immaturo, infiammatorie e lo stato stazionario come il midollo osseo muri...

generation of bone marrow derived murine dendritic cells for use in 2-photon imaging

Diversi metodi per la preparazione di cellule dendritiche murine possono essere trovati in letteratura. Qui, vi presentiamo un metodo che produce più del 85% CD11c cellule dendritiche in alta cultura che ospita il linfonodo drenante dopo iniezione sottocutanea e antigene presente a cellule T antigene specifico (vedi video). Inoltre, utilizziamo strumenti Essen Incucyte per monitorare la maturazione delle cellule dendritiche, dove, al giorno 10, la morfologia delle cellule in coltura è tipico di una cellula matura dendritiche e &lt;85% delle cellule sono CD11chigh. Lo studio di presentazione dell&#39;antigene nei linfonodi periferici da 2-fotone di imaging ha rivelato che ci sono tre fasi distinte di cellule dendritiche e l&#39;interazione delle cellule T 1, 2. Fase I è costituito da brevi contatti seriale tra altamente mobili cellule T antigene specifico antigene e portando le cellule dendritiche 1, 2. La fase due è segnato da contatto prolungato tra antigene-specifica delle cellule T antigene e cuscinetti cellule dendritiche 1, 2. Infine, di fase III è caratterizzata da cellule T distacco dalle cellule dendritiche, recuperare la motilità e l&#39;inizio di dividere 1, 2. Questo è un esempio del tipo di antigene-specifiche interazioni che possono essere analizzati da due fotoni di imaging di antigene-caricati celle dye-inseguitore etichettati cellule dendritiche.

Watch this Scientific Journal Video about Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging at JoVE.com

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Presentazione dell&#39;antigene in organi linfatici secondari da parte delle cellule dendritiche è cruciale per l&#39;avvio della cellula T mediata risposta immunitaria adattativa. Qui mostriamo la cultura del midollo osseo derivate le cellule dendritiche murine, di attivazione e di etichettatura per 2-fotoni imaging.

Generazione di cellule derivate dal midollo osseo dendritiche murini per l&#39;uso in 2-fotone Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

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Research

JoVE Journal

Biology

31.1K Views.  University of California, Irvine (UCI). Presentazione dell'antigene in organi linfatici secondari da parte delle cellule dendritiche è cruciale per l'avvio della cellula T mediata risposta immunitaria adattativa. Qui mostriamo la cultura del midollo osseo derivate le cellule dendritiche murine, di attivazione e di etichettatura per 2-fotoni imaging.

Video: Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response 1. Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 2. Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37&deg;C.

Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.

Dissezione e 2-Photon Imaging di linfonodi periferici nei topi

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the ...

Ricerca

Biologia

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37&deg;C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

Imaging in situ del Timo mouse utilizzando 2-Photon Microscopia

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development.  To follow the ...

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

Un metodo per 2-Photon Imaging del flusso sanguigno nella neocorteccia attraverso una finestra del cranio

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

In Vivo 2-Photon Imaging di calcio a livello 2 / 3 dei topi

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Cultura di cellule dendritiche mieloidi dai precursori del midollo osseo

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

La preparazione di colture primarie di cellule emopoietiche dal midollo osseo murino per elettroporazione

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

Homing delle cellule ematopoietiche del midollo osseo

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Osteoclasti derivazione dal midollo osseo di topo

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Espressione di proteine di fusione fluorescenti in macrofagi e cellule dendritiche derivate da midollo osseo Murine

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...