National Institutes of Health Kirchstein bourse bourse prédoctorale AI-64128 (MPM), le GM-41514 (MDC), le GM-48071 (IP)

1) Retirer les deux os du fémur d&#39;une souris <ol><li> Utilisez des ciseaux pour couper la dissection musculaire et exposent l&#39;os du fémur dessus et en dessous des articulations (genou et hanche). Saisir le centre du fémur avec des pincettes dissection et coupe au-dessus et en dessous des joints de laisser autant de l&#39;épiphyse intacte que possible. </li><li> Nettoyez le muscle le plus possible en utilisant des ciseaux de dissection petits. Transfert du fémur dans un plat de RPMI. </li><li> Toutes les procédures doivent être effectuées dans la hotte partir de ce point, en utilisant uniquement les supports stériles, les instruments, embouts de pipette et des boîtes de culture. </li></ol> 2) Stériliser les os du fémur: <ol><li> Dans la hotte, le transfert des os du RPMI à une boîte de culture de petits remplis avec de l&#39;éthanol à 70% sur la glace. </li><li> Laisser tremper les os dans de l&#39;éthanol pendant 5 minutes sur la glace. </li></ol> 3) Recueillir de moelle osseuse dans des conditions stériles: <ol><li> Transfert des os dans un plat contenant de la culture de petites filtré stérile primaire des milieux de culture DC. </li><li> Avec pinces stériles, maintenez l&#39;os sur un plat jeter ou tube, et avec des ciseaux stériles, couper chaque epiphesis (extrémités de l&#39;os) hors tension. La coupe doit exposer la moelle osseuse qui est rouge vif dans le centre de l&#39;os. </li><li> Avec une seringue stérile (26-28 aiguille de la jauge), sucer jusqu&#39;à environ 100-200 ml de milieu stérile. </li><li> Tenir l&#39;os avec la pince à épiler stérile sur un plat frais de milieux stériles et insérer l&#39;aiguille dans un côté de l&#39;os. Placez le bout de l&#39;aiguille vers le haut de l&#39;os et l&#39;os lentement laver avec les médias stérile. Si vous êtes dans le centre de l&#39;os, l&#39;aiguille doit glisser facilement. </li><li> La moelle osseuse délave, soit en petits morceaux ou en une seule pièce. Il devrait être évacuée de l&#39;os et dans le plat de milieux stériles. </li><li> Répétez cette étape que nécessaire pour laver complètement la moelle de l&#39;os. </li><li> Lorsque l&#39;os est propre, il sera blanc et translucide. </li><li> Répétez cette procédure avec l&#39;os du fémur d&#39;autres. Jeter chaque fois le fémur, il est propre. </li></ol> 4) Re-suspendre et centrifuger les cellules de moelle osseuse: <ol><li> Transfert des cellules dans un tube stérile faucon. </li><li> Si la moelle est encore intact, très doucement la pipette des médias de haut en bas dans le plat, pour briser la moelle dans une suspension cellulaire unique. Cela peut prendre plusieurs minutes et doit être fait lentement pour éviter de tuer les cellules par la force. </li><li> Centrifuger les cellules comme tout les autres cellules de mammifères. </li></ol> Lyse cellulaire 5): <ol><li> Retirer le tube de la centrifugeuse après le spin est faite et verser les médias off (décanter) dans un petits déchets (50 ml tube Falcon) dans la hotte. </li><li> Resuspendre le culot par un léger effleurant le fond du tube. </li><li> L&#39;utilisation de l&#39;eau stérile filtrée et DPBS 10x ou 10x PBS, réaliser une lyse de l&#39;eau pour enlever les globules rouges (hématies) en utilisant les volumes suivants: <ul><li> 900 ml d&#39;eau stérile filtrée </li><li> 100 ml DPBS 10x ou 10x PBS </li></ul></li><li> Ajouter 100 ml de PBS seconde 10x 5-10 après addition de l&#39;eau. Il est très important ce faire immédiatement afin que seuls les globules rouges sont lysés. C&#39;est une bonne idée d&#39;avoir deux pipettes rempli et prêt. </li><li> Ajouter 5 ml - 10 ml de milieux stériles cc de base. </li></ol> 6) Nombre de cellules: <ol><li> Dans la hotte, mélanger le ml 6 à 11 de cellules de moelle osseuse en remuant doucement le tube avec une légère inversion, une inclinaison de 45 degrés (non pas pour que les médias touche le haut du tube) </li><li> Retirer 10 ml de milieu dans un tube stérile pour le comptage. </li><li> Re-bouchon et centrifuger les cellules à nouveau. </li></ol> 7) Les cellules dendritiques Placage: <ol><li> Les cellules doivent être étalées à une densité de 1 x 106/ml. </li><li> Retirer les cellules d&#39;une centrifugeuse, des médias décanter et remettre en suspension les cellules dans la quantité appropriée de Media DC primaire pour le placage. </li><li> Placez dans le tissu de la culture incubateur. </li></ol> 8) Soins de la Culture et de la maturation: Vérifiez toujours les cultures avant d&#39;ajouter plus de médias ou de l&#39;utilisation de toutes les expériences. Vérifiez l&#39;état de santé générale des cellules et regarder de la contamination potentielle. Jour 0: Les cellules dendritiques plaqué. JOUR 3: Suppression de 75% des médias et non adherant cellules et rajouter primaire média DC. JOUR 6: Après le placage cellule initiale: Re-plaque de cellules en utilisant la procédure suivante: <ol><li> Retirez le support, qui devrait contenir quelques PED non adherant à ce point, et les placer dans un tube stérile Falcon de 50 ml, en laissant des plaques très légèrement humide (vous ne voulez pas les cellules adhérentes à sécher) </li><li> Ajouter 3 mM EDTA dans PBS dans chaque assiette et laisser reposer pendant 5 minutes. </li><li> Tenez la plaque à un angle de 45 degrés et délicatement la pipette des médias de haut en bas contre le fond de la plaque en douceur déloger les cellules non adherant. </li><li> EDTA / PBS devraient devenir plus épaisses cellules sont recueillies en indiquant le bas de laassiette. </li><li> Après plusieurs minutes de cela, mélange de cellules peuvent être transférées sur le tube Falcon de 50 ml. </li><li> Spin toutes les cellules après avoir effectué cette procédure pour chaque plaque. </li><li> Comte et de la plaque des cellules à une densité de 1 x 10 ^ 6 plaque par des médias DC secondaire dans de nouvelles boîtes de culture stérile de 10 cm. Retour aux cellules de culture de tissus incubateur. </li></ol> JOUR 10-11 PED Mature prête pour une stimulation / antigène de chargement. 9) la stimulation des cellules dendritiques: <ol><li> Utilisez le LPS (lipopolyscaccharide) en combinaison avec le peptide désiré pour l&#39;activation et le chargement des peptides. En fonction de votre source de LPS, les conditions de stimulation différents peut donner de meilleurs résultats. En outre, la quantité désirée de peptide pour la présentation DC doit être optimisée. Conditions de stimulation dans ce protocole ont été de 100 ng / mL et LPS 100 ug / ml OVA (ovalbumine). </li><li> Antigènes de charge / traiter avec le LPS pendant 18-24 heures avant la récolte / utilisation. </li></ol> Médias cellules dendritiques: filtre stérile après que tout a été ajouté S&#39;il vous plaît voir la section de discussion pour un examen des différentes conditions de culture cellulaire dendritique et pour le phénotype des cellules dendritiques qui en résulte. Ces conditions de culture sont conçus pour produire des cellules dendritiques matures qui a rapidement chez les ganglions lymphatiques drainant, 18-24 heures après injection sous-cutanée et l&#39;antigène présent efficace. Primaire DC médias: filtre stérile après que tout a été ajouté <ul><li> 500 ml IMDM (enlever 55 à 60 ml pour un volume final de 500 ml) </li><li> 50 ml de FBS inactivé de la chaleur </li><li> 5 ml de 200 mM de L-Gln, la concentration finale de 2 mM </li><li> 100 UI / ml de pénicilline et 100 mg / mL de streptomycine (5 ml de 10.000 UI / mL Pen et 10 000 mg / mL de stock STREP) </li><li> 50 uM B-Me (14,3 M B-moi de stock, diluer 1:100 dans hotte chimique, utiliser 35 ul par 100 ml de milieu pendant 50 concentration micromolaire) </li><li> 20-30 ng / ml de GM-CSF </li><li> 100-400 UI / mL d&#39;IL-4 d&#39;environ 10 à 40 ng / ml </li></ul> Les médias SecondaryDC pour la maturation * 100 ng / ml de TNF-alpha (ajouter à DC primaire médias) * Note: le GM-CSF + IL-4 devrait produire des cellules dendritiques immatures. En utilisant le GM-CSF + IL4 (400 UI / ul) + TNF-alpha (100 ug / ml) va créer des cellules qui ressemblent à maturité des cellules dendritiques dérivées de monocytes. Cellules dendritiques matures sont connues pour ne pas prendre les antigènes de manière aussi efficace que les cellules dendritiques immatures et des cultures endogènes peuvent avoir le TNF-a libéré par les cellules stromales 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>

Les cellules dendritiques sont des médiateurs clés de la réponse immunitaire adaptative et la cellule d&#39;antigènes les plus efficaces de présenter caractérisé à ce jour. Méthodes pour l&#39;homme et la culture de cellules dendritiques de souris dans la littérature diffèrent par le type de cytokines utilisées pour influencer le développement de différents types de cellules dendritiques. Notamment, le GM-CSF, flt3L, IL4, IL13, le TNF-alpha et IFN-gamma sont utilisés dans différentes combinaisons pour produire matures, immat...

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

Plusieurs méthodes pour la préparation de cellules dendritiques murines peuvent être trouvés dans la littérature. Ici, nous présentons une méthode qui produit plus de 85% CD11c haute cellules dendritiques en culture que chez les ganglions lymphatiques drainant, après injection sous-cutanée et l&#39;antigène présent à cellules T spécifiques d&#39;antigènes (voir vidéo). De plus, nous utilisons des instruments Essen Incucyte pour suivre la maturation des cellules dendritiques, où, au jour 10, la morphologie des cellules cultivées est typique d&#39;une cellule dendritiques matures et &lt;85% des cellules sont CD11chigh. L&#39;étude de la présentation des antigènes dans les ganglions lymphatiques périphériques par l&#39;imagerie 2-photons ont révélé qu&#39;il existe trois phases distinctes de cellules dendritiques et l&#39;interaction des cellules T 1, 2. La phase I consiste à brève série de contacts entre les cellules antigènes très mobiles T spécifiques d&#39;antigènes et de la réalisation des cellules dendritiques 1, 2. La deuxième phase est marquée par des contacts prolongés entre l&#39;antigène des cellules T spécifiques d&#39;antigènes et des cellules dendritiques portant 1, 2. Enfin, la phase III est caractérisé par des cellules T détachant les cellules dendritiques, retrouver la motricité et le début de diviser 1, 2. Ceci est un exemple du type d&#39;antigène spécifique des interactions qui peuvent être analysés par imagerie à deux photons de l&#39;antigène de cellule-chargé Tracker colorant étiquetés cellules dendritiques.

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

Présentation de l&#39;antigène dans les organes lymphoïdes secondaires par des cellules dendritiques est crucial pour l&#39;initiation de la cellule T médiée réponse immunitaire adaptative. Ici nous démontrons la culture de la moelle osseuse des cellules dendritiques dérivées de souris, l&#39;activation et d&#39;étiquetage pour les deux photons d&#39;imagerie.

Génération de la moelle osseuse des cellules dendritiques dérivées murin pour l&#39;utilisation dans l&#39;imagerie 2-photons

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% ...

generation-bone-marrow-derived-murine-dendritic-cells-for-use-2

Research

JoVE Journal

Biology

31.0K vues.  University of California, Irvine (UCI). Présentation de l'antigène dans les organes lymphoïdes secondaires par des cellules dendritiques est crucial pour l'initiation de la cellule T médiée réponse immunitaire adaptative. Ici nous démontrons la culture de la moelle osseuse des cellules dendritiques dérivées de souris, l'activation et d'étiquetage pour les deux photons d'imagerie.

Vidéo: 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.

Dissection et 2-Photon Imaging des ganglions lymphatiques périphériques chez les souris

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

Recherche

Biologie

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.

Dans l&#39;imagerie in situ de la souris en utilisant deux Thymus microscopie biphotonique

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.

Une méthode pour le 2-Photon Imaging du flux sanguin dans le néocortex travers une fenêtre crânienne

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 calcium dans la couche 2 / 3 de souris

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.

Culture de cellules dendritiques myéloïdes à partir de précurseurs de moelle osseuse

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 préparation des cultures primaires de cellules hématopoïétiques de la moelle osseuse murine pour l&#39;électroporation

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 des cellules hématopoïétiques de la moelle osseuse

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 suivi 4-Dimensional de hématopoïétiques cellules souches et progénitrices adultes dans la voûte du crâne de souris de moelle osseuse

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.

Ostéoclastes Dérivation de moelle osseuse de souris

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.

Expression des protéines de Fusion Fluorescent en Murine dérivés de la moelle osseuse des cellules dendritiques et 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 ...