National Institutes of Health Kirchstein Fellowship Promotionsstipendium AI-64128 (MPM), GM-41514 (MDC), GM-48071 (IP)

1) Entfernen Sie beide Oberschenkel Knochen von einer Maus <ol><li> Verwenden Sie Dissektion Schere wegschneiden Muskel-und aussetzen des Oberschenkelknochens oberhalb und unterhalb der Gelenke (Knie und Hüfte). Fassen Sie die Mitte des Oberschenkels mit Dissektion Pinzette und schneiden oberhalb und unterhalb der Gelenke, so viel von der Epiphyse intakt wie möglich zu verlassen. </li><li> Reinigen Sie so viel Muskelmasse wie möglich mit kleinen Dissektion Schere. Übertragen Sie die Oberschenkel in eine Schüssel mit RPMI. </li><li> Alle Verfahren sollten in der Motorhaube durchgeführt werden ab diesem Zeitpunkt mit nur sterile Medien, Instrumente, Pipettenspitzen und Kulturschalen. </li></ol> 2) sterilisieren Femur Knochen: <ol><li> In der Haube, Transfer Knochen aus dem RPMI zu einer kleinen Petrischale mit 70% Ethanol auf Eis gefüllt. </li><li> Lassen Knochen in Ethanol für 5 Minuten auf Eis genießen. </li></ol> 3) Sammeln Knochenmark unter sterilen Bedingungen: <ol><li> Transfer-Knochen zu einer kleinen Petrischale mit sterilfiltriert Primary DC Kulturmedien. </li><li> Mit einer sterilen Pinzette, halten Sie die Knochen über einen verwerfen Schale oder Rohr, und mit einer sterilen Schere, schneiden Sie jeden epiphesis (Enden der Knochen) aus. Der Schnitt sollte aussetzen Knochenmark, die leuchtend rot in der Mitte des Knochens. </li><li> Mit einer sterilen Spritze (26-28 Gauge-Nadel), saugen ca. 100-200 ml sterile Medien. </li><li> Halten Sie die Knochen mit den sterilen Pinzette über einen frischen Teller sterile Medien und die Nadel in die eine Seite des Knochens. Positionieren Sie die Spitze der Nadel in der Nähe der Spitze des Knochens und langsam waschen den Knochen mit dem sterilen Medien. Wenn Sie in der Mitte des Knochens sind, sollte die Nadel schieben in einfach. </li><li> Das Knochenmark wäscht, entweder in kleinen Stücken oder als ein einziges Stück. Es sollte aus dem Knochen und in die Schüssel von sterilen Medien gespült werden. </li><li> Wiederholen Sie diesen Schritt als notwendig, um vollständig zu waschen das Mark aus den Knochen. </li><li> Wenn die Knochen sauber ist, wird es weiß und durchscheinend. </li><li> Wiederholen Sie diesen Vorgang mit den anderen Oberschenkelknochens. Verwerfen jedes Oberschenkelknochens, wenn es sauber ist. </li></ol> 4) Re-suspend und Spin down Knochenmarkzellen: <ol><li> Transfer-Zellen in ein steriles Falcon-Röhrchen. </li><li> Wenn Knochenmark noch intakt ist, sehr sanft Pipette Medien rauf und runter in die Schale aufzubrechen dem Knochenmark in einem einzigen Zellsuspension. Dies kann einige Minuten dauern und sollte langsam erfolgen, um zu vermeiden Abtötung von Zellen durch schiere Kraft. </li><li> Centrifuge Zellen als jedes andere Säugetier-Zellen. </li></ol> 5) Cell Lysis: <ol><li> Entfernen Sie den Schlauch aus der Zentrifuge nach dem Spin ist getan und gießen Medien aus (dekantieren) in einen kleinen Abfall (50 ml Falcon-Röhrchen) in der Motorhaube. </li><li> Pellet durch vorsichtiges Schnippen der Boden des Röhrchens. </li><li> Mit sterilfiltriert Wasser und 10x DPBS oder 10x PBS, eine Wasseranalyse vornehmen Lyse der roten Blutkörperchen (Erythrozyten) mit den folgenden Bänden zu entfernen: <ul><li> 900 ml sterile gefilterte Wasser </li><li> 100 mL 10x DPBS oder 10x PBS </li></ul></li><li> Add 100 ml 10x PBS 5-10 Sekunden nach der Zugabe des Wassers. Es ist sehr wichtig dies sofort, so dass nur die Erythrozyten lysiert sind. Es ist eine gute Idee, haben beide Pipetten gefüllt und fertig. </li><li> Add 5 ml - 10 ml sterile DC basischen Medien. </li></ol> 6) Graf Cells: <ol><li> In der Haube, mischen Sie die 6 bis 11 ml Knochenmark-Zellen durch vorsichtiges Schwenken des Röhrchens mit einer leichten Inversion, eine 45-Grad-Neigung (nicht so, dass die Medien der Spitze der Röhre berührt) </li><li> Nehmen Sie 10 ml Medium in einem sterilen Röhrchen zum Zählen. </li><li> Re-cap die Zellen und Zentrifuge wieder. </li></ol> 7) Plating Dendritische Zellen: <ol><li> Die Zellen sind bei einer Dichte von 1 x 106/ml verchromt werden. </li><li> Remove-Zellen aus der Zentrifuge, dekantieren Medien und resuspendieren Zellen in der entsprechenden Menge an Primary DC Medien für die Beschichtung. </li><li> Legen Sie in Gewebekultur-Inkubator. </li></ol> 8) Kultur Pflege und Reifung: Prüfen Sie stets Kulturen, bevor Sie mehr Medien oder den Einsatz in allen Experimenten. Überprüfen Sie die allgemeine Gesundheit der Zellen und nach möglichen Kontamination. Tag 0: Dendritische Zellen ausplattiert. TAG 3: Entfernen Sie 75% der Medien-und Nicht-adherant Zellen und wieder hinzuzufügen Primary DC Medien. TAG 6: Nach dem ersten Zelle Plating: Re-Platte die Zellen mit dem folgenden Verfahren: <ol><li> Entfernen Sie das Medium, das einige nicht-adherant DCs an dieser Stelle, und in eine sterile 50 ml Falcon-Röhrchen enthalten, sollten Verlassen Platten sehr leicht feucht (Sie wollen nicht, dass die adhärenten Zellen austrocknen) </li><li> Add 3 mM EDTA in PBS zu jeder Platte und lassen Sie sie 5 Minuten lang sitzen. </li><li> Halten Sie die Platte in einem Winkel von 45 Grad und sanft Pipette Medien rauf und runter gegen die Unterseite der Platte vorsichtig lösen nicht adherant Zellen. </li><li> EDTA / PBS sollte dicker Angabe Zellen werden vom Boden der gesammeltenPlatte. </li><li> Nach einigen Minuten dieses kann Zellgemisch auf die 50 ml-Falcon-Röhrchen überführt werden. </li><li> Spin allen Zellen nach der Durchführung dieses Verfahren für jede Platte. </li><li> Graf und die Platte die Zellen bei einer Dichte von 1 x 10 ^ 6 pro Platte in Secondary DC Medien in neue sterile 10 cm Kulturschalen. Zurück Zellen Gewebekultur-Inkubator. </li></ol> Tag 10-11 Reife DCs bereit zur Stimulation / Antigen-Beladung. 9) dendritische Zellen Stimulation: <ol><li> Verwenden LPS (lipopolyscaccharide) in Kombination mit gewünschten Peptids für die Aktivierung und Peptid-Beladung. Abhängig von Ihrer Quelle der LPS, können unterschiedliche Stimulation Bedingungen zu besseren Ergebnissen führen. Darüber hinaus sollten Sie die gewünschte Menge an Peptid für DC-Präsentation optimiert werden. Stimulation Bedingungen in diesem Protokoll wurden 100 ng / mL LPS und 100 ug / mL OVA (Ovalbumin). </li><li> Laden Antigen / mit LPS für 18-24 Stunden vor der Ernte behandeln / zu verwenden. </li></ol> Dendritische Zellen Media: Sterilfilter nach allem, was hinzugefügt wurde Bitte beachten Sie die Diskussion Abschnitt für eine Überprüfung der verschiedenen dendritischen Zellkultur-Bedingungen und für die daraus resultierenden dendritischen Zell-Phänotyp. Diese Kultur Bedingungen sind so konzipiert, um zu reifen dendritischen Zellen produzieren, die rasch nach Hause, um Lymphknoten, 18-24 Stunden nach subkutaner Injektion und präsentieren Antigen effizient. Primary DC Media: Sterilfilter nach allem, was hinzugefügt wurde <ul><li> 500 ml IMDM (entfernen 55-60 ml für 500 ml Endvolumen) </li><li> 50 ml hitzeinaktiviertem FBS </li><li> 5 ml 200 mM L-Gln, Endkonzentration von 2 mM </li><li> 100 IU / mL Penicillin und 100 mg / ml Streptomycin (5 mL von 10.000 IU / mL Pen und 10.000 mg / mL Strep Lager) </li><li> 50 uM B-Me (14,3 M B-Me Lager, verdünnte 1:100 in chemischen Kapuze, benutzen 35 ul pro 100 ml Medium für 50 mikromolaren Konzentration) </li><li> 20-30 ng / mL GM-CSF </li><li> 100-400 IU / mL IL-4 etwa 10 bis 40 ng / mL </li></ul> SecondaryDC Medien für die Reifung * 100 ng / mL TNF-alpha (add to Primary DC Medien) * Hinweis: GM-CSF + IL-4 sollten unreifen dendritischen Zellen zu produzieren. Mit GM-CSF + IL-4 (400 IU / ul) + TNF-alpha (100 ug / ml) erstellt Zellen, die reifen Monozyten abgeleiteten dendritischen Zellen ähneln. Reifen dendritischen Zellen sind bekanntermaßen nicht nehmen Antigen so effizient wie unreife dendritische Zellen und Kulturen endogenen TNF-a veröffentlicht von Stromazellen 4 haben kann.

<ol>
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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. 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Dendritische Zellen sind wichtige Mediatoren der adaptiven Immunantwort und die effizientesten Antigen-präsentierenden Zelle charakterisiert bis heute. Methoden für die Human-und Maus dendritischen Zellkultur in der Literatur in der Art der Zytokine verwendet werden, um die Entwicklung der verschiedenen dendritischen Zelltypen Einfluss abweichen. Bemerkenswert ist, dass GM-CSF, flt3L, IL4, IL13, TNF-alpha und IFN-gamma in verschiedenen Kombinationen eingesetzt, um zu reifen, unreifen, Entzündungs-und Steady-State-wie murine Knochenmark a...

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

Mehrere Methoden für die Herstellung von murinen dendritischen Zellen sind in der Literatur gefunden werden. Hier präsentieren wir eine Methode, die größer als 85% CD11c hoch dendritischen Zellen produziert in Kultur, die Heimat der Lymphknoten nach subkutaner Injektion und präsentieren Antigen Antigen-spezifischen T-Zellen (siehe Video). Darüber hinaus verwenden wir Essen Instruments Incucyte zu dendritischen Zell-Reifung, wo am Tag 10, ist die Morphologie der kultivierten Zellen typisch für einen reifen dendritischen Zellen und &lt;85% der Zellen sind CD11chigh verfolgen. Das Studium der Antigen-Präsentation in peripheren Lymphknoten durch 2-Photonen-Imaging ergab, dass es drei verschiedene Phasen der dendritischen Zellen und T-Zell-Interaktion 1, 2. Phase I besteht aus kurzen serielle Kontakte zwischen hoch bewegliche Antigen-spezifischen T-Zellen und Antigen tragen dendritische Zellen 1, 2. Phase zwei ist bei längerer Kontakte zwischen Antigen-spezifischen T-Zell-und Antigen-tragende dendritische Zellen 1, 2 markiert. Schließlich ist die Phase III von T-Zellen Ablösen von dendritischen Zellen, Wiedererlangung Motilität und Beginn auf 1, 2 teilen aus. Dies ist ein Beispiel für die Art von Antigen-spezifischen Wechselwirkungen, die durch Zwei-Photonen-Imaging von Antigen-beladenen Zellen tracker Farbstoff markierten dendritischen Zellen analysiert werden können.

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

Antigen-Präsentation in sekundären lymphatischen Organen durch dendritische Zellen ist entscheidend für die Einleitung des T-Zell-vermittelten adaptive Immunantwort. Hier zeigen wir die Kultur von Knochenmark abgeleiteten dendritischen Zellen, Aktivierung und Etikettierung für die 2-Photonen-Imaging.

Generierung von Knochenmark murinen dendritischen Zellen für den Einsatz in 2-Photonen-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% ...

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

Research

JoVE Journal

Biology

31.0K Aufrufe.  University of California, Irvine (UCI). Antigen-Präsentation in sekundären lymphatischen Organen durch dendritische Zellen ist entscheidend für die Einleitung des T-Zell-vermittelten adaptive Immunantwort. Hier zeigen wir die Kultur von Knochenmark abgeleiteten dendritischen Zellen, Aktivierung und Etikettierung für die 2-Photonen-Imaging.

Serie: 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 und 2-Photonen-Imaging der peripheren Lymphknoten in Mäusen

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

Forschung

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.

In situ Imaging der Maus Thymus mit 2-Photonen-Mikroskopie

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.

Eine Methode zur 2-Photonen-Imaging der Blutfluss in den Neocortex durch ein Cranial Fenster

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 Calcium Imaging in Layer 2 / 3 der Mäuse

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.

Kultur myeloide dendritische Zellen aus dem Knochenmark Vorstufen

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.

Die Herstellung von primären hämatopoetischen Zellkulturen aus Knochenmark der Maus für die Elektroporation

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 von hämatopoetischen Zellen im Knochenmark

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-4-Dimensional-Tracking von hämatopoetischen Stamm-und Vorläuferzellen im erwachsenen Maus Schädeldachknochenmark

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.

Osteoklasten Ableitung aus Knochenmark der Maus

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.

Ausdruck von fluoreszierenden Fusionsproteinen in murinen Knochenmark gewonnenen dendritischen Zellen und Makrophagen

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