Dieses Protokoll wurde getestet und optimiert im Labor von Dr. David Scadden. Die Finanzierung erfolgt durch das Dana Farber Cancer Institute / Massachusetts General Hospital Clinical Hämatologie und Onkologie Fellowship Program zur Verfügung gestellt.

<ol><li> Wir beginnen die Homing Experiment durch Extrahieren von Zellen im Experiment verwendet werden. Aus diesem aktuellen Experiment, das wir wollen wissen, interessiert, wenn die Referenzfahrt der gesamte Knochenmark zu den Nischen in den langen Knochen des Empfängers Tieren unterscheidet sich für Knochenmark-Zellen aus WT Tieren wie C57BL/6J (WT) extrahiert und jene, die transgene sind Vorprägungen für Lysophosphatidsäure-Rezeptor 1 (LPAR1) (KO). </li><li> Bevor wir den Versuch starten, die wir sammeln die Materialien, die wir für dieses Experiment. In unserem Labor alle Maus-Sektionen sind in einer laminaren Strömung Gewebekultur Kapuze trug vor allem, wenn die Zellen gehen, um in einen Empfänger Tier statt für ex-vivo-Analyse verwendet transplantiert werden. Wir beginnen damit, eine blaue Folie im Inneren der Kapuze. Andere Objekte benötigt eine Pinzette und eine scharfe Schere, die autoklaviert worden sind, eine sterile Einweg-Skalpell und Alkoholtupfer. Wir modifizierten PBS und eine sterile 6-Well Platte, ein Mörser müssen, gekrönt Stößel, 50cc blau konischen Rohr und einem 70um Einweg-Filter. </li><li> Wir beginnen mit der Einnahme von einer WT und einer KO-Maus. </li><li> Diese Mäuse werden durch CO 2-Inhalation getötet und dann tauchten in 70% für 5 Sekunden isopropranolol. Dieser Schritt verhindert die Kontamination der Zellen von Interesse mit der Maus Fell und in unseren Händen hat zu keinen Kompromiss in einer Ausbeute von Zellen oder experimentellen Ergebnissen geführt. </li><li> Die Mäuse werden dann wiederum mit Hilfe einer Pinzette und einer scharfen Schere durchtrennt. Wir arbeiten normalerweise mit dem WT-Maus als eine Frage der Routine zu starten. Ein kleiner Schnitt in der Bauchhaut der Maus über dem Bauch und die Haut wird dann gestreckt mit den Händen gemacht. Die Haut löst sich die Hinterbeine leicht. Die Ober-und Unterschenkels werden dann extrahiert dabei nicht auf den Kopf des Oberschenkelknochens zu verdrängen, da dies eine große Menge von Knochenmark. Die oberen Extremitäten sind aus dem Tier den Oberkörper durch die einfache Anwendung Traktion, um sie unter Beibehaltung der Torso immobile abgelöst. Dies entfernt die oberen Gliedmaßen in einem Stück. Die humeri werden dann von den oberen Gliedmaßen extrahiert. </li><li> Die entnommenen Knochen sind in modifizierter PBS gelegt, die auf 6-Well-Platten aufgenommen worden. Geändert PBS wird durch Zugabe von 2 ml hitzeinaktiviertem fötalem Rinderserum und 2 ccm der EDTA 0,5 M pH 8 hergestellt. Diese Lösung wird dann durch ein Nalgene 0,45 um-Filter filtriert und bei 4 Grad Celsius für mindestens einen Monat gelagert werden. </li><li> Es ist wichtig, in die Vertiefungen in der Platte Label, um in der Lage sein WT und KO Knochen korrekt zu identifizieren. </li><li> Die Maus Kadaver werden entfernt und entsorgt. </li><li> Ein neues Blatt in der Motorhaube platziert und die Knochen gereinigt werden mit einem Einweg-Skalpell. </li><li> Die gereinigten Knochen sind eine Maus in einer Zeit, in der Mörtel gelegt. Zwei ml modifizierter PBS ist es, den Mörtel hinzugefügt und die Knochen werden zerkleinert mit einem Stößel. Es ist wichtig, erste Fragment den Knochen mit 10 hin und her Bewegungen der Stößel und dann zu pulverisieren sie mit kreisenden Drehbewegungen. Ich in der Regel haben 50 im Uhrzeigersinn, dann 50 gegen den Uhrzeigersinn Bewegungen und so weiter, bis das Material in den Mörtel links ist in der Farbe weiß und dem modifizierten PBS hinzugefügt, um das Material bleibt weiß oder transparent. </li><li> Nach jedem Satz von 50 kreisförmigen Bewegungen Zerkleinerung, übertragen wir die Flüssigkeit aus dem Mörtel zu 70 uM Filter auf einem 50 ml konischen Röhrchen gegeben. Vergessen Sie nicht, bis 2 ml PBS, um den Mörtel add nach jeder Übertragung auf den Filter. Sie wollen nicht zu versuchen und zu pulverisieren trockenen Knochen oder lassen Sie sie trocknen für längere Zeit, wie diese zur Apoptose der Zellen führen. </li><li> Sobald das Material in den Mörtel wird weiß, wird das Material in den Filter, um den Mörtel verlegt und die Zerkleinerung wird erneut durchgeführt. Dadurch wird sichergestellt, dass die Zellen in der Filter nicht verschwendet werden. Normalerweise dauert es ein oder zwei Sätze von kreisförmigen Drehbewegungen wieder das Material in den Mörser in der Farbe weiß, zu welchem ​​Zeitpunkt der Gewinnung der Zellen abgeschlossen ist. </li><li> Die Lösung in dem konischen Rohr wird nun zentrifugiert (bitte denken Sie daran, die Zellen aus dem WT-und KO-Maus getrennt zu halten. Daher werden Sie zwei verschiedene Rohre (eines mit WT-Zellen, die andere mit KO-Zellen). Zentrifugation bei 1200 rpm für 10 ausgewählte Minuten. </li><li> Absaugen des Überstandes mit separaten Aspirator Pipetten für jede Röhre. 2 ccm ACK-Lysepuffer zu jedem Pellet. Diese Lyse-Puffer sollte steril sein. Inkubieren auf Eis für 5 Minuten. </li><li> Add 8 cc modifizierter PBS in jedes Röhrchen. Nehmen Sie sich 10 ul aus jedem Röhrchen und Pipette in zwei getrennten Vertiefungen in einer 96-Well-Platte. Die Röhrchen in der Zentrifuge und Spin sie bei 1200 rpm für 10 Minuten. </li><li> Während die Rohre drehen, fügen Sie 30 ul tryphan blauen Farbstoff und 60 ul der modifizierten PBS in jede Vertiefung der 96-Well-Platte, die die Zellen von Interesse führt. </li><li> Add 10 ul dieser Mischung (bitte gut mischen durch Auf-und Abpipettieren), um eine Zählkammer und zählen Sie die Zellen in den vier äußeren Plätzen. </li><li> Die Zellzahl in jeder Probe pro ml ist die Anzahl der Zellen werden somit durch 4 und multiplie geteiltd von 100 000 Euro. </li><li> Nach der Zentrifugation abgeschlossen ist, entfernen Sie das Röhrchen aus der Zentrifuge und die Zellen in PBS ohne Calcium, Magnesium oder L Glutamin in einer Konzentration von 20 Millionen Zellen pro ml. Sie werden wahrscheinlich etwa 5 ml Volumen für den WT und 5 ml Volumen für die KO Tier. Von diesem Schritt weiter werden wir mit PBS, nicht geändert PBS für das Protokoll. </li><li> Zu jedem Röhrchen hinzufügen DiI, ein Zellfärbung Farbstoff, zu erreichen in einer Endkonzentration von 5um. Vortex sofort für 10 Sekunden. Bei 37 Grad für 10 Minuten und zur Vermeidung von Lichteinstrahlung. </li><li> Füllen Sie das Röhrchen mit PBS und Spin für 10 Minuten bei 1200 Umdrehungen pro Minute. </li><li> Nach der Zentrifugation absaugen Überstand und Zellen in PBS bei 40 Millionen Zellen pro ml. </li><li> Legen Sie 500 ul in 1-ml-Spritzen mit einer 27G Nadel (5 Spritzen für die WT-Maus und 5 Spritzen für die KO-Mäuse). Es kann sein, klug, 6 Spritzen für jede Kohorte vorzubereiten. Nachdem eine zusätzliche Hilfe können Sie wie Schwanzvene Injektionen, die wir als nächstes tun werden kann tückisch sein. </li><li> Decken Sie die Spritzen mit einer Folie, um die DiI von zerfallenden zu halten. </li><li> Inject 500 ul der Zelle Mischung in jeder Empfänger Tier; (5 für jede Kohorte). </li><li> Diese Tiere müssen mit 9 Gy Bestrahlung bestrahlt 4-24 Stunden vor der Injektion werden. </li><li> 48 Stunden nach der Injektion, Ernte Knochenmark aus dem hinteren Gliedmaßen des jeweiligen Empfängers Tier. </li><li> Verfahren wie oben für den Spender beschrieben. Resuspendieren der Zellen von jedem Tier in 1 ccm PBS. Besorgen Sie sich eine Zellzahl unter Verwendung eines Hämozytometers. </li><li> Lesen Sie auf der LSR II Durchflusszytometer von BD oder einem vergleichbaren Gerät. Das Ergebnis kann als Prozentsatz oder absolute Anzahl der Zellen in den analysierten Knochenmark, die DiI positiv ausgedrückt werden. </li></ol>

<ol>
	<li>Adams, G. 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=Stem+cell+engraftment+at+the+endosteal+niche+is+specified+by+the+calcium-sensing+receptor.">Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor.</a> Nature. 439 (7076), 599-603 (2006).</li><li>Broxmeyer, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+in+hematopoiesis.">Chemokines in hematopoiesis.</a> Curr Opin Hematol. 15 (1), 49-58 (2008).</li></ol>

Homing ist der Prozess, durch Knochenmarkszellen wie Stammzellen, Vorläuferzellen und differenzierte Zellen, ihren Weg in das Knochenmark, nachdem sie in die Blutbahn oder Knochenmarkshöhle von Mäusen injiziert. Wie aus der Definition, kann ein Wissenschaftler wählten die Homing von hämatopoetischen Stammzellen, Vorläuferzellen oder differenzierten Zellen durch Immunphänotypisierung identifiziert und isoliert durch Zellsortierung zu studieren. Typischerweise Wissenschaftler injizieren Knochenmarkzellen für Linie...

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<td>DPBS (Dulbecco&#x2019;s Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Mediatech, Inc</td>
<td>21-031-CV</td>
<td>&nbsp;</td>
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<td>Sterile Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Valley Biomedical, Inc</td>
<td>BS3033</td>
<td>&nbsp;</td>
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<td>0.5M EDTA (pH 8.0)</td>
<td>&nbsp;</td>
<td>Boston BioProducts</td>
<td>BM-150</td>
<td>&nbsp;</td>
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<td>Tryphan Blue solution</td>
<td>&nbsp;</td>
<td>Mediatech, Inc</td>
<td>25-900-CI</td>
<td>&nbsp;</td>
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<td>ACK Lysing Buffer</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>10-548E</td>
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<td>Isopropranolol U.S.P.</td>
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<td>Denison Pharmaceuticals, Inc</td>
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<td>Vybrant DiD cell-labelling solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>V22887</td>
<td>&nbsp;</td>
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<td>Vybrant DiI cell-labelling solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>V22885</td>
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homing of hematopoietic cells to the bone marrow

Homing ist das Phänomen, dass transplantierte hämatopoetische Zellen sind in der Lage zu erreichen und einprägen Reise-oder niederlassen in das Knochenmark. Verschiedene chemomkines und Rezeptoren sind in der Homing von hämatopoetischen Stammzellen beteiligt sind. [1, 2] Dieser Beitrag skizziert die klassische Homing-Protokoll in hämatopoetischen Stammzellen Studien verwendet. Im Allgemeinen besteht die Isolierung des Zell-Population, deren Referenzierung muss untersucht werden, Färbung dieser Population mit einem Farbstoff der Zinsen und Injektion dieser Zellen in die Blutbahn des Empfängers Tier. Der Empfänger Tier wird dann bei einer vorbestimmten Zeit nach der Injektion und dem Knochenmark für die prozentuale oder absolute Anzahl der Zellen, die positiv sind für den Farbstoff von Interesse ausgewertet geopfert. In einer der häufigsten experimentellen Systemen, die Homing-Effizienz von hämatopoetischen Zellen von zwei genetisch verschiedene Tiere (ein Wildtyp-Tier und die entsprechenden knock-out) verglichen wird. Dieser Artikel beschreibt das hämatopoetische Zellen Homing-Protokoll im Rahmen der, wie zu experimentieren.

Watch this Scientific Journal Video about Homing of Hematopoietic Cells to the Bone Marrow at JoVE.com

Homing of Hematopoietic Cells to the Bone Marrow

Dieser Artikel beschreibt ein Protokoll, mit dem Homing von hämatopoetischen Zellen, ihre Nischen im Knochenmark zu studieren.

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 ...

homing-of-hematopoietic-cells-to-the-bone-marrow

Research

JoVE Journal

Biology

29.3K Aufrufe.  MGH - Massachusetts General Hospital. Dieser Artikel beschreibt ein Protokoll, mit dem Homing von hämatopoetischen Zellen, ihre Nischen im Knochenmark zu studieren.

Serie: Homing of Hematopoietic Cells to the Bone Marrow

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Isolierung und Analyse von hämatopoetischen Stammzellen aus der Plazenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Forschung

Biologie

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 ...

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon 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% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon 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% ...

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 ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentivirale-vermittelte Knockdown Während Ex Vivo Erythropoese von humanen hämatopoetischen Stammzellen

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

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 ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Isolierung, Reinigung und Differenzierung von Osteoclast-Laufläufern aus der Rattenmarkierung

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the proplatelet formation using in vitro-differentiated megakaryocytes; however, there is an increasing evidence that conventional culture systems do not faithfully recapitulate the differentiation/maturation process that takes places inside the bone marrow. In this manuscript, we present an explant method initially described in 1956 by Thi&#233;ry and Bessis to visualize megakaryocytes which have matured in their native environment, thus circumventing potential artifacts and misinterpretations. Fresh bone marrows are collected by flushing the femurs of mice, sliced into 0.5 mm cross sections, and placed in an incubation chamber at 37 &#176;C containing a physiological buffer. Megakaryocytes become gradually visible at the explant periphery and are observed up to 6 hours under an inverted microscope coupled to a video camera. Over time, megakaryocytes change their shape, with some cells having a spherical form and others developing thick extensions or extending many thin proplatelets with extensive branching. Both qualitative and quantitative investigations are carried out. This method has the advantage of being simple, reproducible, and fast as numerous megakaryocytes are present, and classically half of them form proplatelets in 6 hours compared to 4 days for cultured mouse megakaryocytes. In addition to the study of mutant mice, an interesting application of this method is the straightforward evaluation of the pharmacological agents on the proplatelet extension process, without interfering with the differentiation process that may occur in cultures.

Here, we detail the bone marrow explant method, from sample preparation to microscopic slide analysis, to evaluate the ability of megakaryocytes which have differentiated in their physiological environment to form proplatelets.

Proplatbildungsdynamik von mausfrischen Knochenmark-Explantaten

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the ...