Name: tracking mouse bone marrow monocytes in vivo
Uploaded: 2015-02-27T15:00:00
Description: Watch this Scientific Journal Video about Tracking Mouse Bone Marrow Monocytes In Vivo at JoVE.com

De auteurs willen Anne Daron en Pierre Louis Loyher bedanken voor redactionele ondersteuning, het Plateforme Imagerie Pitié-Salpêtrière (PICPS) voor hulp bij de twee-foton microscoop en het dier faciliteit &quot;NAC&quot; en Camille BAUDESSON voor muizen fokken hulp. Het onderzoek leidde tot deze resultaten heeft de financiering van het zevende kaderprogramma van de Europese Gemeenschap ontvangen (FP7 / 2007-2013) onder subsidieovereenkomst n ° 304.810 - RAID&#39;s, en n ° 241440-Endostem, van INSERM aan de Université Pierre et Marie Curie &quot;Emergence &quot;, van la&quot; Ligue contre le cancer &quot;, van&quot; Association pour la Recherche sur le Cancer &quot;en van&quot; Agence Nationale de la Recherche &quot;Programma Emergence 2012 (ANR-EMMA-050). PH werd ondersteund door la &quot;Ligue contre le cancer&quot;.

OPMERKING: Alle experiment protocollen werden goedgekeurd door de Franse Dierproeven en ethische commissie en gevalideerd door de &quot;Service Protection et Santé Animales, Environnement&quot; met nummer A-75-2065. Steekproefomvang gekozen reproduceerbaarheid van de proeven te waarborgen, en volgens de 3R dieren ethische regelgeving. 1. Voorbereiding van de Muis <ol><li> Anesthesie <ol><li> Voor korte beeldvorming (minder dan 1 uur), verdoven de muis met een intraperitoneale injec...

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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=Identification+of+splenic+reservoir+monocytes+and+their+deployment+to+inflammatory+sites.">Identification of splenic reservoir monocytes and their deployment to inflammatory sites.</a> Science. 325, 612-616 (2009).</li><li>Germain, R. N., Miller, M. J., Dustin, M. L., Nussenzweig, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+the+immune+system:+progress,+pitfalls+and+promise.">Dynamic imaging of the immune system: progress, pitfalls and promise.</a> Nat Rev Immunol. 6, 497-507 (2006).</li><li>Charo, I. F., Ransohoff, R. 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L., 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=Activation+of+bone+marrow-resident+memory+T+cells+by+circulating,+antigen-bearing+dendritic+cells.">Activation of bone marrow-resident memory T cells by circulating, antigen-bearing dendritic cells.</a> Nat Immunol. 6, 1029-1037 (2005).</li><li>Mazo, I. 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=Bone+marrow+is+a+major+reservoir+and+site+of+recruitment+for+central+memory+CD8++T+cells.">Bone marrow is a major reservoir and site of recruitment for central memory CD8+ T cells.</a> Immunity. 22, 259-270 (2005).</li><li>Travlos, G. 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A., 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=In+vivo+imaging+of+specialized+bone+marrow+endothelial+microdomains+for+tumour+engraftment.">In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment.</a> Nature. 435, 969-973 (2005).</li><li>Cariappa, A., 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=Perisinusoidal+B+cells+in+the+bone+marrow+participate+in+T-independent+responses+to+blood-borne+microbes.">Perisinusoidal B cells in the bone marrow participate in T-independent responses to blood-borne microbes.</a> Immunity. 23, 397-407 (2005).</li><li>Junt, T., 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=Dynamic+visualization+of+thrombopoiesis+within+bone+marrow.">Dynamic visualization of thrombopoiesis within bone marrow.</a> Science. 317, 1767-1770 (2007).</li><li>Zoumi, A., Yeh, A., Tromberg, B. 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De kritische punten in vivo beeldvorming methodologie stabiliteit van de focus, teneinde de duur van beeldvorming maximaliseren en het risico van bacteriële besmetting en ontsteking, die de dynamiek van de ontstekingscellen kan van invloed minimaliseren. Beeldvorming van de schedel beenmerg volgt deze doelstellingen als de operatie uitgevoerd om toegang tot het beenmerg krijgen is minimaal. Het gebruik van steriel materiaal en antiseptica is essentieel om het risico van infectie die storing kunnen veroo...

The bone marrow plays a central role in hematopoiesis and represents the main reservoir of monocytes that constitutively recirculate between the blood and the medullar parenchyma, renew the pool of circulating monocytes with a short life span 2,3 and participate in the reconstitution of the steady state tissue-macrophages and dendritic cells 4. During inflammation or after transient aplasia, monocytes are actively mobilized from either the bone marrow or the spleen 5, 6, 7 and colonize inflamed organs. Several chemoattractant axis have been involved in the process of myeloid cell mobilization from the bone marrow 8, 5, 6,9. Beyond the myeloid compartment the bone marrow is also an important site of T lymphocyte priming 10 and a niche of immunological memory 11,12. Thus, this tissue is central for numerous investigations in the field of hematology and immunology. Our knowledge on the structural organization of medullar myeloid cells mainly arises from the analysis of histological section of fixed tissues 13. This static view does not allow for a study of the cellular exchange dynamic between the different compartments of the bone marrow, which is the basis of its functional activity.
Intravital imaging constitutes an important biological input in the study of cell mobility, cell adherence and cell-to-cell interactions, which were previously described only from in vitro systems. Technical challenges for proper intravital imaging include the ability to reach the tissue of interest in an optical point of view, and to maximize its isolation from physiological (breath, muscle or peristaltic contractions) or mechanical drifts (tissue disruption and extension following surgery, and exposure to microscope objective as well as temperature and vascular/oxygenation perturbations). Microscopic drifts may limit the ability to keep the focus a long time and could introduce artifacts in the quantification of cell motility. One alternative, validated for several tissues to reduce these technical difficulties, is to work on explanted tissue incubated in a thermostated and oxygenated medium; however, complete disruption of the lymphatic and vascular circulation may be problematic. Intravital imaging of skull bone marrow has several advantages concerning these issues. Firstly, it requires minimal surgical action. Secondly, thickness of the bone in this region allows direct visualization of bone marrow niches without abrasion, thus reducing physiological perturbations. The medullar network can be imaged in the parasagittal region of the bone; however the sinusoids are more visible in the fronto-parietal area where the bone matrix is thinner12,14. 
Intravital imaging relies on the availability of the most accurate fluorescent reporter tagging the population of interest. In vitro labelling of purified cell population before adoptive transfer led to important characterization of hematopoietic stem cell niches 15 or bone marrow endothelial microdomains favouring tumor engraftment 16, and provided several fundamental inputs on key concepts in immunology 17 . However, this approach usually requires hundreds of thousands of cells to get a chance to detect them afterwards in vivo. This could be explained by the high mortality rate following staining, the dilution in the whole body and the change in the activation state, which might lead to biased homing. Endogenous tagging from transgenic mouse system greatly overcomes these limitations and has allowed to image the behaviour of endogenous osteoblast 8, megakaryocytes 18 or myeloid-lineage subsets 6 . Nevertheless, one has to be cautious when considering the specificity of the fluorescent reporter among the studied subset. 
The Csf1r-Gal4VP16/UAS-ECFP, called MacBlue mouse 1, is a valuable transgenic system to study medullar monocytes with real time imaging 6. Intravenous injection of high molecular weight rhodamin-dextran distinguishes the medullar parenchyma from the vascular sinusoid network of the bone marrow. Using this approach, it is possible to track the monocyte behaviour in the different medullar compartments in a specific physiopathological context of interest. Furthermore, we propose an additional strategy to compare monocyte dynamics with that of neutrophils through in vivo labelling using a specific antibody.

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			<td height="21" style="height:21px;width:297px;">Name of Material/ Equipment</td>
			<td style="width:171px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:237px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Ketamin</td>
			<td style="width:171px;">Merial</td>
			<td style="width:119px;"></td>
			<td>100mg/mL, anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Xylazin</td>
			<td style="width:171px;">Bayer HealthCare</td>
			<td style="width:119px;"></td>
			<td>10mg/mL, anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Isofluran</td>
			<td style="width:171px;">Baxter</td>
			<td style="width:119px;"></td>
			<td>2.5%, anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">O2/NO2</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td>70/30 mixture, anesthetic</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:297px;">Rhodamin-Dextran</td>
			<td style="width:171px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td>2MDa, 10mg/mL, Vascular staining</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:297px;">Ly6G-PE</td>
			<td style="width:171px;">Becton-Dickinson</td>
			<td style="width:119px;"></td>
			<td>clone 1A8, neutrophils staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Stereotactic holder</td>
			<td style="width:171px;">Home made</td>
			<td style="width:119px;"></td>
			<td>surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Ethanol 70%</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td>surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Sterile scissors and nippers</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td>surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Rubber ring</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td>18mm diameter, surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Glubran 2</td>
			<td style="width:171px;">Queryo Medical</td>
			<td style="width:119px;"></td>
			<td>Surgical Glue, rubber ring fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Small gauge needles</td>
			<td style="width:171px;">Terumo</td>
			<td style="width:119px;"></td>
			<td>surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss LSM 710 NLO multiphoton microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td>Microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ti:Sapphire crystal laser&#160;</td>
			<td>Coherent Chameleon Ultra</td>
			<td style="width:119px;"></td>
			<td>140fs pulses of NIR light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zen 2010</td>
			<td>Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td>Acquistion Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaris Bitplane&#160;</td>
			<td style="width:171px;">Bitplane</td>
			<td style="width:119px;"></td>
			<td>Analysis Software, 3D automatic tracking</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">PBS 1X</td>
			<td style="width:171px;">D. Dutscher</td>
			<td style="width:119px;"></td>
			<td>surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Thermostated chamber</td>
			<td>Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td>intravital imaging</td>
		</tr>
	</tbody>
</table>

tracking mouse bone marrow monocytes in vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Muis schedel structuur biedt een goede gelegenheid om het beenmerg fysiologie te bestuderen door intravitale beeldvorming. Het bot dun rond de voorkant-parietale gebied, is het mogelijk om toegang tot medullaire niches slijtvaste van het bot krijgen. Figuur 1 een breed 2D gebied van de schedel van een MacBlue transgene muis. Het bot matrix bestaat hoofdzakelijk uit collageen I gemakkelijk door SHG 19. Injectie van rhodamine-dextran kleurt het vasculaire netwerk van het beenmerg en maakt de id...

Watch this Scientific Journal Video about Tracking Mouse Bone Marrow Monocytes In Vivo at JoVE.com

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Tracking muisbeenmerg Monocyten<em&gt; In Vivo</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Research

JoVE Journal

Immunology and Infection

Tracking Mouse Bone Marrow Monocytes In Vivo

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii Cyste muur Vorming in actieve beenmerg afgeleide Macrofagen en Bradyzoite voorwaarden

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

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Onderscheidende functionele rol van genexpressie van immune als niet-immune cellen in de muis Colitis door beenmergtransplantatie

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

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

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Bone Marrow-afgeleide macrofagen Productie

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

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

Isolatie en intraveneuze injectie van muizen beenmerg afgeleide Monocyten

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

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Murine Hind Limb Long Bone Dissection en Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

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

Generatie en identificatie van GM-CSF Afgeleide alveolaire-achtige macrofagen en dendritische cellen van de muis beenmerg

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

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Dijbeen Window Chamber Model voor<em&gt; In Vivo</em&gt; Cell Tracking in het Muizen Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...