Les auteurs tiennent à remercier Anne et Pierre Daron Louis Loyher d&#39;assistance éditoriale, la Plateforme Imagerie Pitié-Salpêtrière (PICPS) pour l&#39;assistance avec le microscope à deux photons et le Fonds des animaux &quot;CNA&quot; et Camille Baudesson pour les souris assistance élevage. Les recherches menant aux présents résultats ont reçu un financement de septième programme-cadre de la Communauté européenne (FP7 / 2007-2013) sous convention de subvention n ° 304810 - RAID, et n ° 241440-EndoStem, de l&#39;Inserm, de l&#39;Université Pierre et Marie Curie &quot;Emergence &quot;, de la&quot; Ligue contre le cancer », de« Association pour la Recherche sur le Cancer »et de« Agence nationale de la recherche &quot;Programme Emergence 2012 (ANR-EMMA-050). PH a été soutenue par la &quot;Ligue contre le cancer».

REMARQUE: Tous les protocoles d&#39;expérimentation ont été approuvés par le Comité d&#39;éthique de l&#39;expérimentation animale et français et validés par le &quot;Service de Protection et Santé Animales, Environnement&quot; avec le numéro A-75-2065. La taille des échantillons sont choisies pour assurer la reproductibilité des expériences, et selon l&#39;3R éthique de régulation de animal. 1. Préparation de la souris <ol><li> Anesthésie <ol><li> Pour courte période de formation d&#39;image (moins de 1 h), anesthésier les souris avec une injection intraperitoneale de 200 ul d&#39;une solution saline contenant de la kétamine (100 mg / kg) et de xylazine (10 mg / kg). </li><li> En variante, pour une longue période de formation d&#39;image (jusqu&#39;à 4 heures), anesthésier les souris avec une inhalation d&#39;isoflurane 2,5% vaporisé dans un mélange 70/30 de O 2 / N 2 O, à travers un masque adapté. </li><li> NOTE: cette façon prévoit l&#39;inconscience plus stable et évite injection intrapéritonéale série de médicaments. </li&gt; <li> Une fois que la souris est anesthésiée, vérifier l&#39;inconscience par la stimulation de la garniture de pied avec des pinces. </li></ol></li><li> Coloration vasculaire / coloration supplémentaire <ol><li> Pour la coloration vasculaire: injecter par voie intraveineuse dans la veine caudale, 200 ul de rhodamine-dextrane (2 MDA, 10 mg / ml dans un tampon salin). </li><li> Pour la coloration des neutrophiles: 2 ug d&#39;injecter Ly6G-PE (clone 1A8) dans 100 ul de solution saline par voie intraveineuse dans la veine caudale. </li></ol></li><li> Immobilisation <ol><li> Pour l&#39;immobilisation, utiliser un support stéréotaxique sur mesure adaptée à la loupe et l&#39;inhalateur de gaz anesthésique. NOTE: Le titulaire stéréotaxique est un support métallique avec deux supports de plaques de métal, l&#39;un étant fixe et l&#39;autre amovibles et adaptables, pour resserrer la tête de l&#39;animal. </li><li> Installez la tête de la souris entre les plaques de retenue pour assurer l&#39;isolation des mouvements respiratoires. Serrer les oreilles entre les plaques de retenue et de la tête pour étirer le sk. Vérifiez la stabilité et le bien-être de respiration de l&#39;animal. </li></ol></li><li> Retirer le cuir chevelu <ol><li> Soigneusement utiliser l&#39;éthanol à 70% pour mouiller les cheveux du cuir chevelu avec un applicateur stérile, évitant tout contact avec les yeux. </li><li> Couper la peau avec des ciseaux et des pinces stériles pour supprimer complètement le cuir chevelu de l&#39;arrière de l&#39;os pariétal à l&#39;os frontal. Tenir à l&#39;écart jusqu&#39;à 3 mm d&#39;yeux et les oreilles pour éviter un saignement important. Retirer le tissu conjonctif lâche couvrant le crâne (périoste). Laver les cheveux avec chaude restante serviette en papier imbibé PBS. </li></ol></li><li> système d&#39;immersion mis en place <ol><li> Collez un anneau de 18 mm de diamètre avec de la colle chirurgicale en caoutchouc directement sur le crâne à l&#39;aide d&#39;une aiguille de petit calibre de contrôler la distribution. Coller la totalité de la périphérie de l&#39;anneau en caoutchouc pour empêcher la fuite (30 ul devraient être suffisants). </li><li> Nettoyez le crâne sous l&#39;anneau une dernière fois avec PBS-serviette imbibée de papier, puis ajouter 37 ° C PBS pour une immersion complète de lacrâne. Ajouter une goutte de solution de NaCl à 0,9% ou un onguent ophtalmique spécifique sur chaque oeil de la souris afin d&#39;éviter la sécheresse oculaire. Faites glisser le support stéréotaxique titre de l&#39;objectif. </li><li> Environ positionner le terrain en utilisant l&#39;oculaire du microscope par transmission directe. </li></ol></li></ol> 2. Deux photons Acquisition d&#39;imagerie <ol><li> Utilisation d&#39;un microscope multiphoton couplé à un laser Ti: saphir à cristaux, qui fournit des impulsions de lumière 140fs NIR, accordables sélectivement de 680 à 1080 nm, et un modulateur acousto-optique pour le contrôle de la puissance du laser. Utilisez une configuration comprenant trois détecteurs externes non balayée (NDD) (qui permettent l&#39;enregistrement simultané des trois canaux fluorescentes) avec une combinaison de deux miroirs dichroïques (565 nm et 690 nm), 565/610 et 500/550 filtres passe-bande, et un 485 filtre passe courte, avec un apochromat plan × 20 (NA = 1) L&#39;objectif de l&#39;immersion en eau. </li><li> Réglez la chambre de chauffage pour permettre une bonne homéothermie des anessouris thetized., REMARQUE: Température pourrait être réglé une heure avant la session d&#39;imagerie pour une meilleure stabilité. Dans notre cas, 32 ° C a été choisi pour obtenir optimale la température du corps de la souris anesthésié (36-37 ° C) et est maintenue jusqu&#39;à 4 h. Cette température peut être adaptée au système utilisé (sonde numérique peut être utilisé pour vérifier ce point). En outre, la chambre de chauffage utilisé est sombre / noir et couvre une partie du système (objectifs et la plaque motorisé) pour éviter la contamination de la lumière extérieure. </li><li> Allumez le système <ol><li> Allumez le laser Ti: saphir, puis l&#39;ensemble du système de microscope. Lancez le logiciel d&#39;acquisition. Définissez la plage de longueur d&#39;onde laser à 870 nm. Sélectionnez NDD appropriée pour l&#39;enregistrement. NOTE: Dans notre cas, génération de second harmonique (SHG) 19 et la protéine fluorescente cyan (ECFP) sont détectées par le DDN après le court filtre 485 passe. ECFP est également détectée par le DDN après le filtre passe-bande 500/550 et la rhodamine-dextrane est détectée par til DDN après le filtre passe-bande 565/610. </li></ol></li><li> Réglez les paramètres d&#39;acquisition <ol><li> Utilisez &quot;l&#39;acquisition en direct&quot; commande d&#39;imagerie du logiciel et en utilisant la commande directionnelle de microscope, la mise au point sur une zone avec un signal ECFP et la rhodamine. Régler la puissance du laser au minimum afin d&#39;obtenir le meilleur rapport signal sur bruit avec un minimum de photo-dommages. NOTE: Le réglage de la puissance utilisée varie en fonction de la profondeur de la région d&#39;intérêt. Généralement, les paramètres AOM sont installés autour de 20%, ce qui représente une puissance de laser au-delà de l&#39;objectif d&#39;environ 15 mW. Il est préférable d&#39;augmenter la puissance de gain sur les PMT pour chaque NDD pour réduire la photo-blanchiment et photo-dommages. Paramètres d&#39;acquisition typiques sont la numérisation simple avec un temps de séjour de pixels de 1,58 ps, et une résolution de 512 x 512 pixels avec un grossissement de 1,5. </li></ol></li><li> Sélectionnez régions d&#39;intérêt <ol><li> Une fois les paramètres d&#39;enregistrement sont réglés, utilisez de nouveau la commande d&#39;acquisition «en direct»,et étudier le tissu de choisir un (x, y, z) champ désiré à surveiller en temps réel. NOTE: En règle générale, nous avons choisi un champ comprenant à la fois vasculaire et zones parenchymateuses. </li><li> Créer le domaine avec la commande de logiciel &quot;de position&quot;. REMARQUE: Cette commande mémorise x, y, z de différents champs éloignés. </li><li> Sélectionner et enregistrer les champs d&#39;intérêt supplémentaires (jusqu&#39;à 3) en utilisant la même procédure. </li><li> Définir un z-stack 3D avec la commande de logiciel &quot;z-pile&quot; sur la première position mémorisée selon le volume requis avec un grossissement choisi. Mémorisez profond première position, puis la position la plus élevée. Normaliser la puissance du laser avec la profondeur. NOTE: Pour les multiples imagerie de champ concomitante, nous acquérons cinq tranches séparées par 3 um z-étapes pour chaque champ d&#39;acquérir un volume de 12 um d&#39;épaisseur. Ces paramètres peuvent être adaptés au type de cellules étudiées. </li></ol></li><li> acquisition de lancement <ol><li> Sélectionnez le220; séries chronologiques &quot;de commande du logiciel et choisissez la durée de l&#39;intervalle de temps et le nombre de cycles. Commencez imagerie time-lapse selon le laps de temps et la durée nécessaire en sélectionnant le &quot;Début de l&#39;expérience&quot; commande logicielle. NOTE: Dans notre cas, nous acquérons un volume chaque 30 secondes pendant 60 cycles représentant 30 min en temps réel. Plus petit laps de temps peut être effectuée à chemin de roulement cellulaire rapide dans le vaisseau sanguin. Dans ce cas, plus mince z-stack et pas plus de deux domaines différents peuvent être enregistrés en même temps. </li><li> Un suivi régulier de la mise en place. <ol><li> Toujours surveiller l&#39;animal pour se assurer qu&#39;il est inconscient. REMARQUE: Secouer des moustaches ou des jambes de l&#39;animal sont des indicateurs de reprise. En cas de respiration de l&#39;animal est saccadé, la quantité de l&#39;isoflurane peut être légèrement réduite. Dérive de tissus forte lors de l&#39;acquisition est un indicateur de la renaissance de la souris. </li><li> Assurer l&#39;objectif est bien immergé. Renouveler 37 ° C PBS between deux acquisitions (30 min). REMARQUE: disparition progressive des signaux lors de l&#39;acquisition est l&#39;indicateur de fuite PBS. </li></ol></li></ol></li><li> Fin de la procédure <ol><li> Une fois les vidéos enregistrées sont nécessaires, euthanasier l&#39;animal par dislocation cervicale rapidement devant la réanimation. REMARQUE: la cicatrisation conduit à la formation d&#39;une croûte en 24-48 h, ce qui limite la possibilité d&#39;effectuer une imagerie longitudinal. </li></ol></li></ol> 3. Analyse des données <ol><li> correction de la dérive <ol><li> Utilisez un logiciel Imaris Bitplane pour le suivi automatique 3D. </li><li> Ouvrez l&#39;image 3D sur Imaris séquence. Sélectionnez &quot;spot&quot; de commande, et de générer suivi manuel (clic de souris + shift) pour tous les points de temps pour un minimum de 4 points d&#39;ancrage différents, si possible répartis de manière homogène dans le domaine. </li><li> Appliquer la correction de la dérive en utilisant la commande &quot;de dérive correcte&quot; dans l&#39;onglet &quot;édition de piste&quot;. </li><li> Vérifiez til la qualité de la correction en x, y et z en particulier dans flottement pour éviter artefact. NOTE: Si la correction ne est pas satisfaisante, essayez d&#39;autres points d&#39;ancrage ou d&#39;exclure la vidéo à partir de l&#39;analyse. </li></ol></li><li> suivi cellulaire <ol><li> Utilisez &quot;ajouter de nouveaux spots&quot; commande et sélectionnez &quot;taches de suivre dans le temps&quot; de réglage de l&#39;algorithme. Aller à l&#39;étape suivante. </li><li> Appliquer la procédure de suivi automatique de la cellule, soit sur l&#39;ensemble du canal d&#39;intérêt en utilisant &quot;canal source&quot; commande. Set objets (ie, cellules) de diamètre à 10 um. Aller à l&#39;étape suivante. NOTE: Le choix dépendra de la distinction entre les objets et la spécificité du signal mesuré. En bref, l&#39;alignement automatique sur l&#39;ensemble du canal ne est possible que si le signal détecté est spécifique à l&#39;objet d&#39;intérêt. Parce que les deux SHG ECFP et sont détectées par le DDN après le filtre 485 passe-court, suivi sera plus précise et plus efficace en utilisant le signal enregistré par l&#39;ECFP NDD après le 500/550 filtres passe-bande. Groupées ou des objets emballés denses nécessiteront sélection manuelle des objets individuels similaires à l&#39;étape 3.1.2. </li><li> Sélectionnez le type de filtre &quot;de qualité&quot;, définir le seuil d&#39;exclure le suivi d&#39;objets non spécifiques. Aller à l&#39;étape suivante. </li><li> Choisissez &quot;brownien algorithme Motion&quot; avec des paramètres précis de &quot;Max la distance» et «taille de Gap&quot; entre deux points. Valider la génération de piste. </li><li> Une fois les pistes sont calculés automatiquement, sélectionnez &quot;la durée de la piste&quot; type de filtre, fixer le seuil d&#39;éliminer pistes plus courtes que 120 secondes (soit 4 points de temps). </li><li> Obligatoire: Après un suivi automatique, vérifier la qualité des pistes. <ol><li> Déroulez la barre de temps pour vérifier que les objets suivent les chemins de piste. Sinon, utilisez les options de correction de point de trace disponibles dans l&#39;onglet &quot;modifier&quot;: Supprimer les pistes fausses ou inappropriées avec commande &quot;Supprimer&quot; (supprimer ne importe quelle piste point individuellement en utilisant la commande &quot;Déconnecter&quot;). Si le chemin de la piste est discontinue, ajouter les données manquantes (clic de souris + décalage), sélectionner tous les points sur une piste donnée, et d&#39;utiliser &quot;connecter&quot; commande dans l&#39;onglet &quot;édition de piste&quot;. </li></ol></li></ol></li><li> paramètres de quantification <ol><li> Sélectionnez l&#39;onglet &quot;statistique&quot; dans la commande «Préférences», choisissez les paramètres dynamiques d&#39;intérêt dans la liste. Sélectionnez &quot;Exporter toutes les statistiques de déposer&quot; commandement et enregistrez le fichier excel généré. NOTE: Plusieurs paramètres dynamiques sont obtenues directement à partir du logiciel tels que longueurs de piste, vitesse instantanée, vitesse moyenne, et de suivre la rectitude. coefficient de motilité 6, 20 peut être déterminé mais ne est pas directement disponible par le logiciel. </li><li> Pour chaque cellule, calculer la moyenne des déplacements pour chaque période de temps (At) (pour un film laps de temps de 30 sec, AT aura 30, 60, 90 secondes, etc).Tracer la moyenne du déplacement quadratique en fonction de l&#39;intervalle de temps. Obtenir coefficient de motilité en calculant la pente de la partie linéaire de la courbe. </li></ol></li></ol>

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The authors have nothing to disclose.

Les points critiques de la méthode in vivo d&#39;imagerie sont en assurer la stabilité de la mise au point afin de maximiser la durée de formation d&#39;image et de réduire au minimum le risque de contamination bactérienne et l&#39;inflammation, ce qui pourrait avoir un impact de la dynamique des cellules inflammatoires. L&#39;imagerie du crâne moelle osseuse suite à ces objectifs comme la chirurgie effectuée pour obtenir l&#39;accès à la moelle osseuse est minime. L&#39;utilisation de...

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>2MDa, 10mg/mL, Vascular staining</td>
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			<td style="width:171px;">Queryo Medical</td>
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			<td>Surgical Glue, rubber ring fixation</td>
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			<td height="21" style="height:21px;width:297px;">Small gauge needles</td>
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			<td>Carl Zeiss</td>
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			<td>140fs pulses of NIR light</td>
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			<td height="21" style="height:21px;">Zen 2010</td>
			<td>Carl Zeiss</td>
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			<td>Analysis Software, 3D automatic tracking</td>
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			<td height="21" style="height:21px;width:297px;">PBS 1X</td>
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			<td>Carl Zeiss</td>
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			<td>intravital imaging</td>
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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.

La structure du crâne de souris offre une bonne occasion pour étudier la physiologie de la moelle osseuse par imagerie intravitale. L&#39;os étant mince autour de la zone avant-pariétale, il est possible d&#39;avoir accès à des niches médullaires sans abrasion de l&#39;os. La figure 1 représente un champ 2D large du crâne d&#39;une souris transgénique MacBlue. La matrice d&#39;os est composée principalement de collagène de type I facilement détectable par 19 SHG. L&#39;injection ...

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.

Suivi moelle osseuse de souris monocytes<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 ...

tracking-mouse-bone-marrow-monocytes-in-vivo

Research

JoVE Journal

Immunology and Infection

Tracking Mouse Bone Marrow Monocytes In Vivo

9.4K vues.  Sorbonne Universités, UPMC Univ Paris 06, CR7. 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. 

Vidéo: Tracking Mouse Bone Marrow Monocytes In Vivo

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Génération et d&#39;étiquetage des murins de la moelle osseuse des cellules dendritiques dérivées des nanocristaux Qdot pour les études de suivi

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Recherche

Immunologie et infection

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

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 to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

Différencier les rôles fonctionnels de l&#39;expression des gènes de cellules immunitaires et non immunitaires dans l&#39;intestin de souris par greffe de moelle osseuse

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 ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Isolement, la purification et l&#39;étiquetage des neutrophiles osseuse de souris de moelle pour les études fonctionnelles et expériences de transfert adoptif

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Dérivées de moelle osseuse Macrophages production

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

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Isolement et injection intraveineuse de moelle osseuse de souris dérivées monocytes

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

Murine Hind Limb Long Bone Dissection and 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, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

Murine Hind Limb os long Dissection et moelle osseuse Isolation

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

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

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

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

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Fémur Fenêtre Chambre Modèle pour<em&gt; In Vivo</em&gt; Suivi des cellules dans la moelle osseuse murine

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

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-tri cellulaire activé pour la purification de cellules dendritiques plasmacytoïdes de la moelle osseuse de souris

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Identification et isolement des Oligopotent et des progéniteurs myéloïdes Lineage-commis de moelle osseuse de souris

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

An &lt;em&gt;In Vivo&lt;/em&gt; Mouse Model to Measure Na&amp;#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Un modèle de souris In Vivo à l’Activation des cellules T CD4 naïves mesure, la prolifération et la différenciation Th1 induite par les cellules dendritiques dérivées de la moelle osseuse

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...