Gli autori desiderano ringraziare Anne Daron e Pierre Louis Loyher per l&#39;assistenza editoriale, il Plateforme Imagerie Pitié-Salpêtrière (PICPS) per l&#39;assistenza con il microscopio a due fotoni e Facility animale &quot;NAC&quot; e Camille Baudesson per topi allevamento assistenza. La ricerca che ha portato a questi risultati ha ricevuto finanziamenti dal Settimo programma quadro della Comunità europea (FP7 / 2007-2013), contratto di sovvenzione n ° 304810 - RAID, e n ° 241440-Endostem, da Inserm, da Université Pierre et Marie Curie &quot;Emergence &quot;, da la&quot; Ligue contre le cancer &quot;, da&quot; Association pour la Recherche sur le Cancer &quot;e da&quot; Agence Nationale de la Recherche &quot;Programma Emersione 2012 (ANR-EMMA-050). PH è stata sostenuta da la &quot;Ligue contre le cancer&quot;.

NOTA: Tutti i protocolli sperimentali sono stati approvati dalla sperimentazione animale ed etica Comitato francese e validati dal &quot;Protection Service et Santé Animales, Environnement&quot; con il numero A-75-2065. Le dimensioni dei campioni sono scelti in modo riproducibilità degli esperimenti, e secondo la 3R della regolamentazione etica animale. 1. Preparazione del mouse <ol><li> Anestesia <ol><li> Per breve periodo di immagini (meno di 1 ora), anestetizzare il mouse con una iniezione intraperitoneale di 200 ml di una soluzione salina contenente ketamina (100 mg / kg) e xilazina (10 mg / kg). </li><li> In alternativa, per un periodo di più immagini (fino a 4 ore), anestetizzare il mouse con inalazione di isoflurano 2,5% vaporizzato in una miscela di 70/30 O 2 / N 2 O, attraverso una maschera adattato. </li><li> NOTA: In questo modo fornisce per incoscienza più stabile ed evita l&#39;iniezione di serie intraperitoneale di droghe. </li&gt; <li> Una volta che il mouse è anestetizzato, verificare la presenza di incoscienza attraverso la stimolazione del pad piede con pinze. </li></ol></li><li> Colorazione vascolare / colorazione supplementare <ol><li> Per la colorazione vascolare: Iniettare per via endovenosa nella vena della coda, 200 ml di Rhodamin-Destrano (2 MdA, 10 mg / ml in tampone salino). </li><li> Per neutrofili colorazione: iniettare 2 ug di Ly6G-PE (clone 1A8) in 100 ml di soluzione salina per via endovenosa nella vena della coda. </li></ol></li><li> Immobilizzazione <ol><li> Per l&#39;immobilizzazione, utilizzare un supporto stereotassica misura adattato al microscopio e l&#39;inalatore gas anestetico. NOTA: Il supporto Sterotactic è un supporto in metallo con due staffe in lamiera metallica, uno essendo fissato e l&#39;altra estraibili e adattabili, per stringere la testa dell&#39;animale. </li><li> Installare la testa del mouse tra le piastre di fissaggio per assicurare l&#39;isolamento dai movimenti respiratori. Serrare le orecchie tra le piastre di fissaggio e la testa per allungare la skin. Controllare la stabilità e la respirazione benessere degli animali. </li></ol></li><li> Rimuovere il cuoio capelluto <ol><li> Usare cautela etanolo al 70% per bagnare i capelli del cuoio capelluto con un applicatore sterile, evitando il contatto con gli occhi. </li><li> Tagliare la pelle con forbici sterili e tronchesi per rimuovere completamente il cuoio capelluto dal all&#39;indietro dell&#39;osso parietale all&#39;osso frontale. Tenere lontano fino a 3 mm da occhi e orecchie per prevenire vasta emorragia. Rimuovere il tessuto connettivo lasso che copre il cranio (periostio). Lavare i capelli rimanenti con caldo tovagliolo di carta PBS-impregnati. </li></ol></li><li> Sistema Immersion istituito <ol><li> Incollare un anello di gomma di 18 mm di diametro con colla chirurgica direttamente sul cranio con un piccolo ago calibro di controllare la distribuzione. Colla l&#39;intera periferia dell&#39;anello di gomma per evitare perdite (30 microlitri dovrebbero essere sufficienti). </li><li> Pulire il cranio sotto l&#39;anello per l&#39;ultima volta con un tovagliolo di carta imbevuta di PBS e poi aggiungere 37 ° C PBS per una completa immersione delcranio. Aggiungere una goccia di soluzione di NaCl allo 0,9% o specifica pomata oftalmica su ciascun occhio del mouse per evitare secchezza oculare. Trascinare il titolare stereotassica nell&#39;ambito dell&#39;obiettivo. </li><li> Circa posizionare il campo con l&#39;oculare del microscopio attraverso la trasmissione diretta. </li></ol></li></ol> 2. due fotoni Imaging Acquisition <ol><li> Utilizzare un microscopio multifotone accoppiato con un laser Ti: zaffiro, che fornisce 140fs impulsi di luce NIR, selettivamente sintonizzabile da 680 a 1.080 nm, e un modulatore acustico-ottico per il controllo della potenza del laser. Utilizzare una configurazione di cui 3 rilevatori esterni non descanned (NDD) (che consentono la registrazione simultanea di 3 canali fluorescenti) con una combinazione di due specchi dicroici (565 nm e 690 nm), 565/610 e 500/550 filtri passa-banda, e una 485 breve filtro passa, con un piano di apochromat × 20 (NA = 1) Obiettivo immersione in acqua. </li><li> Impostare la camera di riscaldamento per consentire una buona omeotermia delle ANAESMouse thetized., NOTA: temperatura potrebbe essere impostato a 1 ora prima di imaging sessione per una migliore stabilità. Nel nostro caso, 32 ° C è stata scelta per ottenere la temperatura corporea ottimale del mouse anestetizzato (36-37 ° C) ed è mantenuta fino a 4 ore. Questa temperatura può essere adattato al sistema utilizzato (sonda digitale può essere utilizzato per controllare questo punto). Inoltre, la camera di riscaldamento utilizzato è scuro / nero e copre una parte del sistema (obiettivi e piattello motorizzato) per evitare la contaminazione luce esterna. </li><li> Accendere il sistema <ol><li> Accendere il laser Ti: zaffiro, l&#39;intero sistema di microscopio. Avviare il software di acquisizione. Impostare l&#39;intervallo di lunghezze d&#39;onda laser a 870 nm. Selezionare appropriata NDD per la registrazione. NOTA: Nel nostro caso, generazione di seconda armonica (SHG) 19 e proteina fluorescente ciano (ECFP) vengono rilevati dal NDD dopo il filtro passaggio corto 485. ECFP viene anche rilevato dal NDD dopo il filtro passa-banda 500/550 e Rhodamin destrano viene rilevato da tegli NDD dopo il filtro passa-banda 565/610. </li></ol></li><li> Regolare le impostazioni di acquisizione <ol><li> Utilizzare la &quot;acquisizione dal vivo&quot; il comando di imaging del software e con il controllo direzionale del microscopio, regolare la messa a fuoco su una regione con il segnale ECFP e Rhodamine. Impostare la potenza del laser al minimo per ottenere il miglior rapporto segnale-rumore con minima foto-danni. NOTA: L&#39;impostazione di potenza utilizzato varia a seconda della profondità della regione di interesse. In genere le impostazioni AOM sono disposte intorno al 20%, che rappresenta una potenza laser oltre l&#39;obiettivo di circa 15 mW. E &#39;meglio aumentare la potenza plusvalenza PMT per ogni NDD per ridurre fotometabolismo e foto-danni. Impostazioni di acquisizione tipiche sono la scansione singola con un tempo di permanenza di pixel di 1,58 ms e una risoluzione di 512 x 512 pixel con un ingrandimento di 1,5. </li></ol></li><li> Selezionare le regioni di interesse <ol><li> Una volta che i parametri di registrazione sono impostati, usare di nuovo il comando di acquisizione &quot;in diretta&quot;,e rilevare il tessuto di scegliere un (x, y, z) campo desiderato da monitorare in tempo reale. NOTA: in genere, abbiamo scelto un campo che include sia vascolare e aree parenchimali. </li><li> Registrare il campo con il comando software &quot;posizione&quot;. NOTA: Questo comando memorizza x, y, z coordinate di diversi campi lontani. </li><li> Selezionare e registrare ulteriori campi di interesse (fino a 3) utilizzando la stessa procedura. </li><li> Impostare un z-stack 3D con il comando software &quot;z-stack&quot; sulla prima posizione memorizzata secondo il volume richiesto con ingrandimento scelto. Memorizza posizione più profonda, e poi la posizione più alta. Normalizza potenza del laser con la profondità. NOTA: Per più immagini campo concomitante, acquisiamo 5 fette separate da 3 micron z-punti per ogni campo di acquisire un volume di 12 micron di spessore. Queste impostazioni possono essere adattati al tipo di cellula studiati. </li></ol></li><li> Acquisizione di lancio <ol><li> Selezionare il220; serie comando &quot;software e scegliere la durata dell&#39;intervallo di tempo e del numero di cicli di tempo. Inizia time-lapse imaging in base al lasso di tempo e la durata richiesta selezionando il comando software &quot;avviare esperimento&quot;. NOTA: Nel nostro caso, acquisiamo un volume ogni 30 secondi per 60 cicli che rappresentano 30 minuti in tempo reale. Più piccolo lasso di tempo può essere effettuata per tenere traccia di laminazione delle cellule veloce nel vaso sanguigno. In questo caso diluente z-stack e non più di 2 campi differenti possono essere registrati contemporaneamente. </li><li> Monitoraggio regolare del set up. <ol><li> Controllare sempre l&#39;animale per assicurarsi che è inconscio. NOTA: Agitazione di baffi o gambe dell&#39;animale sono indicatori di ripresa. In caso di respirazione dell&#39;animale è a scatti, la quantità di isoflurano può essere leggermente ridotta. Forte deriva tessuti durante l&#39;acquisizione è un indicatore del mouse del risveglio. </li><li> Assicurarsi che l&#39;obiettivo sia correttamente immersa. Rinnova 37 ° C PBS between due acquisizioni (30 min). NOTA: la scomparsa progressiva di segnale durante l&#39;acquisizione è l&#39;indice di perdita PBS. </li></ol></li></ol></li><li> Fine della procedura <ol><li> Una volta che i video richiesti vengono registrati, eutanasia animale da dislocazione cervicale rapidamente prima rianimazione. NOTA: guarigione delle ferite porta alla formazione di una crosta in 24 a 48 ore, che limita la possibilità di effettuare l&#39;imaging longitudinale. </li></ol></li></ol> Analisi 3. Dati <ol><li> Correzione Drift <ol><li> Utilizzare software Imaris Bitplane per il 3D tracking automatico. </li><li> Aprire la sequenza di immagini 3D su Imaris. Selezionare &quot;spot&quot; di comando, e generare tracciamento manuale (mouse + shift) per tutti i punti di tempo per un minimo di 4 diversi punti di ancoraggio, se possibile, omogeneamente distribuito in campo. </li><li> Applicare la correzione della deriva mediante il comando &quot;corretto deriva&quot; nella scheda &quot;Modifica brano&quot;. </li><li> Verificare la presenza di tha qualità della correzione in x, ye z in particolare per evitare esitazioni artefatti. NOTA: Se la correzione non è soddisfacente, provare altri punti di ancoraggio o escludere il video dall&#39;analisi. </li></ol></li><li> Monitoraggio cellulare <ol><li> Utilizzare &quot;aggiungere nuovi spot&quot; di comando e selezionare &quot;macchie traccia nel tempo&quot; Impostazione algoritmo. Andare al passo successivo. </li><li> Applicare procedura automatica di monitoraggio cellulare o su tutto il canale di interesse utilizzando &quot;canale di origine&quot; comando. Set oggetti (cioè, le cellule) diametro 10 micron. Andare al passo successivo. NOTA: La scelta dipenderà dalla distinzione tra oggetti e specificità del segnale misurato. In breve, inseguimento automatico l&#39;intero canale è possibile solo se il segnale rilevato è specifico per l&#39;oggetto di interesse. Poiché sia ​​SHG e ECFP vengono rilevati dal NDD dopo il filtro 485 passaggio corto, il monitoraggio sarà più specifico ed efficace utilizzando il segnale ECFP registrato dal NDD dopo la 500/550 filtri passa-banda. Raggruppati o oggetti confezionati densi richiederanno selezione manuale dei singoli oggetti simili al punto 3.1.2. </li><li> Selezionare &quot;qualità&quot; tipo di filtro, impostare la soglia di escludere il monitoraggio di oggetti non specifici. Andare al passo successivo. </li><li> Scegliere &quot;browniano Algoritmo Motion&quot; con i parametri esatti di &quot;distanza Max&quot; e &quot;size Gap&quot; tra due punti. Convalidare generazione track. </li><li> Una volta che le tracce vengono calcolate automaticamente, selezionare &quot;Durata del brano&quot; tipo di filtro, impostare la soglia di eliminare le tracce più breve di 120 secondi (in rappresentanza di 4 punti di tempo). </li><li> Obbligatorio: Dopo inseguimento automatico, controllare la qualità delle tracce. <ol><li> Srotolare la barra del tempo per verificare che gli oggetti seguono i percorsi della pista. In caso contrario, utilizzare le opzioni di correzione del punto di traccia disponibili nella scheda &quot;edit&quot;: Cancellare le tracce false o improprie con comando &quot;Elimina&quot; (eliminare qualsiasi traccia pmista individualmente utilizzando il comando &quot;disconnect&quot;). Se il percorso tracciato è discontinua, aggiungere momento del dato mancante (click del mouse + SHIFT), selezionare tutti i punti di una determinata traccia, e usare &quot;collegare&quot; il comando nella scheda &quot;Modifica brano&quot;. </li></ol></li></ol></li><li> Parametri di quantificazione <ol><li> Selezionare la scheda &quot;Statistiche&quot; in &quot;Preferenze&quot; il comando, scegliere i parametri dinamici di interesse nella lista. Selezionare &quot;Esporta tutte le statistiche su file&quot; di comando e salvare il file excel generato. NOTA: Diversi parametri dinamici sono ottenuti direttamente dal software, come lunghezza della pista, velocità istantanea, velocità media, e tenere traccia rettilineità. Coefficiente Motilità 6, 20 può essere determinata, ma non è fornita direttamente dal software. </li><li> Per ogni cella, calcolare la media degli spostamenti per ciascun periodo di tempo (T) (per un film lasso di tempo di 30 sec, Dt saranno 30, 60, 90 sec, ecc).Tracciare la media dello spostamento quadrata in funzione dell&#39;intervallo di tempo. Ottenere coefficiente motilità calcolando la pendenza della parte lineare della curva. </li></ol></li></ol>

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The authors have nothing to disclose.

I punti critici della metodologia di imaging in vivo devono garantire la stabilità della messa a fuoco per massimizzare la durata di imaging e di minimizzare il rischio di contaminazione batterica e infiammazione, che potrebbe influenzare le dinamiche delle cellule infiammatorie. Imaging del cranio midollo osseo segue questi obiettivi, come l&#39;intervento chirurgico effettuato per ottenere l&#39;accesso al midollo osseo è minimo. L&#39;uso di materiale sterile e antisettici è essenziale per limitare...

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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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.

Struttura del cranio del mouse offre una buona opportunità di studiare la fisiologia del midollo osseo da imaging intravitale. L&#39;osso essendo sottile intorno alla zona anteriore-parietale, è possibile ottenere l&#39;accesso a nicchie midollari, senza abrasione dell&#39;osso. La figura 1 rappresenta un campo 2D ampio del cranio di un MacBlue topo transgenico. La matrice ossea si compone principalmente di collagene I facilmente rilevabile con SHG 19. Iniezione di Rhodamin-destrano macchia...

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.

Monitoraggio midollo osseo di topo monociti<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 Views.  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. 

Video: 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.

Generazione e l&#39;etichettatura di Murine osseo cellule dendritiche derivate dal midollo con nanocristalli Qdot per gli Studi inseguimento

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

Ricerca

Immunologia e infezioni

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.

Differenziare i ruoli funzionali di espressione genica di cellule immunitarie e non immunitarie in Colite mouse da trapianto di midollo osseo

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.

Isolamento, la purificazione e della dicitura del mouse Bone Marrow neutrofili per studi funzionali ed Esperienze trasferimento adottivo

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.

Derivate dal midollo osseo macrofagi 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).

Isolamento e iniezione endovenosa di Murine derivate dal midollo osseo monociti

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.

Murino arto posteriore osso lungo dissezione e isolamento di midollo osseo

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.

Femore finestra del modello Camera per<em&gt; In Vivo</em&gt; Monitoraggio delle cellule nel midollo osseo murino

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.

Separazione delle cellule fluorescenza-attivato per la purificazione delle cellule dendritiche plasmacitoidi dal midollo osseo di topo

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.

Identificazione e isolamento di Oligopotent e dei progenitori mieloidi Lineage-commesso dal midollo osseo del topo

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 modello di topo In Vivo di attivazione delle cellule T Naïve CD4 di misura, la proliferazione e la differenziazione Th1 indotta dalle cellule dendritiche derivate da midollo osseo

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 ...