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.

<table><tbody><tr><td>Ketamin</td><td>Merial</td><td></td><td>100 mg/ml, anesthetic</td></tr><tr><td>Xylazin</td><td>Bayer HealthCare</td><td></td><td>10 mg/ml, anesthetic</td></tr><tr><td>Isofluran</td><td>Baxter</td><td></td><td>2.5%, anesthetic</td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt;/NO&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>70/30 mixture, anesthetic</td></tr><tr><td>Rhodamin-Dextran</td><td>Invitrogen</td><td></td><td>2 MDa, 10 mg/ml, Vascular staining</td></tr><tr><td>Ly6G-PE</td><td>Becton-Dickinson</td><td></td><td>clone 1A8, neutrophils staining</td></tr><tr><td>Stereotactic holder</td><td>Home made</td><td></td><td>surgery</td></tr><tr><td>Ethanol 70%</td><td></td><td></td><td>surgery</td></tr><tr><td>Sterile scissors and nippers</td><td></td><td></td><td>surgery</td></tr><tr><td>Rubber ring</td><td></td><td></td><td>18 mm diameter, surgery</td></tr><tr><td>Glubran 2</td><td>Queryo Medical</td><td></td><td>Surgical Glue, rubber ring fixation</td></tr><tr><td>Small gauge needles</td><td>Terumo</td><td></td><td>surgery</td></tr><tr><td>Zeiss LSM 710 NLO multiphoton microscope&amp;nbsp;</td><td>Carl Zeiss</td><td></td><td>Microscope</td></tr><tr><td>Ti:Sapphire crystal laser&amp;nbsp;</td><td>Coherent Chameleon Ultra</td><td></td><td>140 fsec pulses of NIR light</td></tr><tr><td>Zen 2010</td><td>Carl Zeiss</td><td></td><td>Acquistion Software</td></tr><tr><td>Imaris Bitplane&amp;nbsp;</td><td>Bitplane</td><td></td><td>Analysis Software, 3D automatic tracking</td></tr><tr><td>PBS 1X</td><td>D. Dutscher</td><td></td><td>surgery</td></tr><tr><td>Thermostated chamber</td><td>Carl Zeiss</td><td></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.

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

9.5K 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

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 ...

Ricerca

Immunologia e infezioni

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 ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Femorale aspirazione del midollo osseo in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Medicina

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Imaging neutrofili e monociti in mesenterica Veins intravitale Microscopia su topi anestetizzati in tempo reale

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

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, ...

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.

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 ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

Preparazione del midollo osseo intero per l'analisi della citometria di massa delle cellule di lignaggio neutrofilo

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Biologia

<meta charset="utf8"></head><body>Iniezione intrafemorale per lo studio del midollo osseo in topi vivi

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. In murine models, this technique has been adapted to enhance the seeding efficiency of transplanted hematopoietic stem and progenitor cells (HSPCs). This paper describes a detailed step-by-step technical procedure of IF injection and the following bone marrow (BM) aspiration in mice that allows for serial characterization of cells present in the BM. This method enables the transplantation of valuable samples with low cell numbers that are particularly difficult to engraft by intravenous injection. This procedure facilitates the creation of xenografts that are critical for pathological analysis. While it is easier to access peripheral blood (PB), the cellular composition of PB does not reflect the BM, which is the niche for HSPCs. Therefore, procedures providing access to the BM compartment are essential for studying hematopoiesis. IF injection and serial BM aspiration, as described here, allow for the prospective retrieval and characterization of cells enriched in the BM, such as HSPCs, without sacrificing the mice.

<meta charset="utf8"></head><body>Le iniezioni intrafemorali semplificate con topi vivi consentono un'analisi continua del midollo osseo

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

Le iniezioni intrafemorali semplificate con topi vivi consentono un'analisi continua del midollo osseo

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. ...

Monitoraggio midollo osseo di topo monociti

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Capitoli in questo video

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

Isolamento e iniezione endovenosa di Murine derivate dal midollo osseo monociti

Femorale aspirazione del midollo osseo in Live Mice

Imaging neutrofili e monociti in mesenterica Veins intravitale Microscopia su topi anestetizzati in tempo reale

Femore finestra del modello Camera per

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

Preparazione del midollo osseo intero per l'analisi della citometria di massa delle cellule di lignaggio neutrofilo

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

Le iniezioni intrafemorali semplificate con topi vivi consentono un'analisi continua del midollo osseo

Le iniezioni intrafemorali semplificate con topi vivi consentono un'analisi continua del midollo osseo