Questo lavoro è stato finanziato da pilota e Borsa di studio di fattibilità da UCLA-CURE Center, il morbo di Crohn e Colite Foundation of Career Development Award America (# 2691) e Istituto Superiore di Sanità NIDDK K01 (DK084256) finanziamenti per Hon Wai Koon. Midollo osseo irradiazione operazione è stata assistita da Bernard Levin e cucina Scott di UCLA Center for Research mouse AIDS / umano Core Facility Chimera. Citometria di flusso operazione è stata assistita da impianto UCLA Nucleo Vector.

1. Prima-voi-iniziare Considerazioni tecniche <ol><li> Si consiglia di utilizzare entrambi i C57/BL6 topi wild-type e topi knockout per l&#39;esperimento perché i topi con le molecole CD corrispondenti si possono acquistare dai fornitori principali del mouse. </li><li> Poiché è difficile tenere traccia dei topi wild-type e topi knockout derivate cellule del donatore dopo il trapianto di midollo osseo, è necessario utilizzare CD45.1 topi per rappresentare topi wild-type. (CD45.1 C57/SJL topi WT magazzino Jackson Laboratories # 002014). </li><li> La maggior parte delle colonie del mouse sono CD45.2. I nostri topi knockout appartengono CD45.2 genotipo (in alternativa un CD45.2 fonte topi WT: Jackson Laboratories # 000664). La determinazione di CD45.1 o CD45.2 genotipo può essere eseguita utilizzando la citometria a flusso del midollo osseo e del sangue periferico come indicato al punto 7 in questo protocollo. </li><li> I topi dopo l&#39;irradiazione hanno compromesso l&#39;immunità. Tenere topi in patogeni servizio gratuito. </li><li> Funzionamento di irradiatore richiede di sicurezza speciale clearance e formazione. Per risparmiare tempo nei processi di formazione e di approvazione, è meglio collaborare con un laboratorio con personale qualificato che è in grado di operare l&#39;irradiatore. </li><li> Molte istituzioni hanno laboratori di citometria a flusso con i tecnici esperti. Per ridurre al minimo i problemi con il funzionamento di citometria a flusso, è meglio per discutere il vostro piano di flusso citometria a sperimentare con il tecnico esperto prima di iniziare. </li><li> I topi sono stati assegnati in questo modo (10 topi per gruppo): </li></ol><table border="1"><tbody><tr><td> Gruppo </td><td></td><td> Donatori di midollo osseo al destinatario </td><td> Trattamento </td></tr><tr><td> La </td><td> BM scambio </td><td> CD45.1 WT a CD45.2 KO </td><td> DSS colite </td></tr><tr><td> B </td><td></td><td></td><td> Acqua normale controllo </td></tr><tr><td> C </td><td> BM scambio </td><td> CD45.2 KO a CD45.1 WT </td><td> DSS colite </td></tr><tr><td> D </td><td></td><td></td><td> Acqua normale controllo </td></tr><tr><td> E </td><td> Finzione </td><td> CD45.1 WT a CD45.1 WT </td><td> DSS colite </td></tr><tr><td> F </td><td></td><td></td><td> Acqua normale controllo </td></tr><tr><td> Sol </td><td> Finzione </td><td> CD45.2 KO a CD45.2 KO </td><td> DSS colite </td></tr><tr><td> H </td><td></td><td></td><td> Acqua normale controllo </td></tr></tbody></table> * Una breve illustrazione del protocollo sperimentale è mostrato in Figura 1. 2. Destinatario Mice irradiazione <ol><li> I topi sono allevati e tenuti in esente da organismi patogeni struttura. Luogo in topi torta irradiazione autoclavato con filtro. Mettere un mouse in ciascuno slot di torta di irradiazione. Mettere la torta in irradiatore e assicurarsi che il giradischi e la torta si rivolgono. </li><li> Accendere la pompa ad ariaper la ventilazione. Chiudere la porta irradiatore e bloccarlo. </li><li> Irradiare i topi con 1000 rad (che è equivalente a 10 Gy) circa 10 minuti (a seconda della fonte di radiazione e emivita). Da questo punto di tempo, i topi irradiati sono immuno-compromessi e hanno l&#39;immunità debole contro le infezioni. Dopo l&#39;irraggiamento, prendere la torta fuori e mettere in un recipiente sterile per il trasporto alla biosicurezza armadio in stabulario. Evitare di esporre i topi all&#39;ambiente esterno per minimizzare possibilità di infezione. </li><li> In armadio biosicurezza, trasferire i topi in gabbia topo autoclave con biancheria da letto autoclavato (4 topi per gabbia Aggiungi nuovo acqua sterile con sospensione sulfatrim -. 3,12 ml per 100 ml di acqua). </li><li> Avvolgere la bottiglia d&#39;acqua con un foglio di alluminio come gli antibiotici sono sensibili luce. Agitare per miscelare il flacone soluzione sulfatrim ben prima del caricamento alla gabbia. </li></ol> 3. Bone Marrow Donor Estrazione <ol><li> Sacrificio topi con anidride carbonica o isoflurzione e poi immergerlo in soluzione disinfettante 1:200. Spruzzare i topi con etanolo 70% per bagnare la pelle e sezionare ossa aperte. </li><li> Immergere gli strumenti di dissezione in etanolo al 70% per la sterilizzazione. </li><li> Eseguire la dissezione in un armadio biosicurezza. Utilizzare gli strumenti di dissezione sterili per esporre l&#39;omero, femore, tibia e perone ossa, libero di attaccarsi muscoli e legamenti. </li><li> Tagliare le due estremità delle ossa per mostrare il midollo osseo rosso. Tenere le ossa con una pinza. Lavare il midollo osseo rosso con PBS freddo con siringa da 3 ml e aghi 25G di una capsula di Petri. Lavare da entrambe le estremità delle ossa per produrre le cellule del midollo osseo più. </li><li> Utilizzare 6 siringhe ml e 18G1 / 2 aghi per rompere le spine rosse del midollo osseo attraverso ripetute aspirazione ed espulsione. Trasferire midollo osseo con 50 ml provette Falcon. </li><li> Girare il midollo osseo nel 1800 rpm per 5 min a 4 ° C. Gettare il surnatante, risospendere pellet vortexando per 5-10 sec. </li><li> Diluire 10 volte tampone di lisi degli eritrociti con acqua sterile. Annunciod 1X tampone di lisi RBC (5 ml per topo donatore), vortex ancora per 5 sec e mantenere esattamente 3 min a temperatura ambiente. </li><li> Dopo 3 minuti, riempire i tubi con 20 ml di PBS freddo per fermare la lisi e mescolare. Versare il contenuto attraverso un colino cellula 40 micron direttamente in un nuovo tubo da 50 ml Falcon. </li><li> Sciacquare i tubi originali con 5 ml di PBS e trasferire al filtro cella pure. Top a 50 ml con PBS e mescolare. </li></ol> 4. Conteggio osseo cellule del donatore di midollo <ol><li> Aggiungere 100 pl di Trypan blu ad un tubo Eppendorf e 100 pl di sospensione di midollo osseo di un tubo Eppendorf e mescolare. Pipettare la miscela macchiato cella per un emocitometro. </li><li> La quantità di cellule è calcolato conta totale delle cellule del midollo osseo che vivono in provette Falcon = numero di cellule non colorate in 16 quadrati x 2 (a causa della diluizione Trypan blu) x 10.000 x 50 ml </li><li> Centrifugare le cellule del midollo osseo in provette Falcon da 50 ml a 2000 rpm per 5 min a 4 ° C. </li><li> Rimuovere il surnatante. Basatosulle conta cellulare totale, risospendere il pellet con PBS per 1 x 10 8 cellule per ml </li></ol> 5. Infusione di topi riceventi <ol><li> Riscaldare le topi riceventi su un tappetino di calore e sotto una lampada di riscaldamento. Mettere i topi riceventi in dispositivo di immobilizzazione. Iniettare 1 x 10 7 cellule per topo in 100-200 microlitri via endovenosa. </li><li> L&#39;iniezione di midollo osseo del donatore deve essere effettuato tra 4-24 ore dopo l&#39;irradiazione. </li><li> Tieniti 4 topi iniettati per gabbia. Mantenere i topi riceventi iniettati in gabbie autoclave con acqua trattata con antibiotici per le prime 4 settimane di patogeni stabulario libero e lasciare che i topi riconquistare l&#39;immunità. Gabbie Change, trattata con antibiotici acqua e cibo ogni 4 giorni per mantenere l&#39;igiene. </li></ol> 6. L&#39;induzione di colite e di valutazione del colite <ol><li> 4 settimane dopo il trapianto di midollo osseo e di irradiazione, passare l&#39;acqua normale senza antibiotici e mantenere i topi per 2 settimane per riguadagnare la loro normalegut microflora. </li><li> 6 settimane dopo l&#39;iniezione di midollo osseo, misurare il peso corporeo iniziale. Alcuni topi sono date 5% destrano solfato in acqua potabile ad libitum per indurre colite. Alcuni topi sono dato acqua potabile regolare solo come gruppi di controllo normali. </li><li> 5 giorni dopo, prelevare il sangue periferico, il trasferimento di eparina provette (Vacutainer) e tenere in ghiaccio. Piscina il sangue da 4 topi a 1 tubo. Combinano 300 microlitri di sangue con 300 pl di tampone di colorazione della cellula = 600 microlitri. Dividere il sangue per 5 provette Eppendorf, ciascuno con 100 ul. </li><li> Sacrifica i topi con l&#39;anidride carbonica o isoflurano. Valutare gli spartiti danni macroscopici (si prega di fare riferimento a un&#39;altra pubblicazione dei video JOVE per i dettagli 1). Misurare modifica di peso corporeo. Misurare la lunghezza dell&#39;intestino e spessore con calibro digitale. Osservare consistenza feci (duro, morbido o di sangue). Determinare sangue occulto nelle feci con Hemooccult. Staccare i tessuti del colon e fissare in formalina per la colorazione H &amp; E. </li><li> Dissect 1 femore bone per topo, estrarre il midollo osseo e lavare il midollo osseo con PBS freddo tramite l&#39;ago 25G e siringa da 1 ml. Girone 4 midollo osseo tappi a 1 tubo e tenere in ghiaccio. </li><li> Versare il midollo osseo a Petri, taglio la spina midollo osseo con l&#39;ago 18G e 5 volte siringa ml più fino a quando non spina midollo osseo è presente. </li><li> Spin down con 1.500 rpm per 5 minuti a 4 ° C. Rimuovere il surnatante e risospendere con 600 microlitri di buffer colorazione delle cellule. Dividere il midollo osseo risospeso a 5 provette Eppendorf, ciascuno con 100 ul. </li></ol> 7. Controllo qualità del trapianto di midollo osseo mediante citometria a flusso <ol><li> Etichettare ogni provette di sangue o di midollo osseo # 1-5: </li><li> Preparare la miscela di anticorpi per ogni rispettivo tubo al buio. </li></ol> # 1 Nessun anticorpo # 2 30 microlitri controllo isotipico FITC + 30 pl PE isotipo di controllo + 15 pl CD16/32 blocco # 3 30 microlitri FITC CD45.1 Ab + 15 pl CD16/32 blocco # 4 30 microlitri PECD45.2 Ab + 15 pl CD16/32 blocco # 5 30 microlitri FITC CD45.1 Ab + 30 microlitri PE CD45.2 Ab + 15 pl CD16/32 blocco <table border="1"><tbody><tr><td> Aggiungi nulla da sangue o del midollo osseo di esempio # 1 </td></tr><tr><td> Aggiungere 5 ml di miscela di anticorpi # 2 per ciascun campione di sangue o midollo osseo # 2 </td></tr><tr><td> Aggiungere 3 ml di miscela di anticorpi # 3 per ciascun campione di sangue o midollo osseo # 3 </td></tr><tr><td> Aggiungere 3 ml di miscela di anticorpi # 4 per ciascun campione di sangue o midollo osseo # 4 </td></tr><tr><td> Aggiungere 5 ml di miscela di anticorpi ° 5 di ciascun campione di sangue o midollo osseo # 5 </td></tr></tbody></table><ol start="3"><li> Conservare in buio in ghiaccio per 30 min. </li><li> Aggiungere 2 ml di 1X RBC Lysis Buffer ad ogni provetta. Conservare al buio in ghiaccio per 15 min. </li><li> Centrifugare le cellule a 1.500 rpm, 5 min a 4 ° C. (GH 3,8 rotore, 1.500 giri = 350 xg) Rimuovere il surnatante. Risospendere il pellet con 500 microlitri di buffer colorazione cella al buio, brevemente e poi vortice. </li&gt; <li> Controllare il CD45.1 (che rappresenta WT) e CD45.2 (rappresentando KO) nel sangue immunocolorate e campioni di midollo osseo mediante citometria a flusso. Selezionare FITC per rappresentare WT CD45.1 e PE per rappresentare KO CD45.2. </li><li> Analizzare i risultati citometria a flusso dal software FlowJo. Calcolare il rapporto di FITC: Rapporto PE o PE: rapporto FITC per determinare se il genotipo dei donatori è genotipo dominante nel sangue e nel midollo osseo. </li></ol>

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Nessun conflitto di interessi dichiarati.

Questo approccio di midollo osseo trapianto è adatto per la ricerca di immunologia colite, infezioni, cancro, obesità e altre malattie. Questo esperimento midollo osseo trapianto è necessaria quando il gene d&#39;interesse è espresso in entrambi derivate dal midollo osseo e non le cellule del midollo osseo derivate e il gene d&#39;interesse si sospetta di mediare malattia da parte delle cellule da entrambe le popolazioni. Per esempio, peptide antimicrobico cathelicidin viene mostrato per modulare colite acuta. Ma è...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Cella colorazione tampone </td><td> Biolegend </td><td> # 420201 </td></tr><tr><td> 10X tampone di lisi RBC </td><td> Biolegend </td><td> # 420301 </td></tr><tr><td> FITC isotipo di controllo del mouse </td><td> Biolegend </td><td> # 400207 </td></tr><tr><td> PE del mouse isotipo di controllo </td><td> Biolegend </td><td> # 400211 </td></tr><tr><td> PE anti-topo CD45.2 </td><td> Biolegend </td><td> # 109807 </td></tr><tr><td> FITC anti-topo CD45.1 </td><td> Biolegend </td><td> # 110705 </td></tr><tr><td> Anti-mouse CD16/32 blocco </td><td> Biolegend </td><td> # 101301 </td></tr><tr><td> 40 filtro cella pm </td><td> FISHERBRAND </td><td> # 22363547 </td></tr><tr><td> PBS 1X </td><td&gt; MP Biomedicals </td><td> # 1860454 </td></tr><tr><td> Eparina </td><td> FISHERBRAND </td><td> # BP2425 </td></tr><tr><td> Sulfatrim (SMZ) </td><td> Qualitest </td><td> NC9242720 (fisher) </td></tr><tr><td> Lysol IC </td><td> E vinci la scientifica </td><td> NC9745686 (fisher) </td></tr><tr><td> Vaccutainer </td><td> BD </td><td> # 8000813 </td></tr><tr><td> Mouse restrainer </td><td> Braintree scientifico </td><td> # TV-150 </td></tr><tr><td> Irradiator </td><td> JL Pastore e Associati </td><td> Mark I 68A </td></tr><tr><td> Citometro di flusso </td><td> BD </td><td> BD FACSCanto II </td></tr><tr><td> Citometro di flusso provetta </td><td> Falco </td><td> # 352052 </td></tr><tr><td> Calibro digitale </td><td> FISHERBRAND </td><td> 14-648-17 </td></tr><tr><td> Hemoccult ICT </td><td> Beckman Coulter </td><td> G0328QW </td></tr&gt; </tbody></table>

differentiating functional roles of gene expression from immune and non-immune cells in mouse colitis by bone marrow transplantation

Per capire il ruolo di un gene nello sviluppo della colite, abbiamo confrontato le risposte dei topi wild-type e geni di interessi topi knockout carenti da colite. Se il gene d&#39;interesse è espressa in cellule di midollo osseo derivate entrambe e non le cellule del midollo osseo derivate dell&#39;ospite, tuttavia, è possibile differenziare il ruolo di un gene di interesse in cellule derivate dal midollo osseo e non midollo osseo cellule derivate da tecnica di trapianto di midollo osseo. Per modificare il midollo osseo derivate genotipo cellule dei topi, il midollo osseo originale di topi riceventi sono stati distrutti mediante irradiazione e poi sostituito da midollo osseo del donatore di nuovo genotipo diverso. Quando wild-type topi donatore di midollo osseo è stato trapiantato in topi knockout, potremmo generare topi wild-type con l&#39;espressione del gene in cellule derivate dal midollo osseo. In alternativa, quando i topi knockout donatore di midollo osseo è stato trapiantato in topi wild-type destinatario, topi wild-type, senza gene d&#39;interesse che esprimeda cellule di midollo osseo derivate sono state prodotte. Tuttavia, il trapianto di midollo osseo non può essere completa al 100%. Pertanto, abbiamo utilizzato cluster di differenziazione (CD) molecole (CD45.1 e CD45.2) come marcatori di cellule del donatore e del ricevente per monitorare la proporzione di cellule del midollo osseo del donatore ottenuti in topi riceventi e il successo del trapianto di midollo osseo. Topi wild-type con CD45.1 topi knockout con genotipo e CD45.2 genotipo sono stati utilizzati. Dopo irradiazione di topi riceventi, le cellule ossee donatore di midollo di diversi genotipi sono stati infusi nei topi riceventi. Quando il midollo osseo nuovo rigenerato di prendere in consegna la sua immunità, i topi sono stati sfidati da agenti chimici (destrano solfato di sodio, 5% di DSS) per indurre colite. Qui abbiamo anche mostrato il metodo per indurre colite in topi e valutare il ruolo del gene di interesse espresso da cellule di midollo osseo derivate. Se il gene d&#39;interesse dalle cellule ossee derivate svolge un ruolo importante nello sviluppo della malattia (come Escherichiatis), il fenotipo dei topi riceventi con trapianto di midollo osseo può essere significativamente modificati. Alla fine della sperimentazione colite, il midollo osseo derivate cellule nel sangue e midollo osseo sono state marcate con anticorpi contro CD45.1 e CD45.2 e il loro rapporto quantitativo di esistenza potrebbe essere utilizzato per valutare il successo del trapianto di midollo osseo mediante citometria a flusso. Riuscito trapianto di midollo osseo dovrebbe mostrare una vasta maggioranza di genotipo dei donatori (in termini di marcatori molecola CD) nel genotipo destinatario sia nel midollo osseo e nel sangue di topi riceventi.

Se il gene d&#39;interesse gioca un ruolo significativo in cellule immunitarie durante lo sviluppo della colite, i topi che hanno ricevuto midollo osseo di genotipo diverso attraverso il trapianto di midollo osseo (WT per KO o KO per WT) dovrebbe avere un&#39;alterata risposta alla colite DSS. Uno dei parametri più importanti per determinare la gravità della colite è la H &amp; E colorazione dei tessuti del colon. Colon cambiamenti della struttura dei tessuti e dei segni di infiammazione può essere quantitativamente...

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

Il trapianto di midollo osseo fornisce un modo per modificare il genotipo delle cellule del midollo osseo derivate. Se il gene di interesse viene espresso in cellule di midollo osseo sia derivati ​​e non le cellule del midollo osseo derivate, trapianto di midollo osseo può modificare le cellule del midollo osseo derivate da un genotipo diverso senza cambiare la non-genotipo delle cellule del midollo osseo derivate.

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 ...

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Research

JoVE Journal

Immunology and Infection

17.3K Views.  The University of California Los Angeles, Los Angeles. Il trapianto di midollo osseo fornisce un modo per modificare il genotipo delle cellule del midollo osseo derivate. Se il gene di interesse viene espresso in cellule di midollo osseo sia derivati ​​e non le cellule del midollo osseo derivate, trapianto di midollo osseo può modificare le cellule del midollo osseo derivate da un genotipo diverso senza cambiare la non-genotipo delle cellule del midollo osseo derivate.

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

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Isolamento di cellule cardiache funzionali immunitario

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Ricerca

Immunologia e infezioni

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 ...

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.

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 ...

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

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Generazione e identificazione di GM-CSF Derivato alveolo-come macrofagi e dendritiche cellule da midollo osseo di topo

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

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 ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

Isolamento delle cellule mononucleari Di Lamina propria dal colon Murine utilizzando Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

Piattaforma di trapianto di midollo osseo per studiare il ruolo delle cellule dendritiche nella malattia di Graft-versus-Host

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

Procedure di trapianto di midollo osseo nei topi per studiare l'ematopoiesi clonale

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

Gene Knock-in di CRISPR/Cas9 e smistamento cellulare in macrofagi e linee cellulari T

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...