Questo lavoro è stato sostenuto dalla concessione NS64571 dal NINDS (CLH), con un premio primi stadi dello sviluppo della carriera della Mayo Clinic (CLH), e da un generoso regalo di Donald e Frances Herdrich (CLH). Vorremmo ringraziare il Core Mayo Clinic Citometria a flusso per l&#39;assistenza.

1. Virus iniezione intracranica: La seguente tecnica è stato modificato ed utilizzato ampiamente dal nostro laboratorio e colleghi. In breve, l&#39;iniezione di tensione intracranica Daniel di Theiler ha murino encefalomielite virus (TMEV) o sham-infezione (1, 10) viene eseguita su topi giovani (preferibilmente 5-6 settimane di età), per sollecitare leucociti cervello infiltrante (Bils). Si prega di notare che i risultati saranno diversi tra il ceppo di Daniel, il ceppo BEAN, e il ceppo GDVII. Ai fini della raccolta Bils, topi ricevere 2x10 5 PFU di TMEV e sono infettate per 24 ore. <ol><li> Applicare l&#39;ago di iniezione (27 gauge, ¼, Kendall), a un automatico siringa da 1 ml Hamilton e impostato a consegnare 10 ml di virus. </li><li> Redigere TMEV a 10 L DMEM nella siringa con particolare attenzione a bolle d&#39;aria. Se del caso, i topi infettati farsa ricevere 10 ml di virus DMEM. </li><li> Poco prima iniezione, versare circa 1 ml di isoflurano in una campana di vetro che contiene una griglia metallica con il substrato di cotone sotto. </li><li> Topi luogo direttamente sui griglia metallica nella campana di vetro fino a quando diventano leggermente anestetizzata, insensibile ai piedi, pizzico e immobilizzato dai inalanti anestetico, circa 10-15 secondi. I topi non devono venire a contatto diretto con isoflurano, come sostanza può essere irritante e caustico. </li><li> Mentre è sotto anestesia, preparare i topi per l&#39;iniezione di trovare velocemente coordinate appropriate nella regione di corteccia frontale del cranio con barba e inchiostrazione. La posizione generale per l&#39;iniezione è di 1 mm anteriore al bregma e 1 mm laterale per la sutura sagittale sul lato destro (Figura 1). Mentre l&#39;iniezione stereotassica può essere appropriato per alcune situazioni sperimentali, abbiamo scoperto che a mano libera iniezione senza barba o inchiostrazione è adatto per la maggior parte degli esperimenti. Ripartito il pelo con la punta dell&#39;ago lungo entrambi gli assi è appropriato per l&#39;iniezione. </li><li> Per fare una iniezione, orientare la perpendicolare ago al cranio e stampa attraverso l&#39;osso a una profondità di circa 3 mm. </li><li> Esprimere virus nel cervello premendo il pulsante siringa Hamilton verso il basso e attendere 2 secondi per il virus di entrare nel cervello prima di ritirarsi. </li><li> Rimuovere con attenzione l&#39;ago e posizionare il mouse in un ambiente pulito, asciutto gabbia. I topi risvegliano dall&#39;anestesia in fretta e riprendere la normale funzione in pochi minuti. Prestare la debita attenzione ai topi che presentano sintomi anomali, come ad esempio un eccessivo sanguinamento dal sito di iniezione, come l&#39;eutanasia degli animali può essere appropriato. Una corretta gestione degli animali 6 e aderenza al National Institutes of Health e cura degli animali e le linee guida istituzionali Comitato uso devono essere seguite. </li></ol> 2. Brain-infiltrazione dei leucociti preparazione delle cellule: <ol><li> Preparare a fondo rotondo provette Oak Ridge che può contenere una capacità di 30 ml con 10 ml di RPMI 1640, 9 ml di Percoll e 1 ml di PBS 10X e tubi dovrebbero sedersi a temperatura ambiente in panchina durante il processo di rimozione del cervello. </li><li> Portare Ficoll-Paque Plus per temperatura ambiente (RT). </li><li> Eutanasia animale modificato isoflurano overdose 5 e rimuovere rapidamente il cervello utilizzando un tessuto in acciaio inox spatola e mettere in un tubo da 15 ml contenente 5 ml RPMI sul ghiaccio. In alcune circostanze, PBS-perfusione può essere opportuno eliminare le cellule circolanti nel sangue periferico dalla preparazione Bils. I topi devono essere adeguatamente e completamente anestetizzato prima di PBS-perfusione. </li><li> Trasferimento del cervello e la soluzione RPMI da ml 7 di vetro Pyrex tessuto marchio Tenbroeck smerigliatrice e delicatamente omogeneizzare con 10-15 colpi. </li><li> Pipettare altri 5 ml in RPMI l&#39;omogeneizzatore e pipetta su e giù due volte. Trasferimento cervello omogenato (circa 10 ml) per precedentemente preparato a fondo rotondo tubo e capovolgere delicatamente 2-3 volte per mescolare. </li><li> Spin omogenato cerebrale ave a 7800g per 30 min a RT in un angolo fisso F0360 in un rotore Beckman Allegra X-22R centrifuga clinica. </li><li> Dopo la centrifugazione, rimuovere i detriti della mielina che ha galleggiato alla parte superiore del gradiente. Raccogliere lo strato di leucociti che fluttua sopra il pellet di cellule rosse del sangue (Figura 2). </li><li> Ceppo strato di leucociti attraverso un filtro a 40 micron di cellule in un 50 ml conica. Portare soluzione a 50 ml di volume con RMPI. </li><li> Spin tubi contenenti sospensione diluita di cellule in una centrifuga clinica a 1500 rpm (600g ave) per 5 minuti a temperatura ambiente. </li><li> Aspirare il supernatante e raccogliere il pellet di cellule. Risospendere il pellet in 1 ml di FACS tampone (1% di sieroalbumina bovina, sodio azide 0,02% di calcio e magnesio senza PBS) e trasferire la sospensione cellulare a 5 ml tubi fondo rotondo FACS. </li><li> Attenzione alla base della sospensione con 1 ml di Ficoll-Paque Plus. </li><li> In provetta a 2500 giri al minuto (1400 diede) in centrifuga clinica per 25 minuti a temperatura ambiente, senza freno. </li><li> Rimuovere bianco, strato soffice a livello di interfaccia (Figura 2) con la pipetta da 1 ml e trasferimento al nuovo 5 ml di tubo di FACS. Lavare cells con 4 ml di tampone FACS. </li><li> Rotazione del tubo a 1500 rpm (600g ave) per 5 min a RT in centrifuga clinica alle cellule pellet. </li><li> Aspirare il surnatante e raccogliere pellet cellulare. Risospendere il pellet di leucociti nel tampone appropriato e tenere sul ghiaccio in nuovi tubi FACS. </li><li> Contare le celle, valutare la vitalità cellulare per esclusione Blu tripano e analizzare mediante citometria a flusso. In generale, gli investigatori si può aspettare di acquisire circa 5 x 10 5 cellule / animale. </li></ol> 3. Citometria a flusso immunofenotipo <ol><li> Dopo aver isolato e il conteggio dei leucociti, le cellule rotazione per 3 minuti a 1500 rpm (600 ha) in una centrifuga clinica. </li><li> Risospendere il pellet in tampone bloccante contenente quanto segue: buffer di FACS, sopranatante 2.4G2 ibridoma (Fc block; anti-CD16/32) e siero fetale bovino con un rapporto di 10:05:01 e incubare per 30 minuti a 4 ° C . </li><li> Preparare e aggiungere anticorpi coniugati contro gli antigeni extracellulari alle cellule bloccate ad una concentrazione di 1:200 e incubare per 30 minuti a 4 ° C al buio. Macchia e incubare cellule in 200 microlitri di una 96-e V-piastra inferiore. Controlli appropriati includono le cellule senza macchia e campioni di compensazione fluorocromo, se necessario. </li><li> Gira la cellule colorate a temperatura ambiente per 3 minuti a 1500 rpm (600 ha) in una centrifuga clinica utilizzando un appropriato rotore per piastre da 96 pozzetti. </li><li> Aspirare il surnatante e lavare le cellule con 200 microlitri di buffer FACS pipettando gentilmente su e giù per 3 volte. </li><li> Ripetere la tecnica di lavaggio sopra due volte. </li><li> Dopo il lavaggio, risospendere le cellule nel 2% paraformaldeide e trasferimento in tubi di FACS. Fissaggio è necessario per citometria a flusso delle cellule infette del livello di biosicurezza 2 reagenti. </li><li> Eseguire le cellule fissato su un BD FACS Calibur seguendo un metodo di citometria a flusso modificato 8 e analizzare i file non in linea con FlowJo, WinMDI o altri software disponibili citometria a flusso di analisi. </li><li> L&#39;analisi di 50-100.000 eventi per campione è generalmente richiesto per immunofenotipizzazione adeguata. </li></ol> Gli anticorpi coniugati direttamente utilizzati in questo esperimento: CD45 è stata rilevata con clone 30-F11. Ly6C / G è stata rilevata con clone Gr1, RB6-8C5. CD11b è stato rilevato con clone M1/70. F4/80 è stata rilevata con clone BM8. 4. Rappresentante dei risultati: Mostriamo i risultati fenotipizzazione cellulare per Bils del mouse a 24 ore dopo l&#39;infezione (Figura 3). Leucociti sono state colorate con PerCP-coniugato anti-CD45 del mouse per rilevare le cellule immunitarie, PE-coniugato anti-topo Ly6C / G per rilevare monociti infiammatorie, APC-coniugato anti-topo CD11b e APC-coniugato anti-topo F4/80 per rilevare le cellule di lignaggio monociti. L&#39;analisi è stata eseguita con uno strumento BD FACS Calibur. <img src="/files/ftp_upload/2747/2747fig1.jpg" alt="Figura 1"/> Figura 1. Localizzazione anatomica del sito di iniezione. Il sito di iniezione si trova 1 millimetro anteriore al bregma e 1 mm laterale per la sutura sagittale sul lato destro. <img src="/files/ftp_upload/2747/2747fig2.jpg" alt="Figura 2"/> Figura 2. Leucociti illustrazione di separazione gradiente di leucociti e Bils. Sono inizialmente raccolti direttamente sotto il livello detriti mielina e sopra il pellet RBC nel gradiente Percoll. Il bianco, strato soffice (Bils) presso l&#39;interfaccia è raccolto dal gradiente di Ficoll. <img src="/files/ftp_upload/2747/2747fig3.jpg" alt="Figura 3"/> Figura 3. Immunofenotipo del cervello infilatrating leucociti a 24 ore dopo l&#39;infezione. Leucociti del mouse sono stati isolati dal cervello per centrifugazione differenziale densità del tessuto omogenato preparati con gli animali a 24 ore dopo l&#39;infezione intracranica. Le cellule sono state colorate con anticorpi coniugati contro la fluorescenza-topo CD45, Ly6C / G, CD11b e F4/80. Gating iniziale è stata eseguita tracciando CD45, un marker per le cellule immunitarie, contro la dispersione in avanti (FSC), un indicatore della dimensione della cella (C). L&#39;hi CD45 (A, D), lo CD45 (B, E), e CD45 neg (F, G), le popolazioni sono stati poi analizzati per i livelli di espressione relativa dei marcatori di monociti e macrofagi Ly6C / G, F4/80 (A, B , F), e CD11b (D, E, G). Ly6C / G + + CD11b F4/80 + monociti infiammatori sono stati trovati quasi esclusivamente nella popolazione CD45 hi (A, D), in linea con l&#39;infiltrazione di queste cellule dalla periferia. Al contrario, lo Ly6C/G-F4/80 CD11b + macrofagi sono stati trovati quasi esclusivamente nel CD45 lo (B, E) e CD45 neg (G) le popolazioni, coerentemente con un fenotipo residente. In numerosi esperimenti, non abbiamo mai osservato le cellule CD45 hi o Ly6C / G + + CD11b F4/80 + monociti infiammatorie nel cervello dei non infetti o sham-infezioniTed topi (dati non riportati).

<ol>
	<li>Buenz, E. J., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picornaviruses+and+cell+death.">Picornaviruses and cell death.</a> Trends Microbiol. 14, 28-36 (2006).</li><li>Buenz, E. J., Limberg, P. J., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+3-parameter+flow+cytometry-based+cell+death+assay.">A high-throughput 3-parameter flow cytometry-based cell death assay.</a> Cytometry A. 71, 170-173 (2007).</li><li>Deb, C., Lafrance-Corey, R. G., Zoecklein, L., Papke, L., Rodriguez, M., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demyelinated+axons+and+motor+function+are+protected+by+genetic+deletion+of+perforin+in+a+mouse+model+of+multiple+sclerosis.">Demyelinated axons and motor function are protected by genetic deletion of perforin in a mouse model of multiple sclerosis.</a> J. Neuropath. Exp. Neurol. 68, 1037-1048 (2009).</li><li>Deb, C., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKG2D+contributes+to+efficient+clearance+of+picornavirus+from+the+acutely+infected+murine+brain.">NKG2D contributes to efficient clearance of picornavirus from the acutely infected murine brain.</a> J. Neurovirol. 14, 261-266 (2008).</li><li>Donovan, J., &amp;amp, P. . B. r. o. w. n., , P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Euthanasia.">Euthanasia.</a> Current Protocols in Neuroscience. , A.4H.1-A.4H.4 (2005).</li><li>Donovan, J., Brown, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Handling+&#38;+restraint.">Handling &#38; restraint.</a> Current Protocols in Immunology. 73, 1.3.1-1.3.6 (2006).</li><li>Donovan, J., Brown, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parenteral+injections.">Parenteral injections.</a> Current Protocols in Neuroscience. , A.4F.1-A.4F.9 (2005).</li><li>Holmes, K. L., Otten, G., Yokoyama, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+analysis+using+Becton+Dickinson+FACS+Calibur.">Flow cytometry analysis using Becton Dickinson FACS Calibur.</a> Current Protocols in Immunology. , 5.4.1-5.4.22 (2002).</li><li>Howe, C. L., Ure, D., Adelson, J. D., LaFrance-Corey, R., Johnson, A., Rodriguez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T+cells+directed+against+a+viral+peptide+contribute+to+loss+of+motor+function+by+disrupting+axonal+transport+in+a+viral+model+of+fulminant+demyelination.">CD8+ T cells directed against a viral peptide contribute to loss of motor function by disrupting axonal transport in a viral model of fulminant demyelination.</a> J Neuroimmunol. 188, 13-21 (2007).</li><li>Lipton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theiler's+virus+infection+in+mice:+An+unusual+biphasic+disease+process+leading+to+demyelination.">Theiler's virus infection in mice: An unusual biphasic disease process leading to demyelination.</a> Infect Immun. 11, 1147-1155 (1975).</li></ol>

Nessun conflitto di interessi dichiarati.

Noi abitualmente uso di citometria a flusso per determinare sia la qualità della preparazione infiltrazione delle cellule cerebrali, e per distinguere le diverse popolazioni di cellule immunitarie 2, 9. Al acuta intervalli di tempo, il nostro metodo Bils rese elevate percentuali di monociti infiammatorio all&#39;interno della popolazione CD45hi così come alte percentuali di macrofagi nella popolazione CD45lo. Ciò indica che una risposta immunitaria riproducibile all&#39;interno del cervello possono essere ...

<table border="1"><tbody><tr><th> Reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> TMEV </td><td> Howe Lab </td><td> N / A </td><td></td></tr><tr><td> Ceppo di Daniel </td><td> Invitrogen </td><td> 21063-029 </td><td></td></tr><tr><td> isoflurano </td><td> Novaplus </td><td> NDC 0409-3292-49 </td><td></td></tr><tr><td> a fondo tondo da Oak Ridge centrifuga tubi </td><td> Nalgene </td><td> 3118-0030 </td><td></td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 11875-093 </td><td></td></tr><tr><td> Percoll </td><td> GE Healthcare </td><td> 17-0891-02 </td><td></td></tr><tr><td> 10X PBS </td><td> Roche </td><td> 11666789001 </td><td></td></tr><tr><td> Ficoll-Paque Plus. </td><td> GE Healthcare </td><td> 17-1440-02 </td><td></td></tr><tr><td> Tricloroetano 0,4% (w / v) </td><td> Mediatech </td><td> 25-900-CI </td><td></td></tr><tr><td> Provette da 15 ml </td><td> BD Falcon </td><td> 352097 </td><td></td></tr><tr><td> 7 ml di vetro Pyrex marchio Tenbroeck tessuto smerigliatrice </td><td> Pescatore </td><td> 08-414-10B </td><td></td></tr><tr><td> 40 micron filtro cellulare </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> 50 ml coniche </td><td> BD Falcon </td><td> 352070 </td><td></td></tr><tr><td> albumina di siero bovino </td><td> Sigma </td><td> A9647 </td><td></td></tr><tr><td> di sodio azide </td><td> Sigma </td><td> S8032 </td><td></td></tr><tr><td> CMF-PBS </td><td> Mediatech </td><td> 21-040-CV </td><td></td></tr><tr><td> FACS tubi </td><td> BD Falcon </td><td> 352054 </td><td></td></tr><tr><td> siero fetale bovino </td><td> Sigma </td><td> F4135 </td><td></td></tr><tr><td> 2.4G2 ibridomi </td><td> ATCC </td><td> HB-197 </td><td></td></tr><tr><td> Costar V-piastra inferiore </td><td> Corning </td><td> 3894 </td><td></td></tr><tr><td> Allegra X-22R centrifuga o equivalente </td><td> Beckman </td><td> N / A </td><td></td></tr><tr><td> 96-bene secchio piastra e rotore </td><td> Beckman </td><td> S2096 </td><td></td></tr><tr><td> Rotore ad angolo fisso </td><td> Beckman </td><td> F0360 </td><td></td></tr><tr><td> paraformaldeide </td><td> Sigma </td><td> P6148 </td><td></td></tr><tr><td> BD FACS Calibur </td><td> BD Biosciences </td><td> N / A </td><td></td></tr><tr><td> FlowJo 7,5 </td><td> Albero Star, Inc. </td><td> N / A </td><td></td></tr><tr><td> CD45 </td><td> BD Biosciences </td><td> 557235 </td><td> clone: ​​30-F11 </td></tr><tr><td> GR1 (Ly6C / G) </td><td> BD Biosciences </td><td> 553128 </td><td> clone: ​​RB6-8C5 </td></tr><tr><td> CD11b </td><td> ebiosciences </td><td> 17-0112-83 </td><td> clone: ​​M1/70 </td></tr><tr><td> F4/80 </td><td> ebiosciences </td><td> 17-4801-82 </td><td> clone: ​​BM8 </td></tr></tbody></table>

isolation of brain-infiltrating leukocytes

Descriviamo un metodo per la preparazione di leucociti cervello infiltrante (Bils) dai topi. Noi dimostrare come infettare topi con encefalomielite Theiler ha murino virus (TMEV) attraverso una tecnica rapida iniezione intracranica e come purificare un leucociti arricchito popolazione di cellule infiltrati dalla intero cervello. In breve, i topi vengono anestetizzati con isoflurano in una camera chiusa e sono a mano libera iniettato con una siringa Hamilton nella corteccia frontale. I topi sono poi uccisi in vari momenti dopo l&#39;infezione da overdose isoflurano e cervelli interi vengono estratti e omogeneizzato in RPMI con una smerigliatrice tessuto Tenbroeck. Omogenati cerebrali sono centrifugate con una pendenza costante Percoll 30% per rimuovere i detriti cellulari mielina e gli altri. La sospensione cellulare viene quindi filtrato a 40 micron, lavato e centrifugato su un discontinuo Ficoll-Paque Plus. gradiente di selezionare e purificare i leucociti. I leucociti vengono poi lavate e risospese in buffer appropriato per immunofenotipizzazione mediante citometria di flusso. Citometria a flusso rivela una popolazione di cellule del sistema immunitario innato nelle fasi precoci dell&#39;infezione nei topi C57BL / 6. A 24 ore dopo l&#39;infezione, più sottoinsiemi di cellule immunitarie sono presenti nel Bils, con una popolazione arricchita del Gr1 +, CD11b F4/80 + + e cellule. Pertanto, questo metodo è utile per caratterizzare la risposta immunitaria alle infezioni acute del cervello.

Watch this Scientific Journal Video about Isolation of Brain-infiltrating Leukocytes at JoVE.com

Isolation of Brain-infiltrating Leukocytes

Un metodo rapido per ottenere leucociti infiltrati dal cervello murino è descritto. Questo metodo utilizza una pendenza costante Percoll e gradiente discontinuo di Ficoll selezionare e purificare i leucociti arricchito strato. Leucociti isolati può quindi essere caratterizzata da misure di citometria a flusso.

Isolamento di Brain-infiltrazione Leucociti

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

isolation-of-brain-infiltrating-leukocytes

Research

JoVE Journal

Immunology and Infection

19.0K Views.  Mayo Clinic College of Medicine. Un metodo rapido per ottenere leucociti infiltrati dal cervello murino è descritto. Questo metodo utilizza una pendenza costante Percoll e gradiente discontinuo di Ficoll selezionare e purificare i leucociti arricchito strato. Leucociti isolati può quindi essere caratterizzata da misure di citometria a flusso.

Video: Isolation of Brain-infiltrating Leukocytes

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 of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Isolamento di cellule polmonari mouse dendritiche

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

Isolamento e analisi di Brain-sequestrati da leucociti<em&gt; Plasmodium berghei</em&gt; ANKA topi infettati

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Bioenergetica e il burst ossidativo: Protocolli per l&#39;isolamento e la valutazione dei leucociti umani e piastrine

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Analizzando gli effetti delle cellule stromali per il reclutamento dei leucociti da portata

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

L&#39;isolamento dei leucociti di Human Interface materno-fetale

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

L&#39;isolamento dei leucociti di tessuti murini all&#39;interfaccia materno-fetale

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Isolamento di leucociti infiltranti da mouse pelle utilizzando enzimatica Digest e Separazione Gradient

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

La raccolta selettiva di Marginating-polmonari leucociti

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

La raccolta selettiva di Marginating-epatiche leucociti

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...