Gli autori desiderano ringraziare la signorina Liana Mackiewicz per l&#39;assistenza tecnica. Questo lavoro è stato reso possibile grazie vittoriana sostegno governativo Infrastrutture Stato operativo e del governo australiano National Health and Medical Research Council IRIISS e Progetto di sovvenzione 1.031.212.

1. L&#39;infezione di topi con P. berghei-ANKA <ol><li> Sbrinamento un&#39;aliquota di crioconservati P. berghei ANKA PRBC. </li><li> Trattenere una malaria cerebrale resistente topo BALB / c donatore (8-12 settimane di età) con la tecnica a due mani di ritenuta. Iniettare il mouse con 100-200 ml di PRBC utilizzando un insulina siringa da 1 ml (28G ago). Di routine, 1-2 topi donatori vengono iniettati. </li><li> In 4-5 giorni post-infezione (pi) rimuovere topo donatore da gabbia e collocarla su una workstation o un tampone monouso di lavoro. </li><li> Delicatamente frenare il mouse alla fine della coda e con le forbici piccole tagliare la punta della coda (circa 1 mm). In alternativa, una foratura piccola coda usando un ago 25 G può essere eseguita. </li><li> Una goccia di sangue dalla vena della coda del topo in prossimità della fine di un frosted-end vetrino da microscopio. </li><li> Posizionare una seconda slitta (spreader) su un angolo di 45 ° e sostenerlo nella goccia di sangue. Una volta che il sangue si estende lungo il bordo, premere il divaricatore-slide vigiliaolo attraverso il vetrino da microscopio per rendere la striscio di sangue. Lasciare lo striscio asciugare all&#39;aria. </li><li> Fissare strisci di sangue in metanolo 100% per 30 sec e poi colorare i vetrini in Giemsa appena preparato per 10 min. </li><li> Sciacquare striscio di sangue con acqua corrente per 1 minuto, permette di asciugare vetrino ed esaminare al microscopio (100x, immersione in olio). Enumerare PRBC nel 1000 eritrociti e calcolare parassitemia per cento. Topi donatori dovrebbero raggiungere livelli parassitemia tra il 2,5-5% prima di poter essere sanguinato per infezione di animali da laboratorio. </li><li> Raccogliere il sangue dal topo donatore da retro-orbitale sanguinamento usando un micro-ematocrito eparinizzata tubo capillare. Tutti gli esperimenti sono eseguiti in conformità con la Sala Walter &amp; Eliza Istituto animali previste dal Comitato Etico. A seconda delle esigenze locali animali Ethic Comitatologia sanguinamento altre potrebbero essere utilizzate. Sciogliere quantità di sangue in mezzo RPMI ad una concentrazione finale di 1x10 6 pRBC/0.2 ml. Si consideri cheun ematocrito normale mouse è ~ 6x10 9 cellule rosse del sangue / 1 ml. </li><li> Infect C57BL sperimentale / 6 topi (8-12 settimane) iniettando per via intraperitoneale (ip) 1x10 6 pRBC/0.2 ml. </li><li> Per monitorare i livelli di parassitemia di topi infettati seguire i passaggi 1,3-1,8. Parassitemia deve essere determinato ogni 2-3 giorni a partire dal giorno 2 o 3 pi </li><li> Insorgenza di malaria grave in C57BL / 6 topi potrebbe manifestarsi come aspetto ingobbito, ruffed pelliccia e bassa attività. Alcuni topi possono recuperare in questa fase, ma la progressione della perdita di auto-raddrizzante riflesso indica malattia irreversibile e gli animali devono essere sottoposti a eutanasia. </li></ol> 2. Perfusione Intracardial di topi e di estrazione del cervello <ol><li> Assemblare un sistema manuale di perfusione gravità assicurando un serbatoio di 500 ml di una colonna di supporto fissato alla parte superiore di una colonna di supporto 60 cm. Collegare tubo di plastica per l&#39;estremità inferiore del serbatoio e fissare un morsetto del tubo per controllare il flusso del buffer. Collegare il tubo di un ago 23 G. Fill il serbatoio con PBS (mantenuto a 22 ° C), aprire la fascetta e permettono di eseguire tampone attraverso il tubo per rimuovere tutte le bolle d&#39;aria. </li><li> Eutanasia P. berghei ANKA-infetto mouse CO 2 inalazione. Confermare la morte per mancanza di pedale, orbitale e le risposte respiratorie. </li><li> Pin del mouse eutanasia dalle zampe posteriori e anteriori dorsalmente su una tavola di dissezione di polistirolo contenute in un vassoio di plastica. A seconda del locale Comitato di Etica degli animali anestesia requisiti possono essere somministrati prima di inizio pefusion intracardial. </li><li> Pulire lato ventrale con il 70% di etanolo. </li><li> Usare le forbici grandi e pinze per aprire la pelle lungo la linea mediana per esporre la cavità toracica. Piegare la pelle e il pin ai lati. </li><li> Tenere sterno con pinza sottile e tagliare il diaframma e lungo i due lati dello sterno tranciamento delle costole. Fare attenzione a non danneggiare i grandi vasi sanguigni. </li><li> Pin la gabbia toracica dalla sterno liberamente accanto alla testa. </li><li> Tenere spirito ventricolih pinza sottile e accuratamente incise atrio destro con le forbici sottili. </li><li> Inserire l&#39;ago 23G collegato al sistema di perfusione gravità in ventricolo sinistro verso l&#39;aorta ascendente, mentre è in esecuzione PBS. Inserire solo 0,5 cm dalla punta dell&#39;ago. </li><li> Perfusione del mouse per 5 minuti o fino a quando effluenti è chiara. </li><li> Sblocca il mouse e lo posi sul suo addome. Pulire la testa con il 70% di etanolo. </li><li> Utilizzando le forbici grandi, fare un taglio appena sopra la zona cervicale del midollo spinale. </li><li> Utilizzare le forbici pregiati per fare una mediana caudale-rostrale taglio a partire dalla base del cranio e la pelle a buccia per scoprire l&#39;cranio. </li><li> Tenendo la testa, posizionare le lame di una forbice piccole in ciascuna cavità orbitale ed effettuare un taglio tra le orbite. </li><li> Praticare un taglio longitudinale lungo la sutura sagittale e con attenzione buccia il teschio su ogni emisfero del cervello verso l&#39;esterno. </li><li> Con una spatola, sollevare delicatamente il cervello e metterlo in una provetta da centrifuga da 10 ml contenente mezzo RPMI. </li></ol>3. Isolamento di Brain-sequestrati leucociti (BSL) <ol><li> Lavorare in una cappa di sicurezza, posto appena prelevato cervello sopra un filtro in acciaio inossidabile cella (40-60 maglia) in una piastra Petri contenente 3-5 ml di mezzo di coltura RPMI tessuto. </li><li> Tagliare il tessuto cerebrale in piccoli pezzi. </li><li> Spingere piccoli pezzi di tessuto cerebrale attraverso il filtro cella utilizzando uno stantuffo cristallo. </li><li> Trasferire omogenato cerebrale in una provetta da 10 ml e centrifugare a 250 xg per 10 min a 4 ° C. </li><li> Sciogliere pellet in 3 ml di mezzo RPMI, contenente 0,05% Collagenase D e 2U/ml DNAsi I. </li><li> Ruotare miscela per 30 min a temperatura ambiente. Rimuovere i detriti cellulari spingendo la miscela attraverso un filtro 70 micron cella nylon e / o mediante incubazione in ghiaccio per 5 min. </li><li> Lay omogenato cerebrale su un cuscino 7 ml di Percoll 30% e centrifugare a 400 xg (senza freni) per 20 min a temperatura ambiente. </li><li> Risospendere il pellet in 1 ml di tampone di lisi cellulare rosso sangue e incubare in ghiaccio per 5 min alisare PRBC aderente, che non può essere rimosso dal cervello mediante perfusione intracardial. </li><li> Aggiungere 9 ml di terreno RPMI, lavare le cellule e centrifugare a 250 xg per 10 min a 4 ° C. </li><li> Risospendere BSL contenenti pellet in 50-100 ml di mezzo RPMI e trasferire le cellule su un tubo Eppendorf. </li><li> Diluire un&#39;aliquota 5-10 microlitri del campione cellulare 1:2 in Trypan blu per l&#39;identificazione di cellule vitali. </li><li> Contare le cellule vitali al microscopio utilizzando un emocitometro. Dal giorno 6 in poi, quando pi P. berghei ANKA topi infettati mostrare segni neurologici, 20,000-100,000 BSLs possono essere recuperati. </li></ol> 4. Tipizzazione di BSL di Citometria a Flusso Multicolor <ol><li> Centrifugare le cellule a 250 xg per 10 min a 4 ° C, aspirare il surnatante e risospendere pellet in 50 pl di tampone colorazione (PBS, 1% di siero fetale bovino (FCS), 2 mM EDTA). </li><li> Aggiungere il 1 ml di anticorpo purificato anti-CD16/32 (Fcblock). </li><li> Incubare le cellule per 10 min in ghiaccio. </li><li> Lavare le cellule con 1 ml di tampone di colorazione. Centrifugare le cellule a 250 xg per 10 min a 4 ° C e aspirare il surnatante. </li><li> Risospendere le cellule in 50 pl di tampone contenente colorazione diluizioni ottimali predeterminate di richieste anticorpi fluorescenti, ovvero anti-CD4, anti-CD8, anti-TCR e anti-NK1.1. </li><li> Incubare per 1 ora su ghiaccio. </li><li> Lavare le cellule con 1 ml di tampone di colorazione. Centrifugare le cellule a 250 xg per 10 min a 4 ° C e aspirare il surnatante. </li><li> Risospendere le cellule in 100 microlitri di PBS. </li><li> Trasferire le cellule a un tubo di plastica di citometria a flusso. </li><li> Utilizzando un citofluorimetro, acquisire almeno 5.000-10.000 eventi. Adeguati controlli e campioni di cellule non marcate con fluorocromi di compensazione dovrebbe essere incluso come richiesto. </li><li> Analizzare i risultati utilizzando il software appropriato citometria a flusso. </li></ol>

<ol>
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Nessun conflitto di interessi dichiarati.

L&#39;isolamento e l&#39;analisi di BSL è un metodo che permette la caratterizzazione e la quantificazione delle cellule infiammatorie migrazione al cervello in risposta al danno tissutale o infezione in modelli murini sperimentali. L&#39;introduzione di una fase di perfusione intracardial per la rimozione del comparto sangue prima dell&#39;estrazione organi e isolamento cellulare successiva è utile per prevenire la contaminazione di cellule infiammatorie con leucociti circolanti non-infiammatorie. Questo potrebbe non...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td></td><td></td><td></td><td> Soluzioni e tamponi </td></tr><tr><td> Giemsa azur eosina blu metilene soluzione </td><td> Merck Millipore </td><td> 1.09204.0500 </td><td> Diluizione 1:10 in acqua distillata </td></tr><tr><td> RPMI media </td><td></td><td></td><td> Mouse tonicità </td></tr><tr><td> Mouse tonicità PBS </td><td></td><td></td><td> 20 mM fosfato di sodio, 0,149 NaCl, pH 7,3 </td></tr><tr><td> 0,4% Trypan Blue </td><td> Sigma Aldrich </td><td> T-8154 </td><td> 1:2 </td></tr><tr><td> Collagenasi D </td><td> Biochimica Worthington </td><td></td><td></td></tr><tr><td> Deoxyribonuclease (DNAsi) I </td><td> Sigma Aldrich </td><td> D4263-5VL </td><td> Da pancreas bovino </td></tr><tr><td> Percoll </td><td> GE Healthcare </td><td> 17-0891-01 </td><td> Soluzione al 30% in PBS </td></tr><tr><td> Ultrapura Tris </td><td> Invitrogen </td><td> 15505-020 </td><td></td></tr><tr><td> Cloruro di ammonio (NH 4 Cl) </td><td> AnalaR </td><td> 10017 </td><td></td></tr><tr><td> Red Cell Lysis Buffer </td><td></td><td></td><td> 17 mM Tris, 14nM NH 4 Cl, pH 7,2 </td></tr><tr><td> FCS </td><td> Gibco </td><td> 1009 </td><td></td></tr><tr><td> EDTA sale disodico </td><td> Merck </td><td> 10093.5V </td><td> 0,1 M, pH 7,2 </td></tr><tr><td></td><td></td><td></td><td> Anticorpi e coniugati </td></tr><tr><td> Anti-mouse CD16/CD32 (Fc Block), clone 2.4G2 </td><td> BD Pharmingen </td> &lt;td&gt; 553142 </td><td> 1 ml in 50 microlitri tampone di colorazione (0.5mg/50 ml) </td></tr><tr><td> FITC-anti-topo CD4, clone H129.19 </td><td> BD Pharmingen </td><td> 553651 </td><td></td></tr><tr><td> PE-anti-topo NK1.1, clone PK136 </td><td> BD Pharmingen </td><td> 553165 </td><td></td></tr><tr><td> PerCPCy5.5-anti-topo CD8, clone 53-6-7 </td><td> BD Pharmingen </td><td> 551162 </td><td></td></tr><tr><td> APC-anti-topo TCR-β, clone H57-597 </td><td> BD Pharmingen </td><td> 553174 </td><td></td></tr><tr><td> PE-anti-topo CXCR3, clone 220803 </td><td> R &amp; D Systems </td><td> FAB1685P </td><td></td></tr><tr><td> Biotinilato anti-topo-CCR5, clone C34-3448 </td><td> BD Pharmingen </td><td> 559922 </td><td></td></tr><tr><td> Steptavidin-PerCP-Cy5.5 </td><td> BD Pharmingen </td><td> 551419 </td><td></td></tr><tr><td></td><td></td><td></td><td> Attrezzature e materiali </td></tr><tr><td> SuperFrost vetrino da microscopio </td><td> Lomb Menzel-Glaser </td><td></td><td></td></tr><tr><td> Pinze, forbici dissezione </td><td> REDA Instrumente </td><td></td><td></td></tr><tr><td> 500 ml PBS serbatoio </td><td> Nalgene </td><td></td><td></td></tr><tr><td> Tubi in gomma vulcanizzata </td><td></td><td></td><td></td></tr><tr><td> Ago 23G </td><td> BD PrecisionGlide </td><td> 302008 </td><td></td></tr><tr><td> Cella dissociazione kit contenenti metalli setaccio </td><td> Sigma Aldrich </td><td> CD-1 </td><td></td></tr><tr><td> 70 micron nylon filtro cella </td><td> BD Falcon </td><td> 352350 </td><td></td></tr><tr><td> Emocitometro </td><td> Neubauer GmbH </td><td> 717810 </td><td></td></tr><tr><td> Citometria a Flusso tubi </td><td> BD Falcon </td><td> 352008 </td><td></td></tr></tbody></table>

isolation and analysis of brain-sequestered leukocytes from plasmodium berghei anka-infected mice

Si descrive un metodo per l&#39;isolamento e la caratterizzazione di cellule infiammatorie aderenti da vasi sanguigni cerebrali di P. berghei ANKA topi infettati. L&#39;infezione di ceppi sensibili del mouse con questo risultati di deformazione parassita nella induzione di sperimentale malaria cerebrale, una sindrome neurologica che riassume alcuni aspetti importanti del Plasmodium falciparum mediata da malaria grave in 1,2 esseri umani. Forme mature di sangue stadi malaria esprimere proteine ​​parassite sulla superficie del eritrociti infetti, che consente loro di legarsi a cellule endoteliali vascolari. Questo processo induce ostruzioni del flusso sanguigno, causando ipossia e 3 emorragie e stimola anche il reclutamento di leucociti infiammatori al sito di sequestro parassita. A differenza di altre infezioni, ad esempio virus neutrotopic 4-6, sia la malaria-parassitati globuli rossi (PRBC), così come associati inflammleucociti stre rimangono sequestrati all&#39;interno dei vasi sanguigni e non infiltrante il parenchima cerebrale. Perciò, per evitare la contaminazione di leucociti sequestrati con cellule circolanti non-infiammatorie, ampia perfusione intracardial di topi infetti-prima dell&#39;estrazione di organi e lavorazione dei tessuti è necessario in questa procedura per rimuovere il comparto sangue. Dopo la perfusione, i cervelli vengono raccolte e sezionati in piccoli pezzi. La struttura del tessuto è ulteriormente disturbato da trattamento enzimatico con collagenasi D e DNAsi I. L&#39;omogenato cerebrale risultante viene poi centrifugato su un gradiente di Percoll che permette la separazione dei cervelli sequestrati leucociti (BSL) mielina e detriti da altri tessuti. Cellule isolate vengono quindi lavate, contati utilizzando un emocitometro e colorate con anticorpi fluorescenti per la successiva analisi mediante citometria a flusso. Questa procedura consente una completa caratterizzazione fenotipica dei leucociti infiammatori migrazione al cervello in response a vari stimoli, compreso il colpo così come le infezioni virali o parassitarie. Il metodo prevede anche un utile strumento per la valutazione di nuovi trattamenti antinfiammatori in pre-clinici modelli animali.

I risultati in Fig. 2 percentuali di spettacoli e di numeri assoluti delle diverse popolazioni BSL recuperati da cervelli di perfusi o unperfused topi di controllo della malaria-infettati e ingenuo. Isolato BSL sono state colorate con PE-anti-NK1.1 e APC-anti-TCR β-anticorpi, come indicato nel testo del protocollo. In linea con i risultati precedenti 7-9, αβTCR + cellule T comprende una percentuale elevata della piscina BSL nel cervello di perfusi malaria topi infettati (giorno 6 pi). Questa pop...

Watch this Scientific Journal Video about Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice at JoVE.com

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

Un metodo per l&#39;isolamento di aderenti leucociti infiammatori dai vasi sanguigni cerebrali dei<em&gt; Plasmodium berghei</em&gt; ANKA topi infettati è descritto. Il metodo consente la quantificazione e caratterizzazione fenotipica di leucociti isolati dopo colorazione con anticorpi fluorescenti e successiva analisi mediante citometria a flusso.

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. ...

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Immunology and Infection

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

12.7K Views.  The Walter and Eliza Hall Institute of Medical Research. Un metodo per l'isolamento di aderenti leucociti infiammatori dai vasi sanguigni cerebrali dei Plasmodium berghei ANKA topi infettati è descritto. Il metodo consente la quantificazione e caratterizzazione fenotipica di leucociti isolati dopo colorazione con anticorpi fluorescenti e successiva analisi mediante citometria a flusso.

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

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

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 ...

Ricerca

Immunologia e infezioni

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Un modello sperimentale per studiare la tubercolosi, la malaria coinfezione sulla trasmissione naturale di Mycobacterium tuberculosis E Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

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 ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Isolamento e analisi citofluorimetrica delle cellule immunitarie dal cervello ischemica mouse

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Mieloide isolamento delle cellule da topo pelle e drenante linfonodi seguito intradermica immunizzazione con vivo attenuato<em&gt; Plasmodium</em&gt; sporozoiti

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

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 ...