This work was supported by EMBO (to Y.E.), Foundation Machaon (to Y.E.) and SNSF (to B.A.I.). We thank the Bioimaging Core Facility for the availability of the Nikon A1r microscope and technical assistance. We thank Mrs. Clarissa Bartley for English correction.

NOTA: le procedure di animali sono stati eseguiti in conformità con la Comitato Etico Istituzionale di Animal Care a Ginevra, in Svizzera e l&#39;Ufficio cantonale di veterinaria. Numero di autorizzazione GE / 63/14. 1. Preparazione di una sospensione singola cella da midollo osseo <ol><li> Mouse Sacrificio (8-12 settimane di età) per dislocazione cervicale. Sterilizzare zampe posteriori con il 70% di etanolo. Togliere la pelle dalle gambe posteriori. </li><li> Sezionare femori e tibie e il mouse rimuovere il tessuto da gambe con un bisturi. Sciacquare gambe con il 90% di etanolo e posto in un piatto cultura piena di PBS. </li><li> Sotto il cofano della cultura, il tessuto pulito da femore e tibia con un bisturi e separato ginocchio. Tagliare ogni estremità delle ossa. </li><li> Lavare midollo osseo da ciascun osso utilizzando un ago 23G e una siringa da 1 ml riempito con media preparazione (fenolo aneritro DMEM / F12, siero di ratto 1%), in 50 ml vite tubo. Disrupt aggregati di cellule da delicatamente passaging attraverso il non 23Gedle e una siringa da 1 ml. Portare il volume totale di 25 ml di media preparazione. </li><li> Centrifuga 250 xg, 5 min, 4 ° C. Surnatante scarto. Risospendere pellet in 1 ml Globulo rosso tampone Lysis rblc (8,3 g di NH 4 Cl, 1 g NaHCO 3, 1 ml di EDTA (100 mm), completo di 1 L con acqua distillata, filtrare 0,22 micron) in 15 ml di vite tubo. Incubare 30 sec su ghiaccio. </li><li> Aggiungere 10 ml di terreno preparato e centrifugare a 250 xg, 5 min, 4 ° C. Gettare il surnatante e risospendere in 2 ml di mezzo di preparazione. </li></ol> 2. neutrofili Arricchimento per selezione negativa <ol><li> Aggiungere 150 ml di mouse cocktail neutrofili arricchimento (isolamento delle cellule kit piattaforma come EasySep). Mescolare e incubare in ghiaccio per 10 min. </li><li> Aggiungere 10 ml di fenolo DMEM rosso gratuito. Invertire una volta e centrifugare 250 xg, 3 min, 4 ° C. </li><li> Surnatante scarto. Risospendere pellet in 1,75 ml di media preparazione in 5 ml di polistirolo tubi tondi fondo. Aggiungere 250 microlitri Biotin Selezione Cocktail. Mescolare e incubare in ghiaccio per 10 min. </li><li> Agitare le particelle magnetiche (dal kit piattaforma di isolamento delle cellule) per 30 secondi per risospendere le particelle. Aggiungere 500 microlitri di particelle magnetiche sospensione cellulare. Mescolare e incubare in ghiaccio per 10 min. </li><li> Mettere la provetta a magnete. Attendere 3 minuti. </li><li> Invertire il magnete su un nuovo 5 ml polistirolo tubi tondi fondo per raccogliere le cellule senza etichetta desiderati. Non prendere l&#39;ultima goccia appeso nel tubo. Cellule indesiderate rimarranno nel tubo all&#39;interno del magnete. Eliminare questo tubo in una rifiuti a rischio biologico. </li><li> Posizionare il nuovo tubo nel magnete. Attendere 3 minuti. </li><li> Invertire il magnete su un nuovo 15 ml vite tubo per raccogliere le cellule senza etichetta desiderati. Non prendere l&#39;ultima goccia appeso nel tubo. </li><li> Completa il volume a 5 ml con rosso fenolo DMEM gratuito. </li></ol> 3. Etichettatura dei neutrofili <ol><li> Aggiungere 0,5 ml di un colorante arancione come il cellulare Tracker Orange (concentrazione di lavoro1 micron, CMRA - clorometil-rodol-acetato, magazzino 10 mM in DMSO). Incubare 2 min a 37 ° C. </li><li> Aggiungere 2,5 ml di terreno preparato. Centrifuga 250 xg, 5 min, 4 ° C. </li><li> Surnatante scarto. Risospendere pellet in 1 ml di media preparazione in una provetta da 1,5 ml. </li><li> Prelevare un &#39;aliquota di contare con hematocytometer. </li><li> Incubare 5 minuti a 37 ° C. Centrifuga 250 xg, 5 min, 4 ° C. </li><li> Surnatante scarto. Risospendere pellet in 1 ml di PBS. Centrifugare 250 xg, 5 min, 4 ° C per lavare. </li><li> Surnatante scarto. Risospendere il pellet come 10x10 6 cellule per 100 l di PBS. Tenere in ghiaccio fino a iniezione endovenosa. NOTA: cellule devono essere iv iniettato il più rapidamente possibile. 6-12x10 6 neutrofili / mouse sono solitamente ottenuti. </li></ol> 4. Preparazione del mouse per intravitale Microscopia NOTA:.. Fase 4.1) e 4.2) deve essere eseguita all&#39;inizio dell&#39;esperimento per evitare tempi di attesa prolungato di neutrofili etichettati sulla ice. <ol><li> Pesare il CX 3 CR1 topo GFP (8-12 settimana di vita). </li><li> Preparare 800 ml di anestetici (ketamina 60 mg / kg, xilazina 4,5 mg / kg, acepromazone 1,75 mg / kg in PBS). </li><li> Anestetizzare il mouse. Ip Iniezione con 200 microlitri / 20 g di mouse. Controllo anestesia da punta pizzicotti e controllando la frequenza respiratoria. </li><li> Iniettare neutrofili (10x10 6 cellule in 100 microlitri di PBS) per via endovenosa utilizzando le vene coda laterali o seno retro-orbitale, alla convenienza dello sperimentatore. </li><li> Ponga il mouse sul tappetino riscaldamento e proteggere gli occhi con il gel oculare. </li><li> Un coprioggetto di vetro (diametro 35 mm) viene messo in un piatto di coltura di tessuto 10 cm, che ha un foro di diametro 30 mm. Usare olio è di mantenere il piatto di coltura di tessuto a contatto con il coprioggetto. </li><li> Incidere la pelle addominale con le forbici per esporre il peritoneo. Incidere peritoneo con le forbici per esporre intestino. </li><li> Aggiungere 200 microlitri di PBS (preriscaldata a 37 ° C) in pcavità eritoneal. Prendete il mouse in mano e girare a faccia in giù, in modo che l&#39;intestino esce dalla cavità peritoneale con una leggera pressione. Posizionare il mouse nel piatto coltura di tessuti. </li><li> Deroll delicatamente l&#39;intestino con bastoncini di cotone sterilizzati sul vetrino, al fine di esporre i vasi mesenterici. </li><li> Tagliate fazzoletti di carta in bande e bagnarli con 37 ° C riscaldata PBS. Immobilizzare l&#39;intestino con i pezzi di fazzoletti di carta per ridurre i movimenti risultanti dal peristalsi. </li><li> Mettere il piatto di coltura tissutale e il mouse all&#39;interno della camera termostatata 37 ° C sul palco del vassoio su misura del microscopio invertito. Introdurre una siringa contenente anestetici sc nella zampa posteriore. NOTA: Inoltre, il mouse può ricevere ossigeno (0,5 L / min) con una maschera. </li><li> Il mouse di controllo per l&#39;anestesia da punta pizzicare e il controllo della frequenza respiratoria ogni 30 min. Se necessario, fornire una spinta di anestetici (20 ml) per mantenere correttamente anestesia. NONE: Essiccamento della vascolarizzazione deve essere evitato. Pertanto, l&#39;umidità è mantenuta bagnando regolarmente i fazzoletti di carta usati per immobilizzare l&#39;intestino con 37 ° C-riscaldato PBS. </li></ol> 5. Misurazione dei neutrofili e monociti comportamento in recipienti infiammate In Vivo con intravitale Microscopia <ol><li> Impostare laser per 488- 561- e alla potenza minima necessaria per evitare la fototossicità indotta da laser. Nei nostri esperimenti, è inferiore a 0,5%. Intensità di GFP in monociti e arancio tintura dei neutrofili è così intensa che di solito lo 0,3% è sufficiente. NOTA: Ciò rappresenta una potenza laser di 0.15 al 0.25 mW con il nostro sistema. </li><li> Acquisire immagini da 512 x 512 pixel (vale a dire, 635 micron) in 2.2 secondi con un foro stenopeico aperto con l&#39;obiettivo 20X secco del microscopio invertito. Utilizzare un z-profondità compresa tra 10 e 20 micron, che garantisce un segnale sufficientemente con le potenze laser bassi. NOTA BENE: Di solito effettuare esperimenti con una z-profondità compresa tra 12 a 1656; m. Nel caso delle cifre presentate e film, la z-profondità è di 20 micron. Contrasto di fase permette l&#39;identificazione di pareti endoteliali e di imaging viene eseguita su vene mesenteriche. Poiché le cellule pattugliano muovono molto lentamente con una velocità di 5-10 micron / min 6, la registrazione di un campo di interesse ogni 20 s è soddisfacente. Con le impostazioni descritte, la fase motorizzato consente di registrare fino a 7 campi di interesse allo stesso tempo. </li><li> Posizionare il mouse e vasi mesenterici nella camera del termostato del microscopio sono consentirne il recupero dalla preparazione per 30 minuti prima dell&#39;acquisizione del primo film. Durante questo tempo, scegliere diversi campi di interesse. Focus sulla superficie luminale della nave vicina all&#39;obiettivo. Se necessario, sfruttare la leggera auto-fluorescenza di endotelio per impostare la messa a fuoco. NOTA: La chirurgia durante la preparazione del mouse sottolinea leggermente l&#39;endotelio. Di conseguenza, a rotazione dei neutrofili e/ o monociti possono essere osservati nei primi 15 minuti dopo la preparazione del mouse. </li><li> Registrare un primo film per 30 min a regime, una immagine ogni 20 secondi. NOTA: Il software acquisisce ogni campo di interesse per 2,2 secondi e lo stadio motorizzato si sposta automaticamente da un campo all&#39;altro. Si ritorna alla prima posizione entro 20 secondi durante i 30 min del film registrazione. </li><li> Per avviare infiammazione dei vasi sanguigni, aggiungere una goccia di 20 l di PBS contenente 100 mg di TLR2 / TLR1 agonista Pam3CSK4 12 direttamente sui vasi imaged. </li><li> Controllare il punto di riferimento per ogni campo di interesse. NOTA: Nel corso di acquisizione, c&#39;è sempre lievi modifiche di focalizzazione su asse z. Nonostante il 10-20 micron z-profondità, questo può provocare una diminuzione dell&#39;intensità di fluorescenza. Pertanto, riorientare manualmente ogni campo di interesse alla fine di ogni film, se necessario. Film record per 30 min-periodi e fino a 3 ore aTal dopo l&#39;inizio di infiammazione. </li><li> Alla fine dell&#39;esperimento, sacrificare topo anestetizzato per dislocazione cervicale. </li></ol> 6. Generazione di file di film e tracciamento dei leucociti NOTA: Il software di acquisizione Nikon genera .nd2 file, ma la seguente procedura sarà simile con altri software e marchi. <ol><li> I file di esportazione come serie tiff. </li><li> Vai a software ImageJ <a href="http://rsb.info.nih.gov/ij/">(http://rsb.info.nih.gov/ij/).</a> Importare la serie tiff. Ogni canale come un file diverso. </li><li> Applicare un filtro mediano con un raggio di 2.0 pixel per i canali verde e rosso per ridurre il rumore di fondo (di processo&gt; Filtri&gt; mediani ...). </li><li> Convertire i file in binario (Processo&gt; Binary&gt; Crea Binary). Attivare il software per calcolare la soglia per ogni immagine. </li><li> Unire i canali in un&#39;unica serie di immagini (Immagine&gt; Colore&gt; Unisci canali ...). Selezionare i monociti del green canali, i neutrofili nel canale rosso e la luce trasmessa nel canale grigio. </li><li> Convertire immagini impilate in colori RGB (Immagine&gt; Colore&gt; Stack per RGB). </li><li> Salvare il file come .avi </li><li> I dati vengono importati e compilati nel software Imaris (Bitplane). Movimenti di monociti e neutrofili vengono poi monitorati tramite il tracking guidata Spot. </li></ol>

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The authors declare that they have no competing financial interests.

Le metodologie descritte in questo manoscritto forniscono un approccio coerente per studiare in modo efficiente monociti e il comportamento dei neutrofili nelle vene mesenteriche in condizioni di stato e infiammatorie costante. La fase cruciale della preparazione è l&#39;immobilizzazione dell&#39;intestino con tessuti PBS-bagnate. Se eseguita correttamente, vasi mesenterici sono ben esposti sul vetrino per l&#39;acquisizione delle immagini. Ciò consente la selezione di vari campi di intere...

<html> Neutrofili e monociti sono cellule del sistema immune innato che circolano continuamente nel sangue. Su lesioni o infezioni, segnali infiammatori inducono leucociti diapedesi in tessuti danneggiati e infetti, qui l&#39;avvio di una risposta immunitaria cellulare 1-3. La rapidità di leucociti mobilizzazione determina l&#39;esito positivo delle risposte immunitarie. Questi processi intricati basano su molecole specifiche (ad esempio, selectine, chemochine endotelio-bound) presenti sull&#39;endotelio infiammata che aiutano per la creazione di contatti tra adesivi leucociti circolanti e l&#39;endotelio 1-3 .Per ottenere approfondimenti sulle molecole implicate nella leucociti assunzione in cascata, è importante visualizzare la cinetica di reclutamento di cellule e per monitorare il comportamento di ciascuna cella / popolazione. Un metodo efficace consiste microscopia intravitale su topi transgenici che esprimono proteine ​​fluorescenti in cellule di interesse. content &quot;&gt; Ad oggi, diversi approcci usando la microscopia intravitale sono stati sviluppati per l&#39;immagine del sistema vascolare 4,5. Ad esempio, l&#39;imaging del derma orecchio vascolarizzazione o vene mesenteriche mediante microscopia confocale intravitale è stato utilizzato per identificare il comportamento pattugliamento delle Ly6C murino monociti bassi e umano CD14 dim CD16 + monociti sul lato luminale dei vasi sanguigni a regime di 6-8. Il modello di vascolarizzazione del mouse cremastere viene spesso utilizzato per monitorare il comportamento di neutrofili o Ly6C infiammatoria elevati monociti in condizioni infiammatorie o ischemiche in topi transgenici. In tale caso, Cremaster è stimolata mediante iniezione intrascrotale di IL1β, CCL2, TNFβ o fMLP. Dopo 2-4 ore, i tessuti vengono poi chirurgicamente esteriorizzate e analizzati da confocale microscopio intravitale 9-11. Il protocollo seguente descrive un metodo per monitorare monociti e neutrofili contemporaneamente con qualsiasifluorescenza invertito microscopio confocale. Inoltre, il nostro metodo descrive come immagine stessa nave prima (condizione di stato stazionario) e dopo l&#39;infiammazione e come seguire la cinetica di reclutamento dei leucociti. A questo scopo, si usa il CX 3 CR1 topo GFP / peso, il cui monociti esprimere eGFP, iv iniettato con un arancio neutrofili murini dye-etichettati. Utilizzando un microscopio confocale invertito, è possibile (1) per monitorare e analizzare le pattugliamento Ly6C basse monociti in condizioni stazionarie e (2) a seguire l&#39;assunzione di entrambi i monociti e neutrofili nella stessa nave dopo infiammazione locale. Qui creiamo le condizioni infiammatorie utilizzando l&#39;agonista TLR2 / TLR1 Pam3CSK4 12. Inoltre, tali immagini può aiutare a determinare il ruolo di specifiche molecole di interesse nelle varie fasi della cascata reclutamento dei leucociti se eseguita su specifici topi knock-out o in presenza di anticorpi bloccanti 6,9,13.

<table>
	<tbody>
		<tr>
			<td>5 mL polystyrene round bottom tubes&#160;</td>
			<td>Beckton Dickinson</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>10x CFI Plan Apochromat 0.45&#160;&#160; DT:4mm&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20x CFI Plan Apochromat VC 0.75&#160;&#160; DT:1mm&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Tracker Orange CMRA Dye</td>
			<td>Life Technologies</td>
			<td>C34551</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>Stem Cell Technologies</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Mouse Neutrophil Enrichement kit</td>
			<td>Stem Cell Technologies</td>
			<td>19762</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>PAA</td>
			<td>A15-042</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion Oil Type A</td>
			<td>Nikon</td>
			<td></td>
			<td>any viscous oil&#160;</td>
		</tr>
		<tr>
			<td>Life Box Temperature Control System</td>
			<td>Life Imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Sigma Aldrich</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R confocal microscope</td>
			<td>Nikon</td>
			<td>A1R</td>
			<td>inverted microscope, motorized x/y/z stage, NIS elements software</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>phenol red free DMEM/F12</td>
			<td>Life Technologies</td>
			<td>21041-025</td>
			<td>any phenol red free medium is suitable</td>
		</tr>
		<tr>
			<td>PAM3CSK4</td>
			<td>Invivogen</td>
			<td>tlrl-pms</td>
			<td>reconstitute in PBS</td>
		</tr>
		<tr>
			<td>Rat serum</td>
			<td>Stem Cell Technologies</td>
			<td></td>
			<td>included in EasySep Mouse Neutrophil Enrichement kit</td>
		</tr>
		<tr>
			<td>tissue culture dish 100</td>
			<td>TPP</td>
			<td>93100</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging neutrophils and monocytes in mesenteric veins by intravital microscopy on anaesthetized mice in real time

Risposta immunitaria efficace dipende da una rapida mobilitazione dei leucociti del sangue al sito di infezione o lesioni. Indagare la migrazione dei leucociti in vivo è di fondamentale importanza per la comprensione delle basi molecolari della migrazione dei leucociti transendoteliale e l&#39;interazione con l&#39;endotelio vascolare. Un approccio potente coinvolge microscopia intravitale su topi transgenici che esprimono proteine ​​fluorescenti in cellule di interesse. Qui vi presentiamo un protocollo per i monociti imaging e neutrofili nel CX 3 CR1 GFP / peso del mouse iv iniettato con arancio neutrofili dye-etichettati con un microscopio confocale invertito. Film time-lapse raccolti da 30 minuti a diverse ore dell&#39;imaging permettere l&#39;analisi del comportamento dei leucociti nelle vene mesenteriche sia in condizioni di stato e infiammatorie costante. Abbiamo inoltre descritti i passi per indurre localmente infiammazione dei vasi sanguigni con TLR2 / TLR1 agonisti Pam3SK4 e monitorare la subsequent reclutamento dei neutrofili e monociti. La tecnica presentata può anche essere utilizzato per monitorare altre popolazioni di leucociti e indagare le molecole implicate nel reclutamento dei leucociti o nel traffico con altri stimoli o topi transgenici.

Il manoscritto descrive un protocollo ottimizzato in modo da controllarne il comportamento di monociti e neutrofili nelle vene mesenteriche di topi anestetizzati in tempo reale. L&#39;uso di una camera 37 ° C-termostatato è obbligatoria per mantenere la temperatura del mouse e anche a causa della temperatura di circolazione dipendente leucociti. Preparazione del mouse viene visualizzato in Figura 1. La Figura 2 mostra tutta la zona visto al microscopio. Luce trasmessa permette l&#39;i...

Watch this Scientific Journal Video about Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time at JoVE.com

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

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

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

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

imaging-neutrophils-monocytes-mesenteric-veins-intravital-microscopy

Research

JoVE Journal

Immunology and Infection

10.4K Views.  University of Geneva. We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

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

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

Monitoraggio Crawling neutrofili intraluminale, migrazione e chemiotassi transendoteliale in tessuto al microscopio video intravitale

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

Ricerca

Immunologia e infezioni

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

In tempo reale di immagine degli eterotipica piastrine-neutrofili interazioni sull&#39;endotelio Attivato durante l&#39;infiammazione vascolare e la formazione di trombi in topi vivi

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

Lo studio delle interazioni di<em&gt; Staphylococcus aureus</em&gt; Con neutrofili mediante citometria a flusso e Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Altamente Risolto intravitale Striped-illuminazione Microscopia dei Centri Germinal

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

In tempo reale Imaging di cellule mieloidi Dynamics in<em&gt; Apc<sup&gt; Min / +</sup</em&gt; Intestinale tumori da Spinning Disk Microscopia confocale

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Long Term intravitale Multiphoton Microscopia Imaging di cellule immunitarie in sani e malati di fegato Uso Mice CXCR6.Gfp Reporter

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

Interazioni intravitale Microscopia di leucociti-endoteliali e piastrine-leucociti in Mesenterial Vene Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Immagini dal vivo di antifungini attività da Human neutrofili e monociti primaria in risposta a<em&gt; A. fumigatus</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

La formazione immagine interazione cellula nella Mucosa trachea durante l'infezione da Virus influenzale usando la microscopia Intravital due-fotone

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease.
We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section.
IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

Infiltrazione leucocita del muscolo Cremaster nei topi valutata dalla microscopia intravitale

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The ...