Questo lavoro è stato sostenuto dal National Institute of Allergy and Infectious Disease di Grant R01AI079253 (a BHS), e dal National Cancer Institute Cancer Support Center Concessione CA016056 al Roswell Park Cancer Institute.

Tutte le procedure eseguite su animali in questo studio sono stati approvati dal Comitato cura degli animali ed uso al Roswell Park Cancer Institute e rispettate tutte le statali, federali e istituti nazionali di regolamentazione di salute. 1. In vivo macrofagi Labeling Nota: PHK26 è un colorante lipofilo che sarà ripreso da fagociti sia in circolazione e nei tessuti quando somministrato per via endovenosa (iv). PKH26 è stato utilizzato per l&#39;etichettatura in vivo per minimizzare la quantità di manipolazione post-raccolta del macrofago. PKH-26 ha il vantaggio di accumulare stabilmente nei macrofagi alveolari per periodi prolungati. Attendere 10 giorni dopo la somministrazione permette per la liquidazione di tutte le cellule marcate dalla circolazione lasciando solo alveolare macrofagi stabile etichettati con PKH26 8, 9. PKH26 è un fluorocromo rosso che ha eccitazione (551 nm) ed emissioni (567 nm) caratteristiche compatibilicon i sistemi di rodamina o di rilevazione ficoeritrina. Qualsiasi procedura di etichettatura può introdurre artefatti, inclusa la perdita di vitalità. Tuttavia, dato che utilizziamo lo stesso approccio per l&#39;etichettatura dei macrofagi in diversi genotipi del mouse (ad esempio, WT vs NADPH ossidasi-carenti), gli effetti di questi manufatti sarebbero uniforme. <ol><li> Diluire soluzione madre di PKH26 (1 x 10 -3 M in etanolo) 1:5 in diluente C fornito dal produttore. </li><li> Carico: 100 ml di diluito PKH26 in una siringa da 1/2 ml insulina (29 G x 1/2 &quot;) per ogni mouse. </li><li> Somministrare a topi mediante iniezione ev (sia vena della coda o retro orbitale) almeno 10 giorni prima del raccolto. </li></ol> 2. Raccolta alveolari macrofagi di lavaggio broncoalveolare (BAL) Il numero di topi da utilizzare per un esperimento può variare. Una resa tipica di un mouse non stimolato è nell&#39;intervallo di 3-5 x 10 5 macrofagi alveolari, ma questo numero può variare notevolmente basEd da fattori quali la dimensione del mouse, esperienza della persona che effettua il lavaggio, e il numero di instillazioni di liquido di lavaggio e il volume prelevato dai polmoni. In questo protocollo lavaggio polmonare viene eseguita con una cavità toracica chiuso con ripetuti lavaggi 1 ml contribuisce ad aumentare la quantità di cellule. <ol><li> Euthanize topo somministrando una dose letale di anestetico avertin (12,5 mg) per via intraperitoneale (ip) utilizzando condizioni asettiche per conservare la soluzione sterile. </li><li> Spray topo accuratamente con etanolo al 70%. </li><li> Rimuovere pelle dall&#39;estremità craniale animale esporre il torace (Figura 1A). <ol><li> Fare un piccolo taglio nella pelle appena sotto il diaframma. </li><li> Inserire dito nel taglio e tirare pelle dall&#39;estremità craniale dell&#39;animale (Figura 1A). </li><li> Premere leggermente sulla testa, estendendo la parte ventrale del collo (Figura 1A). </li></ol></li><li> Tagliare un piccolo foro attraverso la gabbia toracicanel lato destro del torace per sgonfiare i polmoni e anche permettere la visualizzazione dei polmoni durante le seguenti fasi. </li><li> Individuare la trachea, e con attenzione sezionare il tessuto circostante. <ol><li> Rimuovere le ghiandole salivari, muscoli sterno-ioideo e laringe, ecc con pinze curve. </li><li> Sollevare delicatamente con una pinza curve e tagliare con le forbici il tessuto sottile che copre la trachea (avventizia) rivelando la struttura cartilaginea anelli sottostante. </li></ol></li><li> Inserire il catetere nella trachea. <ol><li> Utilizzando pinze curve, posizionare una sutura dietro (dorsale a) la trachea e tirare una lunghezza 10 cm attraverso l&#39;apertura. </li><li> Avvio sopra la cricoide (l&#39;anello ispessito della cartilagine) guida con attenzione un catetere G 22 lungo la trachea fermandosi dove la trachea scompare dietro lo sterno. </li><li> Fermamente legare sutura intorno trachea e il catetere per una perfetta aderenza. Rimuovere l&#39;ago dal catetere. </li></ol></li><li> Montare a 4 vie rubinetto. <ol><li> Riempireuno 6 ml siringa con DPBS, 1% FBS e attribuiscono al 4-way rubinetto come indicato nella figura 1B. </li><li> Collegare un vuoto 6 ml siringa nella posizione indicata sul 4-way rubinetto (Figura 1B). </li><li> Collegare la porta aperta sul 4-way rubinetto per catetere (Figura 1B). Fare attenzione a non rimuovere il catetere dalla trachea. </li></ol></li><li> Raccogliere liquido di lavaggio dai polmoni. Nota: Gli investigatori possono scegliere di utilizzare minori volumi di liquido di lavaggio per ridurre al minimo la possibilità di danni al tessuto. È importante che lo stesso approccio venga applicato coerentemente in tutti i gruppi di topo. <ol><li> Girare la maniglia sul rubinetto in modo che la siringa vuota è chiuso e iniettare lentamente ~ 1 ml DPBS, 1% FBS per gonfiare i polmoni. Osservare l&#39;inflazione polmone attraverso il taglio foro nella fase 2.4. </li><li> Fare attenzione a NON gonfiare eccessivamente i polmoni. </li><li> Girare la maniglia del rubinetto in modo che l&#39;intero siringa è chiuso, e prelevare con cautela l&#39;influenza<html> id dai polmoni. </li><li> Osservare i polmoni si sgonfiano attraverso il foro praticato nel punto 1.4. Il catetere può essere necessario spostare leggermente in avanti o indietro per ottenere un buon pareggio. Prendere precauzioni speciali per mantenere il catetere all&#39;interno della trachea. </li><li> Ripetere i punti 2.8.1-2.8.4 5-6x finché tutto il DPBS, 1% FBS è stata immessa e prelevata dai polmoni. Nota: il recupero del fluido polmonare non sarà 100% del fluido iniettato. </li></ol></li><li> Siringa vuota con il liquido di lavaggio in una provetta da 50 ml. Mantenere le cellule a temperatura ambiente. </li><li> Agglomerare cellule per centrifugazione a 300 xg per 5 min a temperatura ambiente e versare il surnatante. Questo può essere salvata per l&#39;analisi di citochine, se lo desideri. </li><li> Risospendere le cellule in 1-5 ml di RPMI 1640 con 10% di FBS e contare su un emocitometro. Nel topo non stimolato,&gt; 95% di cellule raccolte da BAL sono ritenute macrofagi, che può essere confermata mediante citologia. </li></ol> 3. Coltura e raccolta funghi &quot;&gt; L&#39;Aspergillus fumigatus utilizzato in questo protocollo esprime GFP in tutte le fasi del suo ciclo di vita. Spore, germlings e ife di questo ceppo fungino sono state fornite dal Dott. Margo Moore, Simon Fraser University, Burnaby, BC, Canada. <ol><li> Piatto conidi di un ceppo di Aspergillus fumigatus esprimere GFP su Sabouraud cuore cervello infusione (BHI) slant agar con cloramfenicolo (0,05 g / L) e Gentamicina (0,5 g / L). </li><li> Incubare al buio liberamente ricoperto per 7-10 giorni a temperatura ambiente. </li><li> Harvest conidi mediante lavaggio slant con 10 ml di 0,01% di Tween 20 in DPBS. Leggermente raschiare il fungo con il stripette per allentare. Sciacquare l&#39;inclinazione più volte per aumentare il rendimento. </li><li> Passare sospensione conidi attraverso un colino cella 100 micron in una conica da 50 ml. </li><li> Pellet conidi per centrifugazione a 400 xg per 10 min. </li><li> Risospendere conidi in 50 ml DPBS e contare con un emocitometro. </li><li> Lavare due volte con DPBS e diluire a concentrazione desideratistrazione. </li></ol> 4. Fungina Challenge e Microscopia confocale Per studiare il ruolo dei macrofagi NADPH ossidasi in difesa ospite, la capacità dei macrofagi alveolari isolati da topi di tre genotipi per controllare la crescita delle spore di Aspergillus fagocitati è stata valutata. I genotipi utilizzati in questo studio includono; 1) topi wild-type, 2) Ncf1 * / * Mn-topi con una mutazione naturale Ncf1 (Ncf1 codifica per p47 phox, una componente necessaria del fagociti ossidasi NADPH) lasciando loro NADPH ossidasi carente, e 3) Ncf1 * / * Mn + topi transgenici (MN denota il transgene ospitare tipo selvatico Ncf1 sotto il controllo del promotore di CD68 umano) dove l&#39;attività NADPH ossidasi è stato ricostituito nella popolazione di monociti / macrofagi 10, 11. Microscopia confocale È più adatto per questo test per la presenzae la crescita di conidi non fagocitato sugli scivoli. Il microscopio confocale permetterà la visualizzazione di piani individuali all&#39;interno l&#39;asse Z, e consentire la differenziazione tra fungo cresce intorno piuttosto che all&#39;interno dei macrofagi. Tutte le immagini confocale vengono acquisite con un obiettivo a immersione in olio 63X. Z-stack sono acquisiti con un fattore di zoom elettronico di 2.5. Lo spessore di un Z-stack è determinato utilizzando DAPI e immagini in campo chiaro per individuare la parte superiore e inferiore del campo. Z-stack variava da 14 a 24 micron, con singole sezioni ~ 1 micron di spessore. Per la quantificazione dei macrofagi, singole scansioni di zoom elettronico 1X sono stati eseguiti su 10 campi per genotipo. Macrofagi intatti sono stati identificati sulla base PKH26 e colorazione DAPI della membrana e nucleo rispettivamente. Conidi sono stati identificati in base all&#39;immagine campo chiaro espressione FITC e. <ol><li> Preparare vetrini sterili e piastre <ol><li> Sotto una cappa di sicurezza biologica, immergere 22 x 22 mm coprioggetto di vetro nel 70% ethanol per 5-10 min. </li><li> Sciacquare coprioggetto in PBS sterile. </li><li> Utilizzando pinze sterili, posizionare delicatamente un singolo vetrino sul fondo di ciascun pozzetto di una singolarmente confezionati 6-pozzetti sterile. </li></ol></li><li> Seed ~ 1 x 10 6 raccolto PKH26 etichettato macrofagi alveolari sospesi in 300 ml RPMI 1640, 10% FBS su ogni vetrino. Il numero esatto può variare a seconda del rendimento del raccolto. </li><li> Lasciare macrofagi di aderire incubando a 37 ° C con 5% di CO 2 durante la notte. </li><li> La mattina seguente, aggiungere ~ 1 x 10 7-GFP che esprimono A. fumigatus conidi sospeso in 100 ml di pre-riscaldato (37 ° C) RPMI 1640, 10% FBS. </li><li> Piastra Rientro a 37 ° C incubatore con il 5% di CO 2. </li><li> Le cellule devono essere fissati per un minimo di 2 ore a temperatura ambiente o per una notte a 4 ° C. A 3, 7, e 14 hr cellule fix aggiungendo un volume uguale di 2% di formaldeide in ogni pozzetto (concentrazione finale 1% di formaldeide). Piastre posto per l&#39;punti temporali presto a 4 ° C durante la notte. Per il punto finale di tempo (14 ore), fissare le cellule a temperatura ambiente per 2 ore. </li><li> Dopo la fissazione, rimuovere delicatamente coprioggetto con pinze e sciacquare in DPBS. </li><li> Rimuovere il liquido in eccesso ponendo il bordo del vetrino su un Kimwipe piegato. </li><li> Applicare 6 mezzo di montaggio microlitri fluorescenza con DAPI ad un vetrino da microscopio in vetro. </li><li> Posare un bordo del vetrino sul vetrino e rilasciare delicatamente con lato della cella giù nel mezzo di montaggio. Mezzo di montaggio si diffonderà e formare uno strato anche sotto il vetrino. Non c&#39;è bisogno di premere sul vetrino. </li><li> Sigillare i bordi del coprioggetto con smalto trasparente e lasciare asciugare all&#39;aria. </li><li> Conservare preparati vetrini in una scatola slitta (protetto dalla luce) a temperatura ambiente fino al momento di analizzare mediante microscopia confocale. </li></ol>

<ol>
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Usando questo approccio ex vivo per analisi dell&#39;attività antifungina di macrofagi insieme in vivo sfida fungina, abbiamo precedentemente dimostrato che NADPH ossidasi in macrofagi svolge un ruolo importante nella difesa contro A. fumigatus 4. L&#39;uso di isolati macrofagi alveolari ci permette di concentrarsi sulla loro attività antifungina in assenza di altre cellule immunitarie (ad esempio, neutrofili reclutati) che difendersi Aspergillus in vivo. <p ...

Macrofagi alveolari sono i fagociti di prima linea che incontrano i microbi inalati. I macrofagi riconoscono motivi Aspergillus dai recettori di riconoscimento patogeni, ingerire e limitano la crescita delle spore per via inalatoria (conidi), e avviare le risposte infiammatorie. Il ossidasi dei fagociti NADPH converte l&#39;ossigeno molecolare per anione superossido e valle intermedi reattivi dell&#39;ossigeno (ROI). Malattia granulomatosa cronica (CGD) è una malattia ereditaria della NADPH ossidasi caratterizzata da infezioni batteriche e fungine gravi e le risposte infiammatorie eccessive. Anche se NADPH ossidasi è fondamentale per neutrofilo-mediato difesa ospite 1-3, l&#39;importanza della NADPH ossidasi nei macrofagi non è ben definito. Precedenti studi hanno dimostrato che i macrofagi alveolari ingerire ed uccidere le spore di Aspergillus, considerando che i neutrofili principalmente di mira il palco ife 5. Tuttavia, ci sono stati Resul contrastantits come il ruolo della NADPH ossidasi in macrofagi nel controllare la crescita di A. fumigatus spore 6, 7. L&#39;obiettivo di questo metodo è stato quello di delineare il ruolo specifico della NADPH ossidasi nei macrofagi nel mediare difesa dell&#39;ospite contro A. spore fumigatus. Abbiamo trovato che NADPH ossidasi in macrofagi alveolari controlla la crescita di fagocitato A. fumigatus spore 4. Per modellare più strettamente la risposta dell&#39;ospite ai funghi per via inalatoria, abbiamo usato macrofagi alveolari raccolte da lavaggio broncoalveolare da topi non stimolati subito dopo il sacrificio. L&#39;uso di isolati macrofagi alveolari ci ha consentito di concentrarsi sulla loro attività antifungina in assenza di altre cellule immunitarie (ad esempio, neutrofili reclutati) che difendersi Aspergillus in vivo. In questo metodo, macrofagi alveolari sono state colorate in vivo prima della raccolta in modo da minimizzare la quantità di manipolazione post-raccolta. &lt;/ P&gt; Un altro vantaggio di questo protocollo è l&#39;uso di un GFP che esprimono A. ceppo fumigatus. Questo fungo esprime GFP dalla fase conidi attraverso la fase di ife e non ha bisogno di ulteriori colorazione. Per ottenere immagini con maggiore dettaglio, abbiamo scelto microscopia confocale che permette la ricostruzione di strutture tridimensionali delle immagini ottenute. Ricostruzione tridimensionale dà la capacità di distinguere tra ife crescono intorno piuttosto che in o attraverso un macrofago. Conidi può anche essere precisamente distinto come intracellulare contro extracellulare.

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	<colgroup>
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		<col />
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		<col />
	</colgroup>
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">PKH26GL-1KT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2,2,2 Tribromoethanol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T48402</td>
			<td>Used to make AvertinAnesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-methyl-2-butanol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A-1685</td>
			<td>Used to make AvertinAnesthetic</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dulbecco&#39;s Phosphate Buffered Saline (without calcium and magnesium)</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">NH4CL</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A0171</td>
			<td>Used to make ACK red cell lysis buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">KHCO3</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P91444</td>
			<td>Used to make ACK red cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">E6758</td>
			<td>Used to make ACK red cell lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">22 gauge X 1.00 inch i.v. catheter</td>
			<td style="width:71px;">BD&#160;</td>
			<td align="right" style="width:119px;">381523</td>
			<td>Insyte Autoguard Winged</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">insulin syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6 ml syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">4-way stopcock</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">50-700-077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">suture thread</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 ml conical centrifuge tubes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gauze sponges (4 inch square)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 with L-glutamine</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">10-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:71px;">Hyclone</td>
			<td style="width:119px;">SH30396.03</td>
			<td>heat inactivated</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hema 3 Stain Set</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">22-122-911</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cytoseal 60</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">8310-4</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sabouraud Brain Heart Infusion Agar with Chloramphenicol and Gentamicin Slants</td>
			<td style="width:71px;">BD&#160;</td>
			<td align="right" style="width:119px;">297252</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Aspergillus fumigatous strain expressing green fluorescent protein</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>provided by Dr Margo Moore, Simon Fraser University, Burnaby, BC, Canada</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100 micron Cell Strainers</td>
			<td style="width:71px;">BD Falcon</td>
			<td style="width:119px;">64753-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">scissors</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">forceps</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biological Safety Cabinet Class II</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sorval ST40 Centrifuge with swing bucket rotor</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Leica DM1000 light microscope</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemocytometer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cytospin 2 centrifuge</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Cell Culture Incubator</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>37 &#176;C, 5%&#160; CO2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">22X22 mm micro cover glass</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">48366-227</td>
			<td>glass coverslips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6 well Cell Culture Plates</td>
			<td style="width:71px;">Corning</td>
			<td align="right" style="width:119px;">3506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10% Neutral Buffered Formalin</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">BDH0502-1LP</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Vectashield Mounting medium with DAPI</td>
			<td style="width:71px;">Vector Laboratories</td>
			<td style="width:119px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Leica TCS SP2 system with a laser point scanner</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Mounted on DMIRE2 fluorescence microscope</td>
		</tr>
	</tbody>
</table>

assessing anti-fungal activity of isolated alveolar macrophages by confocal microscopy

Il polmone è un&#39;interfaccia in cui cellule ospiti sono regolarmente esposti ai microbi e prodotti microbici. Macrofagi alveolari sono i fagociti di prima linea che incontrano i funghi per via inalatoria e altri microbi. Macrofagi e altre cellule immunitarie riconoscono motivi Aspergillus dai recettori di riconoscimento patogeno e avviare risposte infiammatorie a valle. Il ossidasi dei fagociti NADPH genera intermedi reattivi dell&#39;ossigeno (ROI) ed è fondamentale per la difesa ospite. Anche se NADPH ossidasi è fondamentale per neutrofilo-mediato difesa ospite 1-3, l&#39;importanza della NADPH ossidasi nei macrofagi non è ben definito. L&#39;obiettivo di questo studio è stato quello di delineare il ruolo specifico della NADPH ossidasi nei macrofagi nel mediare difesa dell&#39;ospite contro A. fumigatus. Abbiamo trovato che NADPH ossidasi in macrofagi alveolari controlla la crescita di fagocitato A. fumigatus spore 4. Qui, descriviamo un metodo per valutare la capacità di un topomacrofagi lveolar (AMS) per controllare la crescita delle spore di Aspergillus fagocitati (conidi). Macrofagi alveolari sono macchiati in vivo e dieci giorni più tardi isolati da topi da lavaggio broncoalveolare (BAL). I macrofagi sono placcati su vetrini, poi seminate con la proteina fluorescente verde (GFP) che esprimono A. spore fumigatus. A volte specificato, le cellule vengono fissate e il numero di macrofagi intatte con spore fagocitati è valutata mediante microscopia confocale.

<html> Raccolta dei macrofagi alveolari da BAL da topi non stimolati si tradurrà in una popolazione pura&gt; 95% in base citologia. Il 3 e 7 ore (Figura 2) punti temporali precedono la fase di ife di crescita fungina e consentono un confronto di efficienza fagocitaria dei conidi tra i genotipi. Il punto di tempo 14 hr è dove genotipi possono essere confrontati per quanto riguarda la capacità di inibire il passaggio di conidi fagocitato alla fase hyphal tessuto invasivo (Figura 3). Mac...

Watch this Scientific Journal Video about Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy at JoVE.com

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

Un metodo per valutare la capacità di isolato macrofagi alveolari del mouse per controllare la crescita di spore di Aspergillus fagocitati mediante microscopia confocale.

Valutare l&#39;attività anti-fungina isolato Alveolar macrofagi dal Microscopia confocale

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

assessing-anti-fungal-activity-isolated-alveolar-macrophages-confocal

Research

JoVE Journal

Immunology and Infection

11.4K Views.  Roswell Park Cancer Institute.  Un metodo per valutare la capacità di isolato macrofagi alveolari del mouse per controllare la crescita di spore di Aspergillus fagocitati mediante microscopia confocale. 

Video: Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Forno a microonde Assist diagnosi rapida di malattie virali piante da microscopia elettronica a trasmissione

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Ricerca

Immunologia e infezioni

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

Microscopia intravitale della Milza: analisi quantitativa della mobilità Parasite e del flusso sanguigno

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

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 ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

Proteomica Profiling dei macrofagi da 2D elettroforesi

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

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

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

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

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

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

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Isolamento, caratterizzazione e purificazione dei macrofagi dai tessuti colpiti da infiammazione obesità

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

Visualizzazione del macrofago trappole extracellulari mediante microscopia confocale

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolamento e coltura In Vitro dei macrofagi alveolari murini ed umani

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Time-lapse Imaging 3D della fagocitosi dai macrofagi del topo

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.