Questa ricerca è stata sostenuta in parte dal NIH premi 5T32AI07506, 5R01AI033774, 5R37AI033142, 5R01AI052733.

Tutto il lavoro animale è stato fatto in conformità con le normative e le linee guida dell&#39;Istituto di studi sugli animali con Albert Einstein College of Medicine. 1. Crescita Condizioni di C. neoformans H99 (sierotipo A) <ol><li> Grow individuale C. neoformans (Cn) colonie su piastre di agar Sabouraud. </li><li> Un giorno prima dell&#39;esperimento, selezionare una colonia e inoculare 10 ml di brodo Sabouraud. </li><li> Lasciare cultura a crescere durante la notte a 37 ° C agitando a velocità di 250 rpm. </li></ol> 2 Isolamento e preparazione di primaria Bone macrofagi da C57 / BL6 Mice <ol><li> Euthanize topi mettendo animale in una camera di CO 2 fino a non respirare più. Come seconda misura, cervicale dislocare il collo prima di iniziare la dissezione. Sterilizzare tutto il corpo con il 70% di alcol etilico. </li><li> Rimuovere entrambi i femori e tibia e posto le ossa in DMEM sterile. </li><li> Rimuovere il tessuto in eccesso e muscolare con delicate asciuga fino ossa sono completamente puliti. </li><li> Posizionare le ossa intatte solo in una capsula di Petri contenente il 70% di alcol etilico e incubare per 3 minuti per uccidere i microrganismi associati. Togliere le ossa e posto in capsula di Petri con DMEM sterile. </li><li> Tagliare con cautela estremità delle ossa. Tenere osso su un tubo conico vuoto 50 ml posti su ghiaccio e lavare accuratamente 10 ml di DMEM freddo utilizzando un ago da 25 G con ogni osso. NOTA: Ogni topo produrrà 4 ossa (2 femori, tibie 2). Pertanto un mouse dovrebbe produrre circa 40 ml di sospensione cellulare. </li><li> Sospensione cellulare Centrifugare a temperatura ambiente per 10 min a 650 x g. </li><li> Durante questo centrifugazione, preparare e filtro sterilizzare midollo osseo macrofagi alimentazione dei media che consiste di DMEM supplementato con il 20% L929, siero di vitello fetale 10%, 10% NCTC-109, 1% Pen-Strep, 1% HEPES, 1% L-glutammina , 1% di aminoacidi non essenziali, 0,1% 2-mercaptoetanolo. </li><li> Risospendere il pellet risultante con 10 ml di alimentazione dei supporti. Passare suspens cellulariion attraverso un filtro 70 micron cella di interrompere grumi di cellule e rimuovere i detriti di grandi dimensioni. </li><li> Tavola 1 ml di sospensione cellulare in 10 ml di media alimentare in capsule di Petri che vengono specificati per l&#39;uso coltura tissutale (per un totale di 10 piatti). </li><li> Consentono alle cellule di aderire a 37 ° C con 10% di CO 2. Aggiungere 5 ml di supporti di alimentazione fresca il giorno 3 Il giorno 7, completamente sostituire il supporto di alimentazione e macrofagi sono pronti per essere utilizzati. </li><li> Il giorno prima dell&#39;esperimento, staccare macrofagi di targa togliendo mezzi di alimentazione e aggiungere 5 ml di una cellula dolce strippaggio reagente alla piastra. </li><li> Incubare per 5-10 min a 37 ° C e rimuovere macrofagi con dolce pipettaggio. </li><li> Cellule pellet per centrifugazione a 650 xg per 10 min. </li><li> Risospendere il pellet in 1 ml di alimentazione dei supporti. Usando una diluizione 1:20, calcolare la concentrazione di macrofagi pipettando 10 ml di sospensione cellulare su un emocitometro. </li><li> Utilizzo di 14 millimetri con fondo trasparente piastre di Petri, un piattoconcentrazione di 1 x 10 5 macrofagi direttamente sul pozzo interno che contiene un massimo di 200 ml, quindi prendere precauzioni per non superare questo volume, come mostrato in Figura 4. </li><li> Consentire alle cellule di aderire alla piastra di Petri di vetro mettendo i piatti in un incubatore a 37 ° C per 1 ora. </li><li> Aggiungere 1-2 ml di alimentazione supporti integrati con LPS a 1 mg / ml e IFNg a 500 U / ml e permettono alle cellule di incubare una notte. </li></ol> 3 co-incubazione dei macrofagi murini primari e C. neoformans <ol><li> Il giorno dell&#39;esperimento, rimuovere 1 ml di cellule di coltura e lievito pellet pernottamento inoculate mediante centrifugazione per 5 min a 420 x g. </li><li> Lavare le cellule 3 volte con PBS sterile per rimuovere il supporto e prodotti criptococco extracellulari come la glucuronoxylomannan polisaccaride (GXM) alla velocità e il tempo di cui sopra. </li><li> Risospendere il pellet di cellule in 1 ml di PBS sterile e fare una diluizione 1: 100. Pipettare 10 ml di tegli diluizione su un emocitometro e contare le cellule. Aggiungi cellule di lievito in una MOI di 1: 5. Ad esempio, se ci sono 1 x 10 5 macrofagi / bene, fare una sospensione cellulare di 5 x 10 6 / ml di Cn e aggiungere 100 ml di questa soluzione per un importo complessivo di 5 x 10 5 Cn. </li><li> Registrare il volume calcolato di sospensione cellulare criptococcica a 1 ml di mezzi di alimentazione con l&#39;anticorpo monoclonale 18B7 ad una concentrazione di 10 mg / ml. Incubare sospensione cellulare a temperatura ambiente per 5 min. </li><li> Rimuovere i supporti di alimentazione dal fondo di vetro piatto di Petri e lavare 1 volta con PBS sterile. Aggiungere 100 ml di opsonizzati Cn direttamente al bene. </li><li> Consentono alle cellule di incubare per 2 ore a 37 ° C con il 10% di CO 2. </li><li> Dopo aver co-incubazione, controllare da microscopio che i macrofagi hanno interiorizzato Cn. Per essere considerati interiorizzato, le cellule criptococco dovrebbero chiaramente essere visto entro i confini del macrofago. </li><li> Lavare 3x Petri piatto con PBS sterile per rimuovere ogni e qualsiasi extracellular Cn. </li><li> Aggiungere 1-2 ml di alimentazione dei supporti, senza l&#39;aggiunta di mAb 18B7, e impostare microscopio. </li></ol> 4. Visualizing non-litica Esocitosi in tempo reale Utilizzando Luce Microscopia Digitale NOTA: Per eseguire questi esperimenti, un microscopio che viene fornito con una camera di incubazione allegato che include CO 2 la consegna è richiesta una unità di riscaldamento. <ol><li> Prima dell&#39;esperimento, pre-riscaldare la camera di incubazione per almeno 30 min a 37 ° C. Assicurarsi che l&#39;unità di CO 2 legge 5% all&#39;inizio dell&#39;esperimento. </li><li> Luogo fondo di vetro piatto di Petri su piattaforma microscopio, coprire con CO 2 coperchio, e chiudere tutte le porte per garantire l&#39;assenza di fughe di calore. </li><li> Utilizzando l&#39;obiettivo 10X con il microscopio a contrasto di fase, concentrarsi su una chiara campo di macrofagi infettati. Sulla base della sperimentazione, determinare l&#39;obiettivo utilizzato da quanti macrofagi necessario studiare. </li><li> Impostare il software microscopio a take un&#39;immagine ogni 4 minuti per un periodo di 24 ore. </li><li> Dopo 24 ore, l&#39;esperimento avrà raggiunto il completamento e sarà stato raccolto un totale di 361 immagini. Esportare queste immagini come .jpegs a compressione 5%. Utilizzando software Image J, visualizzare le immagini come una pila di Visual a 5 fotogrammi al sec. Se il filmato deve essere visto per la pubblicazione o una presentazione, salvarla sia come avi o mov di file a seconda di quale formato funziona meglio per lo spettatore. </li></ol>

<ol>
	<li>Voelz, K., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcal+Interactions+With+the+Host+Immune+System.">Cryptococcal Interactions With the Host Immune System.</a> Eukaryotic cell. 9 (6), 835-846 (2010).</li><li>Johnston, S. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcus+interactions+with+macrophages:+evasion+and+manipulation+of+the+phagosome+by+a+fungal+pathogen.">Cryptococcus interactions with macrophages: evasion and manipulation of the phagosome by a fungal pathogen.</a> Cellular microbiology. 15 (3), 403-411 (2012).</li><li>Sabiiti, W., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+infection+by+the+human+fungal+pathogen+Cryptococcus+neoformans.">Mechanisms of infection by the human fungal pathogen Cryptococcus neoformans.</a> Future microbiology. 7 (11), 1297-1313 (2012).</li><li>Bliska, J. B., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+pathogenic+bacteria+and+fungi+&#8212;+a+case+of+convergent+evolution.">Intracellular pathogenic bacteria and fungi &#8212; a case of convergent evolution.</a> Nature Reviews Microbiology. 7, 165-171 (2008).</li><li>Alvarez, M., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+spread+and+massive+vacuole+formation+after+Cryptococcus+neoformans+infection+of+murine+macrophages.">Cell-to-cell spread and massive vacuole formation after Cryptococcus neoformans infection of murine macrophages.</a> BMC immunology. 8 (1), 16 (2007).</li><li>Ma, H., Croudace, J. E., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+cell-to-cell+spread+of+a+pathogenic+yeast.">Direct cell-to-cell spread of a pathogenic yeast.</a> BMC immunology. 8, 15 (2007).</li><li>Alvarez, M., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagosome+extrusion+and+host-cell+survival+after+Cryptococcus+neoformans+phagocytosis+by+macrophages.">Phagosome extrusion and host-cell survival after Cryptococcus neoformans phagocytosis by macrophages.</a> Curr Biol. 16 (21), 2161-2165 (2006).</li><li>Ma, H., Croudace, J. E., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expulsion+of+live+pathogenic+yeast+by+macrophages.">Expulsion of live pathogenic yeast by macrophages.</a> Curr Biol. 16 (21), 2156-2160 (2006).</li><li>Johnston, S. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+fungal+pathogen+Cryptococcus+neoformans+escapes+macrophages+by+a+phagosome+emptying+mechanism+that+is+inhibited+by+Arp2/3+complex-mediated+actin+polymerisation.">The human fungal pathogen Cryptococcus neoformans escapes macrophages by a phagosome emptying mechanism that is inhibited by Arp2/3 complex-mediated actin polymerisation.</a> PLoS pathogens. 6 (8), (2010).</li><li>Carnell, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+polymerization+driven+by+WASH+causes+V-ATPase+retrieval+and+vesicle+neutralization+before+exocytosis.">Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis.</a> The Journal of cell biology. 193 (5), 831-839 (2011).</li><li>Voelz, K., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+signaling+regulates+the+outcome+of+intracellular+macrophage+parasitism+by+Cryptococcus+neoformans.">Cytokine signaling regulates the outcome of intracellular macrophage parasitism by Cryptococcus neoformans.</a> Infection and immunity. 77 (8), 3450-3457 (2009).</li><li>Bain, M. J., Lewis, E. L., Okai, B., Quinn, J., Gow, A. R. N., Erwig, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-lytic+expulsion/exocytosis+of+Candida+albicans+from+macrophages.">Non-lytic expulsion/exocytosis of Candida albicans from macrophages.</a> Fungal genetics and biology. 49 (9), 677-678 (2012).</li></ol>

Gli autori dichiarano che non abbiamo interessi finanziari concorrenti o altri conflitti di interesse.

Qui abbiamo descritto un metodo con cui le interazioni fungine-macrofagi possono essere registrati e analizzati in tempo reale per un periodo di 24 ore. Il nostro laboratorio ha utilizzato questo protocollo per studiare molti degli aspetti temporali della esocitosi non-litica e continuerà ad utilizzare la microscopia in tempo reale per accertare le componenti morfologiche che circondano questo processo. Per questa tecnica per produrre risultati ottimali, particolare attenzione deve essere d...

Le fasi di una infezione fungina tra le cellule criptococco e macrofagi sono ben documentati 1-3. Dopo cellule di lievito vengono ingeriti dai macrofagi, una varietà di interazioni possono verificarsi: il macrofago può lisare rilasciando il suo carico fungina nello spazio extracellulare, o potrebbe controllare l&#39;infezione mantenendo le cellule di lievito entro i confini della sua membrana cellulare 4. Tuttavia, alcuni anni fa un nuovo risultato è stato descritto indipendentemente da due gruppi: esocitosi non-litico, un processo in cui un macrofago cancellata alcune o tutte le celle criptococco nell&#39;ambiente circostante o una cellula vicina e sia host e patogeno rimangono vitali 5 -8. Diversi studi hanno cercato di comprendere i meccanismi molecolari alla base di questa interazione, ma abbiamo ancora non hanno una conoscenza completa su ciò che guida questo fenomeno. La capacità di catturare immagini in tempo reale e successivamente analizzare infettare piùEd macrofagi permette di rispondere a molte domande sulle proprietà fisiche circostanti esocitosi non-litica per quanto riguarda i tempi, la capacità spaziale, il cambiamento nella morfologia e anche lo stress di macrofagi nel corso di una infezione fungina. Permettendo alle cellule di essere ospitati in un ambiente che è paragonabile a quello di un incubatore, possiamo intravedere la natura dinamica delle interazioni cellulari di macrofagi-fungine. I ricercatori hanno usato questa tecnica per studiare vari elementi di questo processo. Il ruolo del phagosome in questo processo è stato reso evidente con colorazione fluorescente e actina agenti di blocco 9, mentre un altro gruppo ha usato questo metodo per dimostrare che esocitosi non-litica è stata bloccata in cellule che hanno avuto la WASH nucleazione domini proteici cancellato 10. L&#39;effetto di segnalazione delle citochine è stata anche determinata mediante microscopia in tempo reale 11. Questo processo non è limitato a C. neoformans come è stato osservato anche con Candida albicans, another patogeno fungino che ha la capacità di infettare i macrofagi 12. Questi risultati sono esempi di come questo metodo può fornire una serie di informazioni in un&#39;area che sappiamo così poco.

<table border="0" cellpadding="0" cellspacing="0" width="976">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sabouraud Dextrose Agar</td>
			<td style="width:183px;">BD</td>
			<td style="width:227px;">DF0109-17-1</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sabouraud Dextrose Broth</td>
			<td>BD</td>
			<td>DF0382-17-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dubelcco&#39;s Modified Eagle&#39;s Medium(DMEM)</td>
			<td>Corning Cellgro</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Calf Serum ( FCS)&#160;</td>
			<td>Atlanta Biologicals</td>
			<td>S12450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NCTC 109</td>
			<td>Invitrogen</td>
			<td>21340-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES Buffer</td>
			<td>Corning Cellgro</td>
			<td>25-060-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutamax</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-essential Amino Acids</td>
			<td>Corning Cellgro</td>
			<td>25-025-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985-023</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3003 Tissue Culture Petri Dishes&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>08772E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellStripper</td>
			<td>Corning Cellgro</td>
			<td>25-056-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mat-tek Glass Bottom Culture Dishes</td>
			<td>MatTek</td>
			<td>P35GC-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LPS</td>
			<td>Sigma</td>
			<td>L3137</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse IFN-g</td>
			<td>Roche</td>
			<td>NC 9222016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mAb 18B7</td>
			<td colspan="2">&#160;&#160;&#160;&#160;&#160;&#160; Non-commerical antibody produced in our lab</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axiovert 200M Microscope with Incubating Chamber</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing non-lytic exocytosis of cryptococcus neoformans from macrophages using digital light microscopy

Molti aspetti l&#39;infezione di macrofagi da Cryptococcus neoformans sono stati ampiamente studiati e ben definito. Tuttavia, una particolare interazione che non è chiaramente compreso è esocitosi non-litico. In questo processo, le cellule di lievito vengono rilasciati nello spazio extracellulare con un meccanismo poco compreso che lascia entrambi i macrofagi e Cn praticabile. Qui, descriviamo come seguire un gran numero di macrofagi infettati singolarmente per un periodo di 24 ore l&#39;infezione mediante microscopia-tempo trascorso. Macrofagi infetti sono alloggiati in una camera di riscaldamento con un atmosfera di CO 2 collegato ad un microscopio che fornisce le stesse condizioni di un incubatore di coltura cellulare. Microscopio digitale diretta in grado di fornire informazioni sulle interazioni dinamiche tra un host e agente patogeno che non è disponibile da immagini statiche. Essere in grado di visualizzare ogni cellula infettata può fornire indizi su come i macrofagi gestire infezioni fungine, e viceversa. Questa tecnica isa potente strumento per studiare le dinamiche che stanno dietro un fenomeno complesso.

Le immagini che questa tecnica produce possono essere analizzati in una varietà di modi per raccogliere informazioni che circonda ciò che accade dopo che le cellule sono fagocitati C. neoformans. Come si vede nella figura 1, ci sono circa 100 i macrofagi che sono o infetto o non infetto. Ognuno di questi macrofagi dà allo spettatore l&#39;opportunità di studiare le interazioni ospite-patogeno su un livello molto microscopico. Come le figure 2 e 3 mostrano, ...

Watch this Scientific Journal Video about Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy at JoVE.com

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Descriviamo come visualizzare macrofagi C. neoformans (Cn) interazioni in tempo reale, con particolare enfasi sul processo di esocitosi non-litica mediante microscopia ottica digitale. Utilizzando questa tecnica individuale macrofagi infetti può essere studiato per accertare i vari aspetti di questo fenomeno.

Visualizzazione non-litica Esocitosi di<em&gt; Cryptococcus neoformans</em&gt; Dai macrofagi utilizzando la luce Microscopia Digitale

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

visualizing-non-lytic-exocytosis-cryptococcus-neoformans-from

Research

JoVE Journal

Immunology and Infection

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

11.7K Views.  Albert Einstein College of Medicine.  Descriviamo come visualizzare macrofagi C. neoformans (Cn) interazioni in tempo reale, con particolare enfasi sul processo di esocitosi non-litica mediante microscopia ottica digitale. Utilizzando questa tecnica individuale macrofagi infetti può essere studiato per accertare i vari aspetti di questo fenomeno. 

Video: Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

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

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

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 ...

Ricerca

Immunologia e infezioni

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Macrofagi Colesterolo Depletion e il suo effetto sulla fagocitosi di<em&gt; Cryptococcus neoformans</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs&#8217; mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules&#160;(SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

The goal of the present&#160;protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time&#160;analyses of the secretory granule&#39;s morphology.

Indagare Mast Cellula secretoria Granuli; dal Biosintesi a Esocitosi

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

Trasformazione Biolistic di una fluorescente Tagged Gene nel Opportunistic Fungal patogeni<em&gt; Cryptococcus neoformans</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Misurare risposte fisiologiche di<em&gt; Drosophila</em&gt; Neuroni sensoriali a Lipid feromoni utilizza Live Calcio Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Caratterizzazione fenotipica dei macrofagi dal rene di ratto mediante citometria di flusso

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

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 of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolamento di Salmonella typhimurium-contenente fagosomi dai macrofagi

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Visualizing intracellulare rullante traffico da fluorescenza Lifetime Imaging microscopia

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...