Name: phagosome migration and velocity measured in live primary human macrophages infected with hiv-1
Uploaded: 2016-09-05T15:00:00.000+00:00
Duration: 7 min 32 s
Description: Watch this Scientific Journal Video about Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1 at JoVE.com

We thank Dr Jamil Jubrail for reading the manuscript. This work was supported by grants from CNRS, Inserm, Universit&#233; Paris Descartes, Agence Nationale de la Recherche (2011 BSV3 025 02), Fondation pour la Recherche M&#233;dicale (FRM DEQ20130326518 including a doctoral fellowship for GLB) and Agence Nationale de Recherche sur le SIDA et les h&#233;patites virales (ANRS, including a post-doctoral fellowship for CD) to FN. A. Dumas was supported by doctoral fellowships from Universit&#233; Paris Descartes and Sidaction.

Il protocollo deve essere effettuata in stretta conformità con le normative nazionali ed internazionali e le normative locali. Il sangue da donatori sani che hanno dato il loro consenso a donare il sangue per scopi di ricerca è stato ottenuto dal sangue centri trasfusionali con la quale le istituzioni hanno firmato un accordo. protezioni speciali devono essere prese quando si utilizza il sangue umano. Esperimenti con HIV-1 devono essere eseguite in un livello di biosicurezza 3 o 2 (BSL-3 o 2) di laboratorio in base alla legislazione locale. 1. Preparazione di macrofagi umani derivate da monociti (hMDMs) per gradiente di densità centrifugazione e selezione per adesione <ol><li> Inizia con sangue fresco da donatori sani (9 ml). Diluire l&#39;intero volume di sangue fresco con 1x tampone fosfato sterile (PBS) senza Ca 2 + e Mg 2+ a ottenere un volume finale di 70 ml e aggiungere delicatamente il sangue diluito in due provette da 50 ml coniche (35 ml per provetta) , in cima a 15 ml di unaeutral, altamente ramificata, di massa elevata, polisaccaride idrofila in soluzione già in ciascun tubo. </li><li> Centrifuga entrambi i tubi di sangue al 537 xg per 20 minuti a 20 ° C senza freno. Poi raccogliere le cellule mononucleate del sangue periferico (PBMC) contenuti nel ring cellule nuvoloso a livello di interfaccia e trasferirli in un nuovo tubo da 50 ml contenente 15 ml di 1x PBS senza Ca 2+ e Mg 2+. </li><li> Centrifugare le cellule a 218 xg per 5 minuti a 20 ° C e risospendere il pellet in 45 ml di 1x PBS senza Ca 2+ e Mg 2+. </li><li> Centrifugare le cellule a 218 xg per 5 min a 20 ° C, risospendere il pellet in 10 ml di PBS 1x senza Ca 2 + e Mg 2+, e contare le cellule diluendo ad una diluizione finale di 1/200 in Trypan Blue. </li><li> Centrifugare le cellule a 218 xg per 5 min a 20 ° C e risospendere il pellet in RPMI (Roswell Park Memorial Institute) 1640 integrato con 2 mM L-glutammina e 100 ug / ml PenicIllin-streptomicina avere 7 x 10 6 PBMC per pozzetto in 2 ml di mezzo in ogni pozzetto di una piastra a pozzetti 6. </li><li> Incubare le piastre a 37 ° C con 5% di CO 2 per 2 ore. Dopo 2 ore monociti avranno aderito alla plastica. </li><li> Per consentire monociti appena isolati di differenziarsi in hMDMs, aspirare il terreno e sostituirlo con 2 ml hMDM media (RPMI 1640, il 10% decomplemented siero fetale di vitello (FCS), 2 mM L-glutammina e l&#39;1% di penicillina-streptomicina) integrato con ricombinante umano macrofagi fattore stimolante le colonie (RHM-CSF) ad una concentrazione finale di 10 ng / ml. </li><li> Incubare le cellule a 37 ° C con 5% di CO 2 per 11 giorni (Figura 1). </li><li> Al giorno 11 rimuovere il supporto e lavare 2 volte con 2 ml di terreno hMDM freddo per pozzetto. Successivo lavare i pozzetti 2 volte con 1 ml di freddo 1x PBS. </li><li> Per staccare hMDMs completamente differenziate, lavare ogni tempo ben 1 con 1 ml di freddo 1x PBS con 2 mm di acido ethylenediaminetetraacetatic (EDTA) e incubare le cellule in 2 ml di PBS freddo 1x con 2 mM EDTA per pozzetto per 15-60 minuti a 4 ° C. NOTA: Metodi alternativi per staccare esistono le cellule (ad esempio, tripsina o attività tripsina-simile) che potrebbe dare buoni risultati 11. </li><li> Dopo il distacco (completato pipettando gentilmente su e giù nel pozzo), raccogliere le cellule e metterli in un tubo da 50 ml in ghiaccio contenente 10 ml di terreno hMDM freddo. </li><li> Centrifugare le cellule a 218 xg per 5 min a 20 ° C, risospendere il pellet in 10 ml di terreno hMDM fredda, e contare le cellule diluendo 1/20 in Trypan Blue. </li><li> Seme le cellule a 1 x 10 6 hMDMs a 35 millimetri di vetro microscopia grade piatto fondo e incubare le piastre a 37 ° C con 5% di CO 2 per 1 giorni. </li></ol> 2. Produzione e quantificazione di HIV-1 Stocks virali NOTA: NLR4.3 HIV-1 gag-iGFP (Green Fluorescent Protein) che trasportano una busta R5-tropico, dono di M. Schindler 10 viene utilizzato per infettare i macrofagi e le cellule infette vedere in tempo reale. <ol><li> Produrre stock virali mediante trasfezione delle cellule embrionali umane del rene 293T (2 x 10 6 in 100 millimetri piatto) con 6 ug del corrispondente DNA provirale utilizzando un reagente di trasfezione commerciale. </li><li> Quantificare l&#39;infettività delle riserve di virus utilizzando le cellule indicatore HeLa TZM-bl (recanti il ​​gene beta-galattosidasi sotto il controllo di HIV-1 LTR) utilizzando diluizioni seriali dei titoli virali seguita da una colorazione ß-galattosidasi delle cellule e il conteggio le cellule di blu 12. </li></ol> 3. L&#39;infezione di hMDMs con HIV-1 <ol><li> Aggiungere il virus ad una molteplicità di infezione (MOI) 0,02-0,03 in 1 ml di terreno hMDM per hMDMs (cellule placcati in 1.13). Per controllare pozzetti aggiungere solo 1 ml di terreno hMDM e incubare le piastre a 37 ° C con 5% di CO 2 per 2 giorni (Figura 1). </li><li> Al giorno 2 Lavare le cellule con hMDM medIUM 3 volte e aggiungere 1 ml di terreno hMDM fresco per ogni piatto. Incubare le cellule a 37 ° C con 5% di CO 2 per 6 giorni (Figura 1). </li></ol> 4. Opsonizzazione di pecore Globuli rossi <ol><li> Per la preparazione di 7 x 10 6 GRP che per piatto, lavare i GRP che due volte con 100 ml di soluzione contenente 0,1% di sieroalbumina bovina (BSA) in PBS 1x con centrifugazione a 600 xg per 4 min. </li><li> Risospendere le GRP che lavata in 500 microlitri 1x PBS / BSA 0,1% con IgG di coniglio anti-GRP che ad una concentrazione sub-agglutinante per 5 ml di GRP che e incubare con rotazione a temperatura ambiente per 30 min. NOTA: per determinare la concentrazione sub-agglutinante di anti-GRP che IgG, preparare diluizioni seriali di IgG (magazzino di concentramento di 13,1 mg / ml) da 1/50 a 1 / 25.600 in 20 microlitri in una piastra da 96 pozzetti. Aggiungere 2 x 10 6 GRP che in 20 microlitri di ogni bene e mettere il piatto in una stanza buia durante diverse ore. La concentrazione sub-agglutinante è ildiluizione del pozzo poco prima del bene con agglutinazione (IgG + GRP che formano una rete). </li><li> Dopo la rotazione, centrifugare le-GRP che IgG-opsonizzati a 600 xg per 4 minuti e lavare con 100 ml di 1x PBS / BSA 0,1% con centrifugazione a 600 xg per 4 minuti. </li><li> Risospendere le-GRP che IgG-opsonizzati in media pre-riscaldato rosso fenolo-libero RPMI integrato con 2 mM L-glutammina e l&#39;1% di penicillina-streptomicina (1 ml / piatto). </li></ol> 5. cellulare dal vivo microscopio video fagocitosi Assay <ol><li> Utilizzare un sistema di imaging confocale come un sistema disco rotante dotato di una camera di riscaldamento a 37 ° C con CO 2 passa attraverso una bottiglia con acqua per umidificazione. </li><li> Accendere la camera di riscaldamento prima dell&#39;esperimento di avere palco microscopio a 37 ° C prima dell&#39;inizio del saggio fagocitosi. Accendere il microscopio e il computer, e caricare il software di imaging. </li><li> Ottimizzare le impostazioni di imaging come la velocità di scansione, magnification, risoluzione, ecc avere almeno una cella per campo e all&#39;immagine un fotogramma ogni minuto tra 60 a 120 min. NOTA: Qui, il campione viene ripreso un fotogramma ogni minuto durante 60 minuti con 63X lente. </li><li> Porre la capsula di imaging sul palco microscopio. Regolare la messa a fuoco e la posizione per trovare una sola intera HIV-1 macrofagi infettati nel campo. Utilizzare le impostazioni di eccitazione / emissione appropriate in base al sistema di imaging utilizzata e sonda. Includere un canale luminoso campo (BF) per osservare phagosomes (Figura 1ii). Ottimizzare l&#39;aspetto delle diverse immagini del canale regolando la percentuale di tempo di luce e l&#39;esposizione trasmessa. NOTA: Qui, NLR4.3 HIV-1 gag-iGFP è stato eccitato con un laser a 491 nm con 50 msec di tempo di esposizione con il 20% di laser (Figura 1i). </li><li> Rimuovere il piatto di imaging e aggiungere 1 ml di sospensione SRBC a 7 x 10 6 / ml GRP che al piatto (Figura 1). </li><li> Centrifugare a 500 xg per2 min a RT per sincronizzare fagocitosi, registrare il tempo alla fine di questa centrifugazione e tornare il piatto allo stadio. </li><li> Ottimizzare la messa a fuoco e cattura GFP e immagini BF in Z-stack (tutto lo spessore della cella con un passo distanza di 0,3 micron - solitamente 20 aerei) ogni minuto per almeno 1 ora. Salvare il video time-lapse nel file-formato nativo del sistema di imaging utilizzato. NOTA: Qui, filmati time-lapse sono stati salvati nel formato nativo, i file * .stk. </li></ol> 6. L&#39;analisi dei filmati time-lapse <ol><li> Per l&#39;editing video, fare clic sul menu a discesa &quot;Apps&quot; e nella scheda &quot;Review Multi Dimensional Data&quot; nel software di editing video. <ol><li> Per aprire il file, cliccare su &quot;Seleziona file base&quot; e poi su &quot;Seleziona Directory&quot;. Nella casella &quot;Set di dati&quot;, selezionare l&#39;acquisizione da analizzare (in formato .nd) e cliccare su &quot;Visualizza&quot;. I dati sono rappresentati in una tabella nel tempo in una colonnad Z-piano in linea. </li><li> Per rappresentare l&#39;infezione in un Z-proiezione (Figura 2, i pannelli di sinistra), selezionare 491 nm di lunghezza d&#39;onda nella casella &quot;lunghezze d&#39;onda&quot; e fare clic sulla scheda &quot;proiezione Z&quot; con &quot;tutti i piani&quot;. </li><li> Per analizzare una sequenza temporale, selezionare la lunghezza d&#39;onda BF nella casella &quot;lunghezze d&#39;onda&quot; e scegliere il piano ottimale su l&#39;asse Z per distinguere GRP che esterni (Figura 2, punta di freccia rossa), GRP che interni (Figura 2, cerchio rosso) e il nucleo ( Figura 2, cerchio blu). </li><li> Per salvare i montaggi video, cliccare su &quot;Selezione [di X] scheda&quot; e poi su &quot;Load Image (s)&quot;. Infine, salvare le immagini caricate in formato tif e aprirli prossima sul software ImageJ. </li></ol></li><li> Utilizzare il plug &quot;Manual Tracking&quot; sul software ImageJ per misurare la posizione del nucleo e le diverse phagosomes osservate nel canale BF (Figura 3). <ol><li> Download il plugin &quot;Allineamento manuale&quot; sul sito web ImageJ. Su ImageJ, aprire il plugin e la sequenza di immagini da analizzare (Figura 3, punti 1 e 2). </li><li> Inserire le impostazioni quali &quot;Intervallo di tempo&quot;, che rappresenta quantità di tempo tra i fotogrammi adiacenti, e la &quot;taratura x / y&quot;, che rappresenta la distanza per pixel (Figura 3, punto 3). NOTA: Qui, salvare la sequenza di immagini con un intervallo di tempo di 1 min tra ogni fotogramma e x / y calibrazione di 0,205 micron perché lo zoom 63X e una fotocamera con dimensioni pixel di 6,45 x 6,45 micron 2 vengono utilizzati. </li><li> Per avviare il monitoraggio, cliccare su &quot;Add Track&quot; (Figura 3, punto 4) e fare clic su un centro di SRBC a la prima volta quando viene interiorizzato. Il fotogramma successivo appare automaticamente. </li><li> Continuare a fare clic sul centro SRBC in tutti i fotogrammi di avere posizioni diverse nel corso del tempo (figura 3, passo 6 nel riquadro rosso). <ol><li> Iniziare a monitorare il nucleo che la sua posizione in tutti i fotogrammi facendo clic sul suo centro. Traccia successiva le phagosomes (cliccando sul suo centro) uno per uno, al momento (frame) della loro internalizzazione, diversa in funzione della GRP che. Per comodità di vedere SRBC sul canale BF, utilizzare la finestra &quot;Luminosità e contrasto&quot; durante l&#39;inseguimento (Figura 3, punto 5). </li></ol></li><li> Tra ogni monitoraggio SRBC, cliccare su &quot;Add Track&quot; (Figura 3, punto 4) per avere una nuova traccia. Il numero di inseguimento sarà cambiata nella seconda colonna, &quot;Track n °&quot; della tabella dei risultati (Figura 3, punto 6). </li><li> Salvare i dati in un foglio di continuare alla fase successiva dell&#39;analisi. </li></ol></li><li> Utilizzare il software foglio di calcolo per calcolare la distanza percorsa di fagosomi contenenti GRP che verso il nucleo e la velocità dei phagosomes durante i primi 5 minuti dopo l&#39;internalizzazione di GRP che (Fifigura 4). <ol><li> Aprire la tabella foglio di calcolo e un nuovo file di foglio di calcolo. Trasferire in questo nuovo file solo i seguenti parametri, il tempo, gli assi X e Y coordinate del nucleo e tutti i GRP che (figura 4a). </li><li> Per calcolare la distanza tra GRP che il nucleo e con solo loro coordinate, considerare il nucleo e un SRBC in un piano XY coordinata (Figura 4B). NOTA: La distanza tra due punti è la lunghezza del percorso che li collega. Nel piano, la distanza tra SRBC e il nucleo è dato dal teorema di Pitagora. <ol><li> Se c (la distanza tra il nucleo e il SRBC) indica la lunghezza dell&#39;ipotenusa e ae b indicano le lunghezze degli altri due lati, esprimere teorema di Pitagora &#39;come equazione di Pitagora: <img alt="Equazione 1" src="/files/ftp_upload/54568/54568eq1.jpg" /> NOTA: Allo stesso tempo, il ortonormale, la distanza orizzontalea è (x nucleo -x SRBC) e la distanza verticale b è (y nucleo -y SRBC). </li><li> Così, calcolare la distanza tra il nucleo e il SRBC (Figura 4A, scatola viola) da: <img alt="Equazione 2" src="/files/ftp_upload/54568/54568eq2.jpg" /> NOTA: Moltiplicare il valore della distanza ottenuto (in pixel) mediante calibrazione x / y di avere distanza in micron. Qui, la calibrazione x / y è 0,205 micron. </li><li> Ad ogni volta, sottrarre la distanza tra il nucleo e il SRBC alla distanza iniziale (Figura 4C). </li><li> Tracciare la misura contro il tempo per il primo 5 min. Applicare un trend-line lineare (Figura 5A) per la trama e determinare la pendenza della linea di tendenza lineare che rappresenta la velocità phagosome verso il nucleo durante i primi 5 minuti dopo l&#39;internalizzazione SRBC. </li><li> Collazionare i dati per calcolare l&#39;errore medio e statistica dii valori di velocità e li trama in forma adeguata utilizzando il software appropriato (Figura 5B). </li></ol></li></ol></li></ol>

<ol>
	<li>Flannagan, R. S., Jaumouille, V., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+phagocytosis.">The cell biology of phagocytosis.</a> Annu. Rev. Pathol. 7, 61-98 (2012).</li><li>Niedergang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Cell Biology. 2, 751-757 (2016).</li><li>Blocker, A., Griffiths, G., Olivo, J. C., Hyman, A. A., Severin, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+microtubule+dynamics+in+phagosome+movement.">A role for microtubule dynamics in phagosome movement.</a> J Cell Sci. 111 (Pt 3), 303-312 (1998).</li><li>Blocker, A., 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=Molecular+requirements+for+bi-directional+movement+of+phagosomes+along+microtubules.">Molecular requirements for bi-directional movement of phagosomes along microtubules.</a> J Cell Biol. 137, 113-129 (1997).</li><li>Flannagan, R. S., Cosio, G., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+mechanisms+of+phagocytes+and+bacterial+evasion+strategies.">Antimicrobial mechanisms of phagocytes and bacterial evasion strategies.</a> Nat Rev Microbiol. 7, 355-366 (2009).</li><li>Feasey, N. A., Dougan, G., Kingsley, R. A., Heyderman, R. S., Gordon, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+non-typhoidal+salmonella+disease:+an+emerging+and+neglected+tropical+disease+in+Africa.">Invasive non-typhoidal salmonella disease: an emerging and neglected tropical disease in Africa.</a> Lancet. 379, 2489-2499 (2012).</li><li>Mazzolini, J., 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=Inhibition+of+phagocytosis+in+HIV-1-infected+macrophages+relies+on+Nef-dependent+alteration+of+focal+delivery+of+recycling+compartments.">Inhibition of phagocytosis in HIV-1-infected macrophages relies on Nef-dependent alteration of focal delivery of recycling compartments.</a> Blood. 115, 4226-4236 (2010).</li><li>Dumas, A., 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=The+HIV-1+protein+Vpr+impairs+phagosome+maturation+by+controlling+microtubule-dependent+trafficking.">The HIV-1 protein Vpr impairs phagosome maturation by controlling microtubule-dependent trafficking.</a> J Cell Biol. 211, 359-372 (2015).</li><li>Greenberg, S., el Khoury, J., Kaplan, E., Silverstein, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescence+technique+to+distinguish+attached+from+ingested+erythrocytes+and+zymosan+particles+in+phagocytosing+macrophages.">A fluorescence technique to distinguish attached from ingested erythrocytes and zymosan particles in phagocytosing macrophages.</a> J. Immunol. Methods. 139, 115-122 (1991).</li><li>Koppensteiner, H., Banning, C., Schneider, C., Hohenberg, H., Schindler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+internal+HIV-1+is+protected+from+neutralizing+antibodies.">Macrophage internal HIV-1 is protected from neutralizing antibodies.</a> J Virol. 86, 2826-2836 (2012).</li><li>Gartner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+macrophage+and+HIV:+basic+concepts+and+methodologies.">The macrophage and HIV: basic concepts and methodologies.</a> Methods Mol Biol. 1087, 207-220 (2014).</li><li>Wei, X., 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=Emergence+of+resistant+human+immunodeficiency+virus+type+1+in+patients+receiving+fusion+inhibitor+(T-20)+monotherapy.">Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy.</a> Antimicrob Agents Chemother. 46, 1896-1905 (2002).</li><li>Harrison, R. E., Bucci, C., Vieira, O. V., Schroer, T. A., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagosomes+fuse+with+late+endosomes+and/or+lysosomes+by+extension+of+membrane+protrusions+along+microtubules:+role+of+Rab7+and+RILP.">Phagosomes fuse with late endosomes and/or lysosomes by extension of membrane protrusions along microtubules: role of Rab7 and RILP.</a> Mol Cell Biol. 23, 6494-6506 (2003).</li><li>Toyohara, A., Inaba, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+of+phagosomes+in+mouse+peritoneal+macrophages.">Transport of phagosomes in mouse peritoneal macrophages.</a> J Cell Sci. 94 (Pt 1), 143-153 (1989).</li></ol>

The authors have nothing to disclose.

Questa tecnica ha diversi passaggi critici. In primo luogo, la preparazione di hMDMs e l&#39;infezione da HIV-1 è critico poiché la percentuale di infezione è donatore dipendente. Da notare, abbiamo deciso di utilizzare macrofagi che non sono polarizzati in vitro prima dell&#39;infezione, perché lo stato macrofagi potenzialmente incontrate dal virus in vivo non è stata ben caratterizzata finora. Abbiamo controllato l&#39;espressione di diversi marcatori di superficie, il che indica che i macrofagi...

Macrophages play a major role in the innate immune system and in homeostasis. They are professional phagocytes that internalize and eliminate pathogens and debris by a process called phagocytosis 1,2. The phagosome, the closed compartment that forms after the engulfment of particulate material, undergoes a series of fusion and fission events with endocytic compartments, leading to a degradative compartment called the phagolysosome. This compartment has an acidic pH, due to the acquisition of proton-pumping v-ATPases, contains hydrolytic enzymes and is enriched in lysosomal-associated membrane proteins (LAMPs). The maturation of phagosomes is accompanied by their migration on microtubules 3,4 towards the cell center to reach a perinuclear location where lysosomes are accumulated.
Many pathogens have been reported to hijack phagosome maturation, including bacteria with intracellular lifestyles that modify the vacuolar environment where they reside 5. The Human Immunodeficiency Virus (HIV)-1 targets macrophages in addition to T cells. As macrophages are resistant to the cytopathic effects of the virus, unlike T cells, they can be considered as a reservoir for the virus. In addition, macrophages infected with HIV-1 show defective phagocytic functions and contribute to the emergence of opportunistic diseases. In particular, severe invasive non-typhoidal Salmonella disease caused by Salmonella Typhimurium ST313 has been prevalent for the last three decades in sub-Saharan African children or adults infected with HIV 6. It has been estimated that the risk of developing tuberculosis is more than 20 times greater in people living with HIV than among those without HIV infection. 
For all these reasons, it is important to better define the molecular mechanisms underlying the phagocytic defects in HIV-infected macrophages. We have shown that the uptake of particulate material, opsonized particles, bacteria or fungi, was inhibited in HIV-infected macrophages 7. Given that this inhibition is partial, we then set out to analyze the fate of the internalized phagosomes in HIV infected human macrophages 8. Because phagosome maturation is tightly connected with migration to the cell center and fusion with lysosomes, a defect in phagosomal maturation can be due to modifications of the trafficking modalities in the infected cell. The method described here uses IgG-opsonized Sheep Red Blood Cells (IgG-SRBCs) as a model to target receptor-mediated phagocytosis and in particular receptors for the Fc portion of immunoglobulins (FcR). These particles are easier to image in bright field (BF) than latex beads because extracellular and intracellular SRBCs show different refraction properties 9. To measure the velocity of phagosomes moving towards the nucleus in HIV-infected macrophages, we used a fluorescent virus 10 and set up a simple manual tracking method that is described here. The method does not require advanced programming and simply uses ImageJ. It is amenable to adherent cells and any type of particle or pathogen that can be visualized with bright field microscopy or with fluorescent imaging.

<table><tbody><tr><td>Falcon 100 mm TC-Treated Cell Culture Dish</td><td>Corning</td><td>353003</td><td>For viral production</td></tr><tr><td>Glass Bottom Dishes 35 mm uncoated 1.5</td><td>MatTek corporation</td><td>P35G-1.5-14-C Case</td><td>For acquisition</td></tr><tr><td>Falcon Tissue Culture Plates 6-well</td><td>Thermo Fischer Scientific</td><td>Corning. Inc. 353934</td><td>For human monocyte-derived macrophages</td></tr><tr><td>Ficoll-Plaque PLUS</td><td>Dominique Dutscher</td><td>17-1440-03</td><td>a neutral, highly branched, high-mass, hydrophilic polysaccharide in solution for density centrifugation</td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Thermo Fischer Scientific</td><td>14190-094</td><td>RT</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM) 1x, liquid (High Glucose)</td><td>GIBCO, Molecular probes</td><td>31966-021</td><td>Conserved at 4 &amp;deg;C ; for HEK cells culture</td></tr><tr><td>RPMI 1640 medium GLUTAMAX&amp;nbsp; Supplement</td><td>Life technologies</td><td>61870-010</td><td>Conserved at 4 &amp;deg;C; for hMDMs culture</td></tr><tr><td>F&amp;oelig;tal Calf Serum (FCS)&amp;nbsp;</td><td>Eurobio</td><td>CVFSVF0001</td><td>Conserved at -20 &amp;deg;C ; decomplemented</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Thermo Fischer Scientific</td><td>15140-122</td><td>Conserved at -20 &amp;deg;C ; for hMDMs culture</td></tr><tr><td>RPMI 1640 medium, no phenol red (10x 500 ml)</td><td>Life technologies</td><td>11835-105</td><td>Warm in 37 &amp;deg;C water bath before use ; for phagocytosis assay</td></tr><tr><td>FuGENE6 Transfection Reagent</td><td>Promega</td><td>E2692</td><td>Conserved at 4 &amp;deg;C ; for viral production</td></tr><tr><td>Sheep red blood cells (SRBCs)</td><td>Eurobio</td><td>DSGMTN00-0Q</td><td>Conserved in Alsever buffer at 4 &amp;deg;C before use</td></tr><tr><td>Anti-sheep red blood cells IgG</td><td>MP Biomedicals</td><td>55806</td><td>Conserved at 4 &amp;deg;C</td></tr><tr><td>Bovine Serum Albumin heat shock fraction, pH 7, &amp;ge;98%&amp;nbsp;</td><td>Sigma</td><td>A7906</td><td>Conserved at -20 &amp;deg;C</td></tr><tr><td>Inverted microscope DMI600</td><td>Leica</td><td></td><td></td></tr><tr><td>Confocal Spinning Disk Unit&amp;nbsp; CSU-X1M1</td><td>Yokogawa</td><td></td><td></td></tr><tr><td>491 nm 50 mW laser</td><td>COBOLT CALYPSO</td><td></td><td></td></tr><tr><td>HCX PL APO CS Objectif</td><td>Leica</td><td></td><td>Objective lens ; Magnification 100X ; Numerical aperture 1.40 ; Immersion oil</td></tr><tr><td>CoolSnap HQ2 (FireWire) Camera</td><td>Photometrics</td><td></td><td>Pixel size 6.45 &amp;micro;m x 6.45 &amp;micro;m ; Definition 1,392 x 1,040 ; Encoding the image in 14 Bit</td></tr><tr><td>Metamorph 7.7.5 software</td><td>Molecular Devices</td><td></td><td>For the control of the microscope</td></tr><tr><td>GraphPad Prism software</td><td></td><td></td><td>For the statistics analysis</td></tr></tbody></table>

phagosome migration and velocity measured in live primary human macrophages infected with hiv-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

FCR-mediata fagocitosi da parte hMDMs HIV-1-infetti e non infetti è descritto qui utilizzando GRP che IgG-opsonizzati come bersagli modello (Figura 1). I passaggi critici di questo protocollo sono la preparazione del hMDMs e l&#39;infezione da HIV-1. Infatti, la resa e la qualità dei macrofagi differenziati varia tra i donatori, così come il tasso di infezione con efficienze nell&#39;intervallo 10-40%. Inoltre, la preparazione di IgG-opsonizzati-GRP che è anche impor...

Watch this Scientific Journal Video about Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1 at JoVE.com

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

Migrazione phagosome e velocità misurata in Live primarie macrofagi umani infettati con HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

phagosome-migration-velocity-measured-live-primary-human-macrophages

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

8.1K Views.  Inserm U1016, Institut Cochin, CNRS UMR 8104, Université Paris Descartes, Sorbonne Paris Cité. We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes. 

Video: Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

Visualizzazione Cell-per-cell trasferimento del virus HIV usando cloni fluorescenti di HIV e Live Microscopia confocale

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

Ricerca

Immunologia e infezioni

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Live-cellula Microscopia Video di funghi patogeni fagocitosi

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Studio di fagolisosoma Biogenesis nei macrofagi in diretta

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

Alta Tecnica Throughput fluorometrica per la valutazione dei macrofagi fagocitosi e polimerizzazione

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

&quot;Phagosome chiusura Assay&quot; per visualizzare phagosome Formazione in tre dimensioni utilizzando la riflessione totale interna fluorescente Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

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 ...

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).
Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.

Studiare la fagocitosi della Leishmania usando la microscopia confocale

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up ...

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These cells are specialized in microorganism recognition and phagocytosis, but also activate other immune cells and present antigens, as well as promote inflammation and tissue repair. Here, we describe a protocol to obtain mononuclear cells from peripheral blood (PBMC) of healthy donors to separate monocytes that then differentiate into macrophages. These cells can then be infected in vitro at different Lb concentrations to evaluate the ability to control infection, as well as evaluate host cell immune response, which can be measured by several methods. PBMCs were first isolated by centrifuging with Ficoll-Hypaque gradient and then plated to allow monocytes to adhere to culture plates; non-adherent cells were removed by washing. Next, adherent cells were cultured with macrophage-colony stimulating factor (M-CSF) for 7 days to induce macrophage differentiation. We suggest plating 2 x 106&#160;cells per well on 24-well plates in order to obtain 2 x 105&#160;macrophages. Fully differentiated macrophages can then be infected with Lb for 4 or 24 hours. This protocol results in a significant percentage of infected cells, which can be assessed by optical or fluorescence microscopy. In addition to infection index, parasite load can be measured by counting the numbers of parasites inside each cell. Further molecular and functional assays can also be performed in culture supernatants or within the macrophages themselves, which allows this protocol to be applied in a variety of contexts and also adapted to other intracellular parasite species.

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

This protocol describes the process for obtaining human macrophages from monocytes for infection with Leishmania braziliensis. It also allows researchers to evaluate infection rate and parasite viability, ROS production by fluorescence microscopy, and the production of inflammatory mediators in culture supernatants to investigate macrophage response to infection.

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These ...

Real-Time Imaging of Macrophage Phagocytosis of IgG-Opsonized Red Blood Cells

Source: Horsthemke, M., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/57566">Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages.</a> J. Vis. Exp. (2018)
This video demonstrates an assay to monitor independent macrophage-mediated red blood cell (RBC) phagocytosis events. Mouse immunoglobulin G (IgG)-opsonized human RBCs are incubated with mouse macrophages and observed under a confocal microscope. The RBCs are engulfed by macrophages via a phagocytic cup formation and targeted for lysosomal degradation.

Imaging in tempo reale della fagocitosi macrofagica dei globuli rossi opsonizzati IgG

Source: Horsthemke, M., et al. Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages. J. Vis. Exp. (2018)
This video demonstrates an assay to monitor ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

TIRF Microscopy-Based Visualization of Phagosome Formation and Closure

Source: Marie-Ana&#239;s, F., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54470">&#34;Phagosome Closure Assay&#34; to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM).</a> J. Vis. Exp. (2016).
This video demonstrates a high-resolution total internal reflection fluorescence (TIRF) microscopy technique for real-time visualization of phagosome formation and closure during macrophage-mediated phagocytosis of IgG-opsonized red blood cells (RBCs) attached to the surface of a glass bottom dish. The macrophages extend pseudopodia around the RBCs and engulf them inside the phagosomes, detaching them from the glass surface.

Migrazione phagosome e velocità misurata in Live primarie macrofagi umani infettati con HIV-1

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Esplora Altri Video

Capitoli in questo video

Visualizzazione Cell-per-cell trasferimento del virus HIV usando cloni fluorescenti di HIV e Live Microscopia confocale

Live-cellula Microscopia Video di funghi patogeni fagocitosi

Studio di fagolisosoma Biogenesis nei macrofagi in diretta

Alta Tecnica Throughput fluorometrica per la valutazione dei macrofagi fagocitosi e polimerizzazione

"Phagosome chiusura Assay" per visualizzare phagosome Formazione in tre dimensioni utilizzando la riflessione totale interna fluorescente Microscopy (TIRFM)

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

Studiare la fagocitosi della Leishmania usando la microscopia confocale

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

Imaging in tempo reale della fagocitosi macrofagica dei globuli rossi opsonizzati IgG

Visualizzazione basata su microscopia TIRF della formazione e della chiusura dei fagosomi