Gli autori riconoscono notevolmente Pierre Bourdoncle, Thomas Guilbert e Louise Rimbault dello strumento Cochin Imaging. Questo lavoro è stato sostenuto da Inserm, CNRS e Ligue Nationale contre le Cancer (Equipe labellisée).

1. Preparazione di linfociti T <ol><li> Preparare principale del mouse o cellule T umane come descritto 10,11. </li><li> Coltivare cellule T umane in mezzo completo RPMI con 10% siero umano AB ad una densità di 2 - 3 x 10 6 cellule / ml. NOTA: L&#39;uso di siero fetale di vitello (FCS) invece di siero umano può suscitare polarizzazione spontanea di una grande frazione di cellule T umane in coltura. Conservare queste cellule in coltura per 4 - 5 giorni ed ulteriormente processo in qualsiasi momento durante questo periodo. </li><li> Mantenere cellule T del mouse in mezzo completo RPMI supplementato con 10% FCS ad una densità cellulare simile. Utilizzare subito o il giorno successivo, dopo una / cultura O N a 37 ° C, in presenza di 10 ng / ml IL7. </li><li> Coltivare la linea cellulare T CEM umano completo RPMI supplementato con 10% FCS. </li></ol> 2. chemochine Stimolazione <ol><li> Lavare 5 x 10 5 cellule per condizione sperimentale in caldo medio HBSS integrato con Hepe 10 mMs. Per alcuni esperimenti, co-trasfettare cellule T umane primarie il giorno prima con 2 ug pmaxGFP e 8 mg pcDNA3.1 + (vettore vuoto, controllo), PKCζ chinasi morti, o N-terminali plasmidi PAR6. </li><li> Risospendere il pellet di cellule in warm medio HBSS-Hepes (utilizzare 0,3 ml x numero di condizioni sperimentali). </li><li> Distribuire le cellule in provette da 1,5 ml microcentrifuga. NOTA: Pertanto, un tubo corrispondente ad una singola condizione sperimentale conterrà 5 x 10 5 cellule in 0,3 ml di HBSS-Hepes medie. Per alcuni esperimenti, aggiungere 500 nM Latrunculin A o veicolo (DMSO) e incubare le cellule per 30 minuti prima della stimolazione chemochine. </li><li> Aggiungere da 10 a 500 ng / ml CCL19 o CXCL12 chemochine in ciascun tubo. NOTA: Usiamo solitamente 10-200 ng chemochine / ml per le cellule T umane. Cellule CEM non esprimono il recettore CCR7 che lega CCL19. Pertanto, le cellule CEM saranno in grado di rispondere a una stimolazione CXCL12. Inoltre, utilizzare concentrazioni più elevate chemochine (300-500 ng / ml) per polarizzare cellule T del mouse. </li><li> Capovolgere i tubi diverse volte e incubare in un bagno d&#39;acqua a 37 ° per 8 o 10 minuti per le cellule T primarie o CEM, rispettivamente. </li><li> Arrestare il processo di polarizzazione aggiungendo a ciascuna provetta 0,3 ml di una soluzione calda contenente 2% paraformaldeide (PFA) e 10 mM Hepes in PBS. Vibrazione le provette e incubare a 37 ° C per 5 min. </li></ol> 3. colorazioni <ol><li> Trasferire le cellule dai tubi microcentrifuga di citometria a flusso tubi. </li><li> Riempire i tubi completamente con una soluzione contenente RT 1% di albumina sierica bovina (BSA), EDTA 0,5 mM e 10 mM Hepes in PBS. </li><li> Centrifugare le provette a 460 g per 4 min. </li><li> Eliminare il supernatante e risospendere le cellule in 100 microlitri di una soluzione RT contenente 5% FCS in PBS. </li><li> Aggiungere 5 ml di FITC-anti-HLA-ABC in ogni provetta, li vortice, e incubare per 30 min a temperatura ambiente, al riparo dalla luce. </li><li> Lavare le cellule una volta con PBS contenente 5% FCS, unnd una volta con solo PBS. </li><li> A RT: aggiungere 500 microlitri 1% PFA, incubare per 10 min, poi lavare le cellule con una soluzione contenente 1% BSA, EDTA 0,5 mM e HEPES 10 mM in PBS. </li><li> Eliminare il surnatante, riempire completamente i tubi con una soluzione di PBS contenente 0,2% di BSA, 0,1% saponina, 0,5 mM EDTA e 10 mM Hepes (buffer permeabilizzazione). </li><li> Lavare le cellule due volte in questo mezzo. </li><li> Capovolgere i tubi per rimuovere il supporto e vortice li dissociare il pellet. </li><li> Aggiungere 0,25 mg / ml TRITC-falloidina e / o di una diluizione 1: 100 di anticorpi anti-P-ERM a ciascuna provetta, li vortex e incubare per 30 minuti a RT. </li><li> Lavare le cellule con il buffer permeabilizzazione. Incubare con un anticorpo secondario fluorescente se necessario. </li><li> Risospendere le cellule in 200 microlitri di PBS e trasferirli ai tubi microcentrifuga. NOTA: Le cellule sono pronte per l&#39;acquisizione. Alternativamente, pur mantenendo un volume massimo totale di 200 microlitri per provetta, aggiungere PFA concentrata ad una finale 1%concentrazione al fine di preservare le colorazioni se non devono essere utilizzate le cellule lo stesso giorno per ulteriore elaborazione. </li></ol> 4. Immagini Acquisizione e analisi <ol><li> Accendere l&#39;apparecchio (vedi tabella di reagenti specifici / Equipment) e lasciarlo calibrare stesso, in base alle istruzioni del produttore. NOTA: Il software INSPIRE è stato utilizzato con un obiettivo 40X (0,75 NA). Il software IDEAS 3.0 è stato utilizzato per tutte le quantificazioni segnalati. </li><li> Preparare una serie di finestre di acquisizione che possono essere salvate in un modello per futuri esperimenti. Disegnare istogrammi che quantificano i valori di gradiente RMS. Dalla popolazione &quot;In Focus&quot;, selezionati sulla RMS istogramma pendenza, delimitano la popolazione &quot;cella singola&quot; su un istogramma della zona. Una volta che la provetta è stata inserita nella macchina, regolare la potenza del laser, cancello a cellule singole e acquisire 5.000 T linfociti. </li><li> Nelle idee morbideware, aprire il file di acquisizione e creare un istogramma che mostra il valore del valore RMS gradiente per le immagini con sfondo chiaro (Canale 1) ottenuto per ogni singola cella. Disegnare un cancello sulla gradiente istogramma RMS che considera le cellule espositrici RMS valore gradiente sopra 57. NOTA: Il gradiente RMS permette di prendere in considerazione nell&#39;analisi solo i linfociti T che sono nel piano focale. Abbiamo determinato empiricamente che singole cellule che presentano un valore di gradiente RMS superiore a 57 sono nel piano focale, e possono essere considerati accuratamente. </li><li> Nel menu Analysis / Maschere, creare una nuova maschera, chiamato Erode (M01,2) dalla maschera morfologia sulle immagini brightfield M01, eroso di 2 pixel. Poi, nel Analisi / menu Impostazioni, creare una nuova caratteristica della zona, calcolato sulla maschera Erode (M01,2). Dalle celle selezionate nel cancello precedente, aprire un istogramma che mostra l&#39;area di celle che utilizzano questa maschera appena creato. La morfologia maschere che sono disponibili: NOTEpredefinito è molto più grande della dimensione reale della cella. Pertanto, è più preciso di erodere fino a 2 pixel. </li><li> Disegnare una porta sulle singole cellule T, escludendo sferette di calibrazione e detriti (piccola area) e le doppiette (Large Area). Regolare questa porta ad ogni acquisizione. NOTA: Variazioni nella dimensione della cella influenzerà la posizione del cancello. Come cellule T topo sono più piccoli delle cellule T umane che sono a loro volta inferiori cellule CEM, l&#39;area di ogni tipo di cellula sarà proporzionale alle sue dimensioni. </li><li> Considerando i singoli, in-fuoco cellule che sono state gated ai passi precedenti, aprire un diagramma a punti costituito dal grezzo Max Pixel sulla colorazione HLA-ABC (canale 2, asse x) in funzione del grezzo Max Pixel sul falloidina colorazione (Channel 4, l&#39;asse y). Creare una porta per escludere gli eventi fluorescenti senza macchia o saturi. Aperte due istogrammi che mostrano la Raw Max Pixel per HLA-ABC (Canale 2) e falloidina (Channel 4) colorazioni. </li><li> Nel Analysis / menù Maschere, createa nuova maschera, chiamato Erode (M02,2) dalla maschera morfologia delle cellule HLA-ABC macchiati (Canale 2), eroso di 2 pixel. Poi, nel Analisi / menu Impostazioni, creare una nuova funzione che consiste del parametro circolarità, calcolato sulla Erode (M02,2). NOTA: Questo parametro misura la deviazione della forma cellula da un cerchio. Pertanto, una cellula T perfettamente rotonda avrà un valore elevato circolarità che cellule polarizzate allungate esporrà un indice circolarità bassa. </li><li> Successivamente, creare un istogramma che riporterà i valori della funzione Circolarità dalle cellule gated precedenza. Utilizzare questo istogramma per tracciare direttamente la distribuzione di un individuo, popolazione cellulare a fuoco secondo il valore della circolarità per ogni singola cella. </li><li> Guardando la forma dell&#39;istogramma per cellule non stimolate, elaborare un cancello per cellule polarizzate, a partire dal valore circolarità basso fino al valore limite circolarità dove la maggior parte delle cellule non stimolatesono tracciate. Guardate il display statistiche per la percentuale di cellule polarizzate tra la popolazione gated (% Gated). NOTA: Abbiamo impostato questa porta per valori di indice circolarità sotto 13. </li><li> Per guardare la polarizzazione della colorazione actina nelle cellule, tracciare un istogramma del brillante dettaglio valore Similarity R3, confrontando la colorazione HLA-ABC (canale 2) e la colorazione falloidina (Channel 4). NOTA: Più grande è il valore di questa funzionalità, il più sovrapposto le due colorazioni sono. </li><li> Utilizzando la stessa strategia di gating come prima, tracciare una porta e sulle cellule non stimolate per la colorazione segregato che mostra la percentuale di cellule con colorazione polarizzata actina. </li><li> Al termine, la percentuale di cellule che mostrano un segregazione nella distribuzione del MHC di classe I e colorazioni falloidina, la percentuale di cellule polarizzate insieme con i valori medi degli indici di similarità e circolarità di tutte le celle ± SD sono mostrati in correspStatistiche onding pannelli. </li></ol>

<ol>
	<li>Rossi, D., Zlotnik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+chemokines+and+their+receptors.">The biology of chemokines and their receptors.</a> Annu Rev Immunol. 18, 217-242 (2000).</li><li>Thelen, M., Stein, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+chemokines+invite+leukocytes+to+dance.">How chemokines invite leukocytes to dance.</a> Nat Immunol. 9 (9), 953-959 (2008).</li><li>Rougerie, P., Rho Delon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTPases:+masters+of+T+lymphocyte+migration+and+activation.">GTPases: masters of T lymphocyte migration and activation.</a> Immunol Lett. 142 (1-2), 1-13 (2012).</li><li>Sanchez-Madrid, F., del Pozo, 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=Leukocyte+polarization+in+cell+migration+and+immune+interactions.">Leukocyte polarization in cell migration and immune interactions.</a> Embo J. 18 (3), 501-511 (1999).</li><li>Adams, D. H., 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=Hepatocyte+growth+factor+and+macrophage+inflammatory+protein+1+beta:+structurally+distinct+cytokines+that+induce+rapid+cytoskeletal+changes+and+subset-preferential+migration+in+T+cells.">Hepatocyte growth factor and macrophage inflammatory protein 1 beta: structurally distinct cytokines that induce rapid cytoskeletal changes and subset-preferential migration in T cells.</a> Proc Natl Acad Sci U S A. 91 (15), 7144-7148 (1994).</li><li>Rougerie, P., 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=Fam65b+is+a+new+transcriptional+target+of+FOXO1+that+regulates+RhoA+signaling+for+T+lymphocyte+migration.">Fam65b is a new transcriptional target of FOXO1 that regulates RhoA signaling for T lymphocyte migration.</a> J Immunol. 190 (2), 748-7455 (2013).</li><li>Real, E., Faure, S., Donnadieu, E., Delon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+Atypical+PKCs+regulate+T+lymphocyte+polarity+and+scanning+behavior.">Cutting edge: Atypical PKCs regulate T lymphocyte polarity and scanning behavior.</a> J Immunol. 179 (9), 5649-5652 (2007).</li><li>Negulescu, P. A., Krasieva, T. B., Khan, A., Kerschbaum, H. H., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarity+of+T+cell+shape,+motility,+and+sensitivity+to+antigen.">Polarity of T cell shape, motility, and sensitivity to antigen.</a> Immunity. 4 (5), 421-430 (1996).</li><li>Zuba-Surma, E. K., Kucia, M., Abdel-Latif, A., Lillard, J. W., Ratajczak, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ImageStream+System:+a+key+step+to+a+new+era+in+imaging.">The ImageStream System: a key step to a new era in imaging.</a> Folia Histochem Cytobiol. 45 (4), 279-290 (2007).</li><li>Matheu, M. P., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+CD4++T+cells+from+mouse+lymph+nodes+using+Miltenyi+MACS+purification.">Isolation of CD4+ T cells from mouse lymph nodes using Miltenyi MACS purification.</a> J Vis Exp. (9), 409 (2007).</li><li>James, E. A., LaFond, R., Durinovic-Bello, I., Kwok, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+antigen+specific+CD4++T+cells+using+MHC+class+II+tetramers.">Visualizing antigen specific CD4+ T cells using MHC class II tetramers.</a> J Vis Exp. (25), (2009).</li><li>Wabnitz, G. H., 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=L-plastin+phosphorylation:+a+novel+target+for+the+immunosuppressive+drug+dexamethasone+in+primary+human+T+cells.">L-plastin phosphorylation: a novel target for the immunosuppressive drug dexamethasone in primary human T cells.</a> Eur J Immunol. 41 (11), 3157-3169 (2011).</li><li>Freeley, 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=L-plastin+regulates+polarization+and+migration+in+chemokine-stimulated+human+T+lymphocytes.">L-plastin regulates polarization and migration in chemokine-stimulated human T lymphocytes.</a> J Immunol. 188 (12), 6357-6370 (2012).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Usando una tecnologia recente del flusso di imaging citometria, una strategia rapida ed informativo gating per analizzare gli eventi cellulari e molecolari indotti dalla stimolazione chemochine è presentato. Da un unico esperimento, si possono ottenere due tipi principali di informazioni: le variazioni di morfologia cellulare indotta da chemochine stimolazione e la distribuzione subcellulare di proteine ​​differenti durante il processo di polarizzazione. È interessante notare che la prova è prevista anche la poss...

Chemochine sono piccole proteine ​​solubili che attirano le cellule a posizioni specializzate 1. Pertanto, partecipano al corretto posizionamento delle cellule in tessuti, una funzione cruciale per lo sviluppo e la fisiologia. Il sistema immunitario non fa eccezione a questa regola in quanto si basa sull&#39;azione di molti diversi tipi di cellule che agiscono di concerto per montare una risposta immunitaria efficace. Controllando la posizione specifica di un tipo di cellula immunitaria in un dato stato, chemochine sono pre-preliminare antigeni estranei possono essere rilevati e neutralizzati. In linfociti T in particolare, chemochine si legano a specifici recettori di superficie che, dopo l&#39;impegno, suscitano molti segnali intracellulari (aumento di calcio, ERK fosforilazione, attivazione Rho GTPasi, aumento delle integrine affinità e citoscheletro alterazioni) che favoriscono la motilità cellulare T 2,3. A livello cellulare, si possono osservare alterazioni morfologiche indotte dopo stimolazione chemochine.Questi cambiamenti nella forma delle cellule sono particolarmente drammatica in cellule T: cellule T riposo hanno una morfologia rotonda tallone-come quando si viaggia nel flusso sanguigno. Tuttavia, il rilevamento della presenza di chemochine in siti infiammatori o in prossimità degli organi linfoidi sta per cambiare la forma delle cellule T che ora adottano una tipica morfologia &quot;hand-mirror&quot; costituito da un modulo bipolare: un bordo d&#39;attacco al fronte e un bordo di uscita, o uropod, sul retro 4. Inoltre, componenti intracellulari possono dividersi in queste due regioni opposte di una cellula T polarizzata per sostenere la migrazione. Ad esempio, i filamenti di actina polimerizzazione aumenta su chemochine stimolazione 5 e actina polimerizzata accumula nella parte anteriore di una T cellule polarizzate 2. D&#39;altra parte, diverse proteine, come le proteine ​​fosforilate della Ezrin-radixina-Moesin (ERM) famiglia che collegano la membrana plasmatica della corticale citoscheletro F-actina, ri-localizzano al uropod di polarizzataCellule T 6. È interessante notare che, noi e altri hanno dimostrato che questo processo di polarizzazione è necessario per la migrazione delle cellule T. Infatti, qualsiasi trattamento che interferisce con la polarizzazione inibisce la motilità cellulare. Per esempio, l&#39;inibizione dell&#39;attività dei membri della proteina atipica chinasi C (PKC) famiglia, polarizzazione cellulare PKCζ e PKCι blocchi T e il loro processo di scansione migratoria delle cellule dendritiche 7. Polarizzazione delle cellule T è anche regolato da Rho GTPasi. Abbiamo dimostrato che la modulazione dell&#39;attività RhoA dalla proteina Fam65b recentemente descritta interferisce con i cambiamenti nella morfologia delle cellule T e la loro capacità di migrare in un saggio Transwell 6. Come polarizzazione è un passo prerequisito per la motilità cellulare, è quindi essenziale essere in grado di quantificare come lettura principale di risposte chemochine. Alterazioni di forma delle cellule sono stati precedentemente misurati manualmente 8. Tuttavia, questo tipo di quantificazione è molto in termini di tempo, in modo che di solito solo pochidecine di cellule sono presi in considerazione. Qui, un nuovo metodo viene presentato per quantificare rapidamente il grado di alterazioni forma di T linfociti esposti a chemochine stimolazione. Un flusso di imaging citometria tecnologia (vedere Tabella di reagenti specifici / Equipment) viene utilizzato, che combina i vantaggi di un citofluorimetro e un microscopio 9 quantificare efficacemente la quantità di cellule polarizzate in diverse condizioni di stimolazione chemochine. Oltre alla quantificazione delle alterazioni morfologiche che si può misurare robusta con questa tecnologia, è anche possibile valutare le variazioni di localizzazione subcellulare di alcune proteine ​​su segnalazione chemochine.

<table border="0" cellpadding="0" cellspacing="0" width="569">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Name of the material/Equipment</td>
			<td style="width:95px;">Company</td>
			<td style="width:80px;">Catalog number</td>
			<td style="width:255px;">Comments/Description (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">RPMI</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">61870-010</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Human serum AB</td>
			<td style="width:95px;">PAA</td>
			<td style="width:80px;">C11-021</td>
			<td style="width:255px;">Pre-heat to inactivate the complement.</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:140px;">Fetal calf serum</td>
			<td style="width:95px;">PAN Biotech</td>
			<td style="width:80px;">P30-3300</td>
			<td style="width:255px;">Pre-heat to inactivate the complement.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:140px;">Human T cell nucleofactor kit</td>
			<td style="width:95px;">Lonza</td>
			<td style="width:80px;">VCA-1002</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Murine IL7</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">217-17</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:140px;">HBSS</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">14025-050</td>
			<td style="width:255px;">Warm in 37 &#176;C water bath before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Hepes</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">15630-056</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:140px;">Murine CCL19</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">250-27B</td>
			<td style="width:255px;">Aliquots are thawed on ice before adding the chemokine to the cells.</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:140px;">Human CCL19</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">300-29B</td>
			<td style="width:255px;">Aliquots are thawed on ice before adding the chemokine to the cells.</td>
		</tr>
		<tr height="88">
			<td height="88" style="height:88px;width:140px;">Human CXCL12</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">300-28A</td>
			<td style="width:255px;">Aliquots are thawed on ice before adding the chemokine to the cells. This chemokine can also be used on mouse T cells.</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:140px;">PFA</td>
			<td style="width:95px;">Electron Microscopy Sciences</td>
			<td style="width:80px;">157-8-100</td>
			<td style="width:255px;">This is a 8 % PFA solution in water. Mix volume to volume with 2X PBS to obtain a 4 % PFA solution in PBS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">&#160;BSA</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:80px;">A3059</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Saponin</td>
			<td style="width:95px;">Fluka</td>
			<td style="width:80px;">84510</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">Alexa Fluor 594 phalloidin</td>
			<td style="width:95px;">Invitrogen</td>
			<td style="width:80px;">A12381</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">FITC-anti-HLA-ABC antibody</td>
			<td style="width:95px;">Beckman Coulter</td>
			<td style="width:80px;">IM1838</td>
			<td style="width:255px;">clone B9.12.1</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">Anti-P-ERM antibody</td>
			<td style="width:95px;">Cell Signaling Technology</td>
			<td style="width:80px;">3149P</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:140px;">ImageStreamx Mark II</td>
			<td style="width:95px;">Amnis</td>
			<td style="width:80px;"></td>
			<td style="width:255px;"></td>
		</tr>
	</tbody>
</table>

rapid and robust analysis of cellular and molecular polarization induced by chemokine signaling

Le cellule rispondono alla stimolazione chemochine, perdendo la loro forma rotonda in una polarizzazione processo chiamato, e alterando la localizzazione subcellulare di molte proteine. Tecniche di imaging classici sono stati usati per studiare questi fenomeni. Tuttavia, hanno richiesto l&#39;acquisizione manuale di molte cellule seguite da tempo quantificazione della morfologia e la co-localizzazione della colorazione di decine di cellule. Qui, un metodo rapido e potente è descritto per studiare questi fenomeni su campioni costituiti da diverse migliaia di cellule usando un flusso di imaging citometria tecnologia che combina i vantaggi di un microscopio con quelle di un citometro. Utilizzando linfociti T stimolate con CCL19 e colorazione per le molecole MHC di classe I e actina filamentosa, una strategia di gating è presentato per misurare simultaneamente il grado di alterazioni di forma e la misura di co-localizzazione di marcatori che sono affetti da segnalazione CCL19. Inoltre, questa strategia ci ha permesso di gating Observe la segregazione di actina filamentosa (nella parte anteriore) e fosforilata proteine ​​Ezrin-radixina-Moesin (fosfo-ERM) (nella parte posteriore) in cellule T polarizzate dopo la stimolazione CXCL12. Questa tecnica è anche utile per osservare l&#39;effetto di blocco sulla polarizzazione di due elementi differenti: inibizione della polimerizzazione mediante un inibitore farmacologico ed espressione dei mutanti del Par6 / atipica PKC percorso di segnalazione. Così, la prova viene dimostrato che questa tecnica è utile per analizzare sia le alterazioni morfologiche e redistribuzioni di proteine.

Il primo esempio scelto qui riguarda l&#39;uso di linfociti T primari umani stimolati con CCL19. Tuttavia, la stessa strategia può essere utilizzata con le cellule T di topo primaria, linee cellulari T o qualsiasi altro tipo di cellula sensibile alla stimolazione chemochine. La strategia di gating qui presentato comprende una serie di finestre che selezionano gli eventi focalizzato, poi le singole celle. Infine la gamma di intensità di fluorescenza è seguito come esempio su due marcatori: MHC classe I HLA-ABC e actin...

Watch this Scientific Journal Video about Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling at JoVE.com

Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

Analisi rapida e robusta di Cellulare e Molecolare polarizzazione indotta da chemochine Signaling

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many ...

rapid-robust-analysis-cellular-molecular-polarization-induced

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Immunology and Infection

Unit 1

Cell Polarization and Migration

SCIENTISTS IN ACTION

8.0K Views.  Institut Cochin. Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

Video: Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors1. This process leads to bacterial invasion of endothelial cells2. More recently, we observed an interaction of Opc with a 100kDa protein found in whole cell lysates of human cells3. We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as &alpha;-actinin. As no surface expressed &alpha;-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and &alpha;-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with &alpha;-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &amp;#945;-actinin by Confocal Imaging

Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human &alpha;-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular &alpha;-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).

Visualizzazione e quantificazione delle interazioni intracellulari di Neisseria meningitidis E umano α-actinina da Imaging confocale

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

Ricerca

Immunologia e infezioni

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

L&#39;uso del flusso gocciolamento e Reattori disco rotante per Staphylococcus aureus Analisi Biofilm

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

Un saggio di calcio bioluminescenza di analisi funzionale del Mosquito ( Aedes aegypti) E Tick ( Rhipicephalus Microplus) G recettori accoppiati alla proteina

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Imaging di HIV-1 Envelope indotta Synapse virologica e segnalazione sui doppi strati lipidici sintetici

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

Una tecnica di microscopia TIRF per Real-time, Imaging simultanea del TCR e delle sue proteine ​​associate segnalazione

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Analisi del danno epiteliale Prodotto da<em&gt; Entamoeba histolytica</em&gt; Infezione

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Analisi rapida di aberrazioni cromosomiche in linfociti B mouse da PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Rilevazione rapida molecolare e la differenziazione dei virus influenzali A e B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

Dissezione multiproteici segnalazione di purificazione di affinità di complementazione bimolecolare (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is characterized by lethal complications due to plasma leakage, ascites, pleural effusion, respiratory distress, severe bleeding, and organ impairment. A few cases of DENV infection present neurological manifestations; however, studies have not explored DENV-induced neuropathogenesis further. In this study, we present a protocol to use an immunocompetent outbred ICR (Institute of Cancer Research) mouse for investigating the induction of central nervous system (CNS) infection with DENV, followed by the progression of acute viral encephalitis-like disease.

Here, we present a protocol for creating an immunocompetent ICR (Institute of Cancer Research) murine model of central nervous system infection to display the development of neuropathy. Monitoring acute viral encephalitic disorders by identical disease scores could be performed for showing dengue virus-induced neuropathy in vivo.