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>

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	<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. 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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><tbody><tr><td>RPMI</td><td>Gibco</td><td>61870-010</td><td></td></tr><tr><td>Human serum AB</td><td>PAA</td><td>C11-021</td><td>Pre-heat to inactivate the complement.</td></tr><tr><td>Fetal calf serum</td><td>PAN Biotech</td><td>P30-3300</td><td>Pre-heat to inactivate the complement.</td></tr><tr><td>Human T cell nucleofactor kit</td><td>Lonza</td><td>VCA-1002</td><td></td></tr><tr><td>Murine IL7</td><td>Peprotech</td><td>217-17</td><td></td></tr><tr><td>HBSS</td><td>Gibco</td><td>14025-050</td><td>Warm in 37 &amp;deg;C water bath before use.</td></tr><tr><td>Hepes</td><td>Gibco</td><td>15630-056</td><td></td></tr><tr><td>Murine CCL19</td><td>Peprotech</td><td>250-27B</td><td>Aliquots are thawed on ice before adding the chemokine to the cells.</td></tr><tr><td>Human CCL19</td><td>Peprotech</td><td>300-29B</td><td>Aliquots are thawed on ice before adding the chemokine to the cells.</td></tr><tr><td>Human CXCL12</td><td>Peprotech</td><td>300-28A</td><td>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><td>PFA</td><td>Electron Microscopy Sciences</td><td>157-8-100</td><td>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><td>BSA</td><td>Sigma</td><td>A3059</td><td></td></tr><tr><td>Saponin</td><td>Fluka</td><td>84510</td><td></td></tr><tr><td>Alexa Fluor 594 phalloidin</td><td>Invitrogen</td><td>A12381</td><td></td></tr><tr><td>FITC-anti-HLA-ABC antibody</td><td>Beckman Coulter</td><td>IM1838</td><td>clone B9.12.1</td></tr><tr><td>Anti-P-ERM antibody</td><td>Cell Signaling Technology</td><td>3149P</td><td></td></tr><tr><td>ImageStream&lt;sup&gt;x&lt;/sup&gt; Mark II</td><td>Amnis</td><td></td><td></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 ...

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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

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

Indagine di polarizzazione dei macrofagi Utilizzando Bone Marrow macrofagi derivati

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Ricerca

Immunologia e infezioni

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Planar gradiente di diffusione del sistema per Indagare Chemiotassi in un 3D collagene Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Bioingegneria

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

Polidimetilsilossano-policarbonato dispositivi microfluidici per Cell Migration Studies Sotto perpendicolare chimica e gradienti di ossigeno

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Analisi 3D delle risposte multi-cellulari ai gradienti chemoattractant

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

Receptor Imaging proteina G accoppiata (GPCR)-mediata eventi di segnalazione che chemiotassi di controllo di Dictyostelium discoideum</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Biologia

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

Misura della chemiotassi cellulari con ECIS / Taxi

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

Valutazione della migrazione dei linfociti in un sistema di trasmigrazione Ex Vivo

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a chemical. Chemokinesis and chemotaxis are fundamental for the mobilization and deployment of immune cells. For example, chemokines (chemotactic cytokines) can rapidly recruit circulating neutrophils and monocytes to extravascular sites of inflammation. Chemoattractant receptors belong to the large family of G protein-coupled receptors. How chemoattractant (i.e., ligand) gradients direct cell migration via G protein-coupled receptor signaling is not yet fully understood. In the field of immunology, neutrophils are popular model cells for studying chemotaxis in vitro. Here we describe a real-time two-dimensional (2D) chemotaxis assay tailored for mouse resident macrophages, which have traditionally been more difficult to study. Macrophages move at a slow pace of ~1 &#181;m/min on a 2D surface and are less well suited for point-source migration assays (e.g., migration towards the tip of a micropipette filled with chemoattractant) than neutrophils or Dictyostelium discoideum, which move an order of magnitude faster. Widely used Transwell assays are useful for studying the chemotactic activity of different substances, but do not provide information on cell morphology, velocity, or chemotactic navigation. Here we describe a time-lapse microscopy-based macrophage chemotaxis assay that allows quantification of cell velocity and chemotactic efficiency and provides a platform to delineate the transducers, signal pathways, and effectors of chemotaxis.

Here we describe methods using time-lapse, phase-contrast microscopy to image mouse resident peritoneal macrophages in a chemotactic complement C5a gradient. The protocols can be extended to other immune cells.

Immagine time-lapse di Mouse Macrophage Chemotaxis

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Imaging G protein-coupled recettore-mediata chemiotassi ed i suoi eventi di segnalazione nelle cellule HL60 neutrofili-like

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Valutazione della risposta di Dictyostelium discoideum alla stimolazione meccanica acuta

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...