Quest'opera è stata sostenuta da finanziamenti da Sapienza Università di Roma e la Fondazione ricerca fibrosi cistica (progetto FFC #11/2014 adottato dalle delegazioni FFC da Siena, Sondrio Valchiavenna, Cerea Il Sorriso di Jenny e Pavia). Parte di questo lavoro è stata anche sostenuta da FILAS Grant Prot. FILAS-RU-2014-1020.
Siamo grati al Dr. Loretta Ferrera (u.o.c. di Genetica Medica, Istituto Giannina Gaslini, Genova, Italia) per la fornitura di cellule epiteliali bronchiali.

1. cella preparazione
<ol><li>Semi 2.5x106 celle in 10 mL di minimo essenziale medio (MEM) completato con glutamina 2mm (MEMg), più 10% siero bovino fetale (FBS), antibiotici (0,1 mg/mL di penicillina e streptomicina) e con puromicina (0,5 µ g/mL per la selezione e la manutenzione dei linea cellulare) in un matraccio da T75. Incubare la beuta a 37 ° C e 5% CO2 per 2 giorni. Prima di iniziare l'esperimento, verifica la confluenza delle cellule sotto un microscopio invertito. Nota: Le celle utilizzate per l'esperimento sono cellule epiteliali bronchiali umane immortalizzate trasformate con un vettore lentivirale che conferiscono resistenza agli con puromicina. Essi esprimono stabilmente un funzionale CFTR14,15.</li>
	<li>Una volta che ha raggiunto la confluenza delle cellule 90-100%, aspirare il mezzo dal pallone e gettarlo in una bottiglia dei rifiuti nell'ambito di una classe di armadio di sicurezza biologica II. Lavare le cellule con 6 mL di tampone fosfato salino senza calcio e magnesio (CMF-PBS). Delicatamente la boccetta della roccia manualmente ed eliminare CMF-PBS. Nota: Fare attenzione a non toccare il monostrato cellulare con la pipetta.</li>
	<li>Aggiungere 10 mL di PBS CMF e incubare la beuta a 37 ° C e 5% di CO2 per 10 min.</li>
	<li>CMF-PBS di aspirare ed eliminare. Quindi aggiungere 2 mL di tripsina/EDTA al pallone.</li>
	<li>Scuotere delicatamente il matraccio, lasciare che la soluzione completamente ricoprire le cellule ed incubare la beuta a 37 ° C e 5% di CO2 per 10 min, fino a quando le cellule sono visibilmente staccate sotto un microscopio. Nota: Al termine del tempo di incubazione, le cellule dovrebbero apparire arrotondato e non attaccato alla superficie plastica. Se le cellule non sono ben staccate, agitazione manuale può essere necessario.</li>
	<li>Aggiungere 10 mL di MEMg maggiorato del 10% FBS per inattivare tripsina e raccogliere le cellule lavando il fondo del pallone. Trasferire il volume in una provetta conica 50 mL.</li>
	<li>Centrifugare la provetta per 5 min a 80 x g.</li>
	<li>Aspirare il supernatante e risospendere le cellule in 6 mL di MEMg plus 10% FBS. Pipettare ripetutamente per eliminare eventuali grumi.</li>
	<li>Prendere 10 µ l di sospensione cellulare con una micropipetta e iniettare il volume sotto il vetro di copertura precedentemente messo sopra una camera di Burker o Neubauer.</li>
	<li>Contare il numero di cellulare.</li>
</ol>2. semina nella coltura cellulare inserti
<ol><li>In ciascun pozzetto di una piastra 12-pozzetti, trasferire l'inserto di cultura con pinzette sterili (Figura 1). Utilizzare una pinzetta per premere lungo i bordi di inserti per ripararli alla superficie della piastra. Nota: Gli inserti hanno un lato appiccicoso che permette l'adesione.</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56825/56825fig1.jpg"> Figura 1 : Rappresentazione schematica degli inserti in silicone cultura, correttamente messo in pozzetti di una piastra 12-pozzetti. <a href="//cloudflare.jove.com/files/ftp_upload/56825/56825fig1large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
<ol start="2"><li>Diluire correttamente la sospensione cellulare in MEMg maggiorato del 10% FBS. Riempire ogni scomparto dell'inserto con 70 µ l di sospensione cellulare (circa 3.5x104 cellule/camera). Nota: La densità delle cellule applicata in ogni compartimento dipende dal tipo delle cellule. Si consiglia di utilizzare una densità delle cellule che conduce per completare confluenza entro 24 h.</li>
	<li>Sotto il microscopio, verifica che le cellule non sono perdite dai comparti inserto e incubare la piastra 12-pozzetti per 24 h a 37 ° C e 5% CO2.</li>
</ol>3. pseudo-ferita guarigione Assay
<ol><li>Dopo l'incubazione, visualizzare le cellule al microscopio invertito per verificare che sono stati formati strati monomolecolari della cellula confluenti.</li>
	<li>Risospendere le sostanze da testare (ad es., AMPs) in 1 mL di MEMg. Nota: Preparare diluizioni di AMPs freschi a partire dalla soluzione stock conservata a-20 ° C.</li>
	<li>Rimuovere delicatamente gli inserti di pinzette sterili. Fare attenzione a non rompere i monostrati di cellule umane. Trasferire gli inserti sulla carta assorbente. Nota: Per riutilizzare gli stessi inserti, sterilizzarli in etanolo al 70% per almeno 3 ore. Si consiglia di buttarli via in seguito.</li>
	<li>Per rimuovere le cellule non-aderenti, aggiungere 1 mL di MEMg al bene utilizzando una micropipetta. Chiudere la piastra e scuoterla delicatamente. Nota: Non aggiungere il terreno direttamente sulla cima di monostrati di cellule umane per evitare il loro malfunzionamento.</li>
	<li>Aspirare il mezzo e sostituirlo con 1 mL di MEMg per bene. Chiudere la piastra e visualizzare le lacune senza cellula (create dagli inserti) sotto il microscopio invertito con un ingrandimento 4x, dotato di una videocamera. Acquisire immagini a tempo zero (T0) e salvarli in formato. jpg.</li>
	<li>Rimuovere il supporto dai pozzi, lavarli con 1 mL di PBS e scartarla.</li>
	<li>Aggiungere le sostanze da testare (preparati al punto 3.2) ai pozzetti. Per i campioni di controllo non trattato, aggiungere 1 mL di MEMg. Incubare la piastra a 37 ° C e 5% CO2.</li>
	<li>Dopo 15, 20 e 24 trattamenti di h, osservare la migrazione delle cellule sotto il microscopio con un ingrandimento 4x e acquisire immagini. Nota: Durante questo passaggio, tenta di catturare immagini nelle stesse zone per quanto riguarda T0. La scelta di intervalli di tempo in cui le immagini sono acquisite dipende dalla velocità di migrazione delle cellule.</li>
	<li>Per studiare l'effetto di alcuni inibitori selettivi, cioè AG1478, sulla migrazione cellulare, aspirare il mezzo da ogni alloggiamento prima di rimuovere inserti.<ol>
<li>Lavare ogni vano con MEMg fresco e riempirlo con 70 µ l di MEMg completati con AG1478.</li>
		<li>Dopo 30 min di incubazione a 37 ° C e 5% CO2, procedere come descritto dal punto 3.3. Nota: Durante la fase di lavaggio e di pretrattamento di monostrati di cellule umane con inibitori specifici, fare attenzione a non rimuovere gli inserti.</li>
	</ol>
</li>
</ol>4. image Analysis
<ol><li>Al termine dell'esperimento, selezionare le immagini dei campioni più rappresentativi dei vari gruppi sperimentali e creare un file zip contenente immagini singole a T0, T15, T20 e T24 h. Nota: Le singole immagini sono presi intervalli di tempo selezionati per tutti i campioni. Eseguire triplici copie per ogni esperimento, che viene ripetuto almeno tre volte. Alla fine, per gruppi tutti sperimentali, un minimo di 3 immagini ("a", "b", "c", ecc., derivanti da ogni esperimento indipendente) in ogni punto del tempo vengono analizzati.</li>
	<li>Caricare il file zip nel software di analisi di immagine che fornisce automaticamente via e-mail un file di foglio di calcolo di riepilogo contenente i dati sperimentali delle zone rivestite in cella e gratta e Vinci (come percentuale) presso i punti di tempo selezionato. Nota: Il riconoscimento della leading edge e la zona di gap è in gran parte basato su metodo di rilevamento del bordo (volto a identificare i punti in cui la luminosità dell'immagine cambia bruscamente).</li>
	<li>Salvare i dati e raccoglierli. Normalizzare tutti i dati per quanto riguarda il valore medio a tempo zero. Calcolare il valore medio dei dati normalizzati di tutte le repliche a ogni punto del tempo e l'errore standard relativo (SEM). Mediante analisi della varianza (ANOVA) bidirezionale, effettuare analisi statistiche, con un software di statistiche adeguate. Differenze tra gruppi di peptide-trattati e controllo a diversi intervalli di tempo sono considerate essere statisticamente significativo per una p< 0.05.</li>
	<li>Tracciare i dati ottenuti in un grafico come un istogramma che mostra la percentuale di area delle cellule di tutti i campione gruppi contro gli intervalli di tempo selezionati.</li>
</ol>

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Migrazione delle cellule è un processo essenziale in molti eventi fisiologici e patologici, tra cui la guarigione della ferita, lo sviluppo embrionale e metastasi del cancro. La procedura di base per studiare la migrazione delle cellule in vitro comporta: (i) la creazione di un monostrato di cellule, (ii) la produzione di una pseudo-ferita nello strato confluente di cellule, (iii) l'acquisizione di immagini a intervalli di tempo diversi fino a quando ferita chiusura viene raggiunto e (iv) l'analisi della sequen...

È ampiamente noto che la guarigione arrotolata negli animali è un processo fondamentale per ristabilire l'integrità e la funzione normale di strati di tessuto dopo lesioni1. Nonostante le superfici epiteliali esposte all'ambiente esterno (ad es., pelle, vie respiratorie e tratti gastrointestinali) formano una barriera protettiva dagli insulti chimici e fisici, la formazione di ferite può facilmente verificarsi, soprattutto dopo chirurgia o infezioni microbiche2. Ad esempio, la colonizzazione del tessuto polmonare di agente patogeno batterico opportunistico Pseudomonas aeruginosa, soprattutto in chi soffre di fibrosi cistica (CF), porta a danni dell'epitelio airways con conseguente insufficienza respiratoria3, 4. La guarigione della ferita è un meccanismo di riparazione complessi host per ripristinare la normale architettura di un tessuto danneggiato5. È caratterizzata da infiammazione iniziale, seguita da un periodo di rigenerazione che comprende epithelialization, angiogenesi e rimodellamento tissutale con la produzione di collagene e cellule differenziazione6,7,8 . Per garantire l'integrità epiteliale e per controllare la proliferazione microbica, tutti gli organismi viventi producono molecole di difesa, tra cui peptidi antimicrobici (amp)9,10. Il processo di guarigione della ferita è molto difficile da simulare in vitro a causa della mancanza di detriti cellulari e delle complesse interazioni tra diversi tipi di cellule. Tuttavia, la capacità in vitro di un peptide di accelerare la chiusura di una pseudo-ferita di stimolando la migrazione delle cellule epiteliali è indicativa della sua capacità di guarire un epitelio compromesso. Infatti, migrazione delle cellule è un evento di tasso-di limitazione nella guarigione delle ferite, e studiare i fattori che possono influenzare la migrazione cellulare vi aiuterà a terapie target per una migliore cicatrizzazione.
Qui, un'analisi sperimentale altamente riproducibile viene fornita in base su inserti in silicone speciale cultura per valutare di migrazione delle cellule in vitro. Si basa sulla creazione di un 500 μm gap (pseudo-ferita) su un monostrato di cellule confluenti. Le cellule al bordo del campo artificiale "ferito" inizierà la migrazione nell'area senza cellula, formando nuovi contatti cellula-cellula. L'inserto di cultura rappresenta un nuovo strumento per esperimenti di cicatrizzazione veloce. Sono forniti due serbatoi separati da una parete di 500 μm, e possono essere inseriti correttamente in una piastra di 3 cm piatto o nel pozzetto di una piastra 12-pozzetti. Ogni vano dell'inserto di riempimento con una sospensione cellulare permette alle cellule di crescere in ogni area designata fino alla confluenza, mentre la rimozione dell'inserto e concernente un gap pulito privo di cella di circa 500 μm (la stessa larghezza come il muro di separazione). Un mezzo di coltura cellulare adeguata completato con un composto in esame può essere quindi aggiunto nel piatto piatto/pozzetto. In seguito, la chiusura del divario possa essere visualizzata a intervalli di tempo differenti sotto un microscopio invertito, preferibilmente uno dotato di una videocamera per acquisizione di immagini. Misurazione delle variazioni nella zona cella-coperti dal programma di analisi di immagine automatizzata basata su web consentirà, infine, la quantificazione della velocità di migrazione delle cellule e la chiusura della ferita. Nel complesso, questo metodo è un passo avanti per quanto riguarda il dosaggio classico, dove un graffio fatta manualmente dal pungere monostrati confluenti di cellule umane con un ago sterile o una pipetta11suggerimento. Infatti, l'ultima procedura può distruggere il fondo in plastica del piatto piatto/pozzetto ed il rivestimento di superficie, creando le rughe. Inoltre, l'area "ferito" non ha una larghezza ben definita lungo l'intera lunghezza del gap, come questo altamente dipende dalla pressione applicata dai ricercatori alla punta dell'ago. Inoltre, le cellule sloggiate possono formare grumi di cellule vive e morte ai bordi del graffio; Inoltre, la diffusione delle cellule viventi nell'area "ferito" può interferire con la velocità della migrazione cellulare, generando risultati non riproducibili12.
Inoltre, grazie ad una piattaforma di analisi di immagine gratta e Vinci, gli utenti possono rapidamente ricevere (in minuti) dati quantitativi sul comportamento migratorio delle celle selezionate senza la necessità di acquisizione di software aggiuntivo. Questa piattaforma è in grado di analizzare le immagini di microscopia di contrasto di fase di bassa (~ 5 X), media (~ 10x) e alto (~ 20 X) ingrandimento. Dopo aver caricato un file zip di immagini (in *. jpg, *. jpeg, *.jp2, *. png, *. gif, formato TIFF) l'analisi è condotta automaticamente per generare un file di riepilogo che mostra la percentuale di zone coperte di cella e di aree gratta e Vinci, così come la velocità della cella migrazione, a intervalli di tempo distinti.
In questo lavoro, utilizzando un Rana-pelle AMP-derivato, cioè Esc(1-21) e relativo diastereomer Esc(1-21) - 1C13e una linea di cellule bronchiali esprimendo il funzionale CF conduttanza transmembrana regolatore (CFTR)14,15, viene fornito un esempio di migrazione cellulare indotta da peptide in confronto con campioni non trattati (controllo). Si noti che l'epitelio delle vie aeree e la CFTR gioca un ruolo cruciale nel mantenimento della funzione polmonare e la ferita riparazione16. Inoltre, per mezzo di inibitori selettivi (ad es., AG1478)17 del recettore del fattore di crescita epidermico (EGFR), prova che la migrazione di cellule bronchiali indotta dai peptidi di cui sopra comporta l'attivazione di EGFR12, 18 è segnalato.
In sintesi, l'obiettivo di questa procedura è di dimostrare come l'utilizzo di tali inserti di cultura in silicone rappresenta un'analisi veloce e facilmente accessibile per determinare con precisione la migrazione di cellule aderenti (ad es., cellule epiteliali bronchiali) e molecolare meccanismi di controllo di tali eventi.

<table border="0" cellpadding="0" cellspacing="0" width="852">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Minimal essential medium (MEM)&#160;</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECB2071L</td>
			<td style="width:256px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Glutamine</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECB3000D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Heat inactivated Fetal Bovine Serum (FBS)</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECS0180DH&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Penicillin and Streptomycin</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECB3001D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Puromycin</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Trypsin/EDTA 1X in PBS</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECB3052D</td>
			<td>Warm at room temperature before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">DPBS without calcium and magnesium (CMF-PBS)</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">DPBS with calcium and magnesium (PBS)</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8662</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ibidi Culture-Insert 2 well</td>
			<td style="width:179px;">Ibidi&#160;</td>
			<td style="width:119px;">80209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Wimasis Image Analysis</td>
			<td style="width:179px;">Ibidi&#160;</td>
			<td style="width:119px;">30002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PRISM software&#160;</td>
			<td>GraphPad</td>
			<td style="width:119px;">version 6.0</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel in vitro wound healing assay to evaluate cell migration

Lo scopo di questo lavoro è quello di mostrare un nuovo metodo per valutare la capacità di alcune molecole di immunomodulatori, quali peptidi antimicrobici (amp), per stimolare la migrazione cellulare. D'importanza, migrazione delle cellule è un evento limitante durante il processo di guarigione della ferita per ristabilire l'integrità e la funzione normale di strati di tessuto dopo la ferita. Il vantaggio di questo metodo oltre l'analisi classica, che si basa su un graffio fatto manualmente in un monostrato di cellule, è che l'utilizzo della cultura di silicone speciale inserisce fornendo due scomparti per creare un campo di pseudo-ferito senza cellula con una larghezza ben definita (500 μm ). Inoltre, a causa di una piattaforma di analisi di immagine automatizzato, è possibile ottenere rapidamente i dati quantitativi sulla velocità di migrazione delle cellule e la chiusura della ferita. Più precisamente, verrà mostrato l'effetto di due amplificatori di pelle di rana sulla migrazione di cellule epiteliali bronchiali. Inoltre, il pretrattamento di queste cellule con inibitori specifici fornirà informazioni sui meccanismi molecolari alla base di tali eventi.

Questo protocollo è stato usato per determinare l'effetto di Esc(1-21) ed Esc(1-21) - 1C in termini di attività di migrazione delle cellule indotto in cellule epiteliali bronchiali che esprimono la proteina CFTR funzionale di cicatrizzazione. In questo saggio, inserti di cultura sono stati collocati nei pozzetti di una piastra 12-pozzetti, e ogni scomparto è stato seminato con 35.000 cellule in MEMg supplementato con 10% FBS. Le cellule raggiunto confluenza completa entro 24 h. in segu...

Watch this Scientific Journal Video about A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration at JoVE.com

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Qui, presentiamo un protocollo per valutare l'effetto dei peptidi sulla migrazione di cellule epiteliali bronchiali. Questo metodo consente l'ottenimento rapido e altamente riproducibile di dati quantitativi sulla velocità di chiusura della ferita e migrazione cellulare.

Un romanzo In Vitro ferita guarigione test per valutare la migrazione cellulare

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

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Research

JoVE Journal

Immunology and Infection

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

21.6K Views.  Sapienza University of Rome. Qui, presentiamo un protocollo per valutare l'effetto dei peptidi sulla migrazione di cellule epiteliali bronchiali. Questo metodo consente l'ottenimento rapido e altamente riproducibile di dati quantitativi sulla velocità di chiusura della ferita e migrazione cellulare.

Video: A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non invasiva di imaging di homing dei leucociti e la migrazione In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Ricerca

Immunologia e infezioni

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

Competitivo saggi Homing allo Studio Gut-tropico Migrazione cellulare T

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Una valutazione quantitativa della migrazione cellulare con il Test Phagokinetic motilità Traccia

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

In vitro per valutare l&#39;impatto di immunoregolatorio Pathways su HIV-specifica CD4 T effettrici delle cellule di funzione

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

In vitro Test di cocultura per valutare la migrazione transeteliale neutrofila indotta da agenti patogeni

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

Immagine a base di Citometria a flusso Tecnica per valutare le variazioni Granulocyte Funzione<em&gt; In Vitro</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

Un test CFSE-based per studiare la migrazione delle cellule dendritiche murini della pelle in drenanti linfonodi durante l&#39;infezione con<em&gt; Mycobacterium bovis</em&gt; Bacille Calmette-Guérin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

Sviluppo e identificazione di una sottopopolazione romanzo di Human neutrofili di derivazione fagociti Giant<em&gt; In Vitro</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

L&#39;uso di un monociti monostrato test per valutare fagocitosi Fcy recettore-mediata

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Un nuovo sistema privo di alimentatore per la produzione di massa di cellule di assassino naturali murini In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...