The present study was funded in part by a Research Initiation Grant (RIG) from the University of North Texas to Dr. McFarlin. The authors did not receive direct funding for the completion of this study and report no conflict of interest.

NOTA: Tutte le procedure di raccolta del sangue descritte in questo metodo sono state condotte in accordo con la Dichiarazione di Helsinki e approvati dal Comitato Etico UNT (IRB) per i soggetti umani. Tutti i soggetti hanno dato il consenso scritto per la raccolta del sangue, che è stato utilizzato nel metodo attuale per garantire che essi erano apparentemente sani, di peso corporeo normale, e libero da malattia. 1. reagente Fonte &amp; Preparazione <ol><li> Utilizzare CD66b-APC (clone # G10F5; DF = 1: 50) e CD45-APCeFluor780 (clone # 2D1; DF = 1: 50) per questo test. NOTA: Prima dello studio, gli anticorpi CD45 e CD66b sono stati titolati per determinare la diluizione ottimale che granulociti chiaramente risolti (CD45 + / 66b +) da altri leucociti (CD45 + / 66b-) 11. Dopo aggiunta dell&#39;anticorpo diluita per colorazione diluizione finale nel tubo era 875. </li><li> Acquisto e disgelo stock S. aureus bioparticelle etichettato con pHrodo colorante rosso. Sospensione ad una concentrazione di 1 mg di bioparticelle per ml in PBS sterile. Aliquota bioparticelle diluite e conservare congelati a -20 ° C fino al momento del test. </li><li> Sciogliere dihydroethidium (DHE), che viene convertito in etidio bromuro di fluorescenza in presenza di radicali liberi di ossigeno (cioè, burst ossidativo), in DMSO ad una concentrazione finale di 10 g / ml. </li><li> Mescolare N-etilmaleimmide (usato per prevenire fagocitosi supplementare dopo la durata dell&#39;incubazione dosaggio specificato) con PBS sterile in una diluizione 2 step di 200 mg / ml e 17,5 mg / ml. Nel saggio, utilizzare una concentrazione finale di 15 mM. Quando scongelamento N-etilmaleimmide, utilizzare un blocco di calore a 37 ° C, come N-etilmaleimmide non rimane in soluzione. </li><li> Dopo la fase di fissazione finale, utilizzare 7AAD a macchiare DNA nucleare. Prima Inoltre, diluire magazzino 7AAD 1:10 con PBS sterile. </li></ol> 2. Campione di sangue Collection <ol><li> Fare soggetti ad arrivare al laboratorio a seguito di un O / N veloce (&gt; 8 ore) e abstentisu dall&#39;attività fisica (&gt; 12 ore). </li><li> Dopo aver pulito la pelle con un panno imbevuto di alcol preparazione, inserire una sterile raccolta del sangue ago in un braccio vena periferica. </li><li> Prelevare il sangue in tubi sottovuoto che sono in commercio pieni di eparina sodica. </li><li> Dopo la raccolta, applicare un bendaggio adesivo al braccio del soggetto. </li><li> Capovolgere i tubi del sangue di mescolare per 10 volte e poi posto su una sedia a dondolo fino al momento dell&#39;analisi. </li></ol> Tecnica 3. Fagocitosi Assay <ol><li> Bioparticelle Thaw S. aureus (RT), DHE (RT), e N-etilmaleimmide (37 ° C). </li><li> Aggiungere 20 L di bioparticelle S. aureus a 4 singoli 1,2 ml provette durante il lavoro nella cappa sterile. </li><li> Aggiungere 40 L di DHE ad ogni provetta contenente le bioparticelle. </li><li> Battere delicatamente i tubi sul banco di lavoro per raccogliere i reagenti nel fondo della provetta. </li><li> Aggiungere 100 L di sangue intero misto di ogni tubo che è stato riempito con bioparticelle e DHE. </li><li>Pulire il bordo interno dei tubi con un applicatore con punta di cotone per rimuovere il sangue contaminante. </li><li> Mescolare il sangue e reagenti con una pipetta elettronico impostato mescolare il sangue e reagenti per tre cicli dopo aggiunta di sangue. </li><li> Mettere le provette in un secchio di ghiaccio e coprire al riparo dalla luce. </li><li> Incubare le provette per 10, 20, e 40 min. Iniziare con il tubo 40 min a garantire tutte le provette terminano contemporaneamente. </li><li> Scongelare N-etilmaleimmide in 37 ° C bagno tallone. </li><li> Pipettare 15 L di N-etilmaleimmide (descritto in 1.4) in ogni provetta, incubare per 30 minuti. </li><li> Dispensare 10 L di CD66b-APC e 10 L di CD45-APCeFluor780 anticorpi diluiti (sopra descritti in 1.1), incubare per 60 min. </li><li> Dispensare 750 L di fix soluzione Lyse / RBC WBC in ciascuna provetta, incubare per 60 min. </li><li> Centrifuga (10 min a 400 xg) provette per raccogliere il pellet. </li><li> Vacuum aspirare la soluzione lyse WBC / RBC fix, lasciando un residuo flVolume UID (100 L) sopra il pellet di cellule. </li><li> Dispensare 10 L di soluzione diluita 7AAD (descritto in 1.5) e 50 L di PBS in ogni provetta test. </li><li> Aggiungere una goccia di perline di taratura (~ 25 L) a ciascuna provetta, luogo tappi sulle provette, tubi avvolgere in un foglio, e posto in frigorifero. </li><li> Caricare la provetta nel flusso basato su immagini citometro e raccogliere un minimo di 3.000 eventi granulociti utilizzando parametri predefiniti: Autocampionatore, blu (488 nm; 60 mW), rosso (640 nm, 100 nW), SSC (785 nm; 8,5 mW) laser (Figura 1). </li></ol><img alt="Figura 1" src="/files/ftp_upload/52201/52201fig1highres.jpg" /> Figura 1. Metodo &amp; Template Acquisition. I campioni sono stati acquisiti su un flusso basato su immagini citofluorimetro (A). Durante l&#39;acquisizione, dot plots di brillante campo Proporzioni vs. CD66b-APC sono stati generati per granulociti separati dal spillo perline calibrazione usinsoftware g INSPIRE v. 100.2.292.0 (B). Un minimo di 5.000 granulociti sono stati acquisiti per ogni campione utilizzando un campionatore automatico, blu (488 nm; 60 mW), rosso (640 nm, 100 nW), SSC (785 nm; 8,5 mW) laser. Si prega di notare che un certo numero di istogrammi sono presenti durante l&#39;acquisizione di controllare le impostazioni di laser e altri aspetti dei dati raccogliere. Tutti questi istogrammi sono opzionali e necessaria solo se le procedure operative standard di laboratorio li richiede il controllo della qualità. <a href="https://www.jove.com/files/ftp_upload/52201/52201fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> 4. Acquisizione e Sample Analysis <ol><li> Utilizzare la procedura guidata automatica compensazione software del software IDEAS di applicare una matrice di compensazione per i file di immagine grezzi (RIF) e creare compensare i file immagine (CIF). </li><li> Caricare singoli file CIF in software IDEAS e generare i seguenti complotti per identificare i sottoinsiemi granulociti: </li&gt; <li> Stabilire porte iniziali per identificare le cellule che sono state considerate a fuoco utilizzando un istogramma per RMS di gradiente di campo luminosi (Figura 2a). Rendere questa determinazione per ciascun campione usando il controllo non stimolato come standard di riferimento. </li><li> Utilizzare trame secondarie alle cellule singoletto separati da detriti e doppiette utilizzando un diagramma a punti di brillanti proporzioni settore (rapporto tra altezza della cella vs larghezza) vs. zona di campo luminoso (Figura 2B). Rendere questa determinazione per ciascun campione usando il controllo non stimolato come standard di riferimento. </li><li> Una volta che una popolazione di cellule pulita era stato identificato, creare un diagramma a punti di CD45 vs. CD66b (Figura 2C) per identificare positivamente granulociti (CD45 + / 66b +). Creare una trama figlia (Figura 3) della brillante intensità dettaglio per S. aureus (asse x) vs. brillante intensità dettaglio per burst ossidativo (DHE, asse y) per identificare sottogruppi di granulociti attivati. Raccogliere brimmagini campo ight del Canale 1 e 9, bioparticelle a Channel 2, DHE a Channel 4, 7AAD in Canale 5, CD66b in Channel 11, e CD45 in Channel 12. </li></ol><img alt="Figura 2" src="/files/ftp_upload/52201/52201fig2highres.jpg" /> Figura 2. Analisi del modello. Questa figura la serie di trame che sono stati generati per identificare le cellule che erano a fuoco (A), celle singole (B), granulociti (C). Trame supplementari sono stati usati per identificare i tre sottoinsiemi di granulociti attivati ​​contro granulociti inattive. Tutte le analisi di file di immagini acquisite è stato completato utilizzando il software IDEAS v.6. <a href="https://www.jove.com/files/ftp_upload/52201/52201fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a>

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The authors have nothing to disclose.

L&#39;attuale metodo rappresenta un perfezionamento dei metodi esistenti per la valutazione della funzione dei granulociti citometria a flusso 1,3,4,12-14. I passaggi critici di questo test tendono ad essere correlato alla corretta miscelazione del campione di sangue con i bioparticelle e DHE. Miscelazione incompleta si tradurrà in risultati imprecisi. Mentre la completa miscelazione è critica, il metodo di miscelazione deve essere dolce in natura. Si suggerisce che la miscelazione essere realizzato utilizzando una pipetta elettronico con una funzione di miscelazione piuttosto che un vortex. Un altro punto critico nel saggio è garantire sempre non c&#39;è sangue contaminare la metà superiore della provetta. Questo sangue residuo può essere rimosso con un cotton fioc sterili. La rimozione completa è importante perché in caso contrario si potrebbe contaminare la preparazione finale del test con i globuli rossi non-lisato. Prima di utilizzare questo metodo di adeguati controlli di compensazione dovrebbe essere aggiunto al controllo per spectral sovrapposizione tra i reagenti utilizzati per identificare i diversi aspetti della funzione di granulociti. Per questo metodo, controlli di compensazione comportano raccogliere campioni di sangue che sono stati suggeriti 40 min di incubazione dosaggio e poi etichettatura con un singolo marcatore (cioè, E. coli, DHE, ecc). Dopo l&#39;etichettatura, i singoli eventi positivi vengono raccolti e una matrice di compensazione viene generato tramite un wizard automatico nel software di analisi IDEE. E &#39;fondamentale che se si utilizza questo test, adeguati controlli di compensazione sono stati completati per garantire prestazioni adeguate del dosaggio. L&#39;analisi viene eseguita mediante la funzione Finder e co-localizzazione procedure guidate per identificare che brillante intensità dettaglio è il miglior variabile per separare le popolazioni ed anche identificare come esiste molta sovrapposizione tra burst ossidativo e segnali fagocitosi. In particolare, l&#39;uso di immagini basato citometria disponibile la capacità di separare granulociti attivati ​​in tre sottoinsiemi. Tla sua ripartizione sottoinsieme è stato determinato utilizzando luminosa intensità dettaglio di fagocitosi vs. burst ossidativo. Oltre a esaminare queste funzioni cellulari singolari, cellule che mostravano entrambi gli eventi contemporaneamente nella stessa posizione anatomica (co-localizzazione) sono stati identificati. Granulociti che cadde nel sottoinsieme &quot;high-attiva&quot; erano l&#39;unico che ha dimostrato fenotipo coerente co-localizzazione tra fagocitosi e burst ossidativo. Questa identificazione di sottoinsiemi granulociti attivati ​​è la più grande area in cui è necessaria la risoluzione dei problemi. E &#39;molto importante per un nuovo utente di prendere tempo per capire il workflow di processo del campione in idee e per capire i meccanismi di gating le popolazioni cellulari utilizzando il componente di imaging. Altre modifiche che un utente può cercare di rendere comprendono la selezione dei tempi alterni o ulteriori test di incubazione. Il metodo attuale suggerisce l&#39;uso di una durata di 10-40 min; Tuttavia, a seconda del modello sperimentale in cui questo metodo èda utilizzare, può essere necessario selezionare durate dosaggio più. Tale modifica dovrebbe essere valutato su base individuale. Inoltre è stato stabilito che la durata dell&#39;incubazione dosaggio ha un effetto significativo sulla comparsa delle tre sottoinsiemi attivati. L&#39;approccio descritto in questo rapporto rappresenta un ampliamento di quali informazioni possono essere ottenute in precedenza per quanto riguarda la funzione di granulociti 3,4,13,15. Altri laboratori hanno dimostrato l&#39;importanza di valutare i cambiamenti nella funzione dei granulociti nel quadro di una valutazione globale di immunità e malattia 15-17. Nonostante il potenziale di questo test non è senza limitazioni. Una delle maggiori limitazioni è domanda Costi e tempi associati con elevata campione trasformazione produttività. Quando un disegno studio richiede un gran numero di campioni in un dato giorno, questi possono essere difficili da trattare. Il trattamento è semplificato dall&#39;uso di pipette elettroniche e dispenser,ma queste tendono ad essere costosi e non sono necessariamente disponibili in ogni laboratorio. La nostra area di ricerca si concentra su uno studio di come l&#39;esercizio fisico e le abitudini alimentari influenzano la salute del sistema immunitario e la funzione 6,8-10,18-20. Tali obiettivi hanno importanti implicazioni pratiche per una varietà di aree della salute umana. Oltre lo studio di esercizio e nutrizionali effetti il ​​metodo fagocitosi dimostrato in questo manoscritto potrebbe essere utile in altre zone del immunologia clinica dove il monitoraggio delle funzioni fagocitosi è fondamentale per i risultati del trattamento. Il presente saggio è il primo di molti che saggi immunologici con la possibilità di essere reinventate prendendo i vantaggi delle informazioni di imaging unica che può essere in grado da un flusso basato su immagini citometro.

Granulociti costituiscono una componente di sistema immunitario innato del corpo, che fornisce la prima linea di difesa contro gli antigeni invasori. I metodi precedenti per valutare la funzione dei granulociti concentrati sulla capacità di fagocitosi o burst ossidativo con metodi diversi, il che rende difficile accertare collettivamente come granulociti sono cambiati 1-4. I progressi nel campo della citometria a flusso hanno portato alla produzione di strumenti da banco capaci di alta risoluzione, multicolore imaging cellule in maniera high throughput 5. La capacità di coniugare l&#39;imaging con il flusso tradizionale rappresenta citometria un avanzamento che fornisce la piattaforma tecnologica necessaria per innovare nel flusso esistenti citometria metodi ed estrarre nuove informazioni sul sistema immunitario. Negli ultimi dieci anni, il nostro laboratorio, tra gli altri, è stato acutamente focalizzata sull&#39;effetto di vari patte nutrizionale e trattamenti eserciziorns sulla funzione immunitaria innata 6-9. Il metodo illustrato in questo manoscritto ha implicazioni pratiche nel campo della immunologia clinica. Il presente metodo sfrutta la potenza di image-based citometria a flusso per misurare simultaneamente la fagocitosi di particelle batteriche e burst ossidativo. Usando questo approccio, si è in grado di separare granulociti attivati ​​utilizzando variabili forniti dalla parte dell&#39;immagine basata dell&#39;analisi. Questi sottoinsiemi erano identificabili solo dopo aver valutato le immagini cellulari sui singoli granulociti. Ulteriore tempo di incubazione test influenzato la transizione tra i tre sottoinsiemi di attivazione 10. Così, è plausibile che uso di più tempi di incubazione può autorizzare un metodo per testare l&#39;cambiamento nella funzione granulociti seguendo un trattamento sperimentale specifica. Lo scopo di questo manoscritto era di dimostrare un metodo di valutazione della funzione dei granulociti utilizzando un&#39;immagine basata citometria a flusso per misurare simultaneamente fagocitosi con oxidative scoppiare.

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			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:125px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vacutainers</td>
			<td style="width:125px;">BD Life Sciences</td>
			<td style="width:119px;"></td>
			<td>used for blood collection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">WBC Fixative / RBC Lysis solution</td>
			<td style="width:125px;">eBioscience</td>
			<td style="width:119px;">00-5333-57</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD66b-APC</td>
			<td style="width:125px;">eBioscience</td>
			<td style="width:119px;">clone G10F5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD45-APCeFluor780</td>
			<td style="width:125px;">eBioscience</td>
			<td style="width:119px;">clone 2D1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">s.aureus bioparticles</td>
			<td style="width:125px;">Life Technologies</td>
			<td style="width:119px;">A10010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dihydroethidium</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">D7008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N-ethylmalemide</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">4259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">7AAD</td>
			<td style="width:125px;">EMD Millipore</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hematology Analyzer</td>
			<td style="width:125px;">Mindray</td>
			<td style="width:119px;">BC-3200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-channel pipet</td>
			<td style="width:125px;">Integra Biosciences</td>
			<td style="width:119px;">ViaFlo</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bead Bath Incubator</td>
			<td style="width:125px;">LabArmour</td>
			<td style="width:119px;">BeadBath</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Imaging Flow Cytometer</td>
			<td style="width:125px;">EMD Millipore</td>
			<td style="width:119px;">Amnis FlowSight</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">INSPIRE Software</td>
			<td style="width:125px;">EMD Millipore</td>
			<td style="width:119px;">Amnis INSPIRE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IDEAS Software</td>
			<td style="width:125px;">EMD Millipore</td>
			<td style="width:119px;">Amnis IDEAS</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">X-Pierce Piercable Plate Sealer</td>
			<td style="width:125px;">Excel Scientific, Inc.</td>
			<td style="width:119px;">X-Pierce</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dell Precision Workstation</td>
			<td style="width:125px;">Dell Computers</td>
			<td style="width:119px;">Various</td>
			<td>Used for IDEAS analysis</td>
		</tr>
	</tbody>
</table>

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).

Utilizzando basato su immagini citometria a flusso ha permesso di separare una popolazione omogenea di granulociti attivati ​​in tre diversi sottoinsiemi di attivazione (Figura 3). In questo metodo, il modo più efficace per risolvere i tre sottoinsiemi di attivazione è tracciando luminoso intensità dettaglio per fagocitosi (S. aureus) vs. burst ossidativo (DHE) (Figura 3; la figura 4). Inoltre, l&#39;utilizzo della procedura guidata co-localizzazione in software IDEAS consentirà la quantificazione della presenza di fagocitosi simultanea e burst ossidativo, che è un segno distintivo di granulociti altamente attivati. <img alt="Figura 3" src="/files/ftp_upload/52201/52201fig3highres.jpg" /> Figura 3. Identificazione attivati ​​granulociti sottoinsiemi. Dopo l&#39;identificazione di granulociti mediante marcatori della superficie cellulare (CD45 + / 66b +), un complotto figlia è stata generata con brillante dettaglio intensità di fagocitosi vs. brillante intensità dettaglio per burst ossidativo. Usando questo approccio, la percentuale di inattivi (viola), basso-attivo (rosso, A), moderata-attiva (blu, B), e (C, giallo) granulociti alto-attiva è stata determinata. Questa tecnica gating è stato anche utilizzato per valutare l&#39;effetto del tempo di incubazione del test sulla abbondanza relativa dei vari sottogruppi granulociti attivati. La più alta percentuale di granulociti &quot;alto-attiva&quot; è stata presente dopo l&#39;40 min di incubazione. Tutte le analisi di file di immagini acquisite è stato completato utilizzando il software IDEAS v.6. <a href="https://www.jove.com/files/ftp_upload/52201/52201fig3large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> Oltre a dimostrare la presenza di tre sottoinsiemi distinti di granulociti attivati, è stato dimostrato che il tempo di incubazione del test influenzato l&#39;abbondanza relativa di ogni granulociti attivato sottoinsieme. In particolare, il 40 min incubation portato alla maggiore percentuale di granulociti &quot;alto-attiva&quot;. Includendo almeno tre durate incubazione dosaggio può essere possibile determinare come un dato trattamento clinico altera lo stato di attivazione temporale dei granulociti. Questo è il primo metodo pubblicato utilizzando un&#39;immagine basata citometria a flusso per identificare diversi sottoinsiemi granulociti attivati ​​in funzione di misurazione simultanea dello fagocitosi e burst ossidativo. <img alt="Figura 4" src="/files/ftp_upload/52201/52201fig4highres.jpg" /> Figura 4. Immagini rappresentative della Cellular marcatori. Una galleria di immagini di cellule classificate come high-attiva (A), moderata-attiva (B), e basso-attivo (C) viene presentato in questa figura. Bioparticelle S. aureus sono in Ch03 , burst ossidativo è in Ch04, 7AAD per il nucleo è in CH05, scatter lato è in CH06, CD66b è inCH11, e CD45 è in CH12. Inoltre, viene visualizzata un&#39;immagine di unione co-localizzata di fagocitosi (Ch03) vs. burst ossidativo (Ch04). Aree di giallo l&#39;immagine di unione in denotano che la fagocitosi e burst ossidativo stanno verificando nello stesso spazio anatomica nello stesso momento. <a href="https://www.jove.com/files/ftp_upload/52201/52201fig4large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro at JoVE.com

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 ...

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Research

JoVE Journal

Immunology and Infection

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

12.3K Views.  University of North Texas. 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.

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

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Misurazione dell&#39;attività Calpain nelle celle fissi e Living in citometria a flusso

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Ricerca

Immunologia e infezioni

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Umano In Vitro</emSoppressione> come strumento di screening per il riconoscimento di uno Stato precoce di squilibrio immunitario

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

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 ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

L&#39;uso di citometria a flusso per valutare lo stato della cromatina in cellule T

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

La valutazione della retina microglia fagocitaria Funzione<em&gt; In Vivo</em&gt; Usando un test citometria a flusso basato su

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

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 stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

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 ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolamento e coltura In Vitro dei macrofagi alveolari murini ed umani

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

Base di un semplice flusso Cytometry Assay per determinare In Vitro dell'anticorpo dipendente valorizzazione del Virus di febbre rompiossa utilizzando siero convalescente Zika Virus

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Integrate Imaging Flow Cytometry and Transcriptomic Profiling to Evaluate Altered Endocytic CD1d Trafficking

Populational analyses of the morphological and functional alteration of endocytic proteins are challenging due to the demand of image capture at a single cell level and statistical image analysis at a populational level. To overcome this difficulty, we used imaging flow cytometry and transcriptomic profiling (RNA-seq) to determine altered subcellular localization of the cluster of differentiation 1d protein (CD1d) associated with impaired endocytic gene expression in human dendritic cells (DCs), which were exposed to the common lipophilic air pollutant benzo[a]pyrene. The colocalization of CD1d and endocytic marker Lamp1 proteins from thousands of cell images captured with imaging flow cytometry was analyzed using IDEAS and ImageJ-Fiji programs. Numerous cellular images with co-stained CD1d and Lamp1 proteins were visualized after gating on CD1d+Lamp1+ DCs using IDEAS. The enhanced CD1d and Lamp1 colocalization upon BaP exposure was further demonstrated using thresholded scatterplots, tested with Mander&#39;s coefficients for co-localized intensity, and plotted based on the percentage of co-localized areas using ImageJ-Fiji. Our data provide an advantageous instrumental and bioinformatic approach to measure protein colocalization at both single and populational cellular levels, supporting an impaired functional outcome of transcriptomic alteration in pollutant-exposed human DCs.

Imaging flow cytometry provides an ideal approach to detect the morphological and functional alteration of cells at individual and populational levels. Disrupted endocytic function for lipid antigen presentation in pollutant-exposed human dendritic cells was demonstrated with a combined transcriptomic profiling of gene expression and morphological demonstration of protein trafficking.

Integrare la citometria a flusso di Imaging e trascrittomica profilatura per valutare alterata CD1d endocitico traffico

Populational analyses of the morphological and functional alteration of endocytic proteins are challenging due to the demand of image capture at a single ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

Test ELISA in vitro per valutare la potenza del vaccino contro la rabbia

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...