The authors thank the Canadian Blood Services for a Graduate Fellowship Program Award to T.N.T. This research received financial support from the Canadian Blood Services' Centre for Innovation, funded by the federal government (Health Canada) and the provincial and territorial ministries of health. The views herein do not reflect the views of the federal, provincial, or territorial governments in Canada.

Ottenere l&#39;approvazione per l&#39;uso di campioni umani da parte l&#39;etica della ricerca pensione / pensione revisione istituzionale, e ottenere il consenso firmato da tutti i donatori umani. La MMA può essere eseguita anche utilizzando PBMC del mouse in un modo simile come il saggio umana, in seguito all&#39;approvazione uso animale etica. Il MMA utilizza tecniche di coltura dei tessuti asettiche. NOTA: Vedere la Figura 1. NOTA: Anche se si consiglia l&#39;uso di un armadio biocontenimento livello 2 durante il dosaggio per mantenere la tecnica asettica, come il metodo è solo un metodo di coltura &quot;a breve termine&quot;, non c&#39;è il tempo davvero sufficiente per i batteri di contaminare il test. Pertanto, se un cabinet biocontenimento livello 2 non esiste, piuttosto che acquistare uno solo per questo saggio, il saggio può essere eseguito al di fuori di un armadio biocontenimento, su un banco aperto. L&#39;uso di un incubatore a 37 ° con 5% di CO 2, tuttavia, non è un&#39;opzione e deve essere utilizzato perRisultati ottimali. L&#39;isolamento delle cellule 1. sangue periferico mononucleari <ol><li> Ottenere sangue umano intero da un donatore sano o di un paziente tramite venipuntura utilizzando vacutainer contenenti acido citrato destrosio (ACD) anticoagulante (giallo-top tubes). NOTA: sangue intero può essere conservato in ACD a temperatura ambiente (18-22 ° C) per un massimo di 36 ore prima di procedere alla fase successiva 11. Normalmente, 1-2 vacutainer 10 ml di sangue intero sono sufficienti per il dosaggio. </li><li> Diluire il sangue intero 1: 1 v / v in acqua calda completo RPMI (RPMI-1640 supplementato con siero fetale bovino al 10%, 20 mM HEPES, e 0,01 mg / ml gentamicina). </li><li> Isolare le cellule mononucleate del sangue periferico (PBMC) dal sangue intero diluito con densità centrifugazione in gradiente, come raccomandato dal produttore (vedere elenco dei materiali). Strato sangue diluito molto lentamente sopra il gradiente di densità (riscaldato a temperatura ambiente, 18-22 ° C). NOTA: Ridurre al minimo la quantità di Mixing all&#39;interfaccia per la separazione ottimale del sangue attraverso la stratificazione accuratamente la miscela di sangue in un modo a gocce o utilizzando una pipetta. <ol><li> Lasciare la miscela di sangue al layer lentamente sopra la parte superiore del gradiente di densità posizionando la punta della pipetta vicino al gradiente di densità e consentendo la miscela sangue per correre lungo il lato del tubo molto lentamente. </li><li> A seconda della scala dell&#39;esperimento, strato 10 ml di miscela di sangue sulla cima di 3 mL di gradiente di densità (in un tubo da 15 ml) o strato 35 ml di miscela di sangue sulla cima di 15 mL di gradiente di densità (in 50 mL tubo). Tipicamente, 10 ml di sangue intero fornisce 10 milioni di PBMC, con qualche variazione donatore-to-donatore. NOTA: È molto importante che vi sia alcuna miscelazione della miscela di sangue con gradiente di densità. La miscela sangue dovrebbe strato sopra il gradiente di densità e salire lentamente fino a che tutto il sangue è sopra il gradiente di densità. </li><li> Centrifugare la miscela stratificata a 700 g per 30 minuti senza freni. il centrmiscela ifuged dovrebbe separare in 5 strati (dall&#39;alto verso il basso): plasma, buffy coat (contenente PBMC), materiali gradiente di densità, granulociti, e globuli rossi. </li><li> Rimuovere e scartare la maggior parte del plasma e recuperare attentamente il contenuto buffy coat (PBMC) in un nuovo tubo da 15 ml con una pipetta Pasteur e una lampadina di aspirazione. NOTA: La rimozione dello strato di buffy coat è effettivamente fatto applicando aspirazione sulla pipetta Pasteur durante l&#39;esecuzione di un movimento circolare intorno alla parte esterna dello strato, con la punta della pipetta contro il tubo. </li></ol></li><li> Lavare le PBMC isolate tre volte in pH 7,3 tampone fosfato salino (PBS) per centrifugazione a 350 xg per 10 min (con freni pieni) tra lavaggi. Ricostituire il pellet PBMC in terreno completo RPMI. A seconda delle dimensioni del pellet, 3-7 ml di mezzo è sufficiente. </li><li> Contare il PBMC utilizzando trypan blue e un emocitometro. contare solo quelle cellule che non sono macchiati dal blu trypan. Reconstitute le PBMC a 1.750.000 cellule / ml in terreno completo RPMI. </li><li> Seed 400 ml (700.000 cellule) in ciascun pozzetto di una sagoma 8-camera. Incubare il vetrino a 37 ° C, completamente umidificata incubatore di coltura tissutale (supplementato con 5% CO 2) per 1 h per permettere i monociti / macrofagi di aderire. </li></ol> 2. Pre-trattamento dei aderito monociti NOTA: Questo passaggio è necessario solo se alla ricerca di modi per inibire o migliorare la fagocitosi. <ol><li> Pre-treat aderito monociti con qualsiasi farmaco (s) o composti (s) di interesse. Ricostituire il farmaco (s) o altro materiale di prova alla concentrazione desiderata con mezzo completo RPMI. NOTA: per esempio, 200 mg / mL di IVIG è in genere utilizzato per produrre un&#39;inibizione 95-100% della fagocitosi quando si utilizza monociti umani. </li><li> Aspirare e scartare il surnatante contenente le cellule non aderenti dalla slitta 8-camera dopo 1 h di incubazione (dopo il punto 1.6). Sostituirlo con 400ml di un farmaco o altro trattamento e incubare per 1 ora a 37 ° C. <ol><li> Quando aspirazione e sostituendo soluzioni nella slitta 8-camera, assicurarsi di controllare il flusso dei fluidi in modo che le cellule debolmente aderito non tolgono. Inoltre, lavorare con solo 2-3 pozzetti vuoti alla volta ed evitare l&#39;essiccamento dei pozzetti. NOTA: Tipicamente, ogni trattamento viene eseguito in triplicato tecnici. </li></ol></li></ol> 3. Opsonizzazione di R 2 R 2 globuli rossi NOTA: L&#39;R 2 R 2 utilizzati qui sono ottenute dal sangue Collezione Center (Canadian Blood Services), ma sono anche disponibili in commercio. Opsonizzati R 2 R 2 globuli rossi vengono utilizzati come controllo positivo per la fagocitosi FcγR-mediata. Naïve R 2 R 2 deve essere conservato in soluzione di Alsever (per prolungare la shelf life) a 4 ° C per un massimo di 1 mese. La soluzione di Alsever è fatto in casa e composta da 0,8% W v trisodio citrato (diidrato), 1,9% w / v destrosio, 0,42% w / v di cloruro di sodio e 0,05% w / / acido citrico v (monoidrato). Se R 2 R 2 cellule non sono disponibili, altre cellule Rh-phenotyped, come R 1 R 2, R 1 R 1, R 1 R, o R 2 R, può essere utilizzato. <ol><li> Lavare R 2 R 2 (CDE / CDE) globuli rossi nel PBS per un totale di tre volte utilizzando centrifugazione a 350 xg per 5 minuti ciascuna. NOTA: La quantità di globuli rossi necessari dipende dalla dimensione sperimentale e può essere back-calcolato. Lavare sempre una quantità in eccesso di globuli rossi, globuli rossi poiché sono persi durante ogni lavaggio causa di lisi o durante la rimozione del supernatante. Ad esempio, in un esperimento con 5 trattamenti e 2 controlli, tutti condotti in triplicato tecnici, c&#39;è un totale di 21 pozzetti. 21 pozzi con 400 ml / pozzetto di 1,25% miscela RBC indica che 105 ml di sacco, opsonizzati sono necessari RBC. 200 ml di RBC dovrebbe essere inizialmente lavato e 150 &amp; #181; L opportuno opsonizzati per garantire che vi sia una adeguata quantità di globuli rossi per la fase fagocitosi. </li><li> Opsonize il lavato R 2 R 2 pellet con 1: 1 v / v policlonali anti-D anticorpi da siero umano e incubare per 1 ora a 37 ° C, con miscelazione intermittente. NOTA: Se policlonali anticorpi anti-D non sono disponibili, è possibile utilizzare anticorpi monoclonali anti-D, che sono disponibili in commercio, o anti-D che viene normalmente utilizzato per Rh profilassi immunitaria, che può essere ottenuto da banche del sangue o trasfusioni Servizi. test ottimizzazione dovrebbe essere condotta all&#39;inizio, quando si imposta il test, e ogni volta che il passaggio sacco di anticorpi policlonali non-titolato o il passaggio a un anticorpo monoclonale. Questo è di identificare la concentrazione ottimale o volume di anti-D richiesto per un test antiglobulina (IAT) risultato compreso tra 3+ e 4+ e un risultato di fagocitosi tra 70-90 eritrociti fagocitati per 100 monociti contati. </li><li> Lavare il opsonized R 2 R 2 un totale di tre volte in PBS mediante centrifugazione a 350 xg per 5 minuti ciascuna. NOTA: opsonizzazione di successo di R 2 R 2 può essere confermata eseguendo una IAT indiretta. Brevemente, secondarie policlonali anticorpi anti-umani vengono aggiunti per legare anticorpi opsonizzante primarie sulle superfici RBC, e il segnale amplificato possono essere osservati sotto forma di emoagglutinazione. Un dettagliato protocollo produttore può essere trovato nel file materiali supplementari. </li><li> Ricostituire il lavato R 2 R 2 pellet al 1,25% v / v usando mezzo completo RPMI. NOTA: Eccesso opsonizzati 2 R 2 R può essere conservato in soluzione di Alsever a 4 ° C per una settimana. </li></ol> 4. La fagocitosi mediata da recettori Fc <ol><li> Aspirare il farmaco o mezzo supernatante dalla slitta 8-camera e aggiungere 400 ml di 1,25% v / v R 2 R 2 miscela. Incubare a 37 ° C per 2 ore. </li><li> A poppaer 2 h di incubazione, rimuovere le camere utilizzando gli adattatori del produttore. Tamponare l&#39;eccesso di R 2 R 2 su un tovagliolo di carta. Assicurarsi che la diapositiva non si asciughi. </li><li> Riempire un bicchiere da 100 ml con PBS. Immergere e lavare la slitta spostando lentamente la slitta avanti e indietro (circa 30-40 colpi) per rimuovere la maggior parte dei non-fagocitati R 2 R 2. </li><li> Rimuovere il vetrino dal PBS. Tamponare l&#39;eccesso di PBS usando un tovagliolo di carta o tessuto e aria asciugare la diapositiva. </li><li> Fissare la slitta di aria secca in 100% di metanolo per 45 s. Poi, aria asciugare il vetrino fisso. NOTA: diapositive può essere fissato utilizzando un altro metodo che è più compatibile per la colorazione downstream, quali la macchia Grünwald-Giemsa 21 o Wright-Giemsa 22. </li><li> Montare la slitta con un in-house fatta di montaggio elvanol medio (o di un altro mezzo di montaggio disponibile in commercio) e aggiungere lamelle. NOTA: Elvanol mezzo di montaggio è composto da 15% w / v poresina alcool lyvinyl e il 30% v / v in PBS glicerina. La miscela viene riscaldata fino a quando tutta la resina è sciolto e la glicerina è omogeneamente miscelato. Questo può essere sostituito con altro mezzo di montaggio disponibile in commercio. </li><li> Lasciare che il supporto per asciugare durante la notte prima di quantificazione. </li></ol> 5. Quantificazione della fagocitosi <ol><li> Utilizzando un microscopio a contrasto di fase e una lente obiettivo 40X, quantificare manualmente la quantità di eventi fagocitarie contando almeno 200 monociti e il numero dei fagocitati R 2 R 2 all&#39;interno di questi monociti. Avere un contatore in ogni mano per quantificare contemporaneamente il numero dei monociti e il numero di fagocitati R 2 R 2. </li><li> Ottenere l&#39;indice medio di fagocitosi dividendo il numero di fagocitati R 2 R 2 per il numero di monociti e moltiplicando per 100. espresso i dati come la media (indice di fagocitosi media) ± l&#39;errore standard della media (SEM). </li&gt; </ol>

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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+efficient+enzymes+for+the+generation+of+universal+blood+through+structure-guided+directed+evolution.">Toward efficient enzymes for the generation of universal blood through structure-guided directed evolution.</a> J Am Chem Soc. 137 (17), 5695-5705 (2015).</li></ol>

The authors have nothing to disclose.

Il MMA è una tecnica laboriosa che richiede competenze sia nella coltura di tessuti e microscopia. Ci sono diversi passaggi critici per garantire il successo: 1) generazione del monostrato monociti; 2) opsonizzazione dei globuli rossi, e 3) la quantificazione manuale. Il monostrato monociti non aderisce fortemente alla diapositiva camera, in modo pH fisiologico deve essere mantenuta durante tutta la seduta 11 e un numero adeguato di PBMC dovrebbe essere seminato. pipettaggio vigoroso, che potrebbe disturbare le cellule aderito, dovrebbe essere evitato. Un approccio è quello di rimuovere sempre e aggiungere soluzioni dal medesimo angolo della camera e per assicurare che il movimento è lento e costante. Allo stesso modo, durante l&#39;ultima fase di lavaggio per rimuovere l&#39;eccesso di globuli rossi, il movimento deve essere lento e costante. Questo assicura il minimo disturbo al monostrato pur eliminando la maggior parte dei globuli rossi non-fagocitati. lavaggio inadeguata porterà ad un fondo elevato di globuli rossi contaminanti, il che rende qua manualentification difficile. In secondo luogo, i globuli rossi R 2 R 2 devono essere sufficientemente opsonizzati per ottenere un indice medio fagocitosi del 80-120 per il controllo fagocitosi. Questa gamma dei fagociti desiderato un equilibrio tra la facilità di conteggio (ad esempio, monociti con più di 5 fagocitati globuli rossi sono difficili da quantificare con precisione) e il mantenimento di una adeguata quantità di fagocitosi per l&#39;analisi statistica. Il grado di opsonization può essere confermata da un IAT, ed è necessaria una lettura tra 3+ a 4+. L&#39;R 2 R 2 RBC deve essere eliminata quando c&#39;è eccesso lisi durante il lavaggio, quando il supernatante diventa rosso scuro, o quando una diminuzione significativa nella fagocitosi è osservata in esperimenti a causa dell&#39;invecchiamento delle cellule in deposito. Infine, la quantificazione manuale utilizzando il microscopio può essere difficile, soprattutto quando si confrontano i conteggi tra il personale di laboratorio e tra esperimenti. Esaminando stesso campo in ogni pozzetto, o semplicemente contando più cellule, Un conteggio più coerente può essere ottenuta. Si raccomanda di formazione Side-by-side con un tecnico esperto e l&#39;uso di un insieme designato di diapositive formazione. Un importante critica del MMA è la soggettività del passo quantificazione manuale. Tuttavia, con una formazione adeguata, la coerenza può essere ottenuto attraverso diversi contatori. Un altro limite è la intrinseche differenze donatore-to-donatore in monociti capacità fagocitarie e in R 2 R livelli di espressione dell&#39;antigene 2 di superficie, che è una fonte di variazione dei dati quando si tratta di campioni umani. Altre tecniche alternative sono disponibili per l&#39;esame fagocitosi FcγR-mediata. La maggior parte dei kit commerciali utilizzano uscita fluorescente per monitorare la fagocitosi (ad esempio, bioparticelle, sensibile al pH della proteina di fluorescenza, o sfere di lattice fluorescenti IgG-etichettati). L&#39;uso di uscita fluorescente non offrono quantificazione più oggettiva, ma ha anche bisogno di ConSider la disponibilità, costo e formazione associata con l&#39;uso di un microscopio a fluorescenza o un citometro a flusso, nonché la dipendenza conseguente upon kit disponibili in commercio. Infine, questo dosaggio può essere modificato a seconda della domanda di ricerca. Ad esempio, quando si verifica l&#39;inibizione farmaco di fagocitosi, monociti possono sia essere pretrattate o co-incubate con entrambi i farmaci e RBC opsonizzati (cioè, un saggio di competizione). La segnalazione a valle di anticorpi di diversi sottotipi, anticorpi chimerici, o costrutti ricombinanti può essere testato. Con i recenti progressi nello sviluppo di un sangue dell&#39;antigene nullo universale 24, la MMA potrebbe essere utilizzato nelle schermate iniziali di questi globuli rossi antigene-null con diversi anticorpi per valutare se esiste effettivamente un efficacia abbassato nello scatenare la fagocitosi.

The monocyte monolayer assay (MMA) is an in vitro assay originally developed to better predict blood transfusion outcomes in patients with auto- or alloantibodies to red blood cells (RBCs)1-5. By assessing the effect of anti-RBC antibodies in mediating Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis using this in vitro assay, it is possible to predict the clinical outcome in vivo. Indeed, the MMA has been used successfully to avoid immune destruction of antibody-bound RBCs, despite the transfusion of serologically incompatible blood5. The typical pre-transfusion procedure for compatibility testing, also termed crossmatching, involves serological methods that include typing the patient's blood for ABO and Rh antigens and screening for the presence of anti-RBC antibodies in the patient6. Blood matched for ABO/Rh is selected, and if antibodies are present, an attempt to identify them is made so that blood for transfusion can be further selected to avoid these antigens. An ideal crossmatch result occurs when all donor blood is serologically compatible with the patient's blood, which reduces the risk of post-transfusion hemolysis7. However, this system falls short for the small group of patients who have become alloimmunized upon repeated transfusion or pregnancy. These patients produce alloantibodies against specific RBC antigens. Some produce antibodies to antigens of very high frequency in the general population, and thus become progressively more difficult to crossmatch8,9. Adding to the complexity, not all alloantibodies are clinically significant; in other words, the binding of an alloantibody to RBCs detected by a serology test does not necessarily result in hemolysis when antigen-positive, incompatible blood is transfused. The MMA was originally developed to assess the potential clinical significance of serologically incompatible blood in a transfusion setting1-5.
Since extravascular hemolysis of antibody-bound RBCs is known to be mediated by the mononuclear phagocyte system, primary monocytes/macrophages are utilized in the development of diagnostic assays. The first assay to study the interaction of monocytes, RBCs, and antibodies was published in 1975, but the sub-optimal conditions used led only to rosette formation (the binding of RBCs to the periphery of the monocyte), and no phagocytosis was observed10. Significant modifications to the assay were made by several groups, leading to an assay for which the level of phagocytosis of alloantibody-bound RBCs could be correlated to the clinical outcome of hemolysis1-5. Recently, the optimal storage conditions of clinical samples and further optimization of assay conditions were examined to enhance the utility of a clinical MMA crossmatch using autologous patient samples11.
Three other diagnostic techniques have been employed in addition to the MMA in predicting transfusion outcomes: the 51Cr release test, the rosette test, and the chemiluminescence test (CLT). In the 51Cr release test, the patient is injected with 51Cr-labeled donor RBCs, and the half-life of the labeled RBCs is monitored and is predictive of post-transfusion survival or clearance12,13. As this method uses radioactive materials, it is rarely performed anymore. The rosette test involves mixing and incubating monocytes with RBCs and quantifying the level of rosette formation (with no phagocytosis)14. The clinical significance of antibodies in vivo involves active phagocytosis by macrophages found in the spleen and/or the liver; thus, this method does not provide a relevant readout of phagocytosis. The CLT uses luminol to monitor the oxidative burst during monocyte phagocytosis of RBC, since luminol fluoresces blue when oxidized in the phagosome15. This method is good, but contamination by neutrophils can confound the readout. Parallel comparisons have been made to evaluate the sensitivity, practicality, and reproducibility of the four available methods, and both the CLT and MMA were ranked superior16. However, the CLT has been mainly utilized in assessing hemolytic disease of the fetus and newborn (HDFN), and the assay's optimal pH of 8.0 might compromise the level of phagocytosis11.
In addition to its diagnostic and clinical utility, the MMA has been modified for other research purposes. Indeed, the MMA can not only serve as a functional assay to address discrepancies between serology and biology, it has also been used to retrospectively investigate the cause of hemolysis after intravenous immunoglobulin (IVIG) therapy17. It has also been used to examine the structure-function of chemical inhibitors of Fc&#947;R-mediated phagocytosis18-20 and to study the downstream signaling of Fc&#947;R-mediated phagocytosis21. In our laboratory, in addition to using a human MMA, we are developing a murine MMA using primary mouse peripheral blood mononuclear cells (PBMCs) and autologous RBCs. The rationale is to screen antibodies that can induce Fc&#947;R-mediated phagocytosis as an intermediate to developing an in vivo autoimmune hemolytic anemia (AIHA) mouse model (unpublished data). The various modifications focus on different aspects of the IgG antibody and Fc&#947;R interaction that induce phagocytosis.

<table border="0" cellpadding="0" cellspacing="0" width="1148">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Acid citrate dextrose (ACD) vacutainers</td>
			<td style="width:213px;">BD</td>
			<td style="width:136px;">REF364606</td>
			<td style="width:405px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">RPMI 1640</td>
			<td style="width:213px;">Sigma</td>
			<td style="width:136px;">R8758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">HEPES</td>
			<td>Bioshop</td>
			<td>HEP003.100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Fetal bovine serum</td>
			<td style="width:213px;">Multicell&#160;</td>
			<td style="width:136px;">080150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Gentamicin</td>
			<td>Gibco</td>
			<td>15710-64</td>
			<td></td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:395px;">Ficoll-Paque PLUS</td>
			<td style="width:213px;">GE</td>
			<td style="width:136px;">17-1440-03</td>
			<td><a href="https://www.gelifesciences.com/">https://www.gelifesciences.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Phosphate buffered saline</td>
			<td style="width:213px;">Sigma</td>
			<td style="width:136px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">8-chamber slides</td>
			<td style="width:213px;">Lab-Tek-ll</td>
			<td style="width:136px;">154534</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:395px;">R2R2 (cDE/cDE) red blood cells&#160;</td>
			<td style="width:213px;">Canadian Blood Services</td>
			<td style="width:136px;"></td>
			<td>Commercially available (e.g. http://www.bio-rad.com/en-ca/product/reagent-red-blood-cells)&#160;</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:395px;">Polyclonal anti-D from human serum</td>
			<td>Gamma Biologics</td>
			<td>DIN 02247724</td>
			<td>Can be substituted with commercially available monoclonal anti-D or with Rh immune globulin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">100% methanol</td>
			<td style="width:213px;">Caledon</td>
			<td style="width:136px;">6700-1-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Polyvinyl alcohol resin</td>
			<td style="width:213px;">Sigma</td>
			<td style="width:136px;">P8136</td>
			<td>Can be substituted with commercially available mount</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">UltraPure glycerine</td>
			<td style="width:213px;">Invitrogen</td>
			<td style="width:136px;">15514-011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Cover slips</td>
			<td style="width:213px;">VWR</td>
			<td style="width:136px;">48366 067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Novaclone anti-IgG</td>
			<td style="width:213px;">Immucorgamma</td>
			<td style="width:136px;">5461023</td>
			<td>Optional for IAT (http://www.fda.gov/downloads/biologicsbloodvaccines/.../ucm081743.pdf)</td>
		</tr>
	</tbody>
</table>

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.

Seguendo i passaggi cruciali nella figura 1 e la procedura sopra descritta, l&#39;MMA può essere riproducibile eseguita. IVIG è stato utilizzato come un esempio per l&#39;inibizione della fagocitosi in figura 2. IVIG è noto per legare e bloccare i recettori Fc, inibendo quindi il risultato a valle della fagocitosi. Titolando la quantità di IVIG utilizzato, una inibizione dose-dipendente è osservato, nel quale la concentrazione di sopra di 200 mg / mL portato vicino al 100% di inibizione e concentrazioni inferiori a 0,5 mg / mL non ha inibito affatto (Figura 2A). Quando gli indici fagocitarie vengono trasformati e normalizzati per il controllo positivo R 2 R 2 come 0% di inibizione, una curva di inibizione con una IC50 di 3 mg / ml può essere determinato (Figura 2B). Oltre a eseguire il dosaggio costante, quantificazione del saggio a volte può essere difficile. La figura 3 è una raccolta di contrasto di fase immagini al microscopio di varie diapositive MMA. Con l&#39;esperienza microscopia, si può distinguere un monociti da un RBC contaminante (Figura 3D), o un vacuolo da un RBC fagocitato (Figura 3B). Densi ammassi di cellule e / o detriti dovrebbero essere evitati durante la quantificazione (Figure 3E e F). Inoltre, si dovrebbe evitare un&#39;eccessiva opsonizzare R 2 R 2 RBC, che porterebbe alla fagocitosi rafforzata che overcrowds l&#39;interno monociti e interferisce con accurata quantificazione (Figura 3C). <img alt="Figura 1" src="/files/ftp_upload/55039/55039fig1.jpg" /> Figura 1. Schema della MMA. Una rappresentazione step-by-step dei passaggi cruciali nella MMA: isolamento PBMC da sangue intero, che alimenta il monocyt aderitoes con opsonizzati R 2 R 2, e lavando le diapositive da camera. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55039/55039fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src="/files/ftp_upload/55039/55039fig2.jpg" /> Figura 2. endovenoso IgG (IVIG) inibisce la fagocitosi in vitro usando l&#39;MMA. IVIG è noto per inibire la fagocitosi e viene usato come controllo di inibizione nella MMA umana. (A) Titolazione di IVIG determinato una inibizione dose-dipendente di fagocitosi rispetto al greggia R comando R 2 2. I risultati mostrano la media ± errore standard della media (SEM) di n = 3 esperimenti. L&#39;analisi statistica è stata condotta utilizzando Student t-test: ** P≤0.01 e *** p≤0.001. (B) della curva di inibizione IVIG con un IC 50 of 3 mg / mL (linea tratteggiata). I risultati mostrano dati normalizzati per non trattati R 2 R 2 (0% di inibizione); media ± SEM di n = 3 esperimenti. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55039/55039fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/55039/55039fig3.jpg" /> Figura 3. contrasto di fase immagini al microscopio di diapositive campione sotto 40X di ingrandimento. (A) La slitta perfetto in cui i monociti fagocitati 1-2 R 2 R 2 su (frecce nere con un bagliore alone distintivo) media, con pochissimi contaminante, non-fagocitato R 2 R 2 in background (frecce bianche). Vacuoli (B) Come mostrato in questa diapositiva, monociti a volte sono allargata (frecce bianche), che possono essere scambiati per fagocitato R 2 R 2. (C &lt;/ strong&gt;) Quando R 2 R 2 sono stati over-opsonizzati, più di 4-5 fagocitati R 2 R 2 per monociti (frecce nere) renda conteggio preciso difficile, poiché la R 2 R 2 sono affollate ai monociti e cellule distinta i confini non possono essere distinte. (D) di lavaggio inadeguata della slitta porta ad abbondanti R 2 R 2 contaminazione (frecce bianche), che potrebbe essere scambiato per globuli rossi aderito. (E) A volte, monociti e globuli rossi contaminanti possono formare gruppi. I cluster indicano anche la contaminazione batterica, che dovrebbe essere evitato. (F) I monociti può formare aggregati più grandi, che dovrebbero essere evitati durante la quantificazione. Utilizzando selezione casuale dei campi per vedere, pannello A è quello che di solito si osserva se il MMA è stata eseguita correttamente. barra della scala è di 20 micron. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55039/55039fig3large.jpg" target="_blank">Per favore clicca qui</a>per visualizzare una versione più grande di questa figura.

Watch this Scientific Journal Video about Use of a Monocyte Monolayer Assay to Evaluate FcÃŽÂ³ Receptor-mediated Phagocytosis at JoVE.com

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

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 ...

use-monocyte-monolayer-assay-to-evaluate-fc-receptor-mediated

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

17.8K Views.  University of Toronto. The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fcγ receptor (FcγR)-mediated phagocytosis.

Video: Use of a Monocyte Monolayer Assay to Evaluate Fcγ Receptor-mediated Phagocytosis

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

Applicazione di un mouse legatura Patch Peyer Assay intestinale Loop per valutare assorbimento da parte delle cellule batteriche M

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Ricerca

Immunologia e infezioni

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

L&#39;uso di interferone-γ Enzyme-linked Assay Immunospot per caratterizzare Nuovi epitopi T-cellulari di Papillomavirus Umano

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

Un saggio di micropiastre per valutare gli effetti chimici su RBL-2H3 Mast cellule Degranulazione: Effetti del Triclosan senza l&#39;uso di un solvente organico

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

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 ...

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

Misurazione dei granulociti e dei monociti fagocitosi e ossidativo attività Burst nel sangue umano

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Caratterizzazione delle derivate da monociti cellule dendritiche umane di Citometria a Flusso Imaging: un confronto tra due protocolli di isolamento dei monociti

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

L&#39;utilizzo della tecnica di sovrapposizione Soft-agar per lo screening composti inibitori bacterially Prodotto

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

Un test compatibile high-throughput per valutare Drug efficacia contro macrofagi diversi passaggi<em&gt; Mycobacterium tuberculosis</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

We herein describe a phagocytosis assay using the dispersed embryonic cells of Drosophila. It enables us to easily and precisely quantify in vivo phagocytosis levels, and to identify new molecules required for the phagocytosis of apoptotic cells.

Analisi della fagocitosi per le cellule apoptotiche in<em&gt; Drosophila</em&gt; Embrioni

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and ...

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 ...