This work was made possible by funding from the German Federal Ministry of Education and Research (BMBF, 1315883) as well as by the S&#228;chsische Aufbaubank (SAB) project 100116526 from a funding of the European Regional Development Fund (ERDF).

Tutti i campioni di sangue umano sono stati ottenuti da volontari sani, e il protocollo di leucociti arricchimento applicato segue le linee guida di etica commissione della Facoltà di Medicina dell&#39;Università di Lipsia. Gli esperimenti con il sangue di ratto sono stati approvati dal responsabile comitato etico locale (Landesdirektion Sachsen, Referat 24), secondo le linee guida tedesche per la cura degli animali e l&#39;uso. 1. apparato sperimentale NOTA: Poiché la procedura di lisi ipotonica per l&#39;esaurimento di eritrociti da campioni di sangue è una procedura tempi critici, preparare tutte le attrezzature necessarie (ad esempio, buffer) per questa parte del protocollo in anticipo. <ol><li> Etichettare un 15 ml provetta per lisi ipotonica e uno 1,5 ml provetta per la successiva fluoresceina amminofenil (APF) colorazione per campione di sangue. <ol><li> Se l&#39;arricchimento leucociti verrà eseguita in condizioni sterili, eseguire questa etichettatura sotto flussoscatola. </li></ol></li><li> Preparare circa 12 ml di PBS (PBS, 10 mM) per campione. A seconda che i leucociti sono isolati in condizioni sterili o no, preparare PBS sia utilizzando una soluzione pronta sterile da un fornitore o sciogliendo compresse PBS in acqua distillata. Controllare il valore pH e, se necessario, regolare a pH 7,4 utilizzando piccole quantità di 0,1 soluzioni M HCl e NaOH. <ol><li> Sciogliere compresse PBS (vedere Tabella dei materiali) in 200 ml di acqua distillata per ottenere una soluzione tampone non sterile sufficiente per circa 16 campioni. Se lo si desidera, sterilizzare questa soluzione per filtrazione sterile. </li></ol></li><li> Preparare circa 1 ml di soluzione salina bilanciata Hanks (HBSS) supplementato con Ca 2+ per campione. <ol><li> Sciogliere 970 mg di HBSS sale in 100 ml (volume finale) acqua bidistillata. Prima di riempire fino al volume finale, controllare il valore del pH e regolarlo a pH 7.4. Grazie alla sua capacità tampone basso, preparare questo buffer di fresco ogni giorno und eseguire la regolazione del pH con particolare cura, utilizzando piccole quantità di 0,1 soluzioni M HCl e NaOH. NOTA: soluzione sterile può essere utilizzato, a seconda dell&#39;esperimento. La soluzione può essere sterilizzata per filtrazione sterile. </li></ol></li><li> Oltre due buffer, etichette e preparare un tubo (ad esempio, 50 ml tubo di centrifugazione) o pallone (esempio, 250 ml misurino) con acqua bidistillata per la procedura di lisi ipotonica in anticipo. Preparare circa 10 ml di acqua per campione. </li><li> Preparare un contenitore con ghiaccio tritato per la colorazione APF (sezione 3), che viene eseguita su ghiaccio. </li><li> Preparare aliquote di APF in anticipo per consentire la preparazione rapida di soluzioni di lavoro utilizzati durante la colorazione (passo 3.2) e per evitare più volte il congelamento e lo scongelamento della soluzione APF. NOTA: APF è comunemente ottenuto come 5 mg / ml (11.81 mM) soluzione di riserva in acetato di metile. <ol><li> Congelare aliquote di 10-100 ml a 0,5 ml tubi. Per l&#39;APF macchiare una finaleconcentrazione di colorante di 10 pM è utilizzato (passo 3.8). </li></ol></li></ol> 2. ipotonica Lisi degli eritrociti NOTA: Eseguire la procedura di lisi ipotonica (tranne le fasi di centrifugazione) sotto un banco a flusso laminare per evitare la contaminazione. Eseguire l&#39;intera procedura a temperatura ambiente. Come la lisi ipotonica è un processo critico tempo, regolare le pipette per acqua (2 ml) e PBS (5 ml) aggiunta in anticipo e aprire l&#39;acqua e flaconi PBS prima di avviare la procedura. Conservare la soluzione PBS e l&#39;acqua distillata a temperatura ambiente prima dell&#39;uso. <ol><li> Da ogni trasferimento del campione di sangue 100 ml a 15 ml di tubo di centrifugazione appropriata. NOTA: Nei nostri esperimenti campioni di sangue di solito contengono 10 U / ml di eparina per evitare coagulazione. Eppure, a causa di diverse fonti di materiale campione della natura e della concentrazione del anti-coagulanti possono essere diversi. La sua influenza sulla colorazione APF dovrebbe essere testato da Comparzione dei risultati ai risultati ottenuti da campioni di sangue addizionato con altri tipi o concentrazioni di anticoagulante. </li><li> Aggiungere 2 ml di acqua distillata e mescolare i campioni utilizzando un miscelatore vortex impostato su un livello medio. Incubare i campioni per 60 secondi, quindi aggiungere 5 ml di PBS per campione e miscelare con il vortex. NOTA: fino a circa 5 campioni può essere preparato in parallelo. Mantenere l&#39;ordine del campione stesso per l&#39;aggiunta di acqua e PBS per ottenere tempi di incubazione comparabili. </li><li> Dopo la rigenerazione delle condizioni isotoniche di PBS aggiunta (passo 2.2) agglomerare le restanti cellule intatte in tutti i campioni mediante centrifugazione per 6 minuti a temperatura ambiente e 450 x g. </li><li> Rimuovere il surnatante versandolo in un contenitore per rifiuti tavolo. Rimuovere quanto più liquido possibile invertendo il tubo di centrifugazione e toccando l&#39;apertura su un tovagliolo di carta. Assicurarsi che il pellet è il più asciutto possibile. </li><li> Ripetere la procedura di lisi ipotonica come descritto in precedenza (passo 2.2). NOTA: In linea di principio questa procedura lisi ipotonica (passi 2.3 2.5) potrebbe essere ripetuta più volte, a seconda della purezza della frazione leucocitaria mista da ottenere. Dopo due passi lisi la quota rimanente di eritrociti nella frazione cellulare mista ottenuta è di circa il 25%. </li><li> Agglomerare le cellule intatte restanti centrifugando per 6 minuti a temperatura ambiente e 450 x g. Rimuovere il surnatante (vedi punto 2.4). Aggiungere 500 microlitri HBSS al pellet e delicatamente sciogliere fino ad ottenere una soluzione omogenea. Trasferire ciascun campione alla provetta 1,5 ml di conseguenza l&#39;etichetta. </li><li> A seconda del esperimento analizzare direttamente i campioni ottenuti (ad esempio, tramite citometria a flusso) 25, macchia con anticorpi fluorescenza marcati (per l&#39;identificazione di tipi di cellule singole) 32, incubare con stimoli cellulari (per gli esperimenti di attivazione delle cellule) 33 o memorizzati su ghiaccio e l&#39;uso dopo. A seconda dello scopo dello studio, eseguire immediatamente successivi esperimenti sula frazione leucocitaria mista da granulociti hanno un abbastanza breve emivita di circa 20 ore. NOTA: Questo vale anche per la colorazione attività perossidasica descritto di seguito. </li></ol> 3. alogenanti PEROSSIDASICA colorazione NOTA: HOCl- e HOBr-produzione da parte dei perossidasi eme derivati ​​dal sangue umano MPO e EPO è quantificato utilizzando APF, che viene ossidato a fluoresceina dagli acidi ipoalogenati nome. Quindi se l&#39;etichettatura delle cellule con anticorpi fluorescenza marcato viene eseguito in combinazione con APF colorazione, evitare di fluorofori che interferiscono con il segnale di emissione di fluoresceina. <ol><li> Eseguire APF colorazione a 4 ° C in ghiaccio. </li><li> Per la preparazione di una soluzione di lavoro di APF scongelare un&#39;aliquota appropriata del colorante (ad esempio, 10 ml, 11.81 mM) e diluire con HBSS esattamente 1 mM (ad esempio, aggiungere 108,1 tampone microlitri al 10 microlitri). <ol><li> Per ogni campione (500 ml soluzione di cellule) preparare 5 μl di soluzione di lavoro APF descritta. Di conseguenza, utilizzare uno 10 ml di un&#39;aliquota APF (11,81 mm), che produce 118,1 soluzione ul APF di lavoro (1 mm) per preparare circa 20 campioni. A causa della perdita durante il pipettaggio preparare circa il 10% in più di soluzione di lavoro APF quanto calcolato. </li></ol></li><li> Al fine di verificare la specificità perossidasi della colorazione APF, preparare campioni di controllo con l&#39;inibitore perossidasi eme idrazide dell&#39;acido 4-amminobenzoico (4-Abah) 35. <ol><li> Preparare una 1 M soluzione madre di 4-Abah (151.17 g / mol) in dimetilsolfossido (DMSO) diluendo 75,6 mg 4 Abah in 500 ml di solvente. Diluire ulteriormente 4 Abah 1/10 in HBSS. </li></ol></li><li> Per la preparazione di una soluzione un&#39;etichetta H 2 O 2 di lavoro due provette da 1,5 ml di centrifugazione con &quot;70 mM H 2 O 2&quot; e &quot;7 mM H 2 O 2&quot; rispettivamente. <ol><li> Nel tubo con l&#39;etichetta &quot;70 mm H 2 O 2&quot;, appena diluire 10 mldi una soluzione madre al 30% (8.8 mM) di H 2 O 2 con 990 microlitri di acqua distillata per ottenere una concentrazione di circa 88 mM. Conservare su ghiaccio ed utilizzare entro 4 ore. NOTA: H 2 O 2 è tipicamente fornito come una soluzione madre 30% dai fornitori. Se conservato a 4 ° C, è stabile per circa 3 anni. Eseguire diluizioni di H 2 O 2 in acqua distillata per evitare la decomposizione. H 2 O 2 diluizioni possono anche essere preparati in soluzione 0,1 M NaOH. </li><li> Per la determinazione della esatta H concentrazione 2 O 2 preparare una diluizione ulteriore 1 a 10 dalla prima H 2 O 2 soluzione stock (circa 88 mM) aggiungendo 100 microlitri di questa soluzione a 900 ml di acqua distillata nella provetta da 1,5 ml etichettata &quot;7 mm H 2 O 2&quot;. </li><li> Registrare uno spettro nel campo vicino UV (ad esempio, 200-300 nm) utilizzando un photospectrometer UV-Vis e utilizzare l&#39;assorbanza a 240 nm (49; 240 = 34 M -1 cm -1) per determinare il reale H 2 O 2 concentrazione nella seconda H 2 O 2 soluzione madre 34. Assicurarsi che la cuvetta di riferimento contiene solo acqua distillata. Per la misura cuvette uso quarzo come spettro nell&#39;intervallo UV viene registrato. </li><li> Da questo risultato, calcolare le concentrazioni esatte di entrambe le soluzioni madre H 2 O 2. Regolare la concentrazione della prima soluzione di riserva nel &quot;70 mM H 2 O 2&quot; tubo campione esattamente 70 mM aggiungendo una quantità adeguata di acqua distillata. Conservare la soluzione di lavoro ottenuta su ghiaccio e utilizzare entro un&#39;ora. </li></ol></li><li> Aggiungere 5 ml di soluzione di lavoro 4 Abah (100 mM) per campioni appropriati per una concentrazione finale di 1 mM. Incubare i campioni per 15 min a 37 ° C in incubatore prima APF aggiunta. </li><li> Aggiungere 5 microlitri soluzione APF lavoro (1 mM) per ciascun500 microlitri del campione per ottenere una concentrazione di colorante finale di 10 pM. Miscelare i campioni delicatamente (ad esempio, utilizzando il miscelatore a vortice in una posizione media) e incubare per 30 min a 37 ° C. </li><li> Aggiungere 5 microlitri H soluzione di lavoro 2 O 2 (70 mM) per ogni campione microlitri 500 per una concentrazione finale di 700 mM. Mescolare delicatamente i campioni e li incubare per 60 minuti a 37 ° C. Effettuare misure di controllo adeguate in parallelo. NOTA: misurazioni di controllo hanno mostrato che 700 mM H 2 O 2 non sono tossici per le cellule. Né tutte le risposte cellulari significative sono state osservate. NOTA: La colorazione APF può essere eseguita anche omettendo l&#39;aggiunta di H 2 O 2, a seconda della domanda scientifica. In assenza di perossido di idrogeno solo il MPO clorante basale e attività EPO è determinato mentre l&#39;aggiunta di H 2 O 2 permette la rilevazione della massima HOCl e HOBr produzione da parte delle cellule. Tegli quest&#39;ultimo si intende un segnale fluorescente significativamente più alto. </li><li> Per le cellule pellet, centrifugare i campioni per 10 min a temperatura ambiente e 400 x g. Rimuovere accuratamente il surnatante senza distruggere il pellet e aggiungere 250 microlitri HBSS per risospendere le cellule. NOTA: I campioni sono ora pronti per l&#39;analisi mediante citometria a flusso 25. </li><li> Conservare i campioni al buio fino al momento dell&#39;analisi per evitare lo sbiancamento. Dopo eccitazione a 480-490 nm rilevare l&#39;emissione di fluoresceina a circa 525 nm 37. </li></ol>

The authors have nothing to disclose.

Come neutrofili sono i leucociti più abbondanti nel sangue umano l&#39;isolamento delle cellule perossidasi-positive concentra spesso solo su queste cellule e comprende una separazione dei neutrofili di altri leucociti dal gradiente di densità centrifugazione 38. Eppure, come neutrofili sono molto meno abbondanti in campioni di sangue murini 39 per questi ultimi metodi più complessi debbano essere impiegati 40. Inoltre entrambi i metodi portano anche alla rimozione dei monociti peross...

Leucociti polimorfonucleati (PMN, chiamati anche granulociti e monociti) rappresentano importanti componenti cellulari del sistema immunitario innato nel 1,2 sangue. Essi contribuiscono alla difesa primaria contro patogeni nonché l&#39;attivazione del sistema immunitario acquisito e l&#39;avvio di una risposta infiammatoria sistemica 2-4. Eppure, soprattutto neutrofili, il tipo più abbondante di granulociti e monociti anche contribuiscono significativamente alla regolamentazione e alla cessazione di eventi infiammatori acuti 5. Pertanto queste cellule possono anche svolgere un ruolo importante nelle malattie infiammatorie croniche come l&#39;artrite reumatoide 6,7. Infatti, asma, una malattia infiammatoria delle vie aeree cronica, è caratterizzato da una ridotta apoptosi di eosinofili, il secondo tipo più granulociti nel sangue 8. Eppure l&#39;apoptosi dei granulociti e la loro rapida rimozione da parte dei macrofagi sono due passaggi essenziali durante la terminazione cellularedi infiammazione 9-11. Nelle cellule immunitarie denominate due enzimi strettamente correlati, vale a dire mieloperossidasi (MPO, neutrofili e monociti) e perossidasi eosinofila (EPO, eosinofili) possono essere trovati 12,13. Questi perossidasi eme sono classicamente legati alla risposta immunitaria umorale come si ossidano due elettronicamente (pseudo-) alogenuri alla ipo corrispondente (pseudo) acidi halous che sono noti per le loro proprietà battericide 14-16. In condizioni fisiologiche MPO principalmente forme acido ipocloroso (HOCl) e hypothiocyanite (- OSCN) mentre l&#39;acido quest&#39;ultima e ipobromoso (HOBr) sono formate da EPO 17-19. I nuovi risultati suggeriscono che questa (pseudo) l&#39;attività enzimatica alogenazione può anche contribuire alla regolazione delle risposte infiammatorie e alla cessazione di reazioni immunitarie 20,21. Infatti, la produzione HOCl da MPO e suoi derivati ​​hanno mostrato sopprimere T basati su celle risposte immunitarie adattative 22-24. Al fine di ottenere ulteriori delucidazioni sul ruolo immunologico dei leucociti dal sistema immunitario innato a malattie infiammatorie croniche e per determinare il contributo di MPO e EPO a questa funzione fisiologica abbiamo sviluppato un metodo per arricchire rapidamente leucociti da piccoli campioni di sangue per una successiva specifica determinazione dell&#39;attività alogenante perossidasi in queste cellule. Per deplezione degli eritrociti abbiamo scelto un metodo standardizzato compresi due successive fasi di lisi ipotonica con acqua distillata, che porta ad un arricchimento leucociti veloce a costi materiali bassi. Per la successiva determinazione del MPO alogenazione e l&#39;attività EPO la HOCl- e HOBr specifico colorante amminofenil fluoresceina (APF) è stato utilizzato 25-27. In contrasto con l&#39;applicazione di aspecifico perossidasi metodi di colorazione 28,29, questo approccio consente la determinazione selettiva dell&#39;attività alogenante perossidasi, che è spesso compromessa a grave inflinfiammazione 30,31.

<table border="0" cellpadding="0" cellspacing="0" width="738">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">materials/equipment</td>
			<td style="width:203px;"></td>
			<td style="width:120px;"></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">15 ml centrifugation tubes</td>
			<td style="width:203px;">VWR/Corning</td>
			<td style="width:120px;">734-0451</td>
			<td style="width:203px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">1.5 ml sample tubes</td>
			<td style="width:203px;">VWR/Eppendorf</td>
			<td style="width:120px;">211-2130DE</td>
			<td style="width:203px;">-</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:213px;">Pipettes for volumes up to 5 ml</td>
			<td style="width:203px;">Eppendorf</td>
			<td style="width:120px;">e.g. 3120000070</td>
			<td style="width:203px;">We are using Eppendorf Resarch plus pipettes with adjustable volumes in the range 1-10 &#181;l, 10-100&#181;l, 100-1000 &#181;l an 500-5000&#181;l</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">laminar flow bench</td>
			<td style="width:203px;">Thermo Electron Corperation</td>
			<td style="width:120px;">HeraSafe</td>
			<td style="width:203px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Vortex mixer</td>
			<td style="width:203px;">Bender &#38; Hobein AG</td>
			<td style="width:120px;">Vortex Genie 2</td>
			<td style="width:203px;">-</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Tabletop centrifuge</td>
			<td style="width:203px;">Kendro Laboratory Products</td>
			<td style="width:120px;">Laborfuge 400R</td>
			<td style="width:203px;">The centrifuge should be able to be used at 450x g</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Small centrifuge</td>
			<td style="width:203px;">eppendorf</td>
			<td style="width:120px;">5415D</td>
			<td style="width:203px;">The centrifuge should be able to be used at 400x g</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Incubator</td>
			<td style="width:203px;">Heraeus</td>
			<td style="width:120px;">cytoperm 2</td>
			<td style="width:203px;">Settings: 37 &#176;C, 95% humidity, 5 % CO2 content</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">UV-Vis spectrophotometer</td>
			<td style="width:203px;">Varian</td>
			<td style="width:120px;">Cary 50 bio</td>
			<td style="width:203px;">A spectrum between 200 and 300 nm has to be recorded. Thus quartz cuvettes have to be applied</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;">Flow cytometer</td>
			<td style="width:203px;">Becton, Dickinson</td>
			<td style="width:120px;">BD Facs Calibur</td>
			<td style="width:203px;">Any flow cytometer can be used which is equiped with a laser suitable for the excitation of fluorescein (e.g 488 nm argon laser)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td style="width:203px;">Company</td>
			<td style="width:120px;">Catalog Number</td>
			<td style="width:203px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Chemicals</td>
			<td style="width:203px;"></td>
			<td style="width:120px;"></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Phosphate buffered saline (PBS)</td>
			<td style="width:203px;">amresco</td>
			<td style="width:120px;">K812</td>
			<td style="width:203px;">sterile solution, ready to use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;"></td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">P4417</td>
			<td style="width:203px;">tablets for solving in 200 ml millipore water</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:213px;">Hanks balanced salt solution (HBSS) with Ca(2+)</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">H1387</td>
			<td style="width:203px;">970 mg/100 ml, carefully check and adjust the pH value to 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Hydrochloric acid</td>
			<td style="width:203px;">Merck Millipore</td>
			<td style="width:120px;">1.09057.1000</td>
			<td style="width:203px;">1M solution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:213px;">Sodium hydroxide</td>
			<td style="width:203px;">Riedel-deHa&#235;n</td>
			<td style="width:120px;">30620</td>
			<td style="width:203px;">Solid pellets. For a 1M solution solve 4 g/100 ml Millipore water</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:213px;">Aminophenyl fluorescein</td>
			<td style="width:203px;">Cayman</td>
			<td style="width:120px;">10157</td>
			<td style="width:203px;">5 mg/ml solution (11.81 mM) in methyl acetate, aliquotes of e.g. 100 &#181;l should be prepared and stored at -20 &#176;C</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:213px;">Hydrogen peroxide</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">H1009</td>
			<td style="width:203px;">This 30% stock solution corresponds to a concentration of about 8.8 M. Further dilutions have to be freshly prepared in distilled water immediately prior to use and quantified by absorbance measurements</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:213px;">4-aminobenzoic acid hydrazide (4-ABAH)</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">A41909</td>
			<td style="width:203px;">A first stock solution of 1 M should be prepared in DMSO a second one of 100 mM by 1:10 dilution in HBSS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">DMSO</td>
			<td style="width:203px;">VWR chemicals</td>
			<td style="width:120px;">23500.26</td>
			<td style="width:203px;">-</td>
		</tr>
	</tbody>
</table>

fast and specific assessment of the halogenating peroxidase activity in leukocyte-enriched blood samples

In questo lavoro è descritto un protocollo per l&#39;arricchimento rapido e standardizzata dei leucociti da piccoli campioni di sangue intero. Questa procedura si basa sulla lisi ipotonica degli eritrociti e può essere applicato a campioni umani nonché al sangue di origine non umana. Il piccolo volume del campione iniziale di circa 50 a 100 ml rende questo metodo applicabile a prelievo di sangue ricorrenti da piccoli animali da laboratorio. Inoltre, l&#39;arricchimento dei leucociti è raggiunto in pochi minuti e con gli sforzi materiali bassi per quanto riguarda i prodotti chimici e strumentazione, rendendo questo metodo applicabile in molteplici ambienti di laboratorio. Depurazione standardizzata dei leucociti è combinato con un metodo altamente selettiva colorazione per valutare l&#39;attività perossidasica alogenante delle perossidasi eme, mieloperossidasi (MPO) e perossidasi eosinofila (EPO), cioè la formazione di ipocloroso e ipobromoso (HOCl e HOBr). Mentre MPO è fortemente espresso in neutrophils, il tipo di cellule immunitarie più abbondante nel sangue umano come in monociti, il relativo enzima EPO è espresso esclusivamente in eosinofili. L&#39;attività alogenante di questi enzimi sono destinatari utilizzando la quasi HOCl- e HOBr-specifica fluoresceina dye amminofenil (APF) e il perossido di idrogeno substrato perossidasi primaria. Su successiva analisi di citometria di flusso tutte le cellule perossidasi-positive (neutrofili, monociti, eosinofili) sono distinguibili e la loro attività perossidasi alogenazione possono essere quantificati. Poiché APF colorazione può essere combinato con l&#39;applicazione di marcatori di superficie cellulare, questo protocollo può essere esteso ad affrontare specificamente leucociti sotto-frazioni. Il metodo è applicabile per rilevare HOCl e HOBr produzione sia nell&#39;uomo e nei leucociti roditori. Dato il ruolo immunologico ampiamente e diversamente discusso di questi prodotti enzimatici in malattie infiammatorie croniche, questo protocollo può contribuire ad una migliore comprensione dellarilevanza immunologica di perossidasi eme leucociti-derivati.

Come riportato in precedenza il metodo sopra descritto si è rivelato essere applicabile sia ai umana e materiale non-umano 32. Inoltre come mostrato per topi con sintomi asmatici colorazione APF può essere uno strumento atto a rilevare differenze di situazione pro-infiammatoria sistemica. Pertanto, in uno studio successivo abbiamo usato questo protocollo per valutare più volte l&#39;attività alogenazione di MPO (e EPO) in femmine ratti scuro Agouti con artrite pri...

Watch this Scientific Journal Video about Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples at JoVE.com

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

La valutazione rapida e specifica del alogenazione perossidasi attività in campioni di sangue leucociti arricchita

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

fast-specific-assessment-halogenating-peroxidase-activity-leukocyte

Research

JoVE Journal

Immunology and Infection

10.2K Views.  University of Leipzig. This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

Video: Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

L&#39;esaurimento delle popolazioni di cellule specifiche da deplezione del complemento

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

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Immunologia e infezioni

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Purificazione della popolazione di cellule specifiche per fluorescenza separazione delle cellule attivate (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

L&#39;introduzione di shear stress nello studio di adesione batterica

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

Determinazione dell&#39;attività fagocitaria dei Campioni anticorpi clinici

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

Imaging quantitativa di Lineage specifici Toll-like receptor-mediate Signaling in monociti e cellule dendritiche da piccoli campioni di sangue umano

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Generazione di Human alloantigene-specifiche cellule T da sangue periferico

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Flusso analisi di citometria di Particle-bound Bet v 1 allergene in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

Flusso analisi di citometria di Natural Killer cellulare Lytic attività nel Sangue umano intero

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Destrano migliora l'efficienza di trasduzione lentivirale di cellule NK primarie Murine ed umane

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

Metodo dell&#39;agitatore magnetico per la rilevazione di<em&gt; Trichinella</em&gt; Le larve in campioni di muscolo

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...