Questo lavoro è stato sostenuto dal Australian Health and Medical Research Council Nazionale, Star Scientific Foundation (Australia), sindrome di Tourette Association (USA), la sclerosi multipla Trish Fondazione per la Ricerca e la sclerosi multipla Research Australia, Petre Foundation (Australia), la Rebecca L. Cooper Medical Research Foundation (Australia). Ringraziamo tutti i pazienti ei familiari che hanno fornito i campioni per il nostro studio.

<html> 1. Subclonaggio Strategia Per costruire pIRES2-EGFP Vector Encoding D2R o NMDAR <ol><li> Ottenere full-length cDNA clone di D2R umana o NMDAR subunità 1 (NR1). </li><li> Scegliere un vettore di espressione, come pIRES2-EGFP, che è adatto per l&#39;espressione di proteine ​​transmembrana con una proteina migliorata verde fluorescente (GFP) reporter sotto il controllo di un sito di entrata del ribosoma interno (IRES), permettendo sia antigeni e GFP essere coespressi in cellule separatamente. </li><li> Subclone cDNA umano all&#39;interno pIRES2-EGFP vettoriale. <ol><li> Per subclonare cDNA, utilizzare appropriati enzimi di restrizione (ad esempio NheI e XhoI per l&#39;uomo D2R cDNA). </li><li> Inserire legare taglio cDNA entro ristretto pIRES2-EGFP vettoriale. </li><li> Sequenza ligato pIRES2-EGFP D2R o NMDAR vettore per verificare se il vettore contiene la sequenza corretta. </li><li> Purificare e amplificare pIRES2-EGFP D2R o NMDAR vettore per un uso successivo in trasfezione. </li></ol></li></ol>DENOMINAZIONE &quot;&gt; 2. HEK293 Trasfezione cellulare Manifestazione d&#39;neuronale Antigen <ol><li> Cellule Cultura HEK293 in tessuto fiasche di coltura con il fresco di Dulbecco Modified Eagle Medium (1x) (DMEM) completa di 4,5 g / L D-glucosio, L-glutammina e 110 mg / L piruvato di sodio supplementato con 10% di siero fetale bovino (FBS), 2 GlutaMAX mM e 50 pg / ml Gentamicina. </li><li> Staccare cellule HEK293 con tripsina e trasferimento in un tubo conico. <ol><li> Lavare le cellule per centrifugazione a 250 xg per 6 min e risospendere il pellet di cellule in DMEM fresco completo. </li></ol></li><li> Contare le celle e sementi 6 pozzetti a circa 8 x 10 5 cellule / pozzetto e cultura pernottamento in DMEM 2 ml a 37 ° C e 5% di CO 2 in un incubatore. </li><li> Una volta che hanno raggiunto circa il 70% di confluenza, trasfezione le cellule HEK293 con pIRES2-EGFP NR1 (+ cellule HEK293 NMDAR), pIRES2-EGFP D2R (HEK293 D2R + cellule), e (cellule HEK293 CTL; controllo vettoriale) pIRES2-EGFP come segue.Mantenere alcune cellule HEK293 untransfected di risarcimento in seguito. <ol><li> Preparare il volume richiesto di miscela di trasfezione comprendente 2,5 ug di DNA, 200 microlitri 0,9% cloruro di sodio e 4 mg / ml polyethylenimine / pozzetto (ciascuna contenente 2 ml di DMEM fresco completo). </li><li> Vortice immediatamente la miscela per 10 sec e incubare a temperatura ambiente (RT) per 10 minuti, prima di aggiungere volume di miscela di trasfezione ai pozzetti appropriati. </li><li> Coprire la piastra, avvolgere i lati con Parafilm e centrifugare a 280 xg per 5 minuti per aiutare trasfezione cellulare. </li><li> Rimuovere il Parafilm e poi posto in incubatrice alla cultura a 37 ° C. </li><li> Sostituire la cultura dei media 18 ore più tardi con DMEM fresco completo, e mantenere in coltura per un altro 72 hr. </li><li> Facoltativo: 72 ore dopo la trasfezione, le cellule transfettate possono essere selezionati mediante coltura in DMEM completo supplementato con 250 ug / ml Geneticin, al fine di ottenere un policlonale trasfettante stabile. </li></ol></li></ol><pclass = &quot;jove_title&quot;&gt; 3. Citometria a flusso test cellulare per il rilevamento di anticorpi alla superficie antigeni neuronali <ol><li> Staccare HEK293 NMDAR +, HEK293 D2R +, e cellule HEK293 CTL mediante incubazione con 500 microlitri / pozzetto Versene per circa 5 min a 37 ° C. <ol><li> Risospendere in 2ml/well di PBS (-Ca 2 + /-Mg 2 +) integrato con il 2% FBS (PBS / FBS) e trasferimento a 15 ml o 50 ml tubo conico. </li><li> Lavare le cellule per centrifugazione a 250 xg per 6 min e risospendere il pellet di cellule in 15-50 ml di PBS / FBS. Ripetere il lavaggio 2x. </li><li> Contare le cellule e poi risospendere in PBS / FBS a 1 x 10 6 cellule / ml. </li></ol></li><li> Progettare il modello a 96 pozzetti per l&#39;acquisizione citometria a flusso, visto che + cellule HEK293 D2R + o HEK293 NMDAR, così come le cellule HEK293 CTL dovranno essere incubate con ciascun campione clinico o anticorpo primario. </li><li> Cellule seme in una piastra a 96 pozzetti V-bottom a 50.000 cellule / noill secondo la citometria a flusso template di acquisizione. </li><li> Agglomerare le cellule per centrifugazione della piastra a 96 pozzetti a 450 xg per 5 min e rimuovere delicatamente il supernatante utilizzando una pipetta multicanale elettronica o manuale, inclinando la piastra su un angolo e facendo attenzione a non aspirare il pellet di cellule. </li><li> Incubare HEK293 D2R +, HEK293 NMDAR +, e le cellule HEK293 CTL per 1 ora a temperatura ambiente al buio con: <ol><li> Diluizioni seriali di anticorpo primario al fine di valutare l&#39;espressione dell&#39;antigene di superficie. </li><li> I campioni dei pazienti al fine di rilevare gli anticorpi. </li></ol></li><li> Usare adeguato flusso citometria a controlli di compensazione, ad esempio. cellule HEK293 untransfected non colorate, GFP + cellule HEK293 (AF647 -), e untransfected AF647 + HEK293 cellule (GFP). </li><li> Lavare le cellule per centrifugazione a 450 xg per 5 minuti e risospendere il pellet cellulare in 170 microlitri di PBS / FBS. Ripetere la fase di lavaggio altre due volte. </li><li> Incubare le cellule per 1 hr alla RT al buio con diluizione 1:100 di AF647 coniugato anticorpo secondario (anti-IgG umane per il siero del paziente e anti-topo IgG per anticorpi primari anti-NR1 o anti-D2R). </li><li> Lavare le cellule tre volte, come al punto 3.7, e risospendere in 35 microlitri di PBS / FBS per citometria a flusso acquisizione cellulare. </li><li> Impostare il campionatore ad alto rendimento (HTS) sul citofluorimetro (BDLSRII). </li><li> Acquisire 10.000 eventi / bene secondo la citometria a flusso template di acquisizione. </li><li> Export e poi analizzare i dati per l&#39;analisi utilizzando un citometria a flusso pacchetto software, come ad esempio FlowJo v7.5: <ol><li> In primo luogo, porta le cellule HEK293 viventi basata su avanti (dimensione) e laterale (granularità) disperde. </li><li> Ulteriori cancello cellule vive HEK293 basato su alta GFP-positività per analizzare solo le cellule trasfettate. </li><li> Determinare l&#39;intensità di fluorescenza media (MFI) del canale AF647 all&#39;interno delle cellule GFP-positive vivi. </li><li> Esportare dati in Excel o Prism per l&#39;analisi: <ol><li> Plot cconcentrazioni del di anticorpo primario (anti-NMDAR o anticorpo anti-D2R) contro l&#39;MFI ottenuti dalle cellule incubate con questi anticorpi. </li><li> Per ciascun campione e controllo, determinare il ΔMFI sottraendo il MFI delle cellule HEK293 CTL dal MFI del HEK293 D2R + o + NMDAR cellule HEK293, rispettivamente. </li><li> Calcolare la soglia di positività anticorpale con l&#39;aggiunta di tre deviazioni standard sopra la ΔMFI media della coorte di controllo. </li><li> Singoli campioni Plot raggruppati in base al loro tipo di siero su un grafico e rappresentano la soglia come una linea. </li></ol></li></ol></li></ol>

<ol>
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Conflitto di interessi: Un brevetto è stato depositato da FB e RCD (Università di Sydney), sostenendo D2R come obiettivo per autoanticorpi.

Questo articolo descrive una nuova applicazione di citometria a flusso saggio basato su cellule in tempo reale per individuare gli anticorpi specifici mirati proteine ​​neuronali superficie cellulare utilizzando un campionatore high-throughput. Utilizzando questa tecnica, si segnala che un sottogruppo di pazienti affetti da malattie autoimmuni del sistema nervoso centrale hanno anticorpi sierici di superficie NMDAR o alla superficie D2R. Passi essenziali per la rilevazione ottimale degli...

Negli ultimi anni, sono state individuate forme autoimmuni del sistema nervoso centrale (SNC) malattie. E &#39;stato dimostrato che queste malattie sono associate e definiti dalla presenza di autoanticorpi. Questi anticorpi si legano ai recettori neuronali o proteine ​​sinaptiche coinvolte nella neurotrasmissione 1,2. Sono stati rilevati diversi antigeni, come recettore N-metil-D-aspartato (NMDAR) 3-5, recettore γ-amminobutirrico B (GABA B) recettore 6, α-ammino-3-idrossi-5-metil-4- L&#39;acido isoxazolepropionic (AMPA) recettore 7, canale del potassio voltaggio-dipendenti (anti-VGKC) proteine ​​associate: leucina-ricchi glioma attivato 1 proteina (LGL-1) e contattonel-proteina associata 2 (capsr2) 8,9, glutammato recettore 5,10 , e la dopamina-2 recettore (D2R) 11. In passato, questi disturbi autoimmuni del sistema nervoso centrale (principalmente chiamati encefalite) erano spesso non diagnosticata e non trattata. Questi biomarcatori anticorpi romanzo, ad esempio,NMDAR anticorpo o anticorpi D2R, hanno sostanzialmente migliorato la diagnosi e la consapevolezza, e hanno aperto le opzioni di trattamento per i pazienti. Infatti, il trattamento precoce con immunoterapie è associata con una migliore esito 11,12. I metodi tradizionali per rilevare gli anticorpi, come immunoenzimatico assay (ELISA) e Western Blot sono stati utilizzati per la rilevazione di anticorpi nel siero. Tuttavia, essi non consentono facilmente riconoscimento di epitopi extracellulari o di superficie e, invece, possono rivelare immunoreattività verso un epitopo intracellulare. Inoltre, gli anticorpi che si legano al dominio extracellulare di proteine ​​importanti coinvolti nella neurotrasmissione sono suscettibili di essere 2,3,7,13,14 patogeni. Pertanto, lo sviluppo di analisi accurate e sensibili per scoprire superficie cellulare pertinente o bersagli degli anticorpi extracellulare nei pazienti è fondamentale. Il metodo gold standard nel campo si basa sull&#39;utilizzo di cellule vive, nelle cosiddette &quot;cell-basaggi sed &quot;. Questo metodo comporta l&#39;espressione di un antigene sulla superficie di cellule di mammifero (molto spesso renali (293) HEK293 cellule embrionali umane) nella sua forma nativa mediante trasfezione di vettori contenenti la sequenza di cDNA completa l&#39;antigene di interesse. Cellule nonpermeabilized live vengono poi incubate con il siero del paziente diluito e seguiti da fluorocromo coniugato immunoglobulina anti-umana (Ig) anticorpo secondario. Viene quindi rilevata l&#39;intensità o livello di fluorescenza ed è associato al livello di legame autoanticorpi. Questa tecnica è specifico, come un unico antigene è iperespresso nelle cellule. Il ampiamente usato &quot;read-out&quot; più è stata l&#39;analisi di microscopia confocale dopo immunocitochimica 4,5,8-10. Tuttavia, citometria a flusso saggi cellulari sono stati utilizzati con successo per rilevare gli anticorpi nei pazienti con malattie demielinizzanti 15-17. In particolare, Waters et al. 15 rispetto individuazione di anticorpi utilizzando una vaschettael di tecniche di rilevazione degli anticorpi, tra cui saggi cellulari seguite da microscopio o flusso analisi di citometria, e ha mostrato la citometria a flusso saggio basato su cellule ad essere il metodo più sensibile, accurato e affidabile. Pertanto, citofluorimetria saggio basato su cellule è vantaggioso in quanto è quantitativa, non investigatore-dipendente, e non comporta alcun uso di materiale radioattivo. E &#39;anche conveniente in quanto consente grandi coorti di pazienti da analizzare in un breve lasso di tempo. Più di recente, abbiamo ottimizzato un flusso di high-throughput citometria saggio basato su cellule di rilevare NMDAR anticorpi e anticorpi D2R nei pazienti con malattie autoimmuni del sistema nervoso centrale 11. Il nostro gruppo ha recentemente rilevato anticorpi NMDAR nel siero del paziente usando la citometria a flusso saggio basato su cellule. Questi sieri anticorpo-positivi NMDAR sono stati precedentemente analizzati usando la microscopia confocale e sono stati anche trovato positivo 11. Questo protocollo delinea una citofluorimetria saggio basato su cellule per la rilevazione di conformation sensibile anticorpi CNS nel siero del paziente utilizzando un campionatore ad alto rendimento automatizzato.

<table border="0" cellpadding="0" cellspacing="0" width="751">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">pIRES2-EGFP vector</td>
			<td style="width:187px;">Clontech</td>
			<td style="width:136px;">6029-1</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Human HA-Dopamine-2 receptor (D2R) cDNA</td>
			<td style="width:187px;">Missouri S&#38;T cDNA Resource Centre</td>
			<td style="width:136px;">DRD020TN00</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Human subunit 1 of NMDAR (NR1) cDNA</td>
			<td style="width:187px;"></td>
			<td style="width:136px;"></td>
			<td style="width:173px;">Gift from Prof A. Vincent&#160; (Oxford, UK)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Monoclonal purified mouse anti-human NR1 antibody (clone: 54.1)</td>
			<td style="width:187px;">BD Pharmingen</td>
			<td style="width:136px;">556308</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Mouse purified monoclonal anti-DRD2 IgG (clone: 1B11)</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:136px;">WH0001813M1-50UG</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Alexa Fluor 647 donkey anti-mouse IgG (H + L)</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">A31571</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Alexa Fluor 647 goat anti-human IgG (H + L)</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">A21445</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:255px;">Dulbecco&#8217;s Modified Eagle Medium (1x) 4.5 g/L D-glucose, L-Glutamine and 110 mg/L sodium pyruvate</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">11995-065</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Foetal bovine serum</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">10099-141</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Gentamicin</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">15710-072</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">GlutaMAX</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">35050-061</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Penicillin-streptomycin</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">15140-122</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Geneticin</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">10131-035</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:255px;">Dulbecco&#39;s Phosphate-Buffered Saline (1x) (-Ca2+/-Mg2+)</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">14190-144</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">TrypLE Express (1x) with Phenol Red</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">12605-028</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">0.9% Sodium chloride</td>
			<td style="width:187px;">Baxter</td>
			<td style="width:136px;">AHF7975</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Polyethylenimine</td>
			<td style="width:187px;">Polysciences Inc</td>
			<td style="width:136px;">9002-98-6</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Versene</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">15040-066</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">XhoI</td>
			<td style="width:187px;">Roche</td>
			<td style="width:136px;">10899194001</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">NheI</td>
			<td style="width:187px;">Roche</td>
			<td style="width:136px;">10885843001</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PureLink PCR Purification Kit</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">K3100-01</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">JetQuick Gel Extraction Spin Kit</td>
			<td style="width:187px;">Genomed</td>
			<td style="width:136px;">420050</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">T4 DNA Ligase</td>
			<td style="width:187px;">Roche</td>
			<td style="width:136px;">10481220001</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">Plasmid&#160;Plus&#160;Maxi Kit</td>
			<td style="width:187px;">Qiagen</td>
			<td style="width:136px;">12963</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;"></td>
			<td style="width:187px;"></td>
			<td style="width:136px;"></td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">Name of Equipment/Software</td>
			<td style="width:187px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td style="width:173px;">Model/Version</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Flow cytometer with high-throughput sampler system</td>
			<td style="width:187px;">BD Biosciences</td>
			<td style="width:136px;"></td>
			<td style="width:173px;">BDLSRII with HTS</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:255px;">Inverted microscope</td>
			<td style="width:187px;">Olympus</td>
			<td style="width:136px;"></td>
			<td style="width:173px;">CKX41 (with mercury lamp and U-RFLT50 power supply)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Electronic multichannel pipette (15-300 &#956;l)</td>
			<td style="width:187px;">Eppendorf</td>
			<td style="width:136px;">613-2240P</td>
			<td style="width:173px;">12-channel Xplorer Plus</td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:255px;">Excel</td>
			<td style="width:187px;">Microsoft</td>
			<td style="width:136px;"></td>
			<td style="width:173px;">2010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Prism</td>
			<td style="width:187px;">GraphPad Software, Inc.</td>
			<td style="width:136px;"></td>
			<td style="width:173px;">v4</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:255px;">FlowJo</td>
			<td style="width:187px;">Treestar</td>
			<td style="width:136px;"></td>
			<td style="width:173px;">v7.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">50 ml polypropylene conical tubes</td>
			<td style="width:187px;">BD Biosciences</td>
			<td style="width:136px;">352070</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">6-well plate</td>
			<td style="width:187px;">BD Biosciences</td>
			<td style="width:136px;">353046</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tissue culture flask (T75)</td>
			<td style="width:187px;">BD Biosciences</td>
			<td style="width:136px;">353136</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Parafilm</td>
			<td style="width:187px;">Pechiney Plastic Packaging</td>
			<td style="width:136px;">PM-992</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">V-bottom 96-well plate</td>
			<td style="width:187px;">Corning</td>
			<td style="width:136px;">651180</td>
			<td style="width:173px;"></td>
		</tr>
	</tbody>
</table>

high-throughput flow cytometry cell-based assay to detect antibodies to n-methyl-d-aspartate receptor or dopamine-2 receptor in human serum

Nel corso degli ultimi anni, gli anticorpi contro le proteine ​​di superficie e conformazionali coinvolti nella neurotrasmissione sono state rilevate nelle malattie autoimmuni del sistema nervoso centrale nei bambini e negli adulti. Questi anticorpi sono stati utilizzati per guidare la diagnosi e il trattamento. Saggi cellulari hanno migliorato il rilevamento di anticorpi nel siero del paziente. Essi si basano sulla superficie espressione di antigeni cerebrali sulle cellule eucariotiche, che vengono poi incubate con sieri diluiti paziente seguito da anticorpi secondari coniugati con fluorocromi. Dopo il lavaggio, il legame anticorpo secondario viene quindi analizzata mediante citometria a flusso. Il nostro gruppo ha sviluppato un flusso di high-throughput citometria saggio basato su cellule-live per rilevare in modo affidabile gli anticorpi contro recettori dei neurotrasmettitori specifici. Questo metodo di citometria a flusso è dritto in avanti, quantitativa, efficiente, e l&#39;uso di un sistema di campionamento ad alta velocità permette di grandi coorti di pazienti di essere facilmente analizzati in un breve lasso di tempo. Inoltre, questa cella basata comedire che può essere facilmente adattato per rilevare gli anticorpi a molti diversi target antigenici, sia dal sistema nervoso centrale e la periferia. Alla scoperta di biomarcatori supplementari romanzo di anticorpi consentirà una diagnosi tempestiva e accurata e migliorare il trattamento dei disturbi immuno-mediate.

Cellule vive HEK293 D2R + e HEK293 CTL sono stati acquisiti al citofluorimetro utilizzando un campionatore high-throughput. Durante l&#39;analisi, le cellule sono state gated basato su forward scatter (dimensione) e parametri (granularità) (Figure 1A e 1D) side scatter. Cellule HEK293 trasfettate espressi molecola reporter, GFP, nel citoplasma, e le cellule non trasfettate sono stati esclusi dall&#39;analisi (Figure 1B e 1E). All&...

Watch this Scientific Journal Video about High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum at JoVE.com

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Nel corso degli ultimi anni, saggi cellulari vivi sono stati utilizzati con successo per rilevare gli anticorpi contro gli antigeni di superficie e conformazionale. Qui, descriviamo un metodo con elevate throughput citometria consentire l&#39;analisi di grandi coorti di pazienti. Individuazione di anticorpi romanzo migliorare la diagnosi e il trattamento di disturbi immuno-mediata.

High-throughput Citometria a Flusso test cellulare per individuare gli anticorpi per N-metil-D-aspartato Receptor o dopamina-2 recettore nel siero umano

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

high-throughput-flow-cytometry-cell-based-assay-to-detect-antibodies

Research

JoVE Journal

Medicine

16.0K Views.  The University of Sydney.  Nel corso degli ultimi anni, saggi cellulari vivi sono stati utilizzati con successo per rilevare gli anticorpi contro gli antigeni di superficie e conformazionale. Qui, descriviamo un metodo con elevate throughput citometria consentire l'analisi di grandi coorti di pazienti. Individuazione di anticorpi romanzo migliorare la diagnosi e il trattamento di disturbi immuno-mediata. 

Video: High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

La misurazione e trattamento di soppressione in Ambliopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Ricerca

Medicina

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

Stereologica e Citometria a Flusso Caratterizzazione dei leucociti sottopopolazioni in modelli di transitori o permanenti Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

Adattamento umani videofluoroscopia Rondine metodi di studio per individuare e caratterizzare disfagia in modelli di malattia Murine

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective mining of the information in the large-scale datasets that are obtained from gene expression microarrays, deep sequencing experiments or metabolic profiling is essential to uncover and then effectively target the critical regulators of diseased cell phenotypes. Estrogen Receptor &#945; (ER&#945;) is one of the master transcription factors regulating the gene programs that are important for estrogen responsive breast cancers. In order to understand to role of ER&#945; signaling in breast cancer metabolism we utilized transcriptomic, cistromic and metabolomic data from MCF-7 cells treated with estradiol. In this report we described generation of samples for RNA-Seq, ChIP-Seq and metabolomics experiments and the integrative computational analysis of the obtained data. This approach is useful in delineating novel molecular mechanisms and gene regulatory circuits that are regulated by a particular transcription factor which impacts metabolism of normal or diseased cells.

Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol.

Systems Biology di regolazione metabolica da Estrogen Receptor Signaling nel cancro al seno

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Often&#160;potential new biomarkers do not make it&#160;into the clinic&#160;due to limitations&#160;in their development and&#160;high costs.&#160;However,&#160;targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkers&#160;for clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small molecules&#160;in clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4).
In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation.
The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibrate&#160;the MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.

We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.

A High Throughput, Multiplexer e mirata Proteomica CSF test per quantificare neurodegenerative Biomarkers e apolipoproteina E Isoforme Stato

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be ...

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include well-validated targets for therapeutic intervention. Discovery of lead compounds, including small synthetic molecules, that specifically inhibit the receptor&#39;s function, is an important initial step in drug development and relies on sensitive, specific, and robust cell-based assays. Here, we describe a kinetic cellular assay with a fluorescent readout primarily designed to identify receptor-specific antagonists that inhibit the intracellular Ca2+ release evoked upon the activation of the CXC chemokine receptor 4 (CXCR4) by its endogenous ligand, the CXC chemokine ligand 12 (CXCL12). A key advantage of this method is that it also enables screening of compounds endowed with intrinsic agonistic properties (i.e., compounds eliciting an increase in intracellular Ca2+ concentration in the absence of CXCL12) or compounds modulating the receptor&#39;s function via interaction with allosteric binding sites (i.e., positive and negative allosteric modulators (PAMs and NAMs, respectively)). On the down side, autofluorescent compounds might interfere with the assay&#39;s readout, thereby hampering reliable data interpretation. Most likely this assay can be implemented, with minimal adaptations, as a generic drug discovery assay for many other GPCRs of which the activation leads to a release of intracellular Ca2+.

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

The described cellular assay is designed for the identification of CXC chemokine receptor 4 (CXCR4)-interacting agents that inhibit or stimulate, either competitively or allosterically, the intracellular Ca2+ release initiated by CXCR4 activation.

Un'analisi cinetica basata sulla fluorescenza Ca2 + mobilizzazione per identificare il G proteina-ha accoppiato il ricevitore agonisti, antagonisti e modulatori allosterici

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Caratterizzazione di cellule immunitarie nel tessuto adiposo umano mediante citometria a flusso

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment elevated myocardial infarction. However, reperfusion per se can result in further cardiomyocyte death, a phenomenon known as reperfusion injury. The opening of the mitochondrial permeability transition pore (mPTP), with the decrease of the mitochondrial membrane potential (MMP), or mitochondrial depolarization, is universally recognized as the final step of reperfusion injury and is responsible for mitochondrial and cardiomyocyte death. JC-1 is a lipophilic cationic dye that accumulates in mitochondria depending on the value of MMP. The higher the MMP is, the more JC-1 accumulates in the mitochondria. The increasing amounts of JC-1 in mitochondria can be reflected by a fluorescence emission shift from green (~530 nm) to red (~590 nm). Therefore, the reduction of the red/green fluorescence intensity ratio can indicate the depolarization of mitochondria. Here, we take advantage of JC-1 to measure MMP, or the opening of mPTP in human cardiac myocytes after hypoxia/reoxygenation, detected by flow cytometry.

Here, we present a protocol that uses JC-1 dye to assess the mitochondrial membrane potential of cells after being exposed to hypoxia/reoxygenation with or without a protective agent.

Un'analisi basata su citometria a flusso per misurare il potenziale di membrana mitocondriale in miociti cardiaci dopo ipossia/riossigenazione

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Analisi di efflusso di High-throughput Nitrobenzoxadiazole-labeled colesterolo

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in ...

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E (sIgE)

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. However, a specific immunoglobulin E (IgE) measurement system with a high price-performance ratio is lacking in mainland China, especially in the primary care hospitals. This paper describes the principle and operation procedures of using a microfluidic cartridge-based chemiluminescence system to detect allergen-specific IgE in serum. The results were compared with those from ImmunoCAP (System 1), the industrial standard, and the reproducibility of the system to detect patients sensitized to common allergens is evaluated. The results showed that in comparison with ImmunoCAP (System 1), the BioIC System (System 2) has good precision and sensitivity in detecting serum-specific IgE against various inhalant and food allergens but with a significantly lower cost. It can serve as a good alternative to System 1 in primary care hospitals in mainland China who have lower financial affordability.

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E sIgE

This paper presents a protocol for detecting serum-specific immunoglobulin E with a microfluidic cartridge-based chemiluminescence system and the evaluation of its usefulness in allergy diagnoses.

Applicazione della tecnologia microfluidica Biochip per rilevare siero allergene-specifiche dell'immunoglobulina E (sIgE)

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. ...