Este trabajo fue apoyado por el Health and Medical Consejo Australiano de Investigaciones Científicas, la Fundación Científica Star (Australia), el síndrome de Tourette Asociación (EE.UU.), la esclerosis múltiple Trish Fundación para la Investigación y la Investigación de Esclerosis Múltiple Australia, Fundación Petre (Australia), el Rebecca L. Cooper Medical Research Foundation (Australia). Agradecemos a todos los pacientes y familiares que proporcionaron muestras para nuestro estudio.

<html> 1. Subclonación Estrategia para Construir pIRES2-EGFP vector que codifica D2R o NMDAR <ol><li> Obtener ADNc de longitud completa clon de D2R humano o de NMDAR subunidad 1 (NR1). </li><li> Elija un vector de expresión, tal como pIRES2-EGFP, que es adecuado para la expresión de proteínas de transmembrana con una proteína verde fluorescente (GFP) indicador bajo el control de un sitio de entrada interna del ribosoma (IRES), permite que tanto los antígenos y GFP para ser coexpressed en células por separado. </li><li> Subclónese ADNc humano dentro de pIRES2-EGFP del vector. <ol><li> Para subclonar ADNc, utilizar enzimas de restricción apropiadas (por ejemplo NheI y XhoI para D2R ADNc humano). </li><li> CDNA corte Ligar insertar dentro restringido vector pIRES2-EGFP. </li><li> Secuencia ligada pIRES2-EGFP D2R o NMDAR vector para comprobar si el vector contiene la secuencia correcta. </li><li> Purifica y amplificar D2R pIRES2-EGFP o NMDAR vector para su posterior uso en la transfección. </li></ol></li></ol>í tulo &quot;&gt; 2. HEK293 La transfección de células para la expresión de antígeno neuronal <ol><li> Células de cultivo HEK293 en matraces de cultivo de tejidos con medio Eagle modificado fresco de Dulbecco (1x) (DMEM) completo con 4,5 g / l de D-glucosa, L-glutamina y 110 mg / piruvato de sodio L suplementado con suero bovino fetal al 10% (FBS), 2 mM de GlutaMAX, y 50 mg / ml de gentamicina. </li><li> Separar las células HEK293 usando tripsina y la transferencia a un tubo cónico. <ol><li> Lavar las células por centrifugación a 250 xg durante 6 min y resuspender el sedimento de células en DMEM completo fresco. </li></ol></li><li> Contar las células y placas de semillas 6 pocillos a aproximadamente 8 x 10 5 células / pocillo y la cultura durante la noche en DMEM 2 ml a 37 ° C y 5% de CO 2 en una incubadora. </li><li> Una vez que han llegado a cerca del 70% de confluencia, transfectar las células HEK293 con pIRES2-EGFP NR1 (células HEK293 NMDAR +), pIRES2-EGFP D2R (+ células HEK293 D2R), y (células HEK293 CTL; control de vectores) pIRES2-EGFP de la siguiente manera.Mantenga algunas células HEK293 transfectadas de indemnización más adelante. <ol><li> Preparar el volumen necesario de mezcla de transfección que comprende 2,5 g de ADN, 200 l de cloruro de sodio 0,9% y 4 mg / ml de polietilenimina / pocillo (cada uno conteniendo 2 ml de DMEM completo fresco). </li><li> Inmediatamente agite la mezcla durante 10 s y se incuba a temperatura ambiente (TA) durante 10 min, antes de la adición de volumen de mezcla de transfección a los pocillos apropiados. </li><li> Cubrir la placa, envuelva los lados con Parafilm, y centrifugar a 280 xg durante 5 minutos para ayudar a la transfección de células. </li><li> Retire el Parafilm y luego colocar en la incubadora para cultivo a 37 ° C. </li><li> Vuelva a colocar los medios de cultivo de 18 horas más tarde con DMEM completo fresco, y mantener en cultivo durante otras 72 horas. </li><li> Opcional: 72 horas después de la transfección, las células transfectadas se pueden seleccionar mediante el cultivo en DMEM completo suplementado con 250 g / ml de geneticina, con el fin de obtener un transfectante estable policlonal. </li></ol></li></ol><pclass = &quot;jove_title&quot;&gt; 3. La citometría de flujo ensayo basado en células para la detección de anticuerpos contra los antígenos de superficie neuronales <ol><li> Separar HEK293 NMDAR +, HEK293 D2R +, y células HEK293 de CTL mediante incubación con 500 l / pocillo Versene durante aproximadamente 5 min a 37 ° C. <ol><li> Resuspender en 2ml/well de PBS (-Ca 2 + / Mg 2 +) suplementado con FBS al 2% (PBS / FBS) y traslado a unas 15 ml o 50 ml tubo cónico. </li><li> Lavar las células por centrifugación a 250 xg durante 6 min y resuspender el sedimento celular en 15-50 ml de PBS / FBS. Repita lavado 2x. </li><li> Contar las células y luego volver a suspender en PBS / FBS al 1 x 10 6 células / ml. </li></ol></li><li> Diseño de la plantilla de 96 pocillos para la adquisición de citometría de flujo, teniendo en cuenta que + células HEK293 D2R + o HEK293 NMDAR, así como células HEK293 CTL tendrán que ser incubados con cada muestra de paciente o anticuerpo primario. </li><li> Se siembran las células en una placa de 96 pocillos de fondo en V a 50 000 células / nosotrosLL de acuerdo con la citometría de flujo plantilla de adquisición. </li><li> Sedimentar las células por centrifugación de la placa de 96 pocillos a 450 xg durante 5 min y suavemente eliminar el sobrenadante usando una pipeta multicanal electrónica o manual, inclinando la placa en un ángulo y teniendo cuidado de no aspirar el sedimento celular. </li><li> Incubar HEK293 D2R +, HEK293 NMDAR +, y células HEK293 CTL durante 1 hora a temperatura ambiente en la oscuridad con: <ol><li> Diluciones seriadas de anticuerpo primario con el fin de evaluar la expresión del antígeno de superficie. </li><li> Las muestras de pacientes con el fin de detectar anticuerpos. </li></ol></li><li> Utilice flujo apropiado citometría controles de compensación, por ejemplo. células HEK293 no transfectadas no teñidas, GFP + células HEK293 (AF647 -), y AF647 + células HEK293 no transfectadas (GFP). </li><li> Lavar las células por centrifugación a 450 xg durante 5 min y resuspender el sedimento de células en 170 l de PBS / FBS. Repita el paso de lavado dos veces más. </li><li> Se incuban las células durante 1 hr a TA en la oscuridad con una dilución 1:100 de anticuerpo secundario AF647-conjugado (IgG anti-humana para el suero del paciente y anti-IgG de ratón para el anti-NR1 o anti-D2R anticuerpos primarios). </li><li> Lavar las células tres veces, como en el paso 3.7, y volver a suspender en 35 l de PBS / FBS para citometría de flujo de células de adquisición. </li><li> Configure el muestreador de alto rendimiento (HTS) en el citómetro de flujo (BDLSRII). </li><li> Adquirir 10.000 eventos / pocillo de acuerdo con la citometría de flujo plantilla de adquisición. </li><li> Export y luego analizar los datos para el análisis utilizando una citometría de flujo paquete de software, tales como FlowJo v7.5: <ol><li> En primer lugar, puerta de las células HEK293 vivos basada en adelante (tamaño) y lateral (granularidad) dispersa. </li><li> HEK293 células vivas Además de puerta basado en alta GFP-positividad para analizar sólo las células transfectadas. </li><li> Determinar la intensidad de fluorescencia media (MFI) de la canal AF647 dentro de las células positivas para GFP en vivo. </li><li> Exportar datos a Excel o Prism para el análisis: <ol><li> Terreno concentrations de anticuerpo primario (anti-NMDAR o anticuerpo anti-D2R) frente al IMF obtuvieron a partir de las células incubadas con estos anticuerpos. </li><li> Para cada paciente y control de ejemplo, determinar el ΔMFI restando el MFI de las células HEK293 de CTL de la IMF de las células HEK293 NMDAR + D2R + HEK293 o, respectivamente. </li><li> Calcule el umbral de positividad de anticuerpos mediante la adición de tres desviaciones estándar por encima de la ΔMFI media de la cohorte de control. </li><li> Muestras individuales Parcela agrupan de acuerdo a su tipo de suero en un gráfico y representan el umbral como una línea. </li></ol></li></ol></li></ol>

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Conflicto de intereses: Una patente ha sido presentada por el FB y el RCD (Universidad de Sydney), alegando D2R como objetivo para autoanticuerpos.

En este trabajo se describe una nueva aplicación de la citometría de flujo ensayo basado en células vivas para detectar anticuerpos dirigidos a proteínas neuronales específicas de la superficie celular usando un muestreador de alto rendimiento. Usando esta técnica, nos informan de que un subgrupo de pacientes afectados con enfermedades del SNC autoinmunes tienen anticuerpos séricos a aflorar a la superficie NMDAR o D2R. Los pasos esenciales para la detección óptima de anticuerpos ut...

En los últimos años, se han identificado formas autoinmunes del sistema nervioso central (SNC) enfermedades. Se ha demostrado que estas enfermedades están asociadas y definen por la presencia de autoanticuerpos. Estos anticuerpos se unen a los receptores neuronales o proteínas sinápticas implicadas en la neurotransmisión 1,2. Diferentes antígenos se han detectado, tales como receptor de N-metil-D-aspartato (NMDAR) 3-5, receptor de γ-aminobutírico B (GABA B) del receptor 6, α-amino-3-hidroxi-5-metil-4- ácido isoxazolpropiónico (AMPA) 7, el canal de potasio dependiente de voltaje (VGKC) proteínas asociadas: rica en leucina 1 glioma proteínas activadas (LGL-1) y contactina asociada a la proteína 2 (capsr2) receptor de 8,9, glutamato 5,10 , y la dopamina-2 del receptor (D2R) 11. En el pasado, estos trastornos del SNC autoinmunes (principalmente llamados encefalitis) eran a menudo sin diagnosticar y sin tratar. Estos biomarcadores de anticuerpos novedosos, por ejemplo,Anticuerpos NMDAR o anticuerpo D2R, han mejorado sustancialmente el diagnóstico y la sensibilización, y se han abierto las opciones de tratamiento para los pacientes. De hecho, el tratamiento temprano con inmunoterapias se asocia con un mejor resultado 11,12. Los métodos tradicionales para detectar anticuerpos, tales como el ensayo inmunoabsorbente ligado a enzimas (ELISA) y Western Blot se han utilizado para la detección de anticuerpos en el suero. Sin embargo, no permiten fácilmente el reconocimiento de epítopos extracelulares o de superficie y, más bien, pueden revelar inmunorreactividad hacia un epítopo intracelular. Además, los anticuerpos que se unen al dominio extracelular de las proteínas importantes implicadas en la neurotransmisión es probable que sean 2,3,7,13,14 patógena. Por lo tanto, el desarrollo de ensayos precisos y sensibles para descubrir superficie de la célula relevante o los objetivos de anticuerpos extracelulares en los pacientes es de suma importancia. El método estándar de oro en el campo se basa en el uso de células vivas, en la así llamada &quot;célula-BAensayos de sed &quot;. Este método implica la expresión de un antígeno en la superficie de células de mamíferos (de riñón (293) más a menudo células embrionarias humanas HEK293) en su forma nativa por la transfección de vectores que contienen la secuencia completa de ADNc del antígeno de interés. Nonpermeabilized células vivas se incubaron después con suero diluido del paciente y seguidas por-fluorocromo conjugado anti-inmunoglobulina humana (Ig) anticuerpo secundario. Después se detecta la intensidad o nivel de fluorescencia y se asocia con el nivel de unión de autoanticuerpos. Esta técnica es específica, ya que sólo un antígeno se sobreexpresa en las células. El más ampliamente utilizado &quot;lectura&quot; ha sido el análisis de microscopía confocal después de inmunocitoquímica 4,5,8-10. Sin embargo, la citometría de flujo ensayos basados ​​en células se han utilizado con éxito para detectar anticuerpos en pacientes con enfermedades desmielinizantes 15-17. En particular, Waters et al. 15 compararon la detección de anticuerpos usando una cacerolaEL de técnicas de detección de anticuerpos, incluyendo ensayos basados ​​en células, seguido por microscopía o el flujo de análisis de citometría, y ha demostrado citometría de flujo ensayo basado en células para ser el método más sensible, preciso y fiable. Por lo tanto, citometría de flujo ensayo basado en células es ventajosa, ya que es cuantitativo, no investigador-dependiente, y no implica ningún uso de material radiactivo. También es conveniente, ya que permite a las grandes cohortes de pacientes que se sometieron a ensayo en un corto período de tiempo. Más recientemente, hemos optimizado un flujo de alto rendimiento citometría de ensayo basado en células para detectar anticuerpos de NMDAR y D2R anticuerpo en pacientes con enfermedades autoinmunes del sistema nervioso central 11. Nuestro grupo detectado recientemente anticuerpo de NMDAR en sueros de pacientes usando citometría de flujo ensayo basado en células. Estos NMDAR sueros anticuerpos positivos fueron analizados previamente por microscopía confocal y también resultaron ser positivos 11. Este protocolo describe una citometría de flujo ensayo basado en células para la detección de canticuerpo SNC onformation-sensible en el suero del paciente utilizando un muestreador automatizado de alto rendimiento.

<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

Durante los últimos años, los anticuerpos contra las proteínas de superficie y conformacionales implicados en la neurotransmisión se han detectado en las enfermedades autoinmunes del sistema nervioso central en niños y adultos. Estos anticuerpos se han utilizado para guiar el diagnóstico y el tratamiento. Ensayos basados ​​en células han mejorado la detección de anticuerpos en el suero del paciente. Se basan en la expresión de superficie de antígenos del cerebro en las células eucariotas, que luego se incubaron con sueros de paciente diluida seguido por anticuerpos secundarios conjugados con fluorocromo. Después del lavado, el anticuerpo secundario de unión se analizó a continuación por citometría de flujo. Nuestro grupo ha desarrollado un flujo de alto rendimiento de la citometría de ensayo basado en células vivas para detectar de forma fiable anticuerpos contra los receptores de neurotransmisores específicos. Este método de citometría de flujo es sencillo, cuantitativa, eficiente, y el uso de un sistema de toma de muestras de alto rendimiento permite grandes cohortes de pacientes que se sometieron a ensayo fácilmente en un corto espacio de tiempo. Además, esta basado en células comodecir se puede adaptar fácilmente para detectar anticuerpos a muchas dianas antigénicas diferentes, tanto desde el sistema nervioso central y la periferia. El descubrimiento de biomarcadores de anticuerpos novela adicionales permitirá un diagnóstico precoz y preciso y mejorar el tratamiento de los trastornos autoinmunes.

HEK293 células vivas D2R + y CTL HEK293 fueron adquiridas en el citómetro de flujo usando un muestreador de alto rendimiento. Durante el análisis, las células fueron cerrada sobre la base de dispersión frontal (tamaño) y los parámetros de dispersión lateral (granularidad) (Figuras 1A y 1D). Células HEK293 transfectadas expresan la molécula informadora, GFP, en el citoplasma, y las células no transfectadas fueron excluidos del análisis (Figuras 1...

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

Durante los últimos años, los ensayos basados ​​en células vivas se han utilizado con éxito para detectar anticuerpos contra antígenos de superficie y conformacionales. Aquí se describe un método que utiliza el flujo de alto rendimiento citometría permite el análisis de grandes cohortes de pacientes. La detección de nuevos anticuerpos será mejorar el diagnóstico y el tratamiento de trastornos del sistema inmune-mediada.

Ensayo de alto rendimiento basado en células citometría de flujo para detectar anticuerpos a la N-metil-D-aspartato receptor o dopamina-2 del receptor en suero humano

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 vistas.  The University of Sydney.  Durante los últimos años, los ensayos basados ​​en células vivas se han utilizado con éxito para detectar anticuerpos contra antígenos de superficie y conformacionales. Aquí se describe un método que utiliza el flujo de alto rendimiento citometría permite el análisis de grandes cohortes de pacientes. La detección de nuevos anticuerpos será mejorar el diagnóstico y el tratamiento de trastornos del sistema inmune-mediada. 

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 medición y tratamiento de la represión en la ambliopía

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

Investigación

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.

Estereológico y Citometría de Flujo Caracterización de leucocitos subpoblaciones en Modelos de transitorios o isquemia cerebral permanente

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.

Adaptación videofluoroscópicos Swallow Métodos de Estudio Humanos para detectar y caracterizar disfagia en modelos murinos de enfermedades

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.

Biología de Sistemas de regulación metabólica por el estrógeno receptor de señalización en cáncer de mama

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.

Un alto rendimiento, multiplexado y dirigida proteómico CSF ​​ensayo para cuantificar neurodegenerativas Biomarcadores y apolipoproteína E isoformas Estado

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 ensayo de cinética basada en fluorescencia Ca2 + movilización para identificar G proteína-juntó el Receptor agonistas, antagonistas y moduladores alostéricos

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.

Caracterización de células inmunes en el tejido adiposo humano mediante el uso de citometría de flujo

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 análisis basado en la citometría de flujo para medir el potencial de membrana mitocondrial en miocitos cardiacos después de hipoxia/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 ...

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.

Ensayo de eflujo de colesterol marcado con Nitrobenzoxadiazole de alto rendimiento

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.

Aplicación de la tecnología de microfluidos Biochip para detectar suero alergénico-específicos de la inmunoglobulina E (sIgE)

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