This research was supported by a grant from the NSERC Discovery program grant number RGPIN 418522-2013. We thank R. Kilgour for helpful discussions, and Katelin Gresty and Dr. A. Berghdal for providing the mouse muscle sections.

La investigación con muestras de sangre humana fue aprobado por la Universidad Concordia Comité de Ética, número de certificado 10000618. El trabajo en músculo de ratón fue aprobado por la Universidad Concordia Comité de Ética, número de certificado 2010BERG. 1. Aislamiento de PBMC de sangre entera NOTA: Esta operación se realizará en un gabinete de bioseguridad mediante una técnica estéril y equipo fabricante esterilizado. <ol><li> Vierta cuidadosamente 10-15 ml de sangre entera de los tubos (anti-coagulante) de recogida de sangre heparinizados en un tubo cónico estéril de 50 ml. Para derrames sanguíneos menores, limpie con ddH2O y el 70% EtOH utilizando tejido de limpieza. </li><li> Diluir la sangre en el tubo cónico a una proporción de 1: 1 con tampón fosfato salino (PBS 1x) pH 7,4. Asegúrese de que el volumen total máximo no exceda de 30 ml. </li><li> Mezclar suavemente con una pistola de pipeta serológica evitar burbujas. NOTA: No mezclar invirtiendo el tubo para evitar la sangre de unccumulation en la tapa y la posterior filtración durante la centrifugación. </li><li> Añadir 13 ml de Ficoll-Paque (ver Materiales y tabla de Equipo), a un nuevo tubo cónico de 50 ml de capacidad. Mantenga el tubo en posición vertical en el bastidor. </li><li> Con una pipeta de transferencia, tomar sangre diluida, toque la punta de pipeta de transferencia a la pared interior del tubo cerca de la cima. Con la presión lenta y constante, transferir la sangre a lo largo de la pared interior del tubo que forma una capa de sangre en la parte superior de la capa de sacarosa. Realice este paso varias veces hasta que toda la sangre se ha transferido. </li><li> Tapar el tubo herméticamente y se centrifuga el tubo a temperatura ambiente durante 30 min a 700 xg en un rotor de oscilación con el conjunto de aceleración medio a 5, y la deceleración ajustado a cero. </li><li> Poco a poco sacar el tubo y sin molestar a las capas, llevarlo de vuelta a la cabina de bioseguridad. </li><li> Recoger cuidadosamente la capa leucocitaria (fina capa de color blanco turbio), donde se encuentran las PBMC, colocado entre la PBS / plasma unnd capas Ficoll utilizando una pipeta de transferencia. Evitar recolectar capa sacarosa y no molestar a la capa de glóbulos rojos. </li><li> Transferir la capa leucocitaria en un nuevo tubo cónico estéril de 50 ml. Pueden pasar varias veces de repetición del paso 1.8 para recopilar toda la PBMC. </li><li> Añadir PBS pH 7,4 al tubo con la PBMC y rellenar hasta la marca de 45 ml. Agitar el tubo a fondo. No lo hagas vórtice. </li><li> Lavar 1: Centrifugar durante 15 min a 480 xg tanto con la máxima aceleración y deceleración configurado a 9. observar la formación de un pellet de PBMC en la parte inferior del tubo cónico. </li><li> Desechar el PBS (sobrenadante) en un vaso de precipitados de plástico con 10 ml de lejía. </li><li> Afloje sedimento celular suavemente &quot;trasiego&quot; contra una superficie ondulada (es decir, estante vacío). No lo hagas vórtice. </li><li> Añadir 25 ml de PBS pH 7,4 fresco al tubo. Si se está procesando más de un tubo, agrupar los gránulos juntos en este paso. </li><li> Lave 2: Centrifugar el tubo durante 12 minutos a 480 xg with tanto la máxima aceleración y desaceleración ajustado a 9. </li><li> Deseche la PBS (sobrenadante) en el vaso de plástico con un poco de lejía. </li><li> Suavemente &quot;estante&quot; contra una superficie ondulada (es decir, estante vacío). No lo hagas vórtice. </li><li> Añadir 25 ml de PBS fresco al tubo. <ol><li> Sacar 50 l de las células y transferir a un tubo de microcentrífuga para el recuento de viabilidad. </li><li> Añadir una cantidad igual (50 l) de azul de tripano (una mancha de viabilidad) y pipeta de arriba y abajo para mezclar suavemente. </li></ol></li><li> Saque 10 l y transferirlo a un hemocitómetro para comprobar la viabilidad de las células por ml. Anote el número de células / ml. </li><li> Saque la cantidad deseada de células y centrifugar el tubo durante 12 minutos a 480 xg tanto con la máxima aceleración y desaceleración ajustado a 9. </li><li> Deseche la PBS (sobrenadante) en el vaso con lejía. </li><li> Suavemente &quot;estante&quot; contra una superficie ondulada (es decir, estante vacío). Añadir 80 l de PBS en el tubo. </li></ol> 2. Hacer el Slide PBMC <ol><li> Ponga 80 l de las células en un portaobjetos de microscopio. Manchar la gota con la ayuda de otra diapositiva o colocar 2 gotas de 40 l cada uno en ambos extremos de la corredera. </li><li> Deja diapositiva en la cabina de seguridad biológica para secar. Etiqueta de la diapositiva con un lápiz en el lado esmerilado. </li></ol> 3. La fijación de las muestras en las diapositivas <ol><li> Preparar la solución de fijación mediante la mezcla de 0,5 ml de formaldehído al 37% de etanol 4.5 ml de 99%. </li><li> Después los portaobjetos se secan, se llevará a cabo 2 ml de la solución de fijación recién hecho y se vierte en la diapositiva de manera que toda la superficie de la corredera está cubierto. </li><li> Dejar la solución en el portaobjetos durante 1 min. Enjuague el portaobjetos durante 1 minuto con agua del grifo y dejar que se seque al aire. </li></ol> 4. Hacer la Solución amilasa para el Control Negativo <ol><li> Tome 0,25 g de polvo de amilasa y se disuelven en 50 ml de diagua calmado. Vierta la solución en un vaso de 100 ml limpio. </li><li> Sumergir el portaobjetos en el vaso de precipitados de modo que la mitad de la corredera recibe el tratamiento y la otra mitad permanece sin tratamiento y luego se incuba durante 15 min a temperatura ambiente (Figura 1C). </li><li> Tome nota de qué lado de la diapositiva está recibiendo el tratamiento amilasa. Dibujar una línea en la parte posterior de la corredera que indica la frontera entre el tratamiento y control. </li><li> Se lavan los portas con ddH2O para eliminar la solución de amilasa y dejar el portaobjetos se seque al aire. </li></ol> 5. Realice ácido periódico de Schiff (PAS) de tinción e Imagen NOTA: Reactivos de PAS son tóxicos por inhalación y son corrosivos, por lo que los pasos deben realizarse en una campana de humos químicos, y los productos de desecho deben eliminarse de acuerdo con las directrices institucionales. <ol><li> Colocar la placa en una superficie plana y vierta 1,50 a 2,00 ml de solución de ácido periódico sobre la muestra. Incubate durante 5 min a temperatura ambiente. </li><li> Enjuague los portaobjetos en varios cargos de agua destilada. </li><li> Verter 1,50-2,00 ml de reactivo de Schiff en la diapositiva y se incuba a temperatura ambiente durante 15 min. </li><li> Lavar el portaobjetos con agua destilada durante 5 minutos y dejar que se seque al aire. </li><li> Aplicar medio de montaje 10 l en el portaobjetos y cubrir con dos pequeñas cubreobjetos, o aplicar 50 l y utilizar una gran cubreobjetos. </li><li> Aplicar esmalte de uñas transparente en los bordes del cubreobjetos, dejar secar durante la noche. </li></ol> 6. Obtener imágenes con la Luz Microscopio Binocular Utilizando el objetivo 100X

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The authors have nothing to disclose.

Los pasos críticos de este artículo de vídeo fueron durante el lavado y la amilasa tratamiento de las células. Mientras se lava las diapositivas, el paso clave estaba utilizando una botella de lavado squeezable plástico y dejar que el agua corra suavemente a través de la muestra en la diapositiva y no apuntar directamente contra las muestras. Incluso la más mínima presión de agua directa sería hacer que las células para salir de la diapositiva. Otro paso clave fue utilizar la misma diapositiva durante ± cond...

Ácido periódico de Schiff (PAS) tinción es una técnica inmunohistoquímica que se utiliza ampliamente en la investigación y diagnóstico muscular. También se utiliza como una herramienta de diagnóstico en muestras de sangre. La técnica funciona mediante la aplicación de una solución de ácido periódico a la muestra, que se oxida unidades dentro de los polisacáridos crear grupos aldehído que reaccionan con el reactivo de Schiff la incoloro produciendo de este modo un producto magenta profundo. Los pasos de este procedimiento se muestran en la Figura 1. La mancha resulta nada con polisacáridos magenta, incluyendo glucógeno, glicoproteínas, glicolípidos, mucinas, u otras moléculas con restos de polisacáridos. Tinción PAS se utiliza a menudo para medir los niveles de glucógeno en las fibras musculares. Secciones de tejido músculos son ideales para la técnica, ya que se adhieren firmemente a la diapositiva y soportan múltiples etapas de lavado y tinción. El glucógeno es más presente en las fibras musculares de contracción rápida de tipo II, que tienen una gran demandapara la producción de ATP rápido que requiere de glucógeno para el máximo rendimiento de 1,2. El glucógeno es un polímero ramificado de glucosa que se puede dividir en glucosa libre a través de la acción de las enzimas de la glucógeno fosforilasa. En tiempos de descanso y nutricional-suficiencia, el glucógeno se repone a través del proceso de glucogénesis, mientras que en los momentos de alta energía o la insuficiencia de la demanda nutricional; se descompone el glucógeno en glucosa por la glucogenólisis. Ya desde la década de 1950 los científicos clínicos han explorado tinción PAS en muestras de sangre para analizar el contenido de glucógeno en diversas enfermedades 7.3. Por ejemplo, en la enfermedad de Pompe, un almacenamiento de glucógeno de buena fe células blancas de la sangre disease- se acumulan grandes cantidades de glucógeno que difiere significativamente de los controles sanos 8. Este artículo de vídeo demuestra una versión adaptada de la tinción de PAS para su uso en células mononucleares de sangre periférica (PBMC) a partir de muestras de sangre venosa de los sujetos humanos sanos. PBMCs contienen en su mayoría linfocitos de los linfocitos T y las familias de linfocitos B, así como otras células inmunes, tales como células asesinas naturales y monocitos. La primera etapa de purificación elimina los eritrocitos, neutrófilos, granulocitos y otros. Esta técnica proporciona datos sobre una proporción concentrada de linfocitos que permiten para la enumeración más robusta de células PAS positivos en comparación con el uso de enteros frotis de sangre. <img alt="Figura 1" src="/files/ftp_upload/52199/52199fig1highres.jpg" /> Figura 1:. Paso a paso metodología de la tinción PAS en PBMC (A) En primer lugar, el aislamiento de PBMC se logra mediante gradiente de Ficoll, el panel izquierdo muestra la preparación antes de la centrifugación, el panel derecho muestra que después de la centrifugación, donde la capa leucocitaria que contiene la PBMC se observa en el centro del tubo. (B) PBMCs aisladas se fijan sobre el portaobjetos utilizando Solu la adherencia de formalina-etanolción. El portaobjetos se enjuaga suavemente con agua destilada de una botella de lavado de plástico. (C) El portaobjetos se coloca entonces en un vaso de precipitados de 100 ml a mitad de camino lleno de solución amilasa, que se disolverá glucógeno. El portaobjetos se enjuaga suavemente. (D) El portaobjetos se trató con solución de ácido periódico, donde la oxidación de los sacáridos se lleva a cabo. Los portaobjetos se aclararon de forma abundante; se eliminará el exceso de ácido periódico y detener la etapa de oxidación. (E) Cuando se añade el reactivo de Schiff a las diapositivas, es el que reacciona con aldehídos creados durante la etapa de oxidación. Este reactivo incoloro entonces resultar en un producto rojo magenta profundo. Las diapositivas se enjuagan suavemente para eliminar el exceso de reactivo de Schiff.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Periodic Acid Shiff Kit</td>
			<td>Sigma-Aldrich</td>
			<td>395B</td>
			<td>Bring to room temperature prior to use. Materials in this kit are toxic and harmful. Use caution&#160; 
			<a href="http://www.sigmaaldrich.com/catalog/product/sigma/395b?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/395b?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>&#945;-Amylase from porcine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>A3176</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a3176?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/a3176?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>Binocular Microscope&#160;</td>
			<td>Carl Zeiss Microscopy</td>
			<td>Axio Lab A0</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen Assay Kit</td>
			<td>Sigma-Aldrich</td>
			<td>MAK016</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/mak016?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/mak016?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>VWR, GE Healthcare</td>
			<td>17-1440-02</td>
			<td>Nonionic synthetic polymer of sucrose 
			<a href="https://us.vwr.com/store/catalog/product.jsp?product_id=4779441">https://us.vwr.com/store/catalog/product.jsp?product_id=4779441</a></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td>For PBMC isolation, swing buckets were used&#160;</td>
		</tr>
	</tbody>
</table>

detecting glycogen in peripheral blood mononuclear cells with periodic acid schiff staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Para validar los reactivos y la técnica básica, la tinción de PAS se realizó de acuerdo con las instrucciones del fabricante en secciones de músculo sóleo ratón. La tinción se realizó mismo día que el sacrificio y en el último paso de la tinción, las secciones se fijaron por xileno. El músculo sóleo se sabe que contiene ~ 35% de glucógeno-células positivas 9. Las células musculares teñidas muestran dos gránulos puntiformes Características- PAS positivos distintas dentro de la célula, y un...

Watch this Scientific Journal Video about Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining at JoVE.com

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

La detección de glucógeno en las células mononucleares de sangre periférica con ácido periódico de Schiff tinción

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

detecting-glycogen-peripheral-blood-mononuclear-cells-with-periodic

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Immunology and Infection

21.0K vistas.  Centre for Structural and Functional Genomics, PERFORM Centre, Concordia University. Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Video: Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

La inducción de anergia aloantígenos específicos en las células mononucleares de sangre periférica mediante estimulación aloantígeno con bloqueo de señales co-estimuladoras

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

Investigación

Inmunología e Infección

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Expansión de la sangre periférica humana de células T γδ con zoledronato

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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

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

Generación de células T humanas aloantígeno-específicos de la sangre periférica

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

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

El aislamiento y la citometría de flujo análisis de las células mononucleares de sangre periférica Glioma infiltrantes

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Análisis de linfocitos extravasación El uso de una<em&gt; In Vitro</em&gt; Modelo de la barrera sangre-cerebro humano

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

Separación de subpoblaciones de células inmunitarias en muestras de sangre periférica de niños con mononucleosis infecciosa

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

Aislamiento, activación y expansión de células T basadas en flotación a partir de muestras de células mononucleares de sangre periférica humana utilizando microburbujas

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

Recolección y crioalmacenamiento de PBMC para el análisis bioenergético mitocondrial

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.

Autor destacado: Preservación de parámetros bioenergéticos en células mononucleares de sangre periférica después de la criopreservación 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

Criopreservación y Evaluación Bioenergética de Células Mononucleares de Sangre Periférica Humana

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...