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.

A pesquisa com amostras de sangue humano foi aprovado pela Concordia University Ética Review Board, número do certificado 10000618. O trabalho no músculo do rato foi aprovado pela Concordia University Ética Review Board, número do certificado 2010BERG. 1. PBMC Isolamento de Sangue Total NOTA: Realizar este procedimento em uma cabine de segurança biológica utilizando uma técnica estéril e equipamentos esterilizados pelo fabricante. <ol><li> Com cuidado, despeje 10-15 ml de sangue total a partir dos tubos de heparina (anticoagulante) de coleta de sangue em um tubo de 50 ml estéril. Para derramamentos de sangue menores, limpe com DDH 2 O e 70% EtOH usando tecido de limpeza. </li><li> Dilui-se o sangue no tubo cónico para uma proporção de 1: 1 com Solução Salina de Tampão Fosfato (PBS 1x), pH 7,4. Certifique-se de que o volume máximo total não exceda 30 ml. </li><li> Misture delicadamente usando uma pistola de pipeta sorológica evitar bolhas. NOTA: Não misture invertendo o tubo para evitar o sangue de umccumulation na tampa e infiltração subsequente durante a centrifugação. </li><li> Adicionar 13 ml de Ficoll-Paque (ver Materiais e Equipamento tabela), para um novo tubo cónico de 50 ml de capacidade. Mantenha o tubo na posição vertical no rack. </li><li> Usando uma pipeta, tomar sangue diluído, tocar com a ponta da pipeta de transferência para a parede interior do tubo perto do topo. Com pressão constante e lenta, a transferência do sangue ao longo da parede interior do tubo, formando uma camada de sangue no topo da camada de sacarose. Faça este passo várias vezes até que todo o sangue foi transferido. </li><li> Tampe o tubo bem e centrifugar o tubo à temperatura ambiente por 30 minutos a 700 xg em um rotor balanço com set aceleração média a 5, e desaceleração de zero. </li><li> Lentamente, retire o tubo e sem perturbar as camadas, levá-la de volta para a cabine de segurança biológica. </li><li> Recolher cuidadosamente o creme leucocitário (camada branca nublado fina), onde estão localizados PBMC, colocado entre a PBS / plasma umand ficoll camadas utilizando uma pipeta de transferência. Evite coleta camada de sacarose e não perturbe a camada de células vermelhas do sangue. </li><li> Transferir o creme leucocitário para um novo tubo de 50 ml estéril. Pode demorar várias vezes de repetir o passo 1.8 para coletar todo o PBMC. </li><li> Adicionar PBS pH 7,4 para o tubo com a PBMC e encha até à marca de 45 ml. Agitar minuciosamente tubo. Não vortex. </li><li> Lavar 1: Centrifugar durante 15 minutos a 480 xg tanto com aceleração e desaceleração máxima definida para 9. Observar formação de um sedimento de PBMC na parte inferior do tubo cónico. </li><li> Descartar o PBS (sobrenadante) para um copo de plástico com 10 ml de lixívia. </li><li> Solte pellet celular com cuidado &quot;quebrando&quot; contra uma superfície ondulada (ie, rack de vazio). Não vortex. </li><li> Adicionar 25 ml de PBS fresco de pH 7,4 ao tubo. Se mais de um tubo está a ser processado, reunir os peletes juntamente neste passo. </li><li> Lave 2: Centrífuga o tubo por 12 min a 480 xg with tanto aceleração máxima e desaceleração definir a 9. </li><li> Descarte o PBS (sobrenadante) no copo de plástico com um esguicho de água sanitária. </li><li> Gently &quot;cremalheira&quot; contra uma superfície ondulada (ie, rack de vazio). Não vortex. </li><li> Adicionar 25 ml de PBS fresco ao tubo. <ol><li> Retire 50 ul de células e transferir para um tubo de microcentrífuga para contagem de viabilidade. </li><li> Adicionar um montante igual (50 ul) de azul de tripan (a mancha de viabilidade) e pipeta cima e para baixo para misture delicadamente. </li></ol></li><li> Retire 10 ul e transferi-lo para um hemocitômetro para verificar a viabilidade de células por ml. Grave o número de células / ml. </li><li> Retirar a quantidade desejada de células e centrifugar o tubo durante 12 minutos a 480 xg tanto com aceleração e desaceleração máxima definida para 9. </li><li> Descarte o PBS (sobrenadante) no copo com água sanitária. </li><li> Gently &quot;cremalheira&quot; contra uma superfície ondulada (ie, rack de vazio). Adicionar 80 mL de PBS para o tubo. </li></ol> 2. Fazer o slide PBMC <ol><li> Colocar 80 ul de células numa lâmina de microscópio. Manchar a gota, com a ajuda de uma outra corrediça ou colocar duas gotas de 40 ul cada uma em ambas as extremidades da corrediça. </li><li> Deixe slide na cabine de segurança biológica para secar. Rotular o slide com um lápis no lado fosco. </li></ol> 3. Fixação das amostras nos slides <ol><li> Preparar a solução fixadora por mistura de 0,5 ml de formaldeído a 37% de etanol 4,5 ml de 99%. </li><li> Depois as lâminas são secas, tirar 2 ml da solução fixadora frescos e derramar sobre a corrediça de modo que toda a superfície da lâmina é coberta. </li><li> Deixe a solução no slide por 1 min. Lave o slide para 1 min com água corrente e deixe-o secar ao ar. </li></ol> 4. Fazer a Solução Amylase para Controle Negativo <ol><li> Tome 0,25 g de pó de amilase e dissolver em 50 ml de diágua estava calma. Despeje a solução em um ambiente limpo 100 ml. </li><li> Imergir a corrediça no recipiente de forma a que metade da corrediça recebe o tratamento e a outra metade permanece sem tratamento e depois incubar durante 15 min à temperatura ambiente (Figura 1C). </li><li> Tome nota de que lado do slide está recebendo o tratamento amilase. Desenhar uma linha na parte de trás da lâmina que indica a fronteira entre o tratamento e controlo. </li><li> Lavam-se as lâminas com ddH2O para remover a solução de amilase e deixar a lâmina de ar seco. </li></ol> 5. Execute Ácido Periódico de Schiff (PAS) Coloração e Imagem NOTA: reagentes PAS são tóxicas por inalação e são corrosivos, por isso os passos precisam ser feitos em um exaustor química, e os resíduos devem ser eliminadas de acordo com as diretrizes institucionais. <ol><li> Coloque o slide em uma superfície plana e despeje 1,50-2,00 ml de solução de ácido periódico sobre a amostra. Incubate durante 5 min à temperatura ambiente. </li><li> Lavar as lâminas em várias acusações de água destilada. </li><li> Pour 1,50-2,00 ml de reagente de Schiff sobre a lâmina e incubar-la à temperatura ambiente durante 15 min. </li><li> Lava-se a lâmina com água destilada durante 5 minutos e deixa-se secar ao ar. </li><li> Aplicar 10 jul meios de montagem no slide e cubra com duas pequenas lamelas ou aplicar 50 ul e utilizar uma lamela grande. </li><li> Aplique unha polonês claro nas bordas da lamínula, deixe secar durante a noite. </li></ol> 6. obter imagens com a luz do microscópio binocular Usando o Objetivo 100X

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The authors have nothing to disclose.

Os passos críticos deste artigo de vídeo foram amilase durante a lavagem e tratamento das células. Enquanto lavava os slides, o passo fundamental estava usando uma garrafa de lavagem squeezable plástico e deixando a água correr suavemente através da amostra no slide e não visando diretamente sobre as amostras. Mesmo a menor pressão da água direto faria com que as células para sair do slide. Outro passo importante foi usar a mesma lâmina para ± condições de amilase. Após as PBMC foram aderidos à corrediç...

Schiff (PAS) coloração ácido periódico é uma técnica de imuno-histoquímica, que é amplamente utilizado em pesquisas e diagnósticos muscular. Também é utilizada como uma ferramenta de diagnóstico em amostras de sangue. A técnica funciona através da aplicação de uma solução de ácido periódico à amostra, que oxida unidades dentro dos grupos de aldeído de polissacárido criando que reagem com o reagente de Schiff incolor produzindo assim um produto de magenta profunda. Os passos deste processo são mostrados na Figura 1. A mancha transforma nada com polissacarídeos de glicogénio magenta, incluindo, glicoproteínas, glicolipidos, mucinas, ou outras moléculas com porções de polissacáridos. Coloração PAS é muitas vezes usado para medir os níveis de glicogênio nas fibras musculares. Cortes de tecidos músculos são ideais para a técnica como eles firmemente anexar ao slide e suportar múltiplas etapas de lavagem e de coloração. O glicogênio é mais presente em contração rápida fibras musculares tipo II, que possuem uma alta demandapara a produção de ATP rápida exigindo glicogênio para 1,2 desempenho máximo. O glicogénio é um polímero ramificado de glucose que pode ser quebrada em glicose livre através da acção de enzimas de fosforilase de glicogénio. Em tempos de descanso e nutricional-suficiência, o glicogênio é reabastecido através do processo de glycogenesis, enquanto que em tempos de insuficiência nutricional ou de alta demanda de energia; glicogênio é quebrada em glicose por glicogenólise. Já a partir de 1950 os cientistas têm explorado clínico PAS coloração em amostras de sangue para analisar o conteúdo de glicogênio em várias doenças 3-7. Por exemplo, na doença de Pompe-um armazenamento de glicogénio genuíno glóbulos brancos doen- ças acumular grandes quantidades de glicogénio que difere significativamente dos controles saudáveis ​​8. Este vídeo-artigo demonstra uma versão adaptada de coloração PAS para uso em células mononucleares do sangue periférico (PBMC) a partir de amostras de sangue venoso de seres humanos saudáveis. PBMCs contêm principalmente linfócitos do linfócito T e B de linfócitos famílias, bem como outras células imunitárias, tais como as células assassinas naturais e monócitos. O primeiro passo de purificação remove os eritrócitos, neutrófilos, granulócitos e outros. Esta técnica fornece dados sobre uma parte do concentrado para a enumeração de linfócitos que permitem mais robusta de células-PAS positivo em relação ao meio de esfregaços de sangue total. <img alt="Figura 1" src="/files/ftp_upload/52199/52199fig1highres.jpg" /> Figura 1:. Passo a metodologia passo de coloração PAS em PBMC (A) Em primeiro lugar, o isolamento de PBMC é conseguido através de um gradiente de Ficoll, no painel da esquerda mostra a preparação antes da centrifugação, o painel direito mostra que após centrifugação, onde o buffy coat contendo as PBMC Observa-se no centro do tubo. (B) isoladas PBMC são fixadas sobre a lâmina utilizando solu fixador formalina-etanolção. A lâmina é cuidadosamente enxaguada com água destilada a partir de um frasco de lavagem de plástico. (C) A lâmina é então colocada em 100 ml de um meio caminho copo cheio com solução de amilase, que irá dissolver glicogénio. A lâmina é lavada delicadamente. (D) A lâmina é tratado com solução de ácido periódico, onde oxidação de sacarídeos ocorre. As lâminas são lavadas suavemente; isto irá remover o excesso de ácido periódico e parar o passo de oxidação. (E) Quando o reagente de Schiff é adicionada às lâminas, que vai reagir com aldeídos criados durante o passo de oxidação. Este reagente incolor irá então resultar em um produto magenta vermelho escuro. As lâminas são lavadas suavemente para remover o excesso de reagente 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 os reagentes e técnica básica, coloração PAS foi realizada de acordo com as instruções do fabricante sobre seções musculares rato sóleo. A coloração foi feita mesmo dia que o sacrifício e no último passo da coloração, as secções foram fixadas por xileno. O músculo sóleo é conhecido por conter ~ 35% de glicogénio-positiva células 9. As células musculares coradas exibido duas grânulos puntiformes Features- PAS positivo distintas dentro da célula, e uma linha contínua demar...

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.

Detecção de glicogênio no sangue periférico células mononucleares com Periodic Acid Schiff Coloração

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

Research

JoVE Journal

Immunology and Infection

21.0K visualizações.  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.

Vídeo: 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.

Indução de aloantígenos específicos de anergia em células mononucleares do sangue periférico por Estimulação com aloantígenos Co-estimulatórias bloqueio de sinal

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

Imunologia e Infecção

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.

Expansão do sangue periférico humano γδ células T usando 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.

Uma<em&gt; In vitro</em&gt; Modelo para estudar respostas imunes de sangue periférico humano células mononucleares para Vírus Respiratório Sincicial Humano Infecção

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.

Geração de Células T humanas alo-específicos a partir de Sangue Periférico

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.

Isolamento e Análise de Citometria de Fluxo infiltrantes Glioma Células Mononucleares do Sangue Periférico

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.

Análise de Linfócitos extravasamento Usando um<em&gt; In Vitro</em&gt; Modelo do Sangue-Cérebro humano Barreira

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.

Separação de subpopulações de células imunes em amostras de sangue periférico de crianças com mononucleose 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.

Isolamento, ativação e expansão de células T baseadas em flotação de amostras de células mononucleares do sangue periférico humano usando microbolhas

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

Coleta e crioarmazenamento de PBMC para análise bioenergética 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 em destaque: Preservação de parâmetros bioenergéticos em células mononucleares do sangue periférico após criopreservação 

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.

Criopreservação e Avaliação Bioenergética de Células Mononucleares do Sangue Periférico Humano

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