Este trabajo fue apoyado por la National Multiple Sclerosis Society (TA 3021-A1 / T para AEC) y la Universidad de Texas en San Antonio.

Preparación de los reactivos Preparar acciones isotónica Percoll (SIP), 4 ml por el cerebro, mediante la mezcla de 9 partes de Percoll con una parte de HBSS 10 veces sin Ca + + y Mg + +. Prepare SIP en un 70% en 1X HBSS sin Ca + + y Mg + +, 2 ml por cada cerebro se distribuye en un tubo de polipropileno cónico de 15 ml. Nota: Se recomienda que Percoll se debe utilizar a temperatura ambiente, si se utiliza en frío, las células tienden a agruparse y separación celular es menos eficaz. Tejido de recogida y homogeneización Anestesiar a los ratones y perfundir a través del ventrículo izquierdo con helado de HBSS 1X. Diseccionar el cerebro y la médula espinal y mantener en RPMI sin rojo fenol hasta que todos los ratones son sacrificados. Tejidos a cabo en 7 o 15 ml Dounce homogeneizadora que contiene 3 ml de RPMI, con cuidado hacer una suspensión de células con una mano suelta (un tamaño), seguido de un mortero de Tigh-instalación (tamaño B) a disociar aún más los tejidos. Completa el volumen de las suspensiones celulares a 7 ml con RPMI. Gradiente de establecer Añadir 3 ml de la SIP a la suspensión celular para hacer un último sorbo del 30% Poco a poco la capa de la suspensión de células de 10 ml en la parte superior de la SIP el 70%. Este es el paso más crítico del procedimiento, utilice una pipeta de ayuda establecidos en el modo de evitar la mezcla de gravedad el 70% y 30% de las soluciones. Una línea plana muy claro debe ser visible en el cruce de 70% -30%. Centrifugar 30 minutos a 500G 18 ° C. Asegúrese de centrífuga se detiene con un mínimo o ningún freno para que la interfase no se altera. Usando una pipeta, retire suavemente la capa de escombros de la parte superior del tubo y recoger 2,0-3,0 ml de la interfase 70% -30% en un tubo limpio cónico que contiene 8 ml de 1X HBSS. Asegúrese de que el Percoll que contiene la interfase se diluye alrededor de tres veces, mezcla un par de veces por inversión y centrifugre min 7 de 500G a 18 ° C. Resuspender el pellet en 1 ml de tampón de tinción celular y transferirlo a un tubo de 1.5 ml y lavar una vez más utilizando una centrifugadora micro-a 10.000 G 1 min a 4 ° C. Tinción de anticuerpos Pellets Resuspender en 50 l de agua purificada anti-ratón CD16/CD32 diluido 1:200 en buffer de tinción celular para bloquear los sitios de unión Fc. Incubar en hielo durante 10 min. Recuento de células utilizando un hemocitómetro. Añadir 50 l de mezcla de anticuerpos cócteles e incubar en hielo durante 30 minutos. Cada anticuerpo debe ser el primero titulado para evaluar la dilución óptima. Uso adecuado de las combinaciones de fluorocromos elegidos en función de las capacidades del láser y la configuración de los filtros de su citómetro de flujo particular. Lavar las células en un tampón de tinción de células, pellets de volver a suspender en 100-200 l de buffer de tinción celular y de analizar de inmediato en el citómetro, o, alternativamente, las células pueden ser resuspendidas en el 2% de paraformaldehído (PFA), preparado en PBS y se almacenan durante la noche a 4 ° C . Los resultados representativos: Después de girar los gradientes, el 70% -30% de la interfase debe tener definido un halo blanco, y la cuantificación de las células se recuperó de un ratón ingenuo debe producir 5.3 x 10 5 células por ratón (cerebro, además de la médula espinal). Cerebro inflamado podría tener que van del número de células inflamatorias en función de la lesión del SNC o modelo de la enfermedad (Figura 1). <img src="/files/ftp_upload/2348/2348fig1.jpg" alt="Figura 1" /> Figura 1. Aislamiento de células mononucleares, de ingenuo y EAE-cerebro en la enfermedad de pico. Las células del cerebro suspensiones fueron separadas en discontinuo 70% / 30% de los gradientes de Percoll. Manchadas las células fueron incubadas con el FC bloque seguido por anticuerpos anti-CD45 durante 30 minutos sobre el hielo. Las células fueron lavadas en buffer de tinción de células, y se fija en el 2% antes de la adquisición de PFA en un LSR-II. CD45 intensidad se mide en el eje Y, en tres poblaciones distintas se visualizan. Además de la caracterización del fenotipo inmunológico de las células CD45 de alta infiltración se implementa el uso de anticuerpos adicionales y, posteriormente, analizadas por citometría de flujo.

<ol>
	<li>Sedgwick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+direct+characterization+of+resident+microglial+cells+from+the+normal+and+inflamed+central+nervous+system.">Isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system.</a> Proceedings of the National Academy of Sciences of the United States of America. 88, 7438-7442 (1991).</li><li>Ponomarev, E. D., Shriver, L. P., Maresz, K., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+cell+activation+and+proliferation+precedes+the+onset+of+CNS+autoimmunity.">Microglial cell activation and proliferation precedes the onset of CNS autoimmunity.</a> J. Neurosci. Res. 81, 374-389 (2005).</li><li>Ford, A. L., Goodsall, A. L., Hickey, W. F., Sedgwick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+adult+ramified+microglia+separated+from+other+central+nervous+system+macrophages+by+flow+cytometric+sorting.+Phenotypic+differences+defined+and+direct+ex+vivo+antigen+presentation+to+myelin+basic+protein-reactive+CD4++T+cells+compared.">Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared.</a> J Immunol. 154, 4309-4321 (1995).</li><li>Cardona, A. E., Huang, D., Sasse, M. E., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+murine+microglial+cells+for+RNA+analysis+or+flow+cytometry.">Isolation of murine microglial cells for RNA analysis or flow cytometry.</a> Nat. Protoc. 1, 1947-1951 (2006).</li><li>Juedes, A. E., Ruddle, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+and+infiltrating+central+nervous+system+APCs+regulate+the+emergence+and+resolution+of+experimental+autoimmune+encephalomyelitis.">Resident and infiltrating central nervous system APCs regulate the emergence and resolution of experimental autoimmune encephalomyelitis.</a> J. Immunol. 166, 5168-5175 (2001).</li><li>Prinz, M., Priller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tickets+to+the+brain:+Role+of+CCR2+and+CX(3)CR1+in+myeloid+cell+entry+in+the+CNS.">Tickets to the brain: Role of CCR2 and CX(3)CR1 in myeloid cell entry in the CNS.</a> J Neuroimmunol. , (2010).</li><li>Mildner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+in+the+adult+brain+arise+from+Ly-6ChiCCR2++monocytes+only+under+defined+host+conditions.">Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions.</a> Nat. Neurosci. 10, 1544-1553 (2007).</li><li>Djukic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+monocytes+engraft+in+the+brain,+differentiate+into+microglia+and+contribute+to+the+pathology+following+meningitis+in+mice.">Circulating monocytes engraft in the brain, differentiate into microglia and contribute to the pathology following meningitis in mice.</a> Brain. 129, 2394-2403 (2006).</li></ol>

Los análisis de marcadores de superficie celular en los leucocitos del SNC de los tejidos normales de ratón y se inflama se ha utilizado por décadas varios 1.3. Protocolos de aislamiento se basan en la separación de la microglía y leucocitos por cuatro densidad de centrifugación. Aquí se describe un método rápido y eficaz de aislar a los leucocitos del SNC con gradientes de Percoll discontinua. Después del aislamiento de las células, tinción con anticuerpos diferentes, tales como, pero ...

Homogeneización de tejidos y degradados <ul><li> Percoll (Amersham) </li><li> Solución salina equilibrada de Hanks 10X, sin calcio y magnesio (Gibco) </li><li> Medio RPMI 1640 sin rojo fenol (Gibco) </li><li> 7 ml o 15 ml de tejido Dounce Molinos (VWR) </li></ul> Citometría de flujo <ul><li> Celular tinción de amortiguación (Biolegend) </li><li> Rata purificado anti-ratón CD16/CD32 (ratón BD Fc Block), BD Pharmingen </li><li> Hemocitómetro (Fisher) </li><li> Anti-CD45 de ratón (clon 30-F11), CD4 (clon RM4-5), CD19 (clon 6D5), BioLegend </li><li> Fosfato de 10X de solución salina tamponada, sin calcio y magnesio (Thermo Scientific) </li><li> Paraformaldehído (Sigma Aldrich) </li><li> Análisis de citometría de flujo realizado con un LSR II (BD Biosciences) </li></ul>

isolation of brain and spinal cord mononuclear cells using percoll gradients

El aislamiento de las células inmunes que se infiltran en el sistema nervioso central (SNC) durante la infección, traumatismo, enfermedades autoinmunes o la neurodegeneración, es a menudo necesaria para definir su fenotipo y funciones efectoras. Métodos histoquímicos son fundamentales para determinar la ubicación de la infiltración de las células y para analizar el sistema nervioso central patología asociada. Sin embargo, in-situ histoquímica y técnicas de inmunofluorescencia tinción se ven limitados por el número de anticuerpos que se pueden utilizar en un momento único para caracterizar los subtipos de células inmunes en un determinado tejido. Por lo tanto, los enfoques histológicos en relación con el fenotipo inmunológico por citometría de flujo son fundamentales para caracterizar la composición de la infiltración del SNC local. Este protocolo se basa en la separación de suspensiones celulares del SNC más discontinuo gradientes de Percoll. El presente artículo describe un protocolo rápido para aislar eficazmente células mononucleares de tejidos de la médula espinal y el cerebro que puede ser efectivamente utilizada para la identificación de las diferentes poblaciones de células inmunes en una sola muestra por citometría de flujo.

Watch this Scientific Journal Video about Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients at JoVE.com

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

El presente artículo describe un protocolo rápido para aislar eficazmente células mononucleares de tejidos de la médula espinal y el cerebro que puede ser efectivamente utilizado para el análisis de citometría de flujo.

Aislamiento de células del cerebro y la médula espinal mononucleares uso de gradientes de Percoll

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

isolation-brain-spinal-cord-mononuclear-cells-using-percoll

Research

JoVE Journal

Immunology and Infection

40.6K vistas.  University of Texas at San Antonio - UTSA. El presente artículo describe un protocolo rápido para aislar eficazmente células mononucleares de tejidos de la médula espinal y el cerebro que puede ser efectivamente utilizado para el análisis de citometría de flujo.

Video: Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

Aislamiento de células de ratón cavidad peritoneal

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Investigación

Inmunología e Infección

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

Evaluación cuantitativa de las células inmunes en el tejido de la médula espinal lesionada por citometría de flujo: un nuevo uso de un método de purificación celular

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

El aislamiento de la infiltración de leucocitos cerebro-

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Aislamiento de células inmunes funcionales cardíacos

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Aislamiento de células dendríticas del pulmón de ratón

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Aislamiento de los precursores de células B subconjuntos de Sangre del Cordón Umbilical

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

Aislamiento de células dendríticas mieloides y células epiteliales de timo humano

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

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.

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. ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Aislamiento y análisis de citometría de flujo de células inmunes del cerebro isquémico Ratón

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

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 ...