Este trabalho foi financiado pela National Multiple Sclerosis Society (TA 3021-A1 / T para AEC) e da Universidade do Texas em San Antonio.

Preparação de reagentes Prepare estoque isotônica Percoll (SIP), 4 ml por cérebro, através da mistura de 9 partes de Percoll com uma parte de HBSS 10X sem Ca + + e Mg + +. Prepare SIP menos 70% em 1X HBSS sem Ca + + e Mg + +, 2 ml por cérebro dispensado em um tubo de polipropileno 15 ml. Nota: Recomenda-se que Percoll deve ser usado em temperatura ambiente, se usado frio, as células tendem a aglutinar-se e separação celular é menos eficiente. Coleta de tecido e homogeneização Anestesiar ratos e perfundir através do ventrículo esquerdo com gelado HBSS 1X. Dissecar o cérebro ea medula espinhal e manter em RPMI sem vermelho de fenol até que todos os ratos são sacrificados. Tecidos lugar em 7 ou 15 ml Dounce homogeneizador contendo 3 ml de RPMI, gentilmente fazer uma suspensão de células com um pilão largas (A tamanho), seguido de um pilão Tigh-fitting (tamanho B) para continuar a dissociar os tecidos. Completar o volume de suspensões de células a 7 ml com RPMI. Gradiente de set up Adicionar 3 ml de SIP para a suspensão de células para fazer um SIP final de 30% Lentamente, a camada de suspensão de células 10 mL em cima da SIP 70%. Esta é a etapa mais crítica do procedimento, use uma pipeta ajuda-set no modo de gravidade evitando a mistura de 70% e soluções de 30%. Uma linha muito clara flat deve estar visível na junção 70% -30%. Centrifugar 30 minutos a 500G 18 ° C. Certifique-se de centrífuga vai parar com o freio de um mínimo ou nenhum de modo que a interfase não é perturbado. Usando uma pipeta de transferência, remova a camada de detritos a partir do topo do tubo e recolher 2,0-3,0 ml da interfase 70% -30% em um tubo limpo cônico contendo 8 ml de 1X HBSS. Garantir que o Percoll contendo a interfase é diluída cerca de três vezes, misturar algumas vezes por inversão e min centrifugre 7 a 500G a 18 ° C. Ressuspender sedimento em 1 ml de tampão de coloração celular e transferi-lo para um tubo de 1,5 ml e lavar mais uma vez usando uma centrífuga de micro-a 10.000 G 1 min a 4 ° C. Coloração de anticorpos Pellets ressuspender em 50 ul de purificada antimouse CD16/CD32 diluído 1:200 em tampão de coloração celular para bloquear os sites Fc vinculativo. Incubar mais de gelo por 10 min. Contagem de células utilizando um hemocitômetro. Adicionar 50 ul de mistura de anticorpos cocktail e incubar no gelo por 30 min. Cada anticorpo deve ser o primeiro a avaliar titulada de diluição ideal. Uso adequado combinações fluorocromo escolhidos de acordo com as capacidades do laser e configurações de filtro do seu citômetro de fluxo particular. Lave as células em tampão de células coloração, pellets ressuspender em 100-200 mL de tampão de coloração de células e analisar imediatamente no citômetro, ou, alternativamente, as células podem ser suspensas em paraformaldeído 2% (PFA), preparado em PBS e armazenado durante a noite a 4 ° C . Resultados representativos: Depois de girar os gradientes, a interfase 70% -30% deve ter definido um halo branco, e quantificação das células recuperou de um rato ingênuo deve render 3-5 x10 5 células por mouse (cérebro além da medula espinhal). Cérebro inflamado pode ter números que variam de células inflamatórias, dependendo do insulto SNC ou modelo de doença (Figura 1). <img src="/files/ftp_upload/2348/2348fig1.jpg" alt="Figura 1" /> Figura 1. Isolamento de células mononucleares, a partir de ingênuo e EAE-cérebro na doença de pico. Suspensões de células cerebrais foram separados mais de 70% descontínua / gradientes de Percoll 30%. Manchada células foram incubadas com Fc-bloco seguido por anti-CD45 anticorpos por 30 min no gelo. As células foram lavadas em tampão coloração celular e fixa em 2% aquisição PFA antes em um LSR-II. CD45 intensidade é medida no eixo Y, onde três populações distintas são visualizados. Uma melhor caracterização do fenótipo imunológico de células CD45 oi infiltrando é implementado usando anticorpos adicionais e posteriormente analisadas por citometria de fluxo.

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	<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>

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Análises de marcadores de superfície celular no SNC leucócitos a partir de tecidos de rato normal e inflamado tem sido utilizado por várias décadas 1-3. Protocolos de isolamento são baseados na separação de microglia e leucócitos por 4 centrifugação densidade. Aqui nós descrevemos um método rápido e eficaz de isolar CNS leucócitos usando gradientes descontínuos de Percoll. Após o isolamento de células, coloração com anticorpos diversos, tais como, mas não se limitando a-CD45, CD4, CD8, CD11b, CD19, etc permite a identificação de populações imunológico que infiltram o SNC sobre a indução de uma patologia particular. Coloração com anticorpos anti-CD45 revela três populações distintas por citometria de fluxo; (1) CD45 negativo da população constituída por células neuronais, astrócitos e oligodendrócitos entre as células nervosas outros, (2) CD45 lo ou intermediário, que contém principalmente surveillant células da microglia, (3) CD45 população oi consistindo de leucócitos infiltrantes hematogênica. Estudar a contribuição das células mielóides durante patologias do SNC tem sido um desafio. No entanto, a combinação de vários marcadores de células superficiais 5, 6 e uso de camundongos quiméricos medula óssea mostraram insights sobre as distinções funcionais de células mielóides durante o CNS inflamação 2, 7, 8. Após a imunização com peptídeos de mielina, uma reação auto-imune é induzido e um afluxo maciço de células do sangue é recrutado para o cérebro. Após este ponto, há um amplo repertório de anticorpos disponíveis para estudar o fenótipo destas células, quer pela presença de moléculas de superfície de células diferentes, bem como a expressão de proteínas intracelulares, tais como citocinas ou fatores de transcrição. Quando homogeneização dos tecidos, use extrema cautela manusear o homogeneizador com facilidade. Fagócitos mononucleares são altamente suscetíveis a autólise. Se experimentando aglutinação das células, a prática de um protocolo suave homogeneização e adicionar DNase para o buffer durante a etapa de homogeneização. Portanto, avaliar a porcentagem de células mortas na sua população isolada. Este protocolo pode ser facilmente modificado para adicionar várias enzimas digestivas, tais como colagenase, dispase entre outros. O rendimento das células é geralmente mais elevada. No entanto, alguns marcadores de superfície celular são suscetíveis a esse tratamento e, portanto, limitar a sua detecção por citometria de fluxo, devido à sua clivagem por proteases. Em nosso protocolo, que rotineiramente evitar o uso de enzimas digestivas, dissecar tecidos mais rápido possível, sem comprometer a perfusão, e evite deixar os tecidos por longos períodos de tempo no gelo (&gt; 1h) homogeneização prévia. O protocolo descrito tem o potencial de ser aplicado à cultura gene da proteína de expressão e de células da população isolada.

Homogeneização do tecido e Gradients <ul><li> Percoll (Amersham) </li><li> Solução Hanks 10X &#39;Salt equilibrado, sem cálcio e magnésio (Gibco) </li><li> Meio RPMI 1640, sem vermelho de fenol (Gibco) </li><li> 7-ml ou 15 ml Dounce Tissue Grinders (VWR) </li></ul> Citometria de fluxo <ul><li> Tampão de células de coloração (Biolegend) </li><li> Rat purificada Anti-Mouse CD16/CD32 (Mouse BD Fc Block), BD PharMingen </li><li> Hemocitômetro (Fisher) </li><li> Anti-camundongo CD45 (clone 30-F11), CD4 (clone RM4-5), CD19 (clone 6D5), BioLegend </li><li> Phosphate Buffered Saline 10X, sem cálcio e magnésio (Thermo Scientific) </li><li> Paraformaldeído (Sigma Aldrich) </li><li> Citometria de fluxo realizados com um LSR II (BD Biosciences) </li></ul>

isolation of brain and spinal cord mononuclear cells using percoll gradients

Isolamento de células do sistema imunológico que se infiltrar no sistema nervoso central (SNC) durante a infecção, trauma auto-imunidade, ou neurodegeneração, é muitas vezes necessária para definir seu fenótipo e funções efetoras. Abordagens histoquímica são instrumentais para determinar a localização das células infiltrando e analisar o SNC patologia associada. No entanto, in-situ histoquímica e técnicas de coloração de imunofluorescência são limitados pelo número de anticorpos que podem ser usados ​​em uma única vez para caracterizar os subtipos de células imunes em um tecido particular. Portanto, as abordagens histológicas em conjunto com imuno-fenotipagem por citometria de fluxo são fundamentais para caracterizar completamente a composição do CNS infiltração local. Este protocolo é baseado na separação do CNS suspensões celulares mais descontínua Percoll gradientes. O presente artigo descreve um protocolo rápido de forma eficiente isolamento de células mononucleares a partir de tecidos do cérebro e da medula espinhal que pode ser efetivamente utilizado para a identificação de várias populações de células imunes em uma única amostra por citometria de fluxo.

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

O presente artigo descreve um protocolo rápido de forma eficiente isolamento de células mononucleares a partir de tecidos do cérebro e da medula espinhal que pode ser efetivamente utilizado para análises de citometria de fluxo.

Isolamento do Cérebro e da Medula Espinhal Células Mononucleares Usando Percoll Gradients

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

40.6K visualizações.  University of Texas at San Antonio - UTSA. O presente artigo descreve um protocolo rápido de forma eficiente isolamento de células mononucleares a partir de tecidos do cérebro e da medula espinhal que pode ser efetivamente utilizado para análises de citometria de fluxo.

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

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.

Avaliação quantitativa das células imunes no tecido medula lesionada por Citometria de Fluxo: um novo uso para um método de purificação celular

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

Imunologia e Infecção

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.

Isolamento de Brain-infiltrando Leucócitos

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.

Isolamento de células do sistema imunológico funcional cardíaca

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.

Isolamento de células dendríticas pulmonares do rato

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.

Isolamento de precursor de células B subconjuntos de Sangue do Cordão Umbilical

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

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

Isolamento de tecido adiposo de células imunes

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

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.

O isolamento de células dendríticas mielóides e células epiteliais 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.

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. ...

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.

Isolamento e análise de citometria de fluxo de células imunes do cérebro isquêmico Rato

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.

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 ...