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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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, C...

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

Research

JoVE Journal

Immunology and Infection

40.5K 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 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 ...

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 ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

Isolamento de células mononucleares de lamina propria de cólon murino usando Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

Isolamento de células mononucleares tonsiláres para estudar respostas imunes inatas ex vivo em um tecido linfoide mucosal humano

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

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 ...