Os autores gostariam de agradecer Bing Zhang por sua assistência técnica. Este trabalho foi financiado pelos Institutos Canadenses de Pesquisa em Saúde (MOP-86595). TAB é um CIHR novo investigador e estudioso AHFMR. QH é apoiado por uma Bolsa de Pós-Graduação CIHR Canadá - Doutorado e um Studentship tempo inteiro AIHS. SAN é apoiado por uma Rainha Elizabeth II de Bolsas de Estudo de Pós-Graduação. AYWS é apoiado por uma Bolsa de Pós-Graduação NSERC - Doutorado.

Consulte a Figura 1 para um esquema geral do protocolo experimental. 1. Dissecação <ol><li> Coloque tela de malha de aço estéril em 60 15 mm x placa de Petri. Uma unidade é necessária por amostra de tecido. </li><li> Adicionar 5 ml de solução salina equilibrada de Hank (HBSS) para cada prato. Mantenha pratos no gelo. </li><li> Euthanize ratos com CO 2. </li><li> Rato seguro para a superfície, dissecção lado ventral para cima. Pulverização do rato com etanol a 70% para a esterilização e para assegurar que a pele é opaco para baixo. </li><li> Utilizando uma tesoura cirúrgica, dissecção começar por fazer uma incisão ventral na pele abdominal acima da genitália. Estender para cima da incisão para o queixo. </li><li> A partir da linha média, estender a incisão para baixo ao longo de todos os membros. Puxe a pele solta e fixá-lo para a superfície dissecção. </li><li> Colheita do timo: <ol><li> Levante a ponta inferior do esterno para fazer uma incisão.</li><li> Evitando o fígado, o diafragma cortado para separar a caixa torácica e depois cortar a caixa torácica para cada lado. Corte as costelas para cima em cada lado, tendo o cuidado de evitar os pulmões eo coração. </li><li> Com cuidado, puxe a parte de trás da caixa torácica com uma pinça. O timo é um órgão, branco bilobado localizado acima do coração. Usando a ponta plana do fórceps para agarrar o fundo dos lobos, puxe o timo e colocá-lo na tela de malha. </li></ol></li><li> Colher o baço: <ol><li> O baço é um vermelho, o órgão em forma de prancha de surf localizada no lado esquerdo da cavidade abdominal do rato, abaixo do fígado. </li><li> Fazer uma incisão na cavidade abdominal. Retire cuidadosamente o baço com um par de fórceps, usando o segundo par de provocar o tecido conjuntivo. Coloque o baço em uma tela de malha separada. </li></ol></li></ol> 2. Preparação de células <ol><li> Usando um êmbolo de uma seringa de 3 ml, moerórgãos para a tela de malha até que o tecido conjuntivo e apenas restos de gordura. Lavar malha com HBSS do prato três vezes. Usar um êmbolo para cada amostra de tecido. <ol><li> Outras opções para a homogeneização do tecido pode ser usado em lugar deste método. </li></ol></li><li> Contar as células utilizando um hemocitómetro. </li><li> Células de pelotas por centrifugação a 335 xg durante 5 min a 4 ° C. </li><li> Esplenócitos Ressuspender em 500 ul de tampão de lise ACK (0,15 M NH 4 Cl, 10 mM KHC0 3, 0,1 mM Na 2 EDTA, pH 7,2) durante 10 min à temperatura ambiente. Timócitos Ressuspender em 20 x 10 6 células / ml em tampão FACS (PBS, 1% de FCS, 0,02% de azida de sódio) e de lado em gelo. </li><li> Retornar esplenócitos de isotonicidade pela adição de 5 ml de HBSS. Esplenócitos de pelotas por centrifugação e ressuspender em 20 x 10 6 células / ml em tampão de FACS. Se as células precisam permanecer estéril, você pode usar estéril RPMI + FCS 10%. </li></ol> 3.Células coradas para citometria de fluxo O objectivo deste protocolo é traçar estratégias para a análise de seleção positiva e negativa utilizando tipo selvagem (WT) e camundongos CD4 HY. Para considerações gerais sobre a citometria de fluxo experimental design, aquisição, análise e, nos referimos aos leitores uma excelente revisão por Tung et al. 11 <ol><li> Alíquota de 4 x 10 6 timócitos por exemplo citometria de fluxo para cada cavidade de uma placa de 96 poços, bem como 1 x 10 6 por esplenócitos WT controlo de compensação. Se usar camundongos transgênicos, você deve incluir um mouse como controle WT em sua experiência. </li><li> Bloquear os receptores Fc, por incubação com células anti-CD16/32 (clone 2.4G2) durante 10 min em gelo. </li><li> Girar em placas a 335 xg a 4 ° C durante 5 min. Retire a tampa e dissipar líquido de poços mexendo na placa de uma vez, de bruços em uma pia. Ressuspender a cada poço em 200 ul de tampão de FACS. Repetir a lavagem. </li><li> Preparar cocktails de anticorpo tal como enumeradas a seguir, utilizando a concentração óptima de cada anticorpo com base na diluição de 200 ul de tampão de FACS por poço. A concentração óptima de cada um dos anticorpos é definido como a concentração mínima necessária para se obter a maior separação de populações positivas e negativas. Se não houver uma população negativa para um dado anticorpo, um anticorpo isotipo devem ser incluídas. O volume total por poço pode ser reduzida (por exemplo, a 100 ul por poço), se desejado. <ol><li> WT timo: anti-TCRβ, anti-CD4, anti-CD8, anti-CD69 ou anti-CD5, anti-CD24 </li><li> CD4: HY timo anti-TCR HY (T3.70), anti-CD4, anti-CD8, anti-CD69 ou anti-CD5, anti-CD24 Para obter uma melhor separação entre a CD69 e CD69 eis em populações hi timócitos, recomenda-se que um anticorpo biotinilado anti-CD69 primário ser utilizado, seguido de uma mancha secundária contendo estreptavidina conjugada com fluorocromo. Nós use a mesma estratégia para CD5 coloração. </li></ol></li><li> Incubar as células com 200 uL de mistura de anticorpos em tampão de SCAF, durante 30 minutos em gelo no escuro. </li><li> Concomitante com coloração coquetel de anticorpos, cada mancha controlo de compensação com um anticorpo conjugado com fluorocromo único. Idealmente, a coloração compensação envolve os mesmos anticorpos, usado no cocktail de anticorpos. No entanto, a coloração para cada antigénio individual pode não ser praticável para experiências maiores quando muitos antigénios estão a ser ensaiados. Por isso, tanto para a coloração anti-CD4 ou anti-CD8 é recomendado devido à elevada expressão destes antigénios, ou a utilização de esferas de compensação. Mancha em tampão de FACS durante 30 minutos em gelo no escuro. Deixe um controle de compensação imaculada. </li><li> Lave as células duas vezes com tampão FACS. </li><li> Ressuspender as células em tampão de SCAF e transferência para tubos de FACS. </li><li> Adquirir amostras no citômetro de fluxo. Incluem a aquisição de FSC-A e FSC-W para umGUARDE para a discriminação gibão. </li></ol> 4. Análise de citometria de fluxo de dados - Ratos não transgénicos TCR Usamos FlowJo para a análise de dados de citometria de fluxo. Consulte a Figura 2A para a estratégia de propagação para os ratos não-transgênicos TCR. <ol><li> Usando FSC-A por SSC, portão eletrônico sobre a população &quot;linfócitos&quot;. </li><li> Dentro da população &quot;linfócitos&quot;, use FSC-A pelo FSC-W para eletronicamente população portão eis FSC-W para excluir doublets celulares e aggregrates. Este é o &quot;singleto&quot; portão. </li><li> Usando os eventos no &quot;singlet&quot; portão, lote CD8 por CD4. Desenhe portas para DN, maçante DP, brilhante DP, CD4 + CD8 populações eis CD4SP e CD8SP conforme ilustrado na figura 2B. </li><li> Sob os portões e CD4SP CD8SP, criar TCRβ por CD24 parcelas. Utilize a ferramenta gating quadrante para desenhar portas como mostrado na Figura 2C. </li><li> Criar um new enredo da &quot;singlet&quot; porta: TCRβ por CD69. Desenhar portas A, B, C, D, conforme ilustrado na Figura 2D. </li><li> Criar outra trama do &quot;singlet&quot; porta: TCRβ por CD5. Desenhar portões i, ii, iii, iv, como representado na Figura 2E. Esta é uma outra estratégia para a análise de selecção positiva. </li><li> Aplicar DN, DP opaca, DP brilhante, int CD4, CD4SP e CD8SP portões de debaixo &quot;singleto&quot; para portões i, ii, iii, iv A, B, C, D (Figura 2D, E). </li><li> Calcule o número de células em um subconjunto específico multiplicando celularidade órgão pela freqüência de cada portão posterior até chegar ao seu público-alvo. Por exemplo, o número de TCRβ CD69 hi-CD8SP timócitos seria determinado por timo celularidade x% TCR bhi CD69-(fracção D) x CD8SP%. <ol><li> Contando já leva em conta as células vivas e mortas para que não incluem o lymphoc &quot;gate &quot;YTE nos seus cálculos. </li></ol></li></ol> 5. Análise de dados de citometria de fluxo - TCR transgênico Ratos Consulte a Figura 3A para a estratégia de gating para camundongos transgênicos TCR. <ol><li> Siga os passos 4.1 e 4.2 como na seção anterior. </li><li> Sob o título &quot;singleto&quot; gate, criar um T3.70 por SSC trama e portão na T3.70 + população de células para análise de células T específicas de antigénio (Figura 3B). Em algumas circunstâncias, pode ser de interesse para comparar esta população para o antigénio não específico de população celular (T3.70) T. </li><li> Dentro da população + T3.70, desenhar DN, DP, CD4SP e CD8SP portas (Figura 3C). <ol><li> Para as amostras controle WT, desenhar estas portas sob o &quot;singlet&quot; portão ou criar um T3.70 porta-uma vez que haverá muito poucos T3.70 + células. </li></ol></li><li> Sob o portão CD8SP, criar umT3.70 por CD24 trama. Use a ferramenta de propagação do quadrante para dividir as células em quatro populações (Figura 3F). </li><li> Criar um histograma representando sobreposto expressão CD69 no compartimento DP de ratinhos WT e T3.70 + DP compartimento de camundongos CD4 HY (Figura 4A). Repetir para examinar a expressão CD5 (Figura 4B). </li><li> Calcula-se o número absoluto de células em cada subconjunto de interesse como em 4.8. </li></ol>

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O protocolo aqui apresentado pode ser usado para examinar a selecção positiva e negativa, em não-transgénicos TCR e ratinhos transgénicos TCR. Este protocolo descreve a coloração de antigenes de superfície. Para uma análise mais aprofundada dos mecanismos moleculares, é muitas vezes necessário efectuar a coloração intracelular. Nós usamos o BD Biosciences Kit Cytofix / Cytoperm para a maioria das proteínas intracelulares e da BD Biosciences kit de coloração Foxp3 para fatores de transcrição. Nós gera...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> HyClone solução salina balanceada de Hank </td><td> Thermo Scientific </td><td> SH30030.02 </td><td></td></tr><tr><td> Telas de malha de metal </td><td> Cedarlane </td><td> CX-0080-E-01 </td><td></td></tr><tr><td> Placas de Petri (60 x 15 mm) </td><td> Fisher Scientific </td><td> 877221 </td><td></td></tr><tr><td> Seringas (3 ml) </td><td> BD Biosciences </td><td> 309657 </td><td></td></tr><tr><td> Tubos cônicos (15 ml) </td><td> Sarstedt </td><td> 62.554.205 </td><td></td></tr><tr><td> Microscópio </td><td> Zeiss - Primo Star </td><td> 415.500-00XX-000 </td><td></td></tr><tr><td> Hemocitômetro </td><td> HausserCientífico </td><td> 3110 </td><td></td></tr><tr><td> Placa de 96 poços </td><td> Sarstedt </td><td> 82.1582.001 </td><td></td></tr><tr><td> Pipeta multicanal </td><td> Fisherbrand </td><td> 21-377-829 </td><td></td></tr><tr><td> Soro fetal de vitela </td><td> PAA </td><td> A15-701 </td><td></td></tr><tr><td> Salina tamponada com fosfato </td><td> Fisher Scientific </td><td> SH3025802 </td><td></td></tr><tr><td> A azida de sódio </td><td> IT Baker Chemical Co. </td><td> V015-05 </td><td></td></tr><tr><td> Reagente de bloqueio FcR </td><td></td><td></td><td> Clone 2.4G2 </td></tr><tr><td> Anti-rato HY TCR </td><td> eBioscience </td><td> XX-9930-AA * </td><td> Clone T3.70 </td></tr><tr><td> Anti-CD4 de ratinho </td><td> eBioscience </td><td> XX-0042-AA * </td><td> Clone RM4-5 </td></tr><tr><td> Anti-rato CD8u </td><td> eBioscience </td><td> XX-0081-AA * </td><td> Clone 53-6,7 </td></tr><tr><td> Anti-mouse CD24 </td><td> eBioscience </td><td> XX-0242-AA * </td><td> Clone M1/69 </td></tr><tr><td> Anti-rato TCRβ </td><td> eBioscience </td><td> XX-5961-AA * </td><td> Clone H57-597 </td></tr><tr><td> Anti-CD69 rato biotinilado </td><td> eBioscience </td><td> 13-0691-AA * </td><td> Clone H1.2F3 </td></tr><tr><td> Anti-rato CD5 biotinilado </td><td> eBioscience </td><td> 13-0051-AA * </td><td> Clone 53-7,3 </td></tr><tr><td> Estreptavidina </td><td> eBioscience </td><td> XX-4217-AA * </td><td></td></tr><tr><td> Citómetro </td><td> BD Biosciences - FACS Canto </td><td> 338962 </td><td></td></tr><tr><td> FACS tubos </td><td> BD Biosciences </td><td> 352052 </td><td></td></tr><tr><td> Fluxo de software de análise de citometria </td><td> TreeStar - FlowJo </td><td> FlowJo v7 / 9 </td><td></td></tr><tr><td> HyClone RPMI - 1640 </td><td> Thermo Scientific </td><td> SH30027.01 </td><td></td></tr><tr><td></td><td></td><td></td><td> * XX varia de acordo com fluorocromo e YY varia com o tamanho do frasco. </td></tr></tbody></table>

examination of thymic positive and negative selection by flow cytometry

Um sistema imunitário saudável requer que as células T respondem a antigénios estranhos, permanecendo tolerante para auto-antígenos. Rearranjo aleatório do receptor de células T (TCR) e loci α β gera um repertório de células T com grande diversidade de especificidade antigénica, tanto a auto-e estrangeiras. A selecção do repertório durante o desenvolvimento do timo é crítico para a geração segura e úteis células T. Defeitos de selecção tímica contribuir para o desenvolvimento de doenças auto-imunes e da imunodeficiência 1-4. Progenitores de células T do timo entrar como duplamente negativos (DN) timócitos que não expressam CD4 ou CD8 co-receptores. Expressão da αβTCR e ambos os co-receptores ocorre na fase dupla (DP) positivo. Interacção do αβTCR com auto-péptido-MHC (pMHC) apresentado pelas células do timo determina o destino do timócito DP. Interações de alta afinidade levar a seleção negativa e eliminaçãoção de auto-reactivos timócitos. Baixa afinidade interacções resultam na selecção positiva e desenvolvimento de CD4 ou CD8 positivas únicas células (SP) T capazes de reconhecer antigénios estranhos apresentados pelo MHC próprio 5. Selecção positiva pode ser estudada em ratinhos com um anticorpo policlonal repertório de TCR (tipo selvagem), observando a geração de células T maduras. No entanto, eles não são ideais para o estudo de selecção negativa, o que envolve a eliminação de pequenas populações específicas de antigénio. Muitos sistemas de modelos têm sido utilizados para estudar a selecção negativa, mas variam na sua capacidade para recapitular eventos fisiológicos 6. Por exemplo, a estimulação in vitro de timócitos falta o ambiente do timo, que está intimamente envolvido na selecção, ao passo que a administração de antigénio exógeno pode levar à supressão não específica de timócitos 7-9. Atualmente, as melhores ferramentas para estudar na seleção negativa vivo são ratos que expressam uma transgenic específica TCR para endógena antígeno próprio. No entanto, muitos modelos clássicos TCR transgénicos são caracterizados pela expressão prematura da cadeia TCRα transgénico no estádio de DN, resultando em prematuro de selecção negativa. O nosso laboratório desenvolveu o HY modelo CD4, no qual a transgénico HY TCRα condicionalmente está expresso na fase de DP, permitindo a selecção negativa para ocorrer durante a transição para DP SP como ocorre em ratinhos de tipo selvagem 10. Aqui, nós descrevemos um protocolo de citometria de fluxo com base em examinar selecção positiva e negativa do timo no modelo do rato HY CD4. Enquanto a selecção negativa nos ratos CD4 HY é altamente fisiológico, estes métodos podem também ser aplicados a outros modelos de TCR transgénicas. Nós também irá apresentar as estratégias gerais para a análise de seleção positiva em um repertório policlonal aplicáveis ​​a quaisquer camundongos geneticamente manipulados.

Em fisiológicas modelos TCR transgênicos e camundongos WT, a seleção positiva começa na fase DP brilhante antes de passar à fase DP aborrecido após encontro antígeno. Timócitos DP maçantes em seguida, digite um CD4 + fase de transição CD8 lo antes de se tornar CD4SP ou timócitos CD8SP (Figura 2B). Timócitos maduros SP são caracterizados pela expressão de TCR e da elevada perda de CD24 (Figura 2C). Enquanto o CD8 por ...

Watch this Scientific Journal Video about Examination of Thymic Positive and Negative Selection by Flow Cytometry at JoVE.com

Examination of Thymic Positive and Negative Selection by Flow Cytometry

Apresenta-se um método de citometria de fluxo com base para examinar o desenvolvimento das células T<em&gt; In vivo</em&gt; Usando camundongos geneticamente manipulados em um tipo selvagem ou de fundo do receptor de células T transgênico.

Exame de seleção positiva e negativa Tímico por citometria de fluxo

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

examination-thymic-positive-negative-selection-flow

Research

JoVE Journal

Immunology and Infection

21.6K visualizações.  University of Alberta. Apresenta-se um método de citometria de fluxo com base para examinar o desenvolvimento das células T In vivo Usando camundongos geneticamente manipulados em um tipo selvagem ou de fundo do receptor de células T transgênico.

Vídeo: Examination of Thymic Positive and Negative Selection by Flow Cytometry

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Medir a atividade das calpaínas em células fixas e Vivendo por citometria de fluxo

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Imunologia e Infecção

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Humano In Vitro Supressão como ferramenta de triagem para o reconhecimento de um Estado precoce de desequilíbrio imunológico

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Detecção de nanopartículas fluorescentes Interações com Primário imunitário subpopulações de células por citometria de fluxo

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria.

A avaliação in vivo de Rodent Plasmodium parasitemia e Merozoito Invasion por citometria de fluxo

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Caracterização fenotípica dos macrófagos a partir de rim de rato por Citometria de Fluxo

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Classificação de células activadas por fluorescência Para a purificação de células dendríticas plasmocitï¿½des do rato de Medula Óssea

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolamento e cultura In Vitro de macrófagos alveolares murino e humanos

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Caracterização de subconjuntos de monócitos humanos por sangue total Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

Discriminação de sete subconjuntos de células imunes por dois-fluorocromo citometria de fluxo

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

Preparação de medula óssea inteira para análise de citometria de massa de células de linhagem de neutrófilos

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...