Os autores gostariam de agradecer a senhorita Liana Mackiewicz para assistência técnica. Este trabalho foi possível graças vitoriana Estado de Infra-estrutura de Apoio Operacional e Governo australiano Governo Nacional de Saúde e Conselho de Pesquisa Médica IRIISS e Projeto Grant 1031212.

1. Infecção de camundongos com P. berghei ANKA- <ol><li> Descongelação uma alíquota de P. criopreservado berghei ANKA CHA. </li><li> Conter um cerebral malária resistente rato BALB / c dador (8-12 semanas de idade), utilizando a técnica de retenção de duas mãos. Injectar mouse com 100-200 ul de concentrado de hemácias utilizando uma seringa de insulina 1 ml (28G agulha). Rotineiramente, 1-2 camundongos doadores são injetados. </li><li> Em 4-5 dias pós-infecção (pi) remover camundongo doador de gaiola e coloque-o em uma estação de trabalho ou uma almofada descartável de trabalho. </li><li> Suavemente conter rato pela extremidade da cauda e usando uma tesoura pequena corte da ponta da cauda (cerca de 1 mm). Alternativamente, uma pequena cauda punção utilizando uma agulha de 25 G pode ser realizada. </li><li> Colocar uma gota de sangue a partir da veia da cauda do rato, perto da extremidade de uma lâmina de microscópio fosco end. </li><li> Coloque uma segunda corrediça (espalhador) num ângulo de 45 ° e guardá-lo para a gota de sangue. Quando o sangue se espalha ao longo da borda, empurre o véspera spreader slide-omente através da lâmina de microscópio para fazer o exame de sangue. Permitir o esfregaço secar ao ar. </li><li> Fix esfregaços de sangue em metanol a 100% durante 30 segundos e, em seguida, as lâminas em mancha de Giemsa preparada de fresco durante 10 min. </li><li> Enxaguar esfregaço de sangue com água corrente durante 1 minuto, deixa-se secar ao ar e examinar lâmina sob o microscópio (100x imersão em óleo). Enumerar CHA dentro 1.000 eritrócitos e calcular parasitemia por cento. Ratinhos dadores devem atingir níveis de parasitemia entre 2,5-5% antes de serem sangrados para a infecção de animais de experiência. </li><li> Recolher o sangue do rato doador por sangramento retro-orbital com um tubo de micro-hematócrito capilar heparinizado. Todos os experimentos são realizados em conformidade com o Walter &amp; Eliza Hall Institute animal, as exigências do Comitê de Ética. Dependendo locais animal, as exigências do Comitê de Ética procedimentos sangramento outros possam ser usados. Dissolver a quantidade necessária de sangue em meio RPMI a uma concentração final de 1x10 ml 6 pRBC/0.2. Considere queum hematócrito normal de murganho é ~ 6x10 9 células vermelhas do sangue / 1 ml. </li><li> Infect C57BL experimental / 6 machos (8-12 semanas de idade) por injecção intraperitoneal (ip) 1x10 6 ml pRBC/0.2. </li><li> Para monitorar os níveis de parasitemia de camundongos infectados siga os passos de 1,3-1,8. Parasitemia deve ser determinada em cada 2-3 dias, começando no dia 2 ou 3 pi </li><li> O início da malária grave em camundongos C57BL / 6 pode se manifestar como a aparência corcunda, ruffed pele e baixa atividade. Alguns ratos podem se recuperar nesta fase, mas a progressão para a perda de auto-alinhamento reflexo indica doença irreversível e os animais devem ser sacrificados. </li></ol> 2. Perfusão intracardíaca de Ratos e Extração Cérebro <ol><li> Montar um sistema de perfusão manual de gravidade pela obtenção de um reservatório de 500 ml, a um suporte de coluna ligada à parte superior de um suporte de coluna de 60 cm. Conectar tubos de plástico para a extremidade inferior do reservatório e fixar o grampo de tubo para controlar o fluxo de tampão. Anexar tubo a uma agulha G 23. Fill-se o reservatório com PBS (mantida a 22 ° C), abrir e permitir apertar tampão para ser executado através do tubo para remover todas as bolhas de ar. </li><li> Euthanize P. berghei ANKA rato infectado por inalação de CO 2. Confirmar a morte pela ausência de pedal, orbital e as respostas respiratórias. </li><li> Pin do mouse sacrificados pelas patas traseiras e frente dorsal em uma placa de isopor dissecção contida em uma bandeja de plástico. Dependendo locais de Ética Comitê de anestesia animal requisitos podem ser administrados antes de início pefusion intracardíaca. </li><li> Wipe lado ventral com etanol a 70%. </li><li> Use uma tesoura grande e pinças para abrir pele ao longo da linha média para expor a cavidade torácica. Dobras cutâneas e pino para os lados. </li><li> Segure o esterno com uma pinça fina e cortar o diafragma e ao longo de ambos os lados do esterno cortando as costelas. Tome cuidado para não danificar os grandes vasos sanguíneos. </li><li> Pin da caixa torácica pelo esterno frouxamente ao lado da cabeça. </li><li> Segure sagacidade ventrículosfórceps h finos e cuidadosamente inciso átrio direito com tesoura fina. </li><li> Inserir a agulha 23G ligado a sistema de perfusão gravidade em ventrículo esquerdo para a aorta ascendente, enquanto PBS está em execução. Inserir apenas 0,5 cm da ponta da agulha. </li><li> Perfundir rato por 5 minutos ou até o efluente é clara. </li><li> Desafixar mouse e colocá-lo em seu abdômen. Limpe a cabeça com etanol 70%. </li><li> Com uma tesoura de grande porte, fazer um corte logo acima da área da medula espinhal cervical. </li><li> Use uma tesoura fina para fazer um corte caudo-rostral mediana começando na base do crânio e casca da pele para expor o crânio. </li><li> Segurando a cabeça, colocar as lâminas de uma tesoura pequena em cada cavidade orbital e fazer um corte entre as órbitas. </li><li> Faça um corte longitudinal ao longo da sutura sagital e cuidadosamente descascar o crânio em cada hemisfério cerebral para fora. </li><li> Usando uma espátula, levantar suavemente o cérebro e colocá-lo dentro de um tubo de centrífuga de 10 ml contendo meio RPMI. </li></ol>3. Isolamento de Brain-sequestradas Leucócitos (BSL) <ol><li> Trabalhando numa câmara de segurança, lugar recentemente colhido cérebro em cima de uma peneira de aço inoxidável de células (40-60 malhas) em uma placa de Petri contendo 3-5 ml de meio de cultura RPMI tecido. </li><li> Corte o tecido cerebral em pedaços pequenos. </li><li> Empurrar pequenos fragmentos de tecido do cérebro de células através do filtro utilizando um êmbolo de cristal. </li><li> Transferir homogenato de cérebro de um tubo de 10 ml e centrifugar a 250 xg durante 10 min a 4 ° C. </li><li> Dissolver sedimento em 3 ml de meio RPMI, contendo 0,05% de colagenase D e 2U/ml DNAse I. </li><li> Girar mistura durante 30 min à temperatura ambiente. Remover os detritos celulares, empurrando-se a mistura através de um filtro de 70 | iM de células de nylon e / ou através de incubação em gelo durante 5 min. </li><li> Lay homogenato de cérebro em uma almofada de 7 ml de Percoll a 30% e centrifuga-se a 400 xg (sem travões) durante 20 min à temperatura ambiente. </li><li> Ressuspender o sedimento em 1 ml de tampão de lise de sangue vermelho de células e incubado em gelo durante 5 min alisar CHA aderente, a qual não pode ser removido a partir do cérebro por perfusão intracardíaca. </li><li> Adicionar 9 ml de meio RPMI, lavar e centrifugar as células a 250 xg durante 10 min a 4 ° C. </li><li> Ressuspender o pellet contendo BSL em 50-100 ul de meio RPMI e transferir as células para um tubo de Eppendorf. </li><li> Diluir uma alíquota de 5-10 uL da amostra de células 1:2 em azul de tripano para a identificação de células viáveis. </li><li> Contar as células viáveis ​​no microscópio utilizando um hemocitómetro. A partir de dia 6 pi, quando P. berghei ANKA camundongos infectados exibem sinais neurológicos, 20,000-100,000 BSLs pode ser recuperado. </li></ol> 4. Imunofenotipagem de BSL por citometria de fluxo Multicolour <ol><li> Centrifugar as células a 250 xg durante 10 min a 4 ° C, aspirar o sobrenadante e ressuspender o pellet em 50 ul de tampão de coloração (PBS, 1% de soro fetal de vitelo (FCS), 2 mM de EDTA). </li><li> Adicionar a ul de uma purificado anti-CD16/32 anticorpo (Fcblock). </li><li> Incubar as células durante 10 min em gelo. </li><li> Lavar as células com 1 ml de Tampão de Coloração. Centrifugar as células a 250 xg durante 10 min a 4 ° C e aspirar o sobrenadante. </li><li> Ressuspender as células em 50 ul de tampão de coloração contendo pré-determinadas diluições óptimas de anticorpos fluorescentes necessários, isto é, anti-CD4, anti-CD8, anti-TCR e anti-NK1.1. </li><li> Incubar por 1 hora no gelo. </li><li> Lavar as células com 1 ml de Tampão de Coloração. Centrifugar as células a 250 xg durante 10 min a 4 ° C e aspirar o sobrenadante. </li><li> Ressuspender as células em 100 ul de PBS. </li><li> Transferir as células para um tubo de plástico de citometria de fluxo. </li><li> Utilizando um citómetro de fluxo, adquirir pelo menos 5.000-10.000 eventos. Apropriados controlos de células não coradas e amostras de compensação fluorocromo devem ser incluídos, conforme necessário. </li><li> Analisar os resultados usando o software apropriado citometria de fluxo. </li></ol>

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O isolamento e análise de BSL é um método que permite a caracterização e quantificação de células inflamatórias migram para o cérebro, em resposta a uma lesão tecidual ou infecção em modelos de ratos experimentais. A introdução de um passo de perfusão intracardíaca para a remoção do compartimento de sangue antes da extracção orgânica e o isolamento subsequente das células é útil para prevenir a contaminação de células inflamatórias com não-inflamatórios leucócitos circulantes. Isto pode n...

<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></td><td></td><td></td><td> Soluções e tampões </td></tr><tr><td> Azur Giemsa de eosina solução azul de metileno </td><td> Merck Millipore </td><td> 1.09204.0500 </td><td> Diluição de 1:10 em água destilada </td></tr><tr><td> Meio RPMI </td><td></td><td></td><td> Rato tonicidade </td></tr><tr><td> Rato tonicidade PBS </td><td></td><td></td><td> Fosfato de sódio 20 mM, NaCl 0,149, pH 7,3 </td></tr><tr><td> 0,4% Trypan Blue </td><td> Sigma Aldrich </td><td> T-8154 </td><td> Diluição 1:2 </td></tr><tr><td> Colagenase D </td><td> Worthington Biochemical </td><td></td><td></td></tr><tr><td> Deoxyribonuclease (DNAse) I </td><td> Sigma Aldrich </td><td> D4263-5VL </td><td> A partir de pâncreas bovino </td></tr><tr><td> Percoll </td><td> GE Healthcare </td><td> 17-0891-01 </td><td> Solução a 30% em PBS </td></tr><tr><td> Ultrapura Tris </td><td> Invitrogen </td><td> 15505-020 </td><td></td></tr><tr><td> Cloreto de amônio (NH 4 Cl) </td><td> AnalaR </td><td> 10017 </td><td></td></tr><tr><td> Tampão de Lise Red Cell </td><td></td><td></td><td> 17 mM Tris, 14NM NH 4 Cl, pH 7,2 </td></tr><tr><td> FCS </td><td> Gibco </td><td> 1009 </td><td></td></tr><tr><td> EDTA sal dissódico </td><td> Merck </td><td> 10093.5V </td><td> 0,1 M, pH 7,2 </td></tr><tr><td></td><td></td><td></td><td> Os anticorpos e os conjugados </td></tr><tr><td> Anti-rato CD16/CD32 (Fc Block), clone 2.4G2 </td><td> BD Pharmingen </td> &lt;td&gt; 553142 </td><td> 1 ul de tampão de coloração em 50 ul (0.5mg/50 ml) </td></tr><tr><td> FITC anti-ratinho, clone CD4 H129.19 </td><td> BD Pharmingen </td><td> 553651 </td><td></td></tr><tr><td> PE-anti-rato NK1.1, clone PK136 </td><td> BD Pharmingen </td><td> 553165 </td><td></td></tr><tr><td> PerCPCy5.5 anti-rato CD8, clone 53-6-7 </td><td> BD Pharmingen </td><td> 551162 </td><td></td></tr><tr><td> APC-anti-rato de TCR-β clone, H57-597 </td><td> BD Pharmingen </td><td> 553174 </td><td></td></tr><tr><td> PE-anti-rato CXCR3, clone 220803 </td><td> R &amp; D Systems </td><td> FAB1685P </td><td></td></tr><tr><td> Biotinilado anti-CCR5 do mouse, clone C34-3448 </td><td> BD Pharmingen </td><td> 559922 </td><td></td></tr><tr><td> Steptavidin-PerCP-Cy5.5 </td><td> BD Pharmingen </td><td> 551419 </td><td></td></tr><tr><td></td><td></td><td></td><td> Equipamento e material </td></tr><tr><td> SuperFrost lâmina de microscópio </td><td> Lomb Menzel-Gläser </td><td></td><td></td></tr><tr><td> Pinça de dissecção, tesoura </td><td> REDA Instrumente </td><td></td><td></td></tr><tr><td> 500 ml de PBS reservatório </td><td> Nalgene </td><td></td><td></td></tr><tr><td> Tubo de borracha </td><td></td><td></td><td></td></tr><tr><td> Agulha de 23G </td><td> BD PrecisionGild </td><td> 302008 </td><td></td></tr><tr><td> Dissociação celular kit contendo metal peneira </td><td> Sigma Aldrich </td><td> CD-1 </td><td></td></tr><tr><td> 70 filtro de células mM nylon </td><td> BD Falcon </td><td> 352350 </td><td></td></tr><tr><td> Hemocitômetro </td><td> Neubauer GmbH </td><td> 717810 </td><td></td></tr><tr><td> Citometria de fluxo de tubos </td><td> BD Falcon </td><td> 352008 </td><td></td></tr></tbody></table>

isolation and analysis of brain-sequestered leukocytes from plasmodium berghei anka-infected mice

Descreve-se um método para o isolamento e caracterização de células aderentes inflamatórios dos vasos sanguíneos cerebrais de P. berghei ANKA camundongos infectados. Infecção de estirpes de ratinho-sensíveis com estes resultados de deformação parasita na indução da malária cerebral experimental, uma síndrome neurológica que recapitula certos aspectos importantes da malária Plasmodium falciparum mediada grave em seres humanos 1,2. Formas maduras de sangue fase-malária expressar proteínas parasitas sobre a superfície dos eritrócitos infectados, o que lhes permite ligar-se a células endoteliais vasculares. Este processo induz a obstrução do fluxo sanguíneo, resultando em hipóxia e hemorragias 3 e também estimula o recrutamento de leucócitos inflamatórios para o local de fixação do parasita. Ao contrário de outras infecções, isto é, os vírus neutrotopic 4-6, ambos os malária parasitados glóbulos vermelhos (CGV), bem como associado Inflammleucócitos Atory permanecem sequestrados no interior dos vasos sanguíneos, em vez de se infiltrar no parênquima cerebral. Assim, para evitar a contaminação dos leucócitos sequestrados com não-inflamatórios de células circulantes, perfusão intracardíaca extensa de camundongos infectados-antes da extracção e processamento de tecidos de órgãos é necessária neste procedimento para remover o compartimento do sangue. Após a perfusão, os cérebros são colhidas e dissecado em pequenos pedaços. A estrutura de tecido é adicionalmente interrompido por tratamento enzimático com colagenase D e I. DNAse O homogenato de cérebro resultante é então centrifugado num gradiente de Percoll, que permite a separação de cérebro-sequestradas leucócitos (BSL) de mielina e detritos de outro tecido. As células isoladas são então lavadas, contadas utilizando um hemocitómetro e coradas com anticorpos fluorescentes para posterior análise por citometria de fluxo. Este procedimento permite a caracterização fenotípica abrangente de leucócitos inflamatórios migrar para o cérebro em response a vários estímulos, incluindo acidente vascular cerebral, bem como infecções virais ou parasitárias. O método também proporciona um instrumento útil para a avaliação de novos tratamentos anti-inflamatórias em modelos pré-clínicos com animais.

Os resultados na Fig. 2 percentagens de shows e números absolutos de diferentes populações BSL recuperados de cérebros de ratos perfundidos ou não perfundidada controle de infectados com malária e ingênua. BSL isolado foram coradas com PE-anti-NK1.1 e APC-anti-TCR-β anticorpos, tal como indicado no texto Protocolo. Consistente com resultados anteriores 7-9, as células T αβTCR + compreendia uma elevada proporção da piscina BSL perfundidos em cérebros de ratos infectados com malária (d...

Watch this Scientific Journal Video about Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice at JoVE.com

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

Um método para o isolamento de leucócitos aderentes inflamatórios dos vasos sanguíneos do cérebro<em&gt; Plasmodium berghei</em&gt; ANKA camundongos infectados é descrito. O método permite a quantificação, bem como a caracterização fenotípica de leucócitos isolados, após coloração com anticorpos fluorescentes e subsequente análise por citometria de fluxo.

Isolamento e Análise de Cérebro-sequestrados de Leucócitos<em&gt; Plasmodium berghei</em&gt; ANKA infectadas Mice

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

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Immunology and Infection

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

12.7K visualizações.  The Walter and Eliza Hall Institute of Medical Research. Um método para o isolamento de leucócitos aderentes inflamatórios dos vasos sanguíneos do cérebro Plasmodium berghei ANKA camundongos infectados é descrito. O método permite a quantificação, bem como a caracterização fenotípica de leucócitos isolados, após coloração com anticorpos fluorescentes e subsequente análise por citometria de fluxo.

Vídeo: Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

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 ...

Imunologia e Infecção

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Um modelo experimental para estudar a Tuberculose-Malária Coinfection sobre Transmissão Natural de Mycobacterium tuberculosis E Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Bioenergética e os oxidativo estourou: Protocolos para o Isolamento e Avaliação de leucócitos humanos e plaquetas

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Análise dos efeitos de células estromais no recrutamento de leucócitos de Fluxo

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

Isolamento de Leucócitos do Human Interface materno-fetal

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Isolamento de Leucócitos a partir de tecidos de murino na interface materno-fetal

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Isolamento de leucócitos de infiltração de rato pele utilizando digestão enzimática e separação por gradiente

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

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 ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Isolamento de células mielóides de rato Pele e Drenagem Linfonodo Seguindo intradérmica imunização com vivos atenuados<em&gt; Plasmodium</em&gt; Esporozoítos

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Colheita seletiva de Marginating-hepática Leucócitos

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...