Este trabalho foi financiado por bolsa NS64571 do NINDS (CLH), por um prêmio no início de desenvolvimento de carreira da Clínica Mayo (CLH), e por um generoso dom de Donald e Herdrich Frances (CLH). Gostaríamos de agradecer a Clínica Mayo Núcleo Citometria de Fluxo para obter assistência.

1. Injeção intracraniana de vírus: A técnica a seguir foi modificada e utilizada extensivamente por nosso laboratório e colegas. Resumidamente, a injeção intracraniana da estirpe de Daniel de murino Theiler de encefalomielite vírus (TMEV) ou sham-infecção (1, 10) é realizada em ratos jovens (de preferência 5-6 semanas de idade) para obter leucócitos cérebro infiltrando (Bils). Por favor, note que os resultados diferem entre estirpe de Daniel, a cepa feijão e da estirpe GDVII. Para efeitos de colheita Bils, ratos receber 2x10 5 UFP de TMEV e são infectadas por 24 horas. <ol><li> Coloque a agulha de injeção (calibre 27, em ¼, Kendall), a uma seringa de 1 ml automática Hamilton e dê origem a 10 ml de vírus. </li><li> Elaborar TMEV em 10 DMEM mL na seringa com muita atenção a bolhas de ar. Quando for o caso, os ratos sham infectado receber 10 ml de DMEM livre de vírus. </li><li> Pouco antes da injeção, despeje cerca de 1 mL de isoflurano em uma redoma de vidro que contém uma grade de arame com substrato de algodão por baixo. </li><li> Camundongos lugar direclty na grade de arame na redoma até que se tornem ligeiramente anestesiado, insensível aos pés pinch-e imobilizado pelos inalantes anestésico, cerca de 10-15 segundos. Camundongos não deve entrar em contato direto com isoflurano, como substância pode ser irritante e cáustico. </li><li> Enquanto sob anestesia, prepare ratos para injeção através de uma rápida localização de coordenadas apropriado na região do córtex frontal no crânio usando barbear e tinta. A localização geral para injeção é de 1 mm anterior ao bregma e 1 mm lateral à sutura sagital do lado direito (Figura 1). Enquanto a injeção estereotáxica pode ser apropriado para algumas situações experimentais, descobrimos que a mão livre, sem injeção de barbear ou de tinta é adequado para a maioria dos experimentos. Parting a pele com a ponta da agulha ao longo de ambos os eixos é apropriado para injeção. </li><li> Para tomar uma injecção, orientar a agulha perpendicular ao crânio e pressione através do osso para cerca de profundidade de 3 mm. </li><li> Expressar vírus no cérebro, pressionando o botão seringa Hamilton para baixo e esperar 2 segundos para que o vírus entra no cérebro antes de retirar. </li><li> Cuidadosamente retire a agulha e coloque o rato em uma gaiola, limpo e seco. Camundongos despertar da anestesia rapidamente e retomar o funcionamento normal em poucos minutos. Dar a devida atenção para os ratos que apresentam sintomas anormais, como sangramento excessivo no local da injecção, como a eutanásia do animal pode ser apropriado. Animal adequado manuseio 6 e adesão aos Institutos Nacionais de Saúde e Cuidados Animais Institucional e diretrizes Comitê Use devem ser seguidas. </li></ol> 2. Brain-infiltrando preparação celular de leucócitos: <ol><li> Prepare de fundo redondo de Oak Ridge do tubos de centrífuga que pode conter uma capacidade de 30 ml com 10 ml RPMI 1640, 9 ml Percoll e 1 ml de PBS 10X e tubos deve sentar-se na RT no banco durante o processo de remoção do cérebro. </li><li> Traga Ficoll-Paque Plus à temperatura ambiente (RT). </li><li> Eutanásia de animais modificados por overdose isoflurano 5 e remover rapidamente o cérebro com uma espátula de tecidos de aço inoxidável e coloque em um tubo cônico de 15 ml contendo 5 ml RPMI no gelo. Em algumas circunstâncias, PBS-perfusão pode ser apropriado para remover células circulantes do sangue periférico de preparação Bils. Ratos deve ser plena e devidamente anestesiado antes da PBS-perfusão. </li><li> Transferência cérebro e RPMI solução para um copo ml 7 Pyrex marca moedor tecido Tenbroeck e gentilmente homogeneizar com 10-15 derrames. </li><li> Pipeta um adicional de 5 ml RPMI para o homogeneizador e pipeta cima e para baixo duas vezes. Homogenato cerebral transferência (aprox. 10 ml) previamente preparado para tubo de fundo redondo e inverter cuidadosamente 2-3 vezes para misturar. </li><li> Spin-homogenato de cérebro de 7800g av por 30 min a RT em uma F0360 rotor de ângulo fixo em uma centrífuga Beckman Allegra X-22R clínico. </li><li> Após a centrifugação, remover mielina detritos que flutuou para o topo do gradiente. Coletar a camada de leucócitos que flutua acima do pellet de glóbulos vermelhos (Figura 2). </li><li> Strain camada de leucócitos através de um filtro de células 40 mM para a 50 ml. Levar a solução até 50 ml com RMPI. </li><li> Tubos contendo girar suspensão diluída de células em uma centrífuga clínica a 1500 rpm (600g ave) por 5 min à temperatura ambiente. </li><li> Aspirar o sobrenadante e recolha o pellet celular. Ressuspender o sedimento em 1 ml tampão FACS (1% albumina sérica bovina, azida sódica 0,02% em cálcio e magnésio livres PBS) e transferir a suspensão celular a 5 ml FACS rodada tubos de fundo. </li><li> Cuidadosamente suspensão underlay com 1 ml Além disso Ficoll-Paque. </li><li> Tubo giratório a 2500 rpm (1400 deu) em centrífuga clínica por 25 min à temperatura ambiente sem freio. </li><li> Remover camada, branco macio na interface (Figura 2) com uma pipeta ml e transferir para o novo 5 ml tubo FACS. Lavar cells com 4 tampão FACS ml. </li><li> Tubo giratório a 1500 rpm (600g ave) por 5 min à temperatura ambiente em centrífuga clínica para as células da pelota. </li><li> Aspirar o sobrenadante e recolher pellet celular. Ressuspender o pellet de leucócitos em tampão apropriado e manter no gelo em tubo FACS novo. </li><li> Contagem de células, avaliar a viabilidade celular por exclusão do azul Trypan e analisar por citometria de fluxo. Em geral, os investigadores podem esperar para adquirir cerca de 5 x 10 5 células / animal. </li></ol> 3. Imunofenotipagem por citometria de fluxo <ol><li> Após o isolamento e contagem de leucócitos, as células rodada por 3 minutos a 1500 rpm (600 deu) em centrífuga clínica. </li><li> Ressuspender o sedimento em tampão de bloqueio contendo o seguinte: tampão FACS, sobrenadante de 2.4G2 hibridoma (Fc block; anti-CD16/32) e soro fetal bovino na proporção de 10:05:01 e incubar por 30 minutos a 4 ° C . </li><li> Prepare e adicione os anticorpos contra antígenos conjugados extracelular para as células bloqueadas em uma concentração de 1:200 e incubar por 30 minutos a 4 ° C no escuro. Stain e incubar as células em 200 mL de uma placa de 96 poços V-bottom. Controles apropriados incluem células imaculada e amostras de compensação fluorocromo, conforme necessário. </li><li> Girar a células coradas em RT por 3 minutos a 1500 rpm (600 deu) em centrífuga clínica usando um apropriado rotor para placas de 96 poços. </li><li> Aspirar o sobrenadante e lavar as células com 200 mL de buffer FACS cuidado pipetando para cima e para baixo 3 vezes. </li><li> Repita a técnica de lavagem acima duas vezes. </li><li> Após a lavagem, ressuspender as células em paraformaldeído 2% e transferir para tubos FACS. Fixação é necessário para análise de citometria de fluxo de células infectadas do Nível de Biossegurança 2 reagentes. </li><li> Executar as células fixas em um BD FACS Calibur seguir um método modificado de citometria de fluxo 8 e analisar arquivos off-line usando programa FlowJo, WinMDI ou outros disponíveis fluxo de software de análise de citometria. </li><li> A análise dos 50-100,000 eventos por amostra geralmente é necessária para imunofenotipagem adequada. </li></ol> Anticorpos diretamente conjugados utilizados neste experimento: CD45 foi detectado com o clone de 30 F11. Ly6C / G foi detectado com o clone Gr1, RB6-8C5. CD11b foi detectado com o clone M1/70. F4/80 foi detectado com o clone BM8. 4. Resultados representativos: Apresentados os resultados de células para fenotipagem Bils do mouse em 24 horas após a infecção (Figura 3). Leucócitos foram coradas com conjugado anti-PerCP mouse-CD45 para detectar células do sistema imunológico, PE-conjugado anti-mouse Ly6C / G para detectar monócitos inflamatórias, APC-conjugado anti-mouse CD11b e APC-conjugado anti-rato F4/80 para detectar células da linhagem de monócitos. A análise foi realizada com um instrumento BD FACS Calibur. <img src="/files/ftp_upload/2747/2747fig1.jpg" alt="Figura 1"/> Figura 1. Localização anatômica do local da injeção. O local de injecção está localizado a 1 mm anterior ao bregma e 1 mm lateral à sutura sagital do lado direito. <img src="/files/ftp_upload/2747/2747fig2.jpg" alt="Figura 2"/> Figura 2. Leucócitos ilustração de separação por gradiente de leucócitos e Bils. São inicialmente coletados diretamente abaixo da camada de mielina e detritos acima do pellet RBC no gradiente de Percoll. A camada, branco macio (Bils) na interface é coletado do gradiente de Ficoll. <img src="/files/ftp_upload/2747/2747fig3.jpg" alt="Figura 3"/> Figura 3. Imunofenótipo de cérebro-infilatrating leucócitos em 24 horas pós-infecção leucócitos. Rato foram isoladas a partir do cérebro por centrifugação diferencial densidade do tecido homogeneizados preparados a partir de animais em 24 horas após a infecção intracraniana. Células foram marcadas com anticorpos fluorescente-conjugada contra o rato CD45, Ly6C / G, CD11b e F4/80. Gating inicial foi realizada plotando CD45, um marcador de células do sistema imunológico, contra a dispersão para a frente (FSC), um indicador do tamanho da célula (C). O oi CD45 (A, D), CD45 lo (B, E) e CD45 neg (F, G) populações foram então analisados ​​para os níveis de expressão relativa dos marcadores de monócitos e macrófagos Ly6C / G, F4/80 (A, B , F), e CD11b (D, E, G). Ly6C / G + + F4/80 CD11b + monócitos inflamatórias foram encontradas quase que exclusivamente na população oi CD45 (A, D), de acordo com a infiltração destas células a partir da periferia. Em contraste, macrófagos CD11b Ly6C/G-F4/80 lo + foram encontradas quase que exclusivamente na lo CD45 (B, E) e neg CD45 (G) populações, de acordo com um fenótipo residente. Em numerosos experimentos, nós nunca observamos células CD45 oi ou Ly6C / G + + F4/80 CD11b + monócitos inflamatória no cérebro de não infectados ou sham-infecçãoted camundongos (dados não mostrados).

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	<li>Buenz, E. J., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picornaviruses+and+cell+death.">Picornaviruses and cell death.</a> Trends Microbiol. 14, 28-36 (2006).</li><li>Buenz, E. J., Limberg, P. J., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+3-parameter+flow+cytometry-based+cell+death+assay.">A high-throughput 3-parameter flow cytometry-based cell death assay.</a> Cytometry A. 71, 170-173 (2007).</li><li>Deb, C., Lafrance-Corey, R. G., Zoecklein, L., Papke, L., Rodriguez, M., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demyelinated+axons+and+motor+function+are+protected+by+genetic+deletion+of+perforin+in+a+mouse+model+of+multiple+sclerosis.">Demyelinated axons and motor function are protected by genetic deletion of perforin in a mouse model of multiple sclerosis.</a> J. Neuropath. Exp. Neurol. 68, 1037-1048 (2009).</li><li>Deb, C., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKG2D+contributes+to+efficient+clearance+of+picornavirus+from+the+acutely+infected+murine+brain.">NKG2D contributes to efficient clearance of picornavirus from the acutely infected murine brain.</a> J. Neurovirol. 14, 261-266 (2008).</li><li>Donovan, J., &amp;amp, P. . B. r. o. w. n., , P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Euthanasia.">Euthanasia.</a> Current Protocols in Neuroscience. , A.4H.1-A.4H.4 (2005).</li><li>Donovan, J., Brown, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Handling+&#38;+restraint.">Handling &#38; restraint.</a> Current Protocols in Immunology. 73, 1.3.1-1.3.6 (2006).</li><li>Donovan, J., Brown, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parenteral+injections.">Parenteral injections.</a> Current Protocols in Neuroscience. , A.4F.1-A.4F.9 (2005).</li><li>Holmes, K. L., Otten, G., Yokoyama, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+analysis+using+Becton+Dickinson+FACS+Calibur.">Flow cytometry analysis using Becton Dickinson FACS Calibur.</a> Current Protocols in Immunology. , 5.4.1-5.4.22 (2002).</li><li>Howe, C. L., Ure, D., Adelson, J. D., LaFrance-Corey, R., Johnson, A., Rodriguez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T+cells+directed+against+a+viral+peptide+contribute+to+loss+of+motor+function+by+disrupting+axonal+transport+in+a+viral+model+of+fulminant+demyelination.">CD8+ T cells directed against a viral peptide contribute to loss of motor function by disrupting axonal transport in a viral model of fulminant demyelination.</a> J Neuroimmunol. 188, 13-21 (2007).</li><li>Lipton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theiler's+virus+infection+in+mice:+An+unusual+biphasic+disease+process+leading+to+demyelination.">Theiler's virus infection in mice: An unusual biphasic disease process leading to demyelination.</a> Infect Immun. 11, 1147-1155 (1975).</li></ol>

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Rotineiramente uso de citometria de fluxo para determinar a qualidade da preparação das células cerebrais se infiltrando, e para distinguir diferentes populações de células do sistema imunológico 2, 9. Aguda em tempo de pontos, o nosso método Bils rendimentos elevados percentuais de monócitos inflamatória dentro da população CD45hi, bem como altas porcentagens de macrófagos na população CD45lo. Isto indica que uma resposta imune reprodutíveis dentro do cérebro pode ser robustamente caracteriz...

<table border="1"><tbody><tr><th> Reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> TMEV </td><td> Howe Lab </td><td> N / A </td><td></td></tr><tr><td> Estirpe de Daniel </td><td> Invitrogen </td><td> 21063-029 </td><td></td></tr><tr><td> isoflurano </td><td> Novaplus </td><td> NDC 0409-3292-49 </td><td></td></tr><tr><td> de fundo redondo de Oak tubos de centrífuga de Ridge </td><td> Nalgene </td><td> 3118-0030 </td><td></td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 11875-093 </td><td></td></tr><tr><td> Percoll </td><td> GE Healthcare </td><td> 17-0891-02 </td><td></td></tr><tr><td> PBS 10X </td><td> Roche </td><td> 11666789001 </td><td></td></tr><tr><td> Ficoll-Paque Plus </td><td> GE Healthcare </td><td> 17-1440-02 </td><td></td></tr><tr><td> Trypan Blue 0,4% (w / v) </td><td> Mediatech </td><td> 25-900-CI </td><td></td></tr><tr><td> 15 ml tubos cônicos </td><td> BD Falcon </td><td> 352097 </td><td></td></tr><tr><td> 7 mL de vidro Pyrex marca moedor de tecido Tenbroeck </td><td> Pescador </td><td> 08-414-10B </td><td></td></tr><tr><td> 40 coador célula M </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> 50 ml </td><td> BD Falcon </td><td> 352070 </td><td></td></tr><tr><td> albumina de soro bovino </td><td> Sigma </td><td> A9647 </td><td></td></tr><tr><td> azida de sódio </td><td> Sigma </td><td> S8032 </td><td></td></tr><tr><td> CMF-PBS </td><td> Mediatech </td><td> 21-040-CV </td><td></td></tr><tr><td> FACS tubos </td><td> BD Falcon </td><td> 352054 </td><td></td></tr><tr><td> soro fetal bovino </td><td> Sigma </td><td> F4135 </td><td></td></tr><tr><td> 2.4G2 hibridoma </td><td> ATCC </td><td> HB-197 </td><td></td></tr><tr><td> Costar placa V-bottom </td><td> Corning </td><td> 3894 </td><td></td></tr><tr><td> Allegra centrífuga X-22R ou equivalente </td><td> Beckman </td><td> N / A </td><td></td></tr><tr><td> De 96 poços balde placa e rotor </td><td> Beckman </td><td> S2096 </td><td></td></tr><tr><td> Rotor de ângulo fixo </td><td> Beckman </td><td> F0360 </td><td></td></tr><tr><td> paraformaldeído </td><td> Sigma </td><td> P6148 </td><td></td></tr><tr><td> BD FACS Calibur </td><td> BD Biosciences </td><td> N / A </td><td></td></tr><tr><td> Programa FlowJo 7,5 </td><td> Árvore Star, Inc. </td><td> N / A </td><td></td></tr><tr><td> CD45 </td><td> BD Biosciences </td><td> 557235 </td><td> clone: ​​30-F11 </td></tr><tr><td> Gr1 (Ly6C / G) </td><td> BD Biosciences </td><td> 553128 </td><td> clone: ​​RB6-8C5 </td></tr><tr><td> CD11b </td><td> ebiosciences </td><td> 17-0112-83 </td><td> clone: ​​M1/70 </td></tr><tr><td> F4/80 </td><td> ebiosciences </td><td> 17-4801-82 </td><td> clone: ​​BM8 </td></tr></tbody></table>

isolation of brain-infiltrating leukocytes

Nós descrevemos um método para preparar leucócitos cérebro infiltrando (Bils) de camundongos. Demonstramos como para infectar ratos com encefalomielite murina de Theiler vírus (TMEV) através de uma técnica de injeção intracraniana e rápida como purificar uma população de leucócitos enriquecido de células infiltrando a partir do cérebro inteiro. Resumidamente, os ratos são anestesiados com isoflurano em uma câmara fechada e são free-hand injetado com uma seringa de Hamilton para o córtex frontal. Ratos são, então, matou em vários momentos após a infecção por overdose isoflurano e cérebros inteiros são extraídos e homogeneizados em RPMI com um moedor de tecido Tenbroeck. Homogeneizados de cérebro são centrifugação em um gradiente de Percoll contínua de 30% para remover os restos celulares de mielina e outros. A suspensão de células é então forçada a 40 mM, lavado e centrifugado em um descontínua gradiente de Ficoll-Paque Plus para selecionar e purificar os leucócitos. Os leucócitos são então lavadas e ressuspendidas em tampão apropriado para imunofenotipagem por citometria de fluxo. Citometria de fluxo revela uma população de células do sistema imunológico inato nas fases iniciais da infecção de camundongos C57BL / 6. Menos 24 horas após a infecção, subconjuntos múltiplas de células do sistema imunológico estão presentes no Bils, com uma população enriquecida de Gr1 +, CD11b + e + F4/80 células. Portanto, esse método é útil na caracterização da resposta imune à infecção aguda no cérebro.

Watch this Scientific Journal Video about Isolation of Brain-infiltrating Leukocytes at JoVE.com

Isolation of Brain-infiltrating Leukocytes

Um método rápido para a obtenção de leucócitos infiltrando a partir do cérebro de camundongos é descrito. Este método utiliza um gradiente de Percoll contínua e descontínua gradiente de Ficoll para selecionar e purificar a camada de leucócitos enriquecido. Leucócitos isolados podem então ser caracterizada por medidas de citometria de fluxo.

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-brain-infiltrating-leukocytes

Research

JoVE Journal

Immunology and Infection

19.0K visualizações.  Mayo Clinic College of Medicine. Um método rápido para a obtenção de leucócitos infiltrando a partir do cérebro de camundongos é descrito. Este método utiliza um gradiente de Percoll contínua e descontínua gradiente de Ficoll para selecionar e purificar a camada de leucócitos enriquecido. Leucócitos isolados podem então ser caracterizada por medidas de citometria de fluxo.

Vídeo: Isolation of Brain-infiltrating Leukocytes

Isolation of Mouse Peritoneal Cavity Cells

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

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

Isolamento de células de camundongo Peritoneal Cavity

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

Imunologia e Infecção

Isolation of Functional Cardiac Immune Cells

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

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

Isolamento de células do sistema imunológico funcional cardíaca

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

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

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

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

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

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. ...

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 ...

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Colheita seletiva de Marginating-pulmonar Leucócitos

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

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 ...