Este trabalho foi financiado por um UNCF-Merck de Pós-Graduação Pesquisa em Ciências Dissertation Fellowship (JLM), National Heart, Lung, and Blood Institute concede RO1-HL-62228 (JSJ) e RO1-HL-073990 (JSJ).

1. Preparação de sal de Hanks Balance Solution (HBSS) <ol><li> Autoclave um copo de vidro L, 1000 mL proveta graduada, e mexa bar. </li><li> Reconstituir a solução HBSS pó no copo de vidro autoclavados 1 L para a marca de 900 mL com água destilada duas vezes. </li><li> Misturar a solução HBSS com 3,4 g de sal Hepes Na + e ajustar o pH para um valor entre 7,35-7,41 usando HCL concentrado ou NaOH. </li><li> Uma vez que o pH desejado foi atingido e permanece estável, adicionar 5 g de soro albumina bovina para a solução à temperatura ambiente até que a albumina é completamente dissolvido. </li><li> Adicionar 10 mL da água antibiótico, antimyotic, para a solução e duplamente destilada até o volume total é de 1000 marca mL. </li><li> Agitar a solução completamente. </li><li> Filtrar a solução HBSS e separá-lo em 50 mL tubos Falcon para uso posterior. Tubos adicionais podem ser congelados e utilizados em uma data posterior. </li></ol> 2. PREPARAÇÃO animaisn - Inserção de tubo traqueal <ol><li> Prepare uma bandeja com instrumentos necessários e material para inserção de um tubo traqueal (fórceps ou seja, tesoura, tubo traqueal, 3-0 sutura trançada preta, ventilador de pequenos animais, etc.) </li><li> Quando o rato é completamente anestesiado com um cocktail de cetamina / xilazina na proporção de 2 para 1 quetamina xilazina, raspar o abdômen, tórax e garganta do rato. Para garantir que um estado anestésico profundo foi alcançado, pinch dedo do rato e verificar se há retirada pedal do membro. Certifique-se que o rato está profundamente anestesiados antes de fazer todas as incisões. </li><li> Coloque o rato com a cabeça levantada em uma placa de corte que é inclinada em um ângulo de 30 ° </li><li> Faça uma incisão na pele a partir de meados do abdômen para abaixo do maxilar inferior. Dissecar longe os músculos do triângulo anterior do pescoço para expor a traquéia. </li><li> Coloque um 3-0 sutura trançada preta sob a traquéia. </li><li> Faça uma incisão na traquéia entre os anéis cartilaginosos umd inserir o tubo traqueal. Amarre o fio firmemente para proteger o tubo traqueal até a traquéia. </li><li> Conecte o tubo traqueal a um ventilador de pequenos animais. </li></ol> 3. Preparação instrumento para isolamento de células imunes <ol><li> Prepare uma bandeja com os instrumentos para o processo imunológico cela de isolamento (fórceps ou seja, tesouras, HBSS, aparados macio Teflon calibre 24 Baxter intravascular sobre a agulha quick-cath) </li><li> Prepare duas seringas 10cc, encha uma seringa com HBSS para a introdução do tampão dentro do saco pericárdio e reservar a outra para aspirar as células contendo HBSS. 3.2.1. Reduzir a ponta de teflon do calibre 24 Baxter intravascular sobre a agulha quick-cath (1,6 cm) por 1 / 2 a 3 / 4 de sua extensão original para o procedimento de isolamento. </li><li> Coloque todas as seringas, buffers, e um vazio 15 mL do tubo Falcon (para conter o fluido extraído uma vez o procedimento esteja concluído) no gelo. </li></ol> &lt;class = p &quot;jove_title&quot;&gt; 4. Isolamento de células imunes <ol><li> Faça uma incisão mediana na parede abdominal (ao longo da linha alba) até chegar ao apêndice xifóide. </li><li> Começando um pouco lateralmente ao processo xifóide, fazer incisões bilaterais através da caixa torácica contínua em um ângulo em direção à axila. Tenha cuidado para não perfurar o pulmão, coração ou pericárdio. </li><li> Uma vez que as incisões em ambos os lados do esterno são completos, dissecar afastado do diafragma das costelas inferiores, o corte lateral para medial. Nota: Esteja sempre ciente de onde o pericárdio se conecta ao diafragma e esterno para evitar danos. Use o ligamento falciforme como um marco para observar onde o pericárdio é anexado no lado oposto do diafragma. Além disso, se o pericárdio é cortado, a capacidade de isolar as células é perdido. </li><li> Levantar delicadamente um dos lados da caixa torácica para expor a cavidade torácica e do coração (ainda encapsulados pelo pericárdio).</li><li> Pegue na seringa preenchida e HBSS encontrar um ponto no saco pericárdico, aproximadamente a meio caminho entre o esterno eo coração (de preferência mais rostral do caudal) e insira a ponta de teflon da encurtado quick-cath através das duas camadas do pericárdio. </li><li> Uma vez que o cateter está em vigor, proceder gentilmente preencher o espaço pericárdico com 2 a 3 ml de tampão HBBS. Você saberá imediatamente se o cateter é colocado corretamente quando você percebe o espaço pericárdico em expansão. Se não houver preenchimento é testemunhado, retirar e reposicionar o quick-cath. </li><li> Quando o saco pericárdico é preenchido, tirar a seringa outros 10 cc e colocar a ponta de volta no pericárdio e começar a aspiração do buffer. O buraco mesma entrada pode ser usado ou de outra pode ser criada. Certifique-se que a ponta não vem muito perto das paredes do saco pericárdico, proporcionando sucção, especialmente na parte de baixo do coração. Isto poderia levar a punção do saco pericárdico e perda de células contendo célulass. </li><li> Enquanto aspiração do buffer, a ponta do cateter pode ser movido dentro do saco pericárdico, a fim de coletar tampão, tanto quanto possível. </li></ol> Nota: Se ao fazê-lo um pouco rasgar o pericárdio e fazer o furo na parte superior do coração maior que o buraco original, não entre em pânico. Enquanto o pericárdio ainda cerca o suficiente do coração, você ainda pode realizar outra lavagem sobre o coração, embora com uma menor quantidade de buffer. Para a coleta de uma quantidade ideal de células, 3-4 lavagens adicionais são necessários. <ol start="9"><li> Coloque o tampão extraído em tubos de Falcon que foram colocados no gelo. </li><li> Remover coração, pulmões, e / ou qualquer outro tecido, se necessário para estudos posteriores. Após a coleta de células do sistema imunológico e outros tecidos, o rato deve ser rápida e humanitariamente sacrificados. </li><li> Centrifugar a coletaram amostras de células imunes por 10 minutos a 200g a 4 ° C. Mantenha sempre os tubos no geloquando não em uso. </li><li> Remover o sobrenadante e reconstituir o sedimento em 1 mL de tampão ou Hyclone HBSS. </li></ol> Nota: É importante contar o número de células imunes obtidos em cada coração após o procedimento imunológico cela de isolamento. 5. Resultados representativos: Um exemplo isolado mastócitos cardíacos obtidos com esta técnica e corados com azul de toluidina e safranina O / alcian blue são apresentados nas figuras 2A e 2B, respectivamente. Como pode ser visto na Figura 3, O mastro cardíaca, mantiveram a sua capacidade de liberação de histamina após o tratamento com ativadores conhecidos, como composto 48/80 e substância P, conforme descrito no Morgan et al. 9 parcelas Representante dispersão de citometria de fluxo de células CD4 + e CD8 + são apresentados nas figuras 4 e 5, respectivamente. Os linfócitos T são ainda identificadas e fenotipadas como uma população de células a partir dessa técnica. Figura Fluxograma 1. Procedimento de isolamento das células imunes cardíaca. <img alt="Figura 2" src="/files/ftp_upload/3020/3020fig2.jpg" /> . Figura 2 imagens representativas histológica de mastócitos cardíacos estão manchadas como se segue: (A) com azul de toluidina e (B) são safranina O / alcian blue. <img alt="Figura 3" src="/files/ftp_upload/3020/3020fig3.jpg" /> Figura 3. Estudo funcional de mastócitos cardíacos isolados da região epicárdica do miocárdio e estimulados com composto 48/80 (10μg/mL) e substância P (10μM). <img alt="Figura 4" src="/files/ftp_upload/3020/3020fig4.jpg" /> Figura 4. Representante fluxo parcelas citometria de dispersão de CD4 + isoladas células marcadas com Substância P (1:100 diluindoion). <img alt="Figura 5" src="/files/ftp_upload/3020/3020fig5.jpg" /> Figura 5. Citometria de fluxo Representante do isolado CD8 + células marcadas com Substância P (1:100).

<ol>
	<li>Brower, G. L., Chancey, A. L., Thanigaraj, S., Matsubara, B. B., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cause+and+effect+relationship+between+myocardial+mast+cell+number+and+matrix+metalloproteinase+activity.">Cause and effect relationship between myocardial mast cell number and matrix metalloproteinase activity.</a> Am. J. Physiol. Heart. Circ. Physiol. 283, H518-H525 (2002).</li><li>Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+inhibition+of+mast+cell+degranulation+prevents+left+ventricular+remodeling+induced+by+chronic+volume+overload+in+rats.">Pharmacologic inhibition of mast cell degranulation prevents left ventricular remodeling induced by chronic volume overload in rats.</a> Journal of Cardiac Failure. 11, 548-556 (2005).</li><li>Chancey, A. L., Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+mast+cell-mediated+activation+of+gelatinase+and+alteration+of+ventricular+diastolic+function.">Cardiac mast cell-mediated activation of gelatinase and alteration of ventricular diastolic function.</a> Am. J. Physiol. Heart. Circ. Physiol. 282, H2152-H2158 (2002).</li><li>Levick, S. P., Gardner, J. D., Holland, M., Hauer-Jensen, M., Janicki, J. S., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+from+adverse+myocardial+remodeling+secondary+to+chronic+volume+overload+in+mast+cell+deficient+rats.">Protection from adverse myocardial remodeling secondary to chronic volume overload in mast cell deficient rats.</a> Journal of Molecular and Cellular Cardiology. 43, 56-61 (2008).</li><li>Levick, S. P., McLarty, J. L., Murray, D. B., Freeman, R. M., Carver, W. E., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Mast+Cells+Mediate+Left+Ventricular+Fibrosis+in+the+Hypertensive+Rat.">Cardiac Mast Cells Mediate Left Ventricular Fibrosis in the Hypertensive Rat.</a> Hypertension. 53, 1041-1047 (2009).</li><li>Batista, M. L., Santos, R. V., Cunha, L. M., Mattos, K., Oliveira, E. M., Seelander, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+pro-inflammatory+cytokine+production+and+peritoneal+macrophage+function+in+rats+with+chronic+heart+failure.">Changes in the pro-inflammatory cytokine production and peritoneal macrophage function in rats with chronic heart failure.</a> Cytokine. 34, 284-290 (2006).</li><li>Galli, S. J., Tsai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+Cells:+Versatile+regulator+of+inflammation,+tissue+remodeling,+host+defense+and+homeostasis.">Mast Cells: Versatile regulator of inflammation, tissue remodeling, host defense and homeostasis.</a> Journal of Dermatological Science. 49, 7-19 (2008).</li><li>Yndestad, A., Holm, A. M., Muller, F., Simonsen, S., Froland, S. S., Gullested, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+expression+of+inflammatory+cytokines+and+activation+markers+in+T-cells+from+patients+with+chronic+heart+failure.">Enhanced expression of inflammatory cytokines and activation markers in T-cells from patients with chronic heart failure.</a> Cardiovasc. Res. 60, 141-146 (2003).</li><li>Bozkurt, B., Kribbs, S. B., Clubb, F. J., Michael, L. H., Didenko, V. V., Hornsby, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiologically+Relevant+Concentrations+of+Tumor+Necrosis+Factor-&#x0251;+Promote+Progressive+Left+Ventricular+Dysfunction+and+Remodeling+in+Rats.">Pathophysiologically Relevant Concentrations of Tumor Necrosis Factor-&#x0251; Promote Progressive Left Ventricular Dysfunction and Remodeling in Rats.</a> Circulation. 97, 1382-1391 (1998).</li><li>Frangogiannis, N. G., Lindsey, M. L., Michael, L. H., Youker, K. A., Bressler, R. B., Mendoza, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+Cardiac+Mast+Cells+Degranulate+and+Release+Preformed+TNF-&#x0251;,+Initiating+the+Cytokine+Cascade+in+Experimental+Canine+Myocardial+Ischemia/Reperfusion.">Resident Cardiac Mast Cells Degranulate and Release Preformed TNF-&#x0251;, Initiating the Cytokine Cascade in Experimental Canine Myocardial Ischemia/Reperfusion.</a> Circulation. 98, 699-710 (1998).</li><li>Jobe, L. J., Melendez, G. C., Levick, S. P., Du, Y., Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-&#945;+inhibition+attenuates+adverse+myocardial+remodeling+in+a+rat+model+of+volume+overload.">TNF-&#945; inhibition attenuates adverse myocardial remodeling in a rat model of volume overload.</a> Am. J. Physiol. Heart. Circ. Physiol. 297, H1462-H1468 (2009).</li><li>Forman, M. F., Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+histamine+secretion+during+isolation+of+rat+cardiac+mast+cells.">Spontaneous histamine secretion during isolation of rat cardiac mast cells.</a> Inflammation Research. 53, 453-457 (2004).</li><li>Levick, S. P., Murray, D. B., Janicki, J. S., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sympathetic+nervous+system+modulation+of+inflammation+and+remodeling+in+the+hypertensive+heart.">Sympathetic nervous system modulation of inflammation and remodeling in the hypertensive heart.</a> Hypertension. 55, 270-276 (2010).</li><li>Morgan, L., Levick, S., Voloshenyuk, T., Murray, D., Forman, M., Brower, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+isolating+functional+mast+cells+from+the+heart.">A novel technique for isolating functional mast cells from the heart.</a> Inflammation Research. 57, 241-246 (2008).</li></ol>

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Este método de isolamento de células do sistema imunológico cardíaca permite a análise de sua função, o fenótipo, e maturidade, sem a ativação indesejados que normalmente ocorre durante a técnica de isolamento enzimático de digestão tradicional. O número médio total de células obtidas com esta técnica é de cerca de 400.000 por coração de rato, e coloração diferencial e análise de citometria de fluxo destas células indica população mista de linfócitos T (~ 70%), macrófagos (~ 12%) e mastócito...

<table border="1"><tbody><tr><td> Nome do reagente e Equipamentos </td><td> Companhia </td><td> Número Catálogo </td><td> Comentários </td></tr><tr><td> Salina balanceada de Hanks </td><td> Sigma </td><td> H4891 </td><td> Uma garrafa faz 1 L de Salt Hanks Balance Solução. Sal reconstituir Hanks Balance Solução, como parte do procedimento. </td></tr><tr><td> Antibióticos Antimyotic </td><td> Gibco </td><td> 15240-062 </td><td></td></tr><tr><td> HEPES </td><td> Sigma </td><td> H3784 </td><td></td></tr><tr><td> Albumina de soro bovino </td><td> Sigma </td><td> A3912 </td><td></td></tr><tr><td> 250 mL Nalogene Bottle Filtro </td><td> Thermoscientific </td><td> 157-0020 </td><td></td></tr><tr><td> 12 N HCL </td><td> Fisher Scientific </td><td> CAS 7647-01-0 </td><td> Por 6 N, adicionar volumes iguais de HCL para deionizadada água. </td></tr><tr><td> 10 N NaOH </td><td> Fisher Scientific </td><td> SC267 </td><td> Por 5 N, adicionar volumes iguais de NaOH à água deionizada. </td></tr><tr><td> Água Destilada dupla </td><td></td><td></td><td></td></tr></tbody></table> Tabela de equipamentos específicos: <table border="1"><tbody><tr><td> Nome do reagente e Equipamentos </td><td> Companhia </td><td> Número Catálogo </td><td> Comentários </td></tr><tr><td> 3-O fio de seda trançada </td><td> Ethicon Produtos </td><td> SA64H </td><td></td></tr><tr><td> Ventilador de Pequenos Animais </td><td> CWE Incorporated </td><td> SAR-830 </td><td> Defina a taxa de respiração para 40,5 respirações por min. </td></tr><tr><td> Bisel da agulha de calibre 18 </td><td> BD Biosciences </td><td> 305199 </td><td> Guarnição da agulha 1 / 2 a 3 / 4 de seu comprimento original para ser usado como um tubo traqueal. </td></tr><tr><td> Dica Teflon (com agulha removida) de calibre 24 Baxter cateter quick-cath </td><td> Baxter </td><td> 2N111 </td><td> Reduzir o comprimento Dica Teflon por 1 / 2 a 04/03. </td></tr><tr><td> Seringa de 10 cc </td><td> Fisher Scientific </td><td> 14-823-16B </td><td></td></tr><tr><td> 15 mL Falcon Tubes </td><td> BD Biosciences </td><td> 352097 </td><td></td></tr><tr><td> Centrífuga Refrigerada Sorvall RT6000B </td><td> Fisher Scientific </td><td> 75-004-377 </td><td> Lembre-se de pré-cool e manter a 4 ° C. </td></tr><tr><td> Hyclone </td><td> Thermoscientific </td><td> S300030.02 </td><td></td></tr><tr><td> Hemocitômetro </td><td> Hausser Scientific </td><td> HS-LB-1483 </td><td></td></tr></tbody></table>

isolation of functional cardiac immune cells

Cardíacas células do sistema imunológico estão ganhando interesse para os papéis que desempenham no remodelamento patológico em muitas doenças cardíacas 05/01 Estas células do sistema imunológico, que incluem os mastócitos, células T e macrófagos;. Armazenar e liberar uma variedade de mediadores biologicamente ativos, incluindo citocinas e proteases, como triptase. 6-8 Esses mediadores têm se mostrado os jogadores-chave no metabolismo da matriz extracelular pela ativação de metaloproteinases de matriz ou causando acúmulo de colágeno por função modular os fibroblastos cardíacos. 9-11 No entanto, as técnicas disponíveis para isolar células do sistema imunológico têm cardíaca sido problemática, porque eles usam a colagenase bacteriana para digerir o tecido do miocárdio. Esta técnica faz com que a ativação das células imunes e, portanto, uma perda de função. Por exemplo, cardíaco mastócitos tornam-se significativamente menos sensíveis aos compostos que causam degranulação. 12 Por isso, desenvolvemos uma técnica que permite a tele isolamento de células do sistema imunológico funcional cardíaca que levaria a uma melhor compreensão do papel destas células na doença cardíaca. 13, 14 Este método requer uma familiaridade com a localização anatômica do rato processo xifóide axila, e ligamento falciforme, e pericárdio do coração. Estes marcos são importantes para aumentar o sucesso do procedimento e garantir uma maior produção de células do sistema imunológico cardíaca. Estes isolados cardíaca células do sistema imunológico podem então ser utilizados para a caracterização do fenótipo, funcionalidade, maturidade e co-cultura experiências com outras células cardíacas para obter uma melhor compreensão de suas interações.

Watch this Scientific Journal Video about Isolation of Functional Cardiac Immune Cells at JoVE.com

Isolation of Functional Cardiac Immune Cells

Este método para isolar células do sistema imunológico funcional do coração fornece uma alternativa aos métodos convencionais de digestão da colagenase, que causa a ativação de células imunes indesejadas, resultando em uma capacidade de resposta diminuiu dessas células. Nosso método de isolamento de rendimentos funcional cardíaca células do sistema imunológico, evitando os problemas associados com a digestão enzimática.

Isolamento de células do sistema imunológico funcional cardíaca

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

isolation-of-functional-cardiac-immune-cells

Research

JoVE Journal

Immunology and Infection

14.9K visualizações.  University of South Carolina- School of Medicine. Este método para isolar células do sistema imunológico funcional do coração fornece uma alternativa aos métodos convencionais de digestão da colagenase, que causa a ativação de células imunes indesejadas, resultando em uma capacidade de resposta diminuiu dessas células. Nosso método de isolamento de rendimentos funcional cardíaca células do sistema imunológico, evitando os problemas associados com a digestão enzimática.

Vídeo: Isolation of Functional Cardiac Immune Cells

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

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Sete Passos para Stellate Células

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

Diferenciando papéis funcionais da expressão do gene de células imunes e não-imunes na colite Rato por Transplante de Medula Óssea

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Isolamento de citometria de fluxo de células primárias Murino Tipo II epiteliais alveolares de Estudos funcionais e Molecular

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation of Adipose Tissue Immune Cells

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

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

Isolamento de tecido adiposo de células imunes

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

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Detecção de glicogênio no sangue periférico células mononucleares com Periodic Acid Schiff Coloração

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

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

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Isolamento, caracterização e exploração funcional da célula de rede gengival Imune

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

Isolamento de células dendríticas do tracto reprodutivo feminino humano para estudos fenotípica e funcionais

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

Isolamento do peritônio-derivado de mastócitos e sua caracterização funcional com Ca2 +-imagens e ensaios de degranulação

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...