Dr. Pramod Nehete e Sra. Bharti Nehete de sangue e amostras de PBMC macaco; Este trabalho foi parcialmente financiado com recursos do NIH AI 46969 conceder, e todos os meios de cultura de células foram produzidos pelo Laboratório Central de Mídia, que é financiado pelo NIH conceder CA 16672.

Os procedimentos para a análise detalhada fenotípica e funcional do total, bem como subconjuntos de memória das células T pode ser dividido em quatro partes: 1) a preparação de células e estimulação antigênica, 2) coloração da superfície marcador, 3) coloração de citocinas intracelulares e 4) citometria de fluxo análises. 1. Preparação celular e estimulação antigênica <ol><li> Ambos os recém-isoladas, bem como crio-preservado células mononucleares do sangue periférico (PBMC) foram utilizados neste protocolo. O PBMC foram isoladas de amostras venosas heparinizado ou citratado sangue de macacos rhesus por sedimentação gradiente de densidade Ficoll-Hypaque (Histopaquen-1077, Sigma-Aldrich, St. Louis, MO). Alíquotas de PBMC foram armazenadas congeladas em FCS a 90% e DMSO 10% em nitrogênio líquido. </li><li> Ao usar o crio-preservado PBMC, os frascos de PBMC congelados foram retirados do nitrogênio líquido e descongeladas rapidamente em banho-maria 37 C, delicadamente misturados, lavadas com RPMI-1640 (HyClone laboratórios, logan, UT) para remover o meio de congelamento e re-suspensas em meio completo [CM; RPMI-1640 suplemento com 10% inativado pelo calor FCS (HyClone laboratórios), 2mM de L-glutamina (Sigma-Aldrich), 100U/ml penicilina / estreptomicina (Invitrogen), e cultivadas em 6 - placas de cultura de tecido bem durante a noite a 37 ° C em 5% CO 2 umidificado atmosfera. Na manhã seguinte, contagens de células viáveis ​​foram determinados pelo método de exclusão de tripan azul-dye e re-suspenso no CM. </li><li> As células foram tratadas de forma diferente para não-específica contra antígenos específicos estímulo seguido por análises de citocinas: (a) Para os não-específicas de estimulação com forbol 12-miristato 13-acetato (PMA) e Ionomicina (I) (Sigma-Aldrich, St. Louis, MO), alíquotas de células em um volume de 0,1 ml (1,0 x 10 6 células / poço) foram plaqueadas em poços individuais de placas de 96 poços de cultura de tecidos (BD Biosciences, Franklin lago, NJ). O PMA e Ionomicina foram usados ​​na concentração final de 50ng/mL e 500ng/ml, respectivamente. O volume final total de 96 poços da placa de cultura de tecidos é 0.2ml/well: (b) Para a estimulação antígeno específico, 1.0 x 10 6 células em um volume de 1,0 ml foram semeadas em poços individuais de placas de 24 poços de cultura de tecidos (BD Biosciences , Franklin lago, NJ), onde os poços foram pré-tratados conforme descrito anteriormente com modificações 9. Resumidamente, a placas de 24 poços de cultura de tecido foram revestidas com 2,5 mg / ml / poço de cabra anti-IgG de rato (H + L) (Kierkegaard e Perry Laboratory, Gaithersburg, MD) em solução Tris 50 mM (pH 8,6) para 4 ° C durante a noite. Na manhã seguinte, as placas foram lavadas duas vezes com PBS estéril e co-estimulatórias mAb CD49d, clone 9F10 (BD Biosciences, San Jose, CA) foram adicionados a 10 mcg / ml / poço seguido de incubação durante uma hora a 37 ° C. Após a incubação, as placas foram lavadas duas vezes com PBS estéril à temperatura ambiente. Os antígenos de interesse (incluindo cocktail de seis peptídeos envelope HIV 8 foram adicionados a uma concentração final de 10μg/ml de cada peptídeo. Poços adicionais com as células de CM só foram preparadas como controle negativo. O volume final total para cada poço dos 24 bem-placa de cultura de tecidos foi de 1,0 ml. </li><li> As células foram cultivadas por seis horas a 37 ° C em 5% CO 2 umidificado atmosfera. Brefeldin A (Sigma-Aldrich, St. Louis, MO) foi adicionada à cultura no 10μg/ml para a final 4,5 horas de estimulação. Posteriormente, as células foram transferidas para 5 tubos de polipropileno ml e lavados com água fria (4 ° C) tampão de lavagem de fluxo (PBS Dulbecco (DPBS, Ca 2 / Mg 2-livre, tecnologias de Vida, Rockville, MD) com 2% inativado pelo calor FCS), e em seguida processados ​​para a coloração de fluorescência com os vários anticorpos marcados. </li></ol> 2. Imunofluorescência marcadores de superfície coloração <ol><li> Células estimuladas foram inicialmente coradas com 1ul fixable aquático vivo / morto corante fluorescente reativa a 4 ° C no escuro por 30 minutos, lavado uma vez com tampão frio fluxo de lavagem. </li><li> Então, o seguinte adequadamente titulada fluorescência marcado anticorpos monoclonais foram adicionados às células: anti-CD3 PE-Cy7 (SP34-2), anti-CD8 Alexa700 (RPA-T8), CD28-anti-PerCP cy5.5 (L293) , anti-CD95 APC (DX2) e anti CD4 Pacific Blue (OKT4), todos da BD Biosciences (Franklin lago, NJ), exceto CD4 Pacific Blue (OKT4) de eBioscience (San Diego, CA). As células foram incubadas por 30 minutos a 4 ° C no escuro. </li><li> Para cada experimento compensação de controles e de fluorescência menos um (FMO) controla 10,11 foram utilizados. Controles de compensação foram utilizados para determinar a coeffecients spillover de cada indivíduo mancha em outros detectores. Para os controles de compensação, um conjunto completo de tubos contendo suspensões de células de um dos macacos foram coradas com conjugado fluorescente mAb individualmente como manchas de cor única. FMO é uma combinação coloração multicolor que contém todos os reagentes, mas a de interesse e é utilizada para determinar o limite entre um positivo umand populacional negativo, duplicando auto-fluorescência nível e os dados presentes na amostra totalmente manchado. </li><li> Células coradas foram então lavadas com tampão de fluxo de lava fria, e fixado em fixação / permeabilização solução (BD Biosciences, San Jose, CA) para pelo menos 10 minutos (nesta fase as células podem ser armazenadas durante a noite a 4 ° C no escuro) antes de realizar a coloração de citocinas intracelulares. </li></ol> 3. Coloração de citocinas intracelulares <ol><li> Células fixadas foram lavadas com tampão de lavagem de fluxo, e depois Perm / Tampão de lavagem (BD Biosciences, San Jose, CA) foi adicionado de acordo com as instruções do fabricante. Resumidamente, as células foram incubadas em 0,1 ml do Perm 1X / Tampão de lavagem para 10 minutos a 4 ° C no escuro. </li><li> Posteriormente, as células foram incubadas com o adequadamente titulada FITC-rotuladas anti-IFN-γ (B27) e PE-rotuladas anti-IL-2 (MQ1-17H12) durante 60 minutos a 4 ° C no escuro. </li><li> Em seguida, as células foram lavadas mais duas vezes com a solução de permeabilização </li><li> Após a última lavagem, as células foram re-suspensas em paraformaldeído 1% em DPBS e submetidas a análise de citometria de fluxo em 24 horas. </li></ol> 4. Citometria de fluxo <ol><li> As células coradas foram adquiridos em um fluxo de LSR citómetro II (BD Biosciences, San Jose, CA) ou ADP Ciano (Dako, Carpinteria, CA). FACS dados foram analisados ​​usando programa FlowJo (Árvore Star, Ashland, OR). </li><li> Figura. 1 mostra o esquema de propagação utilizados para as análises dos subconjuntos diferentes de células T de um animal representativo. Os linfócitos foram os primeiros gated via dispersão para a frente (FSC) versus dispersão lateral (SSC), e, em seguida, linfócitos vivos foram identificados com base em SSC e aqua-negativos população. As células T foram positivamente identificados pela expressão CD3 seguido pela detecção do CD4 + CD8-(CD4 + T cells) e CD4-CD8 + (células T CD8 +) populações, dentro do CD3 + T população celular. O CD4 + e CD8 + T foram ainda distinguidos em subconjuntos diferentes com base em CD28 e CD95 expressão como ingênuo (Tn, CD28 + CD95-), a memória central (Tcm, CD28 + CD95 +) e memória efetora (Tem, CD28-CD95 +) células como descrito na literatura 5, 10. </li><li> Figura. 2 mostra como os diferentes subgrupos de células T CD4 + e CD8 + T foram avaliados para a capacidade funcional em termos da produção de citocinas (INF-γ e / ou IL-2) em resposta à estimulação com PMA e ionomicina (PMA + I). </li></ol> 5. Representante FACS dados PBMC composição em macacos rhesus foi determinada usando análise FACS. A Figura 1 mostra o esquema de propagação utilizados para as análises dos subconjuntos diferentes de células T de um animal representativo. Os linfócitos foram os primeiros gated via dispersão para a frente (FSC) versus dispersão lateral (SSC), e, em seguida, linfócitos vivos foram identificados com base em SSC e aqua-negativos população. As células T foram identificadas por CD3 expressão. CD4 + CD8-(as células CD4 + T) e CD4-CD8 + (células T CD8 +) populações, dentro do CD3 + T população celular também foram determinados. Conforme mostrado na Figura 2, o CD4 + e CD8 + T foram ainda distinguidos em subconjuntos diferentes com base em CD28 e CD95 expressão como ingênuo (CD28 Tn, + CD95), a memória central (Tcm, CD28 + CD95 +) e memória efetora (Tem, CD28-CD95 +) células. Figura 2A mostra INF-γ e IL-2 de produção em CD4 unstimulated e PMA / ionomicina estimulado + células T e subconjuntos, e Figura 2B mostra INF-γ e IL-2 de produção em células não estimuladas e PMA / ionomicina estimulado CD8 + T e subconjuntos Nota dos perfis de citocinas distintas eliciada pela estimulação em cada subconjunto. <img src="/files/ftp_upload/1743/1743fig1tmb.jpg" alt="Figura 1" border="0" /> Figura 1. Gating estratégia utilizada para análise de citometria de fluxo de composição rhesus em PBMC macqaues. Por favor, <a href="https://www.jove.com/files/ftp_upload/1743/1743fig1.jpg">clique aqui</a> para ver uma versão ampliada da figura 1. <img src="/files/ftp_upload/1743/1743fig2atmb.jpg" alt="Figura 2a" border="0" /> <img src="/files/ftp_upload/1743/1743fig2btmb.jpg" alt="Figura 2b" border="0" /> Figura 2. (A) perfil de produção de citocinas CD4 + total de células T e subconjuntos. (B) perfil de produção de citocinas do total células CD8 + T e subconjuntos. Por favor, <a href="https://www.jove.com/files/ftp_upload/1743/1743fig2a.jpg">clique aqui</a> para ver uma versão maior da 2a, <a href="https://www.jove.com/files/ftp_upload/1743/1743fig2b.jpg">ou aqui</a> para uma versão ampliada da figura 2b.

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	<li>Ogg, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+HIV-1-specific+cytotoxic+T+lymphocytes+and+plasma+load+of+viral+RNA.">Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA.</a> Science. 279, 2103-2106 (1998).</li><li>Borrow, P., Lewicki, H., Hahn, B. H., Shaw, G. M., Oldstone, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-specific+CD8++cytotoxic+T-lymphocyte+activity+associated+with+control+of+viremia+in+primary+human+immunodeficiency+virus+type+1+infection.">Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection.</a> J Virol. 68, 6103-6110 (1994).</li><li>Koup, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+association+of+cellular+immune+responses+with+the+initial+control+of+viremia+in+primary+human+immunodeficiency+virus+type+1+syndrome.">Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome.</a> J Virol. 68, 4650-4655 (1994).</li><li>Walker, J. M., Maecker, H. T., Maino, V. C., Picker, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+flow+cytometric+analysis+in+SIV-infected+rhesus+macaque.">Multicolor flow cytometric analysis in SIV-infected rhesus macaque.</a> Methods Cell Biol. 75, 535-557 (2004).</li><li>Pitcher, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+homeostasis+of+T+cell+memory+in+rhesus+macaque.">Development and homeostasis of T cell memory in rhesus macaque.</a> J Immunol. 168, 29-43 (2002).</li><li>Jung, T., Schauer, U., Heusser, C., Neumann, C., Rieger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+intracellular+cytokines+by+flow+cytometry.">Detection of intracellular cytokines by flow cytometry.</a> J Immunol Methods. 159, 197-207 (1993).</li><li>Keeney, T. S., Nomura, L. E., Maecker, H. T., Sastry, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+macaque+whole+blood+for+antigen-specific+intracellular+cytokine+production+by+T+lymphocytes.">Flow cytometric analysis of macaque whole blood for antigen-specific intracellular cytokine production by T lymphocytes.</a> J Med Primatol. 32, 23-30 (2003).</li><li>Gauduin, 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=Optimization+of+intracellular+cytokine+staining+for+the+quantitation+of+antigen-specific+CD4++T+cell+responses+in+rhesus+macaques.">Optimization of intracellular cytokine staining for the quantitation of antigen-specific CD4+ T cell responses in rhesus macaques.</a> J Immunol Methods. 288, 61-79 (2004).</li><li>Baumgarth, N., Roederer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+approach+to+multicolor+flow+cytometry+for+immunophenotyping.">A practical approach to multicolor flow cytometry for immunophenotyping.</a> J Immunol Methods. 243, 77-97 (2000).</li><li>De Rosa, S. C., Herzenberg, L. A., Roederer, M. 1. 1. -. c. o. l. o. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=13-parameter+flow+cytometry:+identification+of+human+naive+T+cells+by+phenotype,+function,+and+T-cell+receptor+diversity.">13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity.</a> Nat Med. 7, 245-248 (2001).</li><li>Tung, J. W., Parks, D. R., Moore, W. A., Herzenberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+to+fluorescence+compensation+and+visualization+of+FACS+data.">New approaches to fluorescence compensation and visualization of FACS data.</a> Clin Immunol. 110, 277-283 (2004).</li></ol>

Solução e Buffers <ul><li> Ficoll-Hypaque (Histopaquen-1077, Sigma-Aldrich, St. Louis, MO) </li><li> RPMI-1640 meio (HyClone laboratórios, Logan, UT) </li><li> Soro fetal bovino (FBS), (HyClone laboratórios, Logan, UT) </li><li> L-glutamina (Sigma-Aldrich, St. Louis, MO) </li><li> Penicilina estreptomicina / (Invitrogen, Carlsbad, CA) </li><li> Trypan azul reagente (Sigma-Aldrich, St. Louis, MO) </li><li> 50 mM Tris, pH 8,6; filtrada e armazenar a 4 ° C </li><li> PBS Dulbecco (DPBS, Ca2/Mg2-free; Life Technologies, Rockville, MD) </li><li> Buffer de fluxo de lavagem: suplemento DPBS com 2% de SFB, armazenar a 4 ° C </li><li> BD Fixação Cytofix / Cytoperm / permeabilização Solution Kit (BD Biosciences, San Jose, CA) </li></ul> Agentes de ativação <ul><li> Forbol 12-miristato 13-acetato (PMA) (Sigma-Aldrich, St. Louis, MO) </li><li> Ionomicina (Sigma-Aldrich, St. Louis, MO) </li><li> Co-estimulação mAb CD49d, clone 9F10 (BD Biosciences, San Jose, CA) </li><li> Afinidade de anticorpos purificados F (ab) 2 fragmentos de cabra anti-camundongo IgG (H + L) (Kierkegaard e Perry Laboratory, Gaithersburg, MD) </li><li> Agentes específicos de ativação poderia incluir piscinas peptídeo, único peptídeo ou proteína inteira </li></ul> Secreção de inibidores de citocinas <ul><li> Brefedin A (BFA, Sigma-Aldrich, St. Louis, MO) </li><li> Ligue Golgi pode ser usado alternativamente (BD Biosciences, San Jose, CA) </li></ul> Anticorpos <ul><li> Vivo / morto fixable aquática kit fluorescente mancha (Invitrogen, Carlsbad, CA) </li><li> CD3 PE-Cy7 (SP34-2), (BD Biosciences, San Jose, CA) </li><li> CD4 Pacific Blue (OKT4), (eBiosciences (San Diego, CA) </li><li> CD8 Alexa700 (RPA-T8), (BD Biosciences, San Jose, CA) </li><li> CD28-PerCP cy5.5 (L293), (BD Biosciences, San Jose, CA) </li><li> CD95 APC (DX2), (BD Biosciences, San Jose, CA) </li><li> IFN-FITC (B27), (BD Biosciences, San Jose, CA) </li><li> IL-2 PE (MQ1-17H12), (BD Biosciences, San Jose, CA) </li></ul>

multicolor flow cytometry analyses of cellular immune response in rhesus macaques

O modelo de macaco rhesus é atualmente o melhor modelo disponível para o HIV-AIDS com respeito à compreensão da patogênese, bem como para o desenvolvimento de vacinas e terapêutica<sup&gt; 1,2,3</sup&gt;. Aqui, descrevemos um método para a análise detalhada fenotípica e funcional das respostas imunes celulares, especificamente a produção de citocinas intracelulares por células CD4 + e CD8 + T, bem como os subconjuntos de memória individual. Obtivemos precisas medidas quantitativas e qualitativas para a produção de interferon gama (INF-) e interleucina (IL) -2 em ambos os CD4 + e CD8 + T células do PBMC rhesus estimuladas com PMA mais ionomicina (PMA + I). Os perfis das citocinas foram diferentes nos diferentes subgrupos de células de memória. Além disso, este protocolo forneceu-nos a sensibilidade para demonstrar, mesmo frações menores de CD4 antígeno específico + e CD8 + subpopulações de células T dentro do PBMC amostras de macacos rhesus imunizadas com uma vacina contra o HIV cocktail envelope peptídeo desenvolvido em nosso laboratório. O multicolor técnica de citometria de fluxo é uma ferramenta poderosa para identificar precisamente as diferentes populações de células T<sup&gt; 4,5</sup&gt; Com a produção de citocinas, capacidade<sup&gt; 6</sup&gt; Após a estimulação não-específica ou antígeno específico<sup&gt; 5,7</sup&gt;.

Watch this Scientific Journal Video about Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques at JoVE.com

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

Demonstramos a utilidade da multicolor citometria de fluxo para caracterização fenotípica e funcional detalhada do total, bem como subconjuntos memória de CD4 + e CD8 + T em macacos rhesus, o modelo ideal para estudos de HIV / AIDS vacina.

Fluxo multicolor de Citometria de Análises de resposta imune celular em macacos rhesus

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

multicolor-flow-cytometry-analyses-cellular-immune-response-rhesus

Research

JoVE Journal

Immunology and Infection

21.0K visualizações.  MD Anderson Cancer Center - University of Texas. Demonstramos a utilidade da multicolor citometria de fluxo para caracterização fenotípica e funcional detalhada do total, bem como subconjuntos memória de CD4 + e CD8 + T em macacos rhesus, o modelo ideal para estudos de HIV / AIDS vacina.

Vídeo: Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

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 ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Análise por Citometria de fluxo das células imunológicas Dentro aortas murino

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

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

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

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

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

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

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

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

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

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

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

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

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

In Vitro Análise do mediada por Myd88 Cellular resposta imune a vírus do Nilo Ocidental Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

Análise de células T CD8 específicas do vírus da imunodeficiência símia<sup&gt; +</sup&gt; Células-T em Rhesus Macaques por Peptide-MHC-I tetrâmero coloração

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

Análise qualitativa e quantitativa de sinapse imunológica no sistema humano usando a imagem latente de citometria de fluxo

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

Isolamento e cultura In Vitro de macrófagos alveolares murino e humanos

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

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

Célula única quantificação de mRNA e expressão de proteínas de superfície em CD4 infectados por vírus de imunodeficiência simiana+ T células isoladas de macacos Rhesus