Dr. Pramod Nehete y la señora Bharti Nehete de sangre y muestras de PBMC macacos; Este trabajo fue financiado parcialmente con fondos del NIH AI 46969, y todos los medios de cultivo celular fueron producidos por el Laboratorio Central de los medios de comunicación, que es financiado por el NIH subvención CA 16672.

Los procedimientos para el análisis detallado fenotípica y funcional de la total, así como subconjuntos de células T de memoria se puede dividir en cuatro partes: 1) la preparación celular y la estimulación antigénica, 2) tinción de la superficie marcador, 3) tinción intracelular de citoquinas y 4) La citometría de flujo análisis. 1. Preparación de células y la estimulación antigénica <ol><li> Ambos recién aisladas, así como la crio-preservado las células mononucleares de sangre periférica (CMSP) se utilizaron en este protocolo. El PBMC fueron aisladas de muestras de sangre venosa heparinizada o con citrato de macacos rhesus por sedimentación en gradiente de densidad con Ficoll-Hypaque (Histopaquen-1077, Sigma-Aldrich, ST. Louis, MO). Alícuotas de CMSP se almacenan congelados en el 90% de FCS y DMSO al 10% en nitrógeno líquido. </li><li> Cuando se utiliza la crio-preservado PBMC, las copas de helado PBMC fueron retirados de nitrógeno líquido y se descongelan rápidamente en un baño de agua a 37 C, se mezclan suavemente, se lavaron con RPMI-1640 (Hyclone Laboratories, Logan, UT) para eliminar el medio de congelación y resuspendidas en medio completo [CM; RPMI-1640 con suplemento del 10% FCS inactivado por calor (Hyclone Laboratories), 2 mM L-glutamina (Sigma-Aldrich), 100U/ml penicilina / estreptomicina (Invitrogen), y se cultivan en 6 - y placas de cultivo de tejido durante la noche a 37 ° C en un humidificado 5% de CO2 la atmósfera. A la mañana siguiente, los recuentos de células viables se determinó por el método de exclusión tripano-colorante azul y re-suspendido en el CM. </li><li> Las células fueron tratadas de forma diferente para no específica contra antígenos específicos de estimulación seguido por análisis de citoquinas: (a) Por no específicos de la estimulación con forbol 12-miristato 13-acetato (PMA) e ionomicina (I) (Sigma-Aldrich, St. Louis, MO), alícuotas de las células en un volumen de 0,1 ml (1,0 x 10 6 células / pocillo) se colocaron en los distintos pozos de 96 placas de cultivo tisular (BD Biosciences, Franklin Lake, Nueva Jersey). El PMA e ionomicina se utilizaron a una concentración final de 50ng/ml y 500ng/ml, respectivamente. El volumen total final de 96 y placa de cultivo de tejidos es 0.2ml/well: (b) Para la estimulación antígeno específico, 1,0 x 10 6 células en un volumen de 1,0 ml se colocaron en los distintos pozos de 24 y placas de cultivo de tejidos (BD Biosciences , Franklin Lake, Nueva Jersey), donde los pozos fueron pre-tratados como se ha descrito anteriormente con la modificación de 9. En resumen, las placas de tejido de 24 pocillos se recubrieron con 2,5 mg / ml / pocillo IgG de cabra anti-ratón (H + L) (Kierkegaard y Perry laboratorio, Gaithersburg, MD) en 50 mM Tris solución (pH 8,6) para los 4 ° C durante la noche. A la mañana siguiente las placas fueron lavadas dos veces con PBS estéril y se co-estimuladoras CD49d MAB, clon 9F10 (BD Biosciences, San Jose, CA) se añadieron a 10 mg / ml / seguido bien mediante la incubación durante una hora a 37 ° C. Después de la incubación, las placas fueron lavadas dos veces con PBS estéril a temperatura ambiente. Los antígenos de interés (incluye cóctel de seis péptidos sobre el VIH 8 se añadieron a una concentración final de 10μg/ml de cada péptido. Pozos adicionales con células en CM sólo se prepararon como control negativo. El volumen total final de cada pozo de los 24 y placa de cultivo de tejidos fue de 1,0 ml. </li><li> Las células fueron cultivadas durante 6 horas a 37 ° C en un humidificado 5% de CO2 la atmósfera. Brefeldin A (Sigma-Aldrich, St. Louis, MO) se añadió a la cultura en 10μg/ml para la final de 4,5 horas de estimulación. Posteriormente, las células fueron transferidas a tubos de 5 ml de polipropileno y se lavan con agua fría (4 ° C) el flujo de tampón de lavado (PBS de Dulbecco (DPBS, Ca 2 / Mg 2 libres, Life Technologies, Rockville, MD) con 2% inactivado por calor FCS), y luego se procesa para la tinción con los distintos anticuerpos de fluorescencia etiquetados. </li></ol> 2. Marcadores de inmunofluorescencia tinción de la superficie <ol><li> Las células estimuladas se tiñeron primero con 1 ul en vivo / muerto corregible tinte aqua reactivos fluorescentes a 4 ° C en la oscuridad durante 30 minutos, se lava una vez con tampón de lavado en frío de flujo. </li><li> A continuación, las siguientes apropiadamente titulada fluorescencia anticuerpos monoclonales marcados se han añadido a las células: anti-CD3 PE-Cy7 (SP34-2), anti-CD8 Alexa700 (RPA-T8), anti-CD28-PerCP Cy5.5 (L293) , anti-CD95 APC (DX2) y anti-CD4 Pacific Blue (OKT4), todos de BD Biosciences (Franklin Lake, Nueva Jersey), con excepción de CD4 Pacific Blue (OKT4) de eBioscience (San Diego, CA). Las células fueron incubadas durante 30 minutos a 4 ° C en la oscuridad. </li><li> Para cada experimento tanto la compensación de los controles y de fluorescencia de menos uno (FMO) 10,11 controles se utilizaron. Controles de compensación se utilizaron para determinar la coeffecients desbordamiento de cada individuo mancha en otros detectores. Para los controles de compensación, un conjunto completo de tubos que contienen células en suspensión de uno de los monos fueron teñidas con fluorescente MAb conjugado de forma individual como manchas de color. FMO es una combinación de manchas multicolores que contiene todos los reactivos, pero la que le interese y se utiliza para determinar el límite entre un resultado positivo unpoblación ª negativos mediante la duplicación de autofluorescencia nivel y los datos presentes en la muestra completa de colores. </li><li> Células teñidas fueron lavadas con buffer de lavado en frío de flujo, y se fijaron en solución de fijación / permeabilización (BD Biosciences, San Jose, CA) durante al menos 10 minutos (en esta etapa las células se pueden almacenar durante la noche a 4 ° C en la oscuridad) antes de realizar la tinción intracelular de citoquinas. </li></ol> 3. Tinción intracelular de citoquinas <ol><li> Las células fijadas fueron lavadas con buffer de flujo de lava, y luego de Perm / Wash buffer (BD Biosciences, San Jose, CA) se añadió de acuerdo a las instrucciones del fabricante. Brevemente, las células fueron incubadas en 0,1 ml de la ondulación permanente 1X / Solución de lavado durante 10 minutos a 4 ° C en la oscuridad. </li><li> Posteriormente, las células fueron incubadas con la adecuada valora marcado con FITC anti-IFN-γ (B27) y PE-anticuerpo anti-IL-2 (MQ1-17H12) durante 60 minutos a 4 ° C en la oscuridad. </li><li> Luego se lavaron las células dos veces con la solución de permeabilización </li><li> Después del último lavado, las células volvieron a ser suspendidas en el 1% de paraformaldehído en DPBS y sometidos a análisis de citometría de flujo dentro de las 24 horas. </li></ol> 4. Análisis por citometría de flujo <ol><li> Las células teñidas fueron adquiridas en un citómetro de flujo LSR II (BD Biosciences, San Jose, CA) o cian ADP (Dako, Carpinteria, CA). FACS datos fueron analizados mediante el uso de software FlowJo (Árbol Star, Ashland, Oregón). </li><li> Figura. 1 muestra el esquema utilizado para la activación periódica análisis de los subconjuntos de células T diferentes de un animal representativo. Los linfocitos fueron los primeros cerrada a través de dispersión frontal (FSC) versus dispersión lateral (SSC), y luego linfocitos vivos fueron identificadas en base a cooperación Sur-Sur y la población de aqua-negativo. Las células T eran entonces identificado positivamente por CD3 expresión seguida de la detección de las células CD4 + CD8 (células T CD4 +) y CD4-CD8 + (células T CD8 +) dentro de la población CD3 + población de células T. Las células CD4 + y CD8 + T se distinguen más en diferentes subconjuntos de la base de CD28 y la expresión de CD95 como ingenuo (Tn, CD28 + CD95-), la memoria central (MTC, CD28 + CD95 +) y efectoras de memoria (Tem, CD95-CD28 +) las células, descritos en la literatura 5, 10. </li><li> Figura. 2 muestra cómo los diferentes subgrupos de linfocitos CD4 + y CD8 + T se evaluó la capacidad funcional en términos de producción de citoquinas (INF-γ y / o IL-2) en respuesta a la estimulación con PMA e ionomicina (PMA + I). </li></ol> 5. Representante FACS datos Composición PBMC en macacos rhesus se determinó mediante análisis de FACS. La figura 1 muestra el esquema utilizado para la activación periódica análisis de los subconjuntos de células T diferentes de un animal representativo. Los linfocitos fueron los primeros cerrada a través de dispersión frontal (FSC) versus dispersión lateral (SSC), y luego linfocitos vivos fueron identificadas en base a cooperación Sur-Sur y la población de aqua-negativo. Las células T fueron identificados por CD3 expresión. CD4 + CD8 (linfocitos T CD4 +) y CD4-CD8 + (células T CD8 +) dentro de la población CD3 + población de células T se determinó también. Como se muestra en la Figura 2, las células CD4 + y CD8 + T se distinguen más en diferentes subgrupos sobre la base de CD28 y la expresión de CD95 como ingenuo (Tn, CD28 + CD95-), la memoria central (MTC, CD28 + CD95 +) y efectoras de memoria (Tem, CD28-CD95 +) las células. Figura 2A muestra INF-γ y de IL-2 en las células CD4 no estimulada y estimulada PMA / ionomicina + células T y subconjuntos, y la Figura 2B muestra INF-γ y de IL-2 en estimulada y PMA / ionomicina estimula las células CD8 + T y subconjuntos Tenga en cuenta los perfiles de citoquinas distintas provocados por la estimulación en cada subgrupo. <img src="/files/ftp_upload/1743/1743fig1tmb.jpg" alt="Figura 1" border="0" /> Figura 1. Estrategia de apertura de puerta para el análisis de citometría de flujo de la composición de PBMC de rhesus macqaues. Por favor, <a href="https://www.jove.com/files/ftp_upload/1743/1743fig1.jpg">haga clic aquí</a> para ver una versión ampliada de la 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) el perfil de producción de citocinas de los linfocitos CD4 + total de las células T y subconjuntos. (B) el perfil de producción de citocinas del total de las células T CD8 + y subconjuntos. Por favor, <a href="https://www.jove.com/files/ftp_upload/1743/1743fig2a.jpg">haga clic aquí</a> para ver una versión más grande de la 2a, <a href="https://www.jove.com/files/ftp_upload/1743/1743fig2b.jpg">o aquí</a> para una versión ampliada de la 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>

Solución y Buffers <ul><li> Ficoll-Hypaque (Histopaquen-1077, Sigma-Aldrich, St. Louis, MO) </li><li> RPMI-1640 (Hyclone Laboratories, Logan, UT) </li><li> Suero fetal bovino (FBS), (Hyclone Laboratories, Logan, UT) </li><li> L-glutamina (Sigma-Aldrich, St. Louis, MO) </li><li> Penicilina / estreptomicina (Invitrogen, Carlsbad, CA) </li><li> El azul tripán reactivo (Sigma-Aldrich, St. Louis, MO) </li><li> 50 mM Tris, pH 8,6; filtrar y almacenar a 4 ° C </li><li> Dulbecco PBS (DPBS, Ca2/Mg2-free, Life Technologies, Rockville, MD) </li><li> Flujo de tampón de lavado: suplementos DPBS con FBS al 2%, se almacenan a 4 ° C </li><li> BD Cytofix / Cytoperm fijación / permeabilización solución Kit (BD Biosciences, San Jose, CA) </li></ul> Agentes de la activación <ul><li> Forbol 12-miristato 13-acetato (PMA) (Sigma-Aldrich, St. Louis, MO) </li><li> Ionomicina (Sigma-Aldrich, St. Louis, MO) </li><li> Coestimuladoras AcM CD49d, clon 9F10 (BD Biosciences, San Jose, CA) </li><li> Purificado por afinidad del anticuerpo F (ab &#39;) 2 fragmentos de cabra anti-ratón IgG (H + L) (Kierkegaard y Perry laboratorio, Gaithersburg, MD) </li><li> Agentes específicos de activación podría incluir piscinas péptido, solo péptido o proteína entera </li></ul> Inhibidores de la secreción de citoquinas <ul><li> Brefedin A (BFA, Sigma-Aldrich, St. Louis, MO) </li><li> Plug Golgi se puede utilizar como alternativa (BD Biosciences, San Jose, CA) </li></ul> Anticuerpos <ul><li> Live / Dead corregibles aqua fluorescentes kit de tinción (Invitrogen, Carlsbad, CA) </li><li> PE-CD3 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

El modelo de macaco rhesus es actualmente el mejor modelo disponible para el VIH-SIDA con respecto a la comprensión de la patogénesis, así como para el desarrollo de vacunas y terapias<sup&gt; 1,2,3</sup&gt;. Aquí se describe un método para el análisis detallado fenotípica y funcional de la respuesta inmune celular, específicamente la producción de citoquinas intracelulares de las células CD4 + y CD8 + T, así como los subconjuntos de la memoria individual. Se obtuvieron precisa medidas cuantitativas y cualitativas para la producción de interferón gamma (IFN-) y la interleuquina (IL) -2 en ambos CD4 + y CD8 + T las células de los macacos rhesus PBMC estimuladas con PMA más ionomicina (PMA + I). Los perfiles de citoquinas fueron diferentes en los diferentes subconjuntos de células de memoria. Además, este protocolo nos dio la sensibilidad para demostrar incluso fracciones menores de CD4 + antígeno específico y CD8 + subconjuntos de células T en las muestras de PBMC de macacos rhesus inmunizados con una vacuna contra la envoltura del VIH cóctel péptido desarrollado en nuestro laboratorio. El flujo multicolor técnica de citometría es una poderosa herramienta para identificar con precisión las diferentes poblaciones de células T<sup&gt; 4,5</sup&gt; Con la capacidad de producción de citoquinas<sup&gt; 6</sup&gt; Después de la estimulación no específica o antígeno-específicas<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

Demostrar la utilidad de la citometría de flujo multicolor para la caracterización detallada fenotípica y funcional de la total, así como la memoria subconjuntos de células CD4 + y CD8 + T en macacos rhesus, el modelo ideal para estudios de vacunas contra el VIH / SIDA.

Multicolor análisis de citometría de flujo de la respuesta inmune celular en 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 vistas.  MD Anderson Cancer Center - University of Texas. Demostrar la utilidad de la citometría de flujo multicolor para la caracterización detallada fenotípica y funcional de la total, así como la memoria subconjuntos de células CD4 + y CD8 + T en macacos rhesus, el modelo ideal para estudios de vacunas contra el VIH / SIDA.

Video: 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.

Medición de la actividad calpaína en células fijadas y vivir por citometría de flujo

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

Investigación

Inmunología e Infección

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.

Siete pasos para células estrelladas

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.

Análisis por citometría de flujo de células inmunes dentro de 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 Supresión como herramienta de cribado para el reconocimiento de un estado inicial de desequilibrio inmunológico

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.

La detección de nanopartículas fluorescentes Interacciones con inmunodeficiencia primaria subpoblaciones celulares por citometría de flujo

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.

El análisis in vitro de la respuesta inmune celular Myd88 mediada por el Virus del Nilo Occidental cepa mutante Infección

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.

Análisis de Inmunodeficiencia CD8 Simian Virus-específica<sup&gt; +</sup&gt; Células T en macacos rhesus por el péptido-MHC-I de tetrámeros La tinción

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.

Análisis cualitativo y cuantitativo de la sinapsis inmune en el sistema humano utilizando imágenes de citometría de flujo

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.

Aislamiento y cultivo In Vitro de los macrófagos alveolares murinos y 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.

Sola célula cuantificación del mRNA y la expresión de la proteína de superficie de CD4 infectados por el Virus de la inmunodeficiencia de los simios+ T las células aisladas de macaques del macaco de la India

Methods Article

Multicolor análisis de citometría de flujo de la respuesta inmune celular en macacos rhesus