Dr Pramod Nehete et Mme Bharti Nehete pour le sang et les échantillons de PBMC de macaques; Ce travail a été partiellement financé par des fonds de NIH IA 46969, et tous les milieux de culture cellulaire ont été produites par le Laboratoire central des médias, qui est financé par le NIH octroi CA 16672.

Les procédures pour les analyses détaillées phénotypiques et fonctionnels du total des sous-ensembles ainsi que la mémoire des lymphocytes T peut être divisé en quatre parties: 1) la préparation des cellules et la stimulation antigénique, 2) coloration marqueur de surface, 3) coloration des cytokines intracellulaires et 4) La cytométrie en flux analyses. 1. Préparation des cellules et la stimulation antigénique <ol><li> Les deux fraîchement isolées ainsi que la cryo-préservé les cellules mononucléées du sang périphérique (PBMC) ont été utilisés dans ce protocole. Les PBMC ont été isolées à partir hépariné ou citraté échantillons de sang veineux des macaques rhésus par sédimentation en gradient de densité utilisant Ficoll-Hypaque (Histopaquen-1077, Sigma-Aldrich, ST. Louis, MO). Des aliquotes de PBMC ont été conservés congelés dans 90% de SVF et 10% de DMSO dans l&#39;azote liquide. </li><li> Lorsque vous utilisez la cryo-préservé PBMC, les flacons de produits congelés PBMC ont été retirés de l&#39;azote liquide et rapidement décongelés dans un bain d&#39;eau à 37 C, mélanger doucement, lavée avec du RPMI-1640 (HyClone laboratoires, Logan, UT) pour éliminer le milieu de congélation et remis en suspension dans des milieux complets [CM; RPMI-1640 de compléter avec 10% de la chaleur-FCS inactivé (HyClone laboratoires), 2 mM L-glutamine (Sigma-Aldrich), 100U/ml pénicilline / streptomycine (Invitrogen), et cultivées dans 6 - ainsi des plaques de culture tissulaire nuit à 37 ° C dans une atmosphère humidifiée 2 5% atmosphère. Le lendemain matin, les cellules viables ont été déterminés par la méthode de trypan bleu exclusion de colorant et re-suspendu dans CM. </li><li> Les cellules ont été traitées différemment des non-spécifique contre l&#39;antigène spécifique de stimulation suivie par des analyses de cytokine: (a) Pour les non-spécifique de stimulation par le phorbol 12-myristate 13-acétate (PMA) et de l&#39;ionomycine (I) (Sigma-Aldrich, Saint- Louis, MO), des aliquotes de cellules dans un volume de 0,1 ml (1,0 x 10 6 cellules / puits) ont été étalées dans des puits individuels de plaques de 96 puits de culture tissulaire (BD Biosciences, Franklin Lake, New Jersey). Le PMA et ionomycine ont été utilisés à une concentration finale de 50ng/ml et 500ng/ml, respectivement. Le volume final total pour plaque de 96 puits de culture de tissus est 0.2ml/well: (b) Pour la stimulation spécifique de l&#39;antigène, 1,0 x 10 6 cellules dans un volume de 1,0 ml ont été étalées dans des puits individuels de plaques de 24 puits de culture tissulaire (BD Biosciences , Franklin Lake, New Jersey), où les puits ont été pré-traitées, comme décrit précédemment avec modification 9. En bref, les plaques de 24 puits de culture de tissus ont été recouvertes avec 2,5 ug / ml / puits de chèvre anti-souris IgG (H + L) (Kierkegaard et Perry laboratoire, Gaithersburg, MD) dans du Tris 50 mM solution (pH 8,6) pour 4 ° C pendant la nuit. Le lendemain matin les plaques ont été lavées deux fois avec du PBS stérile et de co-stimulation mAb CD49d, clone 9F10 (BD Biosciences, San Jose, CA) ont été ajoutés à 10 ug / ml / puits suivie par une incubation d&#39;une heure à 37 ° C. Après l&#39;incubation, les plaques ont été lavées deux fois avec du PBS stérile à température ambiante. Les antigènes d&#39;intérêt (incluant le cocktail de six peptides d&#39;enveloppe du VIH 8 ont été ajoutés à une concentration finale de 10μg/ml de chaque peptide. Puits avec des cellules supplémentaires au CM seuls ont été préparés comme contrôle négatif. Le volume total final pour chaque puits sur les 24 bien-plaque de culture de tissus a été de 1,0 ml. </li><li> Les cellules ont été cultivées pendant 6 heures à 37 ° C dans une atmosphère humidifiée 2 5% atmosphère. Bréfeldine A (Sigma-Aldrich, St. Louis, MO) a été ajoutée à la culture à 10μg/ml pour la finale 4,5 heures de stimulation. Par la suite, les cellules ont été transférées à 5 tubes en polypropylène ml et lavé avec de froid (4 ° C) de tampon de lavage de flux (PBS Dulbecco (DPBS, Ca 2 / Mg 2-libres; Life Technologies, Rockville, MD) avec 2% inactivé à la chaleur FCS), puis traitées pour la coloration avec les différents anticorps marqués par fluorescence. </li></ol> 2. Marqueurs de surface immunofluorescence coloration <ol><li> Cellules stimulées ont d&#39;abord été colorés avec 1UL vivants / morts fixable Aqua colorant réactif fluorescent à 4 ° C dans l&#39;obscurité pendant 30 minutes, lavé une fois avec le tampon de flux lavage à froid. </li><li> Ensuite, les éléments suivants appropriée titré marqué par fluorescence des anticorps monoclonaux ont été ajoutés à des cellules: l&#39;anti-CD3 PE-Cy7 (SP34-2), anti-CD8 Alexa700 (RPA-T8), anti-CD28-PerCP Cy5.5 (L293) , anti-CD95 APC (DX2) et anti CD4 Pacific Blue (OKT4), tous de BD Biosciences (Franklin Lake, New Jersey), à l&#39;exception de CD4 bleues du Pacifique (OKT4) de eBioscience (San Diego, Californie). Les cellules ont été incubées pendant 30 minutes à 4 ° C dans l&#39;obscurité. </li><li> Pour chaque expérience à la fois la rémunération de contrôle et de fluorescence moins un (FMO) contrôle 10,11 ont été utilisés. Contrôle d&#39;indemnisation ont été utilisés pour déterminer les retombées coeffecients de chaque individu tache dans d&#39;autres détecteurs. Pour les commandes de compensation, un ensemble complet de tubes contenant les suspensions cellulaires de l&#39;un des singes ont été colorés avec fluorescents conjugués AcM individuellement comme des taches de couleur unique. FMO est une combinaison coloration multicolore qui contient tous les réactifs, mais celui de l&#39;intérêt et est utilisé pour déterminer la frontière entreune population positive et négative en dupliquant auto-fluorescence niveau et des données présentes dans l&#39;échantillon entièrement colorées. </li><li> Les cellules colorées ont ensuite été lavées avec du tampon de flux lavage à froid, et fixé dans la fixation / perméabilisation solution (BD Biosciences, San Jose, CA) pour au moins 10 minutes (à ce stade, les cellules peuvent être stockés une nuit à 4 ° C dans le noir) avant d&#39;effectuer coloration des cytokines intracellulaires. </li></ol> 3. Coloration des cytokines intracellulaires <ol><li> Les cellules fixées ont été lavées avec un tampon de lavage de débit, puis de Perm / Wash buffer (BD Biosciences, San Jose, CA) a été ajoutée selon les instructions du fabricant. Brièvement, les cellules ont été incubées dans 0,1 ml de Perm 1X / tampon de lavage pendant 10 minutes à 4 ° C dans l&#39;obscurité. </li><li> Par la suite, les cellules ont été incubées avec la façon appropriée titré marqué FITC anti-IFN-γ (B27) et PE-anticorps anti-IL-2 (17h12-MQ1) pendant 60 minutes à 4 ° C dans l&#39;obscurité. </li><li> Puis les cellules sont lavées deux fois avec la solution de perméabilisation </li><li> Après le lavage final, les cellules ont été remises en suspension dans du paraformaldéhyde à 1% dans DPBS et soumis à une analyse par cytométrie de flux dans les 24 heures. </li></ol> 4. L&#39;analyse par cytométrie en flux <ol><li> Les cellules colorées ont été acquises sur un flux de LSR II cytomètre (BD Biosciences, San Jose, CA) ou cyan ADP (Dako, Carpinteria, CA). Les données ont été analysées par FACS en utilisant le logiciel FlowJo (Arbre Star, Ashland, OR). </li><li> Figure. 1 montre le schéma de déclenchement utilisé pour les analyses des différents sous-ensembles de lymphocytes T provenant d&#39;un animal représentant. Les lymphocytes ont d&#39;abord été fermée par l&#39;intermédiaire diffusion vers l&#39;avant (FSC) par rapport à diffusion latérale (SSC), puis les lymphocytes vivants ont été identifiés sur la base de PVC et d&#39;aqua-négative de la population. Les cellules T ont ensuite été positivement identifié par le CD3 expression suivie par la détection des cellules CD4 + CD8-(cellules T CD4 +) et CD4-CD8 + (cellules T CD8 +), les populations dans le CD3 + population de cellules T. Les cellules CD4 + et CD8 + ont encore été distingués dans différents sous-ensembles sur la base de CD28 et CD95 expression naïve (Tn, CD28 + CD95-), la mémoire centrale (TCM, CD28 + CD95 +) et la mémoire effecteur (TEM, CD28-CD95 +) des cellules que décrits dans la littérature 5, 10. </li><li> Figure. 2 montre comment les différents sous-ensembles de cellules CD4 + et CD8 + T ont été évalués pour la capacité fonctionnelle en termes de production de cytokines (INF-γ et / ou l&#39;IL-2) en réponse à une stimulation par PMA et ionomycine (PMA + I). </li></ol> 5. Représentant des données FACS Composition de PBMC en macaques rhésus été déterminée en utilisant une analyse FACS. La figure 1 montre le schéma de déclenchement utilisé pour les analyses des différents sous-ensembles de lymphocytes T provenant d&#39;un animal représentant. Les lymphocytes ont d&#39;abord été fermée par l&#39;intermédiaire diffusion vers l&#39;avant (FSC) par rapport à diffusion latérale (SSC), puis les lymphocytes vivants ont été identifiés sur la base de PVC et d&#39;aqua-négative de la population. Les cellules T ont ensuite été identifiés par CD3 expression. CD4 + CD8-(cellules T CD4 +) et CD4-CD8 + (cellules T CD8 +), les populations dans le CD3 + population de cellules T ont également été déterminées. Comme le montre la figure 2, les CD4 + et CD8 + ont encore été distingués dans différents sous-ensembles sur la base de CD28 et CD95 expression naïve (Tn, CD28 + CD95-), la mémoire centrale (TCM, CD28 + CD95 +) et la mémoire effecteur (TEM, CD28-CD95 +) des cellules. Figure 2A montre l&#39;INF-γ et IL-2 de production de CD4 non stimulées et PMA / ionomycine stimulé les cellules T, et sous-ensembles, et la figure 2B montre INF-γ et d&#39;IL-2 dans les cellules T non stimulées et PMA / ionomycine stimulé CD8 +, et sous-ensembles Notez les profils de cytokines distincts provoqué par la stimulation de chaque sous-ensemble. <a href="https://www.jove.com/files/ftp_upload/1743/1743fig1.jpg"><img src="/files/ftp_upload/1743/1743fig1tmb.jpg" alt="Figure 1" border="0" /></a> Figure 1. Gating la stratégie utilisée pour l&#39;analyse par cytométrie en flux de composition PBMC rhésus macqaues <a href="https://www.jove.com/files/ftp_upload/1743/1743fig2a.jpg"><img src="/files/ftp_upload/1743/1743fig2atmb.jpg" alt="Figure 2a" border="0" /></a> <a href="https://www.jove.com/files/ftp_upload/1743/1743fig2b.jpg"><img src="/files/ftp_upload/1743/1743fig2btmb.jpg" alt="Figure 2b" border="0" /></a> Figure 2. (A) le profil de production de cytokines de cellules CD4 + au total des cellules T et des sous-ensembles. (B) Profil de la production des cytokines du total des cellules T CD8 + et des sous-ensembles.

<ol>
	<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>

Solution et tampons <ul><li> Ficoll-Hypaque (Histopaquen-1077, Sigma-Aldrich, St. Louis, MO) </li><li> RPMI-1640 (HyClone laboratoires, Logan, UT) </li><li> Sérum de veau fœtal (FBS), (HyClone laboratoires, Logan, UT) </li><li> L-glutamine (Sigma-Aldrich, St. Louis, MO) </li><li> Pénicilline / streptomycine (Invitrogen; Carlsbad, CA) </li><li> Le bleu trypan réactif (Sigma-Aldrich, St. Louis, MO) </li><li> Tris 50 mM, pH 8,6; filtrée et stocker à 4 ° C </li><li> PBS Dulbecco (DPBS, Ca2/Mg2-free; Life Technologies, Rockville, MD) </li><li> Tampon de lavage de débit: supplément DPBS avec 2% de FBS, conserver à 4 ° C </li><li> BD Cytofix / Cytoperm fixation / perméabilisation Solution Kit (BD Biosciences, San Jose, CA) </li></ul> Agents d&#39;activation <ul><li> Phorbol 12-myristate 13-acétate (PMA) (Sigma-Aldrich, St. Louis, MO) </li><li> Ionomycine (Sigma-Aldrich, St. Louis, MO) </li><li> Costimulation mAb CD49d, clone 9F10 (BD Biosciences, San Jose, CA) </li><li> L&#39;affinité des anticorps purifiés F (ab) 2 fragments de chèvre anti-souris IgG (H + L) (Kierkegaard et Perry laboratoire, Gaithersburg, MD) </li><li> Agents d&#39;activation spécifiques pourraient inclure des piscines peptide, le peptide seul ou protéine entière </li></ul> Inhibiteurs de la sécrétion des cytokines <ul><li> Brefedin A (BFA, Sigma-Aldrich, St. Louis, MO) </li><li> Branchez Golgi peuvent être utilisées alternativement (BD Biosciences, San Jose, CA) </li></ul> Anticorps <ul><li> Live / Dead fixable Aqua Kit colorant fluorescent (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

Le modèle macaque rhésus est actuellement le meilleur modèle disponible pour le VIH-sida à l&#39;égard de la compréhension de la pathogenèse, ainsi que pour le développement de vaccins et de thérapeutiques<sup&gt; 1,2,3</sup&gt;. Ici, nous décrivons une méthode pour l&#39;analyse détaillée phénotypique et fonctionnelle des réponses immunitaires cellulaires, la production de cytokine spécifiquement intracellulaire par CD4 + et CD8 + ainsi que les sous-ensembles de la mémoire individuelle. Nous avons obtenu des mesures précises quantitatifs et qualitatifs pour la production d&#39;interféron gamma (IFN-) et l&#39;interleukine (IL) -2 dans les deux cellules CD4 + et CD8 + T des cellules du macaque rhésus PBMC stimulées par le PMA, plus ionomycine (PMA + I). Les profils de cytokines ont été différentes dans les différents sous-ensembles de cellules mémoire. Par ailleurs, ce protocole nous a fourni la sensibilité à démontrer, même des fractions mineures de l&#39;antigène spécifique de CD4 + et CD8 + sous-ensembles de cellules T dans les échantillons PBMC de macaques rhésus vaccinés avec un vaccin d&#39;enveloppe du VIH cocktail de peptides développés dans notre laboratoire. La technique de cytométrie en flux multicouleur est un outil puissant pour identifier précisément les différentes populations de cellules T<sup&gt; 4,5</sup&gt; Avec la cytokine capacités de production<sup&gt; 6</sup&gt; Suite à une stimulation non-spécifique ou spécifique de l&#39;antigène<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

Nous démontrons l&#39;utilité de la cytométrie en flux multicouleur pour la caractérisation phénotypique et fonctionnelle détaillée du total des sous-ensembles ainsi que la mémoire des lymphocytes CD4 + et CD8 + T dans les macaques rhésus, le modèle idéal pour les études de vaccin contre le VIH / SIDA.

Analyses Multicolor cytométrie de flux de réponse immunitaire cellulaire dans les macaques rhésus

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 vues.  MD Anderson Cancer Center - University of Texas. Nous démontrons l'utilité de la cytométrie en flux multicouleur pour la caractérisation phénotypique et fonctionnelle détaillée du total des sous-ensembles ainsi que la mémoire des lymphocytes CD4 + et CD8 + T dans les macaques rhésus, le modèle idéal pour les études de vaccin contre le VIH / SIDA.

Vidéo: 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.

Mesure de l&#39;activité calpaïne dans des cellules fixées et de la Vie par cytométrie en flux

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

Recherche

Immunologie et infection

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.

Sept étapes pour Stellate Cellules

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.

Débit analyse par cytométrie de cellules immunitaires dans aortes murin

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.

Homme In Vitro</emRépression> comme outil de dépistage pour la reconnaissance d&#39;un Etat précoce de déséquilibre immunitaire

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.

Détection de nanoparticules fluorescentes Interactions avec immunitaire primaire sous-populations de cellules par cytométrie en flux

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.

Analyse in vitro de la réponse immunitaire cellulaire Myd88 médiation au virus du Nil occidental souche mutante 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.

Analyse de l&#39;immunodéficience simienne CD8 spécifiques Virus<sup&gt; +</sup&gt; T-cellules dans rhésus macaques par Peptide-CMH-I tétramère Coloration

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.

Analyse qualitative et Quantitative de la Synapse immunitaire dans le système humain à l’aide d’imagerie de cytométrie en flux

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.

Isolement et la Culture In Vitro de Macrophages alvéolaires murines et humaines

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.

Unicellulaire quantification des ARNm et Expression de protéines de Surface CD4 infectées par le Virus d’immunodéficience simien+ T les cellules isolées de macaques rhésus