Les auteurs tiennent à remercier Bing Zhang pour son aide technique. Ce travail a été financé par les Instituts de recherche en santé (MOP-86595). TAB est un nouveau chercheur des IRSC et chercheur AHFMR. QH est soutenu par une bourse d&#39;études supérieures du Canada des IRSC - Doctorat et une AIHS à temps plein bourse. SAN est soutenu par une bourse de la Reine Elizabeth II d&#39;études supérieures. AYWS est soutenu par une bourse d&#39;études supérieures du CRSNG - Doctorat.

Reportez-vous à la figure 1 pour un schéma d&#39;ensemble du protocole expérimental. 1. Dissection <ol><li> Lieu écran stérile en maille d&#39;acier plat 60 x 15 mm Petri. Une unité est nécessaire pour chaque échantillon de tissu. </li><li> Ajouter 5 ml de solution saline équilibrée de Hank (HBSS) à chaque plat. Garder les plats sur la glace. </li><li> Euthanasier les souris avec du CO 2. </li><li> Souris fixe sur une surface de dissection, face ventrale vers le haut. Souris de pulvérisation avec de l&#39;éthanol 70% pour la stérilisation et à faire en sorte que la fourrure est emmêlée vers le bas. </li><li> Avec des ciseaux chirurgicaux, commencer la dissection en faisant une incision ventrale dans la peau de l&#39;abdomen juste au-dessus des organes génitaux. Étendre vers le haut incision sous le menton. </li><li> De la ligne médiane, de prolonger l&#39;incision le long de tous les membres. Tirez la peau lâche et épingler à la surface de dissection. </li><li> Récolter le thymus: <ol><li> Relever l&#39;extrémité inférieure du sternum pour faire une incision.</li><li> Éviter le foie, couper la membrane de détacher la cage thoracique, puis couper la cage thoracique de chaque côté. Couper vers le haut la cage thoracique de chaque côté, en prenant soin d&#39;éviter les poumons et le cœur. </li><li> Tirez doucement sur le dos cage thoracique avec une pince. Le thymus est un blanc, un organe bilobé situé au-dessus du cœur. En utilisant le côté plat de la pince pour saisir le fond des lobes, tirez doucement sur le thymus et le poser sur le tamis. </li></ol></li><li> Récolter la rate: <ol><li> La rate est un rouge, en forme de planche de surf organe situé sur le côté gauche de la cavité abdominale de la souris, en dessous du foie. </li><li> Faire une incision dans la cavité abdominale. Tirez doucement sur la rate avec une paire de pinces, en utilisant la deuxième paire de taquiner l&#39;écart du tissu conjonctif. Placer la rate sur un tamis à mailles séparé. </li></ol></li></ol> 2. Préparation des cellules <ol><li> L&#39;utilisation d&#39;un piston d&#39;une seringue de 3 ml, meulerorganes dans le tamis jusqu&#39;à ce que le tissu conjonctif et les restes de graisse. Rincer avec du HBSS maille de la capsule trois fois. Utilisez un piston neuf pour chaque échantillon de tissu. <ol><li> D&#39;autres options pour l&#39;homogénéisation du tissu peut être utilisé à la place de cette méthode. </li></ol></li><li> Compter les cellules à l&#39;aide d&#39;un hémocytomètre. </li><li> Cellules par centrifugation à 335 xg pendant 5 min à 4 ° C. </li><li> Splénocytes remettre en suspension dans 500 ul de tampon de lyse ACK (0,15 M NH 4 Cl, 10 mM KHC0 3, 0,1 mM Na 2 EDTA, pH 7,2) pendant 10 min à température ambiante. Remettre les thymocytes à 20 x 10 6 cellules / ml dans du tampon FACS (PBS, 1% de FCS, de l&#39;azoture de sodium à 0,02%) et mis de côté sur la glace. </li><li> Retour splénocytes à l&#39;isotonie par addition de 5 ml de HBSS. Splénocytes par centrifugation et remettre en suspension à 20 x 10 6 cellules / ml dans du tampon FACS. Si les cellules ont besoin pour demeurer stérile, vous pouvez utiliser stérile du RPMI + 10% FCS. </li></ol> 3.Les cellules de coloration pour cytométrie en flux Le but de ce protocole est de définir des stratégies pour l&#39;analyse de la sélection positive et négative à l&#39;aide de type sauvage (WT) et cd4 HY souris. Pour des considérations générales sur la cytométrie en flux expérimental, l&#39;acquisition et l&#39;analyse, nous renvoyons le lecteur à une excellente revue par Tung et al 11. <ol><li> Aliquoter 4 x 10 6 thymocytes par échantillon cytométrie en flux à chaque puits d&#39;une plaque de 96 puits, ainsi que 1 x 10 6 splénocytes par WT commande de compensation. Si vous utilisez des souris transgéniques, vous devez inclure une souris WT comme témoin dans votre expérience. </li><li> Bloquer les récepteurs Fc par incubation des cellules avec anti-CD16/32 (clone 2.4G2) pendant 10 min sur la glace. </li><li> Faites tourner la plaque à 335 xg à 4 ° C pendant 5 min. Retirer le couvercle et dissiper liquide des puits en tapotant la plaque une fois, face vers le bas dans un évier. Resuspendre chaque puits dans 200 ul de tampon FACS. Répéter le lavage. </li><li> Préparer des cocktails d&#39;anticorps indiquées ci-dessous, en utilisant la concentration optimale de chaque anticorps en fonction de la dilution dans 200 pi de tampon FACS par puits. La concentration optimale de chaque anticorps est définie comme la concentration minimale nécessaire pour obtenir la plus grande séparation des populations positives et négatives. S&#39;il n&#39;y a pas de population négative pour un anticorps donné, un anticorps isotype doit être inclus. Le volume total par puits peut être réduite (par exemple à 100 l par puits), si désiré. <ol><li> WT thymus: anti-TCRβ, anti-CD4, anti-CD8, anti-CD69 ou anti-CD5, anti-CD24 </li><li> Cd4 HY thymus: anti-HY TCR (T3.70), anti-CD4, anti-CD8, anti-CD69 ou anti-CD5, anti-CD24 Pour obtenir une meilleure séparation des lo CD69 et CD69 hi populations de thymocytes, il est recommandé un anticorps biotinylé anti-CD69 primaire servir, suivie d&#39;une tache secondaire contenant conjugué à un fluorochrome streptavidine. Nous usoi la même stratégie pour CD5 coloration. </li></ol></li><li> Incuber les cellules avec 200 pi de cocktail d&#39;anticorps dans un tampon de FACS pendant 30 min sur la glace dans l&#39;obscurité. </li><li> Parallèlement à coloration cocktail d&#39;anticorps, de la teinture chaque commande de compensation avec un seul anticorps conjugué à un fluorochrome. Idéalement, la coloration compensation implique que les mêmes anticorps utilisés dans le cocktail d&#39;anticorps. Cependant, la coloration pour chaque antigène individuel peut ne pas être possible pour les grandes expériences lors de nombreux antigènes sont en cours d&#39;analyse. Par conséquent, la coloration soit pour les anticorps anti-CD4 ou anti-CD8 est recommandé en raison de la forte expression de ces antigènes, ou l&#39;utilisation de billes de rémunération. Colorer dans une mémoire tampon FACS pendant 30 min sur la glace dans l&#39;obscurité. Laissez une commande de compensation non colorées. </li><li> Laver les cellules deux fois avec du tampon FACS. </li><li> Resuspendre les cellules dans du tampon FACS et transfert à tubes FACS. </li><li> Prélever des échantillons sur cytomètre de flux. Comprennent l&#39;acquisition de FSC-A et FSC-W à unllow de discrimination doublet. </li></ol> 4. Analyse des données de cytométrie en flux - Non-souris transgéniques TCR Nous utilisons FlowJo pour la cytométrie en flux analyse des données. Reportez-vous à la figure 2A de la stratégie de déclenchement pour les souris transgéniques non-TCR. <ol><li> Utilisation FSC-A par le SSC, portail électronique sur le &quot;lymphocytes&quot; de la population. </li><li> Dans le cadre du «lymphocytes» de la population, utiliser FSC-A par le FSC-W électroniquement la porte du FSC-W lo population d&#39;exclure doublets cellulaires et aggregrates. Il s&#39;agit de la «singulet» du portail. </li><li> Utilisation des événements dans le &quot;singlet&quot; porte, l&#39;intrigue CD8 par le CD4. Dessinez portes pour DN, DP terne, DP lumineux, CD4 + CD8 populations lo, CD4SP et CD8SP comme le montre la figure 2B. </li><li> Sous les portes CD4SP et CD8SP, créer TCRβ par CD24 parcelles. Utilisez l&#39;outil de déclenchement quadrant de tirer portes tel que représenté sur la figure 2C. </li><li> Créer un new complot de la &quot;singlet&quot; portail: TCRβ par CD69. Tirer portes A, B, C, D comme représenté sur la figure 2D. </li><li> Créer un nouveau complot de la &quot;singlet&quot; portail: TCRβ par CD5. Dessinez portes i, ii, iii, iv comme le montre la figure 2E. C&#39;est une autre stratégie pour l&#39;examen de sélection positive. </li><li> Appliquer DN, DP terne, DP lumineux, int CD4, CD4SP et CD8SP portes à partir de la rubrique «singulet» aux portes i, ii, iii, iv, A, B, C, D (figure 2D, E). </li><li> Calculer le nombre de cellules dans un sous-ensemble particulier en multipliant cellularité orgue de la fréquence de chaque porte subséquente jusqu&#39;à ce que vous atteignez votre population cible. Par exemple, le nombre de TCRβ hi-CD69 CD8SP thymocytes serait déterminée par le thymus cellularité x% TCR bhi CD69-(fraction D) CD8SP% x. <ol><li> Compter prend déjà en compte les cellules vivantes et mortes afin de ne pas inclure le lymphoc &quot;YTE «porte dans vos calculs. </li></ol></li></ol> 5. Analyse des données de cytométrie en flux - TCR des souris transgéniques Reportez-vous à la figure 3A pour la stratégie de déclenchement pour les souris transgéniques TCR. <ol><li> Suivez les étapes 4.1 et 4.2 comme dans la section précédente. </li><li> Dans le cadre du «singulet» porte, créez un T3.70 par parcelle SSC et porte sur le T3.70 + population de cellules à analyser spécifiques de l&#39;antigène des cellules T (figure 3B). Dans certaines circonstances, il peut être intéressant de comparer cette population à la non-specific antigen (T3.70-) population de cellules T. </li><li> Sein de la population T3.70 +, tirage DN, DP, CD4SP et CD8SP portes (figure 3C). <ol><li> Pour les échantillons de contrôle WT, dessiner ces portes sous la rubrique &quot;singulet&quot; portail ou créer un T3.70 porte car il y aura très peu de cellules T3.70 +. </li></ol></li><li> Sous la porte CD8SP, créer unT3.70 par CD24 parcelle. Utilisez l&#39;outil de déclenchement quadrant de diviser les cellules en quatre populations (figure 3F). </li><li> Créer un histogramme représentant superposée expression de CD69 dans le compartiment DP de souris WT et T3.70 + DP compartiment de souris cd4 HY (figure 4A). Répétez l&#39;opération pour examiner CD5 expression (figure 4B). </li><li> Calculer le nombre absolu de cellules dans chaque sous-ensemble d&#39;intérêt que dans 4,8. </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+analysis+of+clonal+deletion+in+vivo.">Transcriptional analysis of clonal deletion in vivo.</a> J. Immunol. 179, 837-844 (2007).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Le protocole présenté ici peut être utilisé pour examiner sélection positive et négative dans le non-TCR transgénique et TCR des souris transgéniques. Ce protocole décrit la coloration des antigènes de surface. Pour une analyse plus approfondie des mécanismes moléculaires, il est souvent nécessaire d&#39;effectuer une coloration intracellulaire. Nous utilisons la BD Biosciences Cytofix / Cytoperm Kit pour la plupart des protéines intracellulaires et le kit de BD Biosciences coloration pour des facteurs de ...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> HyClone Hank solution saline équilibrée </td><td> Thermo Scientific </td><td> SH30030.02 </td><td></td></tr><tr><td> Tamis à mailles métalliques </td><td> Cedarlane </td><td> CX-0080-E-01 </td><td></td></tr><tr><td> Des boîtes de Pétri (60 x 15 mm) </td><td> Fisher Scientific </td><td> 877221 </td><td></td></tr><tr><td> Seringues (3 ml) </td><td> BD Biosciences </td><td> 309657 </td><td></td></tr><tr><td> Tubes coniques (15 ml) </td><td> Sarstedt </td><td> 62.554.205 </td><td></td></tr><tr><td> Microscope </td><td> Zeiss - Primo Star </td><td> 415 500-00XX-000 </td><td></td></tr><tr><td> Hémocytomètre </td><td> HausserScientifique </td><td> 3110 </td><td></td></tr><tr><td> Plaque de 96 puits </td><td> Sarstedt </td><td> 82.1582.001 </td><td></td></tr><tr><td> Pipette multicanaux </td><td> Fisherbrand </td><td> 21-377-829 </td><td></td></tr><tr><td> Sérum de veau foetal </td><td> AAP </td><td> A15-701 </td><td></td></tr><tr><td> Tampon phosphate salin </td><td> Fisher Scientific </td><td> SH3025802 </td><td></td></tr><tr><td> L&#39;azoture de sodium </td><td> IT Baker Chemical Co. </td><td> V015-05 </td><td></td></tr><tr><td> Réactif bloquant FcR </td><td></td><td></td><td> Clone 2.4G2 </td></tr><tr><td> Anti-souris HY TCR </td><td> eBioscience </td><td> XX-9930-AA * </td><td> Clone T3.70 </td></tr><tr><td> Anti-souris CD4 </td><td> eBioscience </td><td> XX-0042-AA * </td><td> Clone RM4-5 </td></tr><tr><td> Anti-souris CD8a </td><td> eBioscience </td><td> XX-0081-AA * </td><td> Clone 53-6.7 </td></tr><tr><td> Anti-souris CD24 </td><td> eBioscience </td><td> XX-0242-AA * </td><td> Clone M1/69 </td></tr><tr><td> Anti-souris TCRβ </td><td> eBioscience </td><td> XX-5961-AA * </td><td> Clone H57-597 </td></tr><tr><td> Anti-souris biotinylé CD69 </td><td> eBioscience </td><td> 13-0691-AA * </td><td> Clone H1.2F3 </td></tr><tr><td> Anti-souris biotinylé CD5 </td><td> eBioscience </td><td> 13-0051-AA * </td><td> Clone 53-7.3 </td></tr><tr><td> Streptavidine </td><td> eBioscience </td><td> XX-4217-AA * </td><td></td></tr><tr><td> Cytomètre en flux </td><td> BD Biosciences - FACS Canto </td><td> 338962 </td><td></td></tr><tr><td> FACS tubes </td><td> BD Biosciences </td><td> 352052 </td><td></td></tr><tr><td> Logiciel d&#39;analyse de cytométrie en flux </td><td> Treestar - FlowJo </td><td> FlowJo v7 / 9 </td><td></td></tr><tr><td> HyClone RPMI - 1640 </td><td> Thermo Scientific </td><td> SH30027.01 </td><td></td></tr><tr><td></td><td></td><td></td><td> * XX varie selon le fluorochrome et YY varie selon la taille du flacon. </td></tr></tbody></table>

examination of thymic positive and negative selection by flow cytometry

Un système immunitaire sain nécessite que les cellules T répondent aux antigènes étrangers tout en restant tolérant aux antigènes du soi. Réarrangement aléatoire du récepteur des cellules T (TCR) α et β loci génère un répertoire de lymphocytes T avec la grande diversité dans la spécificité d&#39;antigènes, à la fois pour soi et étranger. Sélection du répertoire au cours du développement dans le thymus est essentiel pour générer sûrs et utiles lymphocytes T. Les défauts de la sélection thymique contribuer au développement de maladies auto-immunes et de l&#39;immunodéficience 1-4. Progéniteurs des cellules T entrez le thymus que double négatifs (DN) thymocytes qui n&#39;expriment pas CD4 ou CD8 co-récepteurs. L&#39;expression de la αβTCR et les deux co-récepteurs se produit à la double positif (DP) scène. L&#39;interaction de la αβTCR avec l&#39;auto-peptide-CMH (pMHC) présenté par les cellules thymiques détermine le sort de la thymocytes DP. Interactions de haute affinité conduire à une sélection négative et éliminationtion des matières autoréactives thymocytes. Faible affinité des interactions se traduisent par une sélection positive et le développement de CD4 ou CD8 simples positives (SP) des lymphocytes T capables de reconnaître des antigènes étrangers présentés par CMH du soi 5. La sélection positive peut être étudiée chez la souris avec un anticorps polyclonal (type sauvage) répertoire de TCR en observant la génération de cellules T matures. Cependant, ils ne sont pas idéales pour l&#39;étude de la sélection négative, ce qui implique la suppression de petites populations spécifiques de l&#39;antigène. De nombreux systèmes modèles ont été utilisés pour étudier la sélection négative, mais varient dans leur capacité de récapituler les événements physiologiques 6. Par exemple, la stimulation in vitro des thymocytes manque de l&#39;environnement thymique qui est intimement impliqué dans la sélection, tandis que l&#39;administration de l&#39;antigène exogène peut conduire à la non-spécifique suppression des thymocytes 7-9. À l&#39;heure actuelle, les meilleurs outils pour l&#39;étude de la sélection négative vivo sont des souris qui expriment une transgenic TCR spécifique pour endogène auto-antigène. Toutefois, de nombreux modèles classiques TCR transgéniques sont caractérisées par prématurée expression transgénique de la chaîne TCRα au stade DN, entraînant prématuré de sélection négative. Notre laboratoire a mis au point le modèle HY cd4, dans lequel le transgénique HY TCRα est conditionnellement exprimé au stade DP, permettant la sélection négative se produire lors de la DP à la transition SP comme c&#39;est le cas chez les souris de type sauvage 10. Ici, nous décrivons une cytométrie de flux à base de protocole pour examiner thymique sélection positive et négative dans le modèle HY souris CD4. Bien que la sélection négative chez la souris cd4 HY est très physiologique, ces méthodes peuvent également être appliquées à d&#39;autres modèles transgéniques TCR. Nous présenterons également les stratégies générales pour l&#39;analyse de la sélection positive dans un répertoire polyclonal applicable à toutes les souris manipulées génétiquement.

En physiologiques modèles transgéniques TCR et les souris WT, la sélection positive commence à l&#39;étape DP lumineux avant de passer à l&#39;étape DP terne après la rencontre antigène. Thymocytes DP ternes puis entrez un nombre de CD4 + CD8 étape de transition avant de devenir lo CD4SP ou thymocytes CD8SP (figure 2B). Thymocytes matures SP se caractérise par une forte expression du TCR et la perte du CD24 (figure 2C). A...

Watch this Scientific Journal Video about Examination of Thymic Positive and Negative Selection by Flow Cytometry at JoVE.com

Examination of Thymic Positive and Negative Selection by Flow Cytometry

Nous présentons une cytométrie de flux basé sur la méthode pour examiner le développement des cellules T<em&gt; In vivo</em&gt; En utilisant des souris génétiquement manipulées sur un fond de type sauvage ou récepteur des cellules T transgéniques.

L&#39;examen de sélection positive et négative thymique par cytométrie en flux

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

examination-thymic-positive-negative-selection-flow

Research

JoVE Journal

Immunology and Infection

21.6K vues.  University of Alberta. Nous présentons une cytométrie de flux basé sur la méthode pour examiner le développement des cellules T In vivo En utilisant des souris génétiquement manipulées sur un fond de type sauvage ou récepteur des cellules T transgéniques.

Vidéo: Examination of Thymic Positive and Negative Selection by Flow Cytometry

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

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 Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria.

Dans l&#39;évaluation in vivo de rongeurs Plasmodium Parasitémie et Merozoite Invasion par cytométrie en flux

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Caractérisation phénotypique des macrophages du Rat Kidney par cytométrie de flux

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-tri cellulaire activé pour la purification de cellules dendritiques plasmacytoïdes de la moelle osseuse de souris

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

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 ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Caractérisation des sous-ensembles de Monocyte humain de sang Flow Cytometry analyse

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

Discrimination des sept sous-ensembles de cellules immunitaires par deux-fluorochrome cytométrie en flux

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

Préparation de la moelle osseuse entière pour l'analyse de la cytométrie de masse des cellules de lignée neutrophile

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...