D.E. and M.B.D. are members of a multidisciplinary research platform (MRP) of Ghent University, and the Ghent Researchers on Unfolded Proteins in Inflammatory Disease (GROUP-ID) consortium.

Dans cette étude, des adultes (6-8 semaines) femelles C57BL / 6 ont été utilisées. Les souris ont été logées et élevés selon les directives du vivarium Université de Gand. Toutes les procédures animales ont été approuvés par le Comité de protection des animaux et de l&#39;éthique institutionnelle. 1. Préparation des cellules mononucléaires (MNC) de Spleen souris <ol><li> Sacrifiez la souris par dislocation cervicale. </li><li> Placez la souris vers le bas sur le plateau de dissection et fixer la biche et les pattes avec des épingles. </li><li> Stériliser la surface de la peau avec 70% (vol / vol) d&#39;éthanol / H 2 O. </li><li> Couper la peau le long de la ligne médiane abdominale au thorax et d&#39;exposer la rate en utilisant des instruments chirurgicaux stériles. </li><li> Après la coupe des tissus adipeux autour, placer la rate dans une plaque 24 puits contenant 1 ml 1x tampon phosphate salin (PBS) à température ambiante. </li><li> On dissèque la rate à l&#39;aide d&#39;un scalpel stérile et transférer les morceaux dans un filtre en nylon de 70 pm placée dans un bain de 50 ml,e. </li><li> En utilisant une seringue de 2 ml plongeur, écraser délicatement les morceaux de la rate à travers le tamis de la cellule et laver avec 5 ml de PBS 1X. </li><li> Ajouter 3 ml de Ficoll-Paque dans un tube de 15 ml et recouvrir le milieu à gradient de densité avec le 5 ml de suspension cellulaire. </li><li> Centrifuger à 400 g pendant 20 min à température ambiante sans frein. </li><li> Transférer l&#39;interphase dans un nouveau tube de 15 ml, ajouter 10 ml froid 1x PBS et centrifuger à 300 g pendant 10 min à 4 ° C. </li><li> Remettre en suspension les cellules dans 2 ml de PBS 1X contenant 0,5% d&#39;albumine de sérum bovin (BSA) et 1 mM d&#39;acide éthylènediaminetétraacétique (EDTA) (tampon FACS). Transfert 10 ul de la suspension cellulaire à un petit tube, mélanger avec 10 ul de 0,4% </li></ol> 2. Enrichissement, la détection et la purification des cellules de souris iNKT <ol><li> L&#39;enrichissement de la rate de la souris CD5positive (CD5 +) lymphocytes. <ol><li> (Facultatif) Stain 10 5 cellules avec FITC conjugué anti-CD5 (4 pi de la dilution 1:30 / 10 6 cellules)pendant 30 min à 4 ° C dans l&#39;obscurité, on lave avec 200 pi de tampon FACS et remettre en suspension dans 500 pi de tampon FACS. Préparer une solution de travail de 20 uM de 4 &#39;, 6-diamidino-2-phénylindole (DAPI) dans 1 x PBS, ajouter 1 pl de celui-ci pour 10 5 cellules dans du tampon FACS et incuber à température ambiante pendant 5 min. Acquérir 10 4 cellules pré-enrichis sur le cytomètre de flux. <ol><li> Utilisation FCS-H et W-FCS, portail électronique sur les cellules lo FSC-W à exclure doublets de cellules et d&#39;agrégats (figure 1A). Puis électronique porte sur DAPI + (morts), les cellules au sein de la FSC-W lo population (figure 1B), et utilisent SSC-A et FCS-A pour sélectionner électroniquement la population de lymphocytes par la taille (figure 1C). </li><li> Utilisation de cellules à l&#39;intérieur de la porte de lymphocytes, tracer SSC-A par rapport CD5 et électroniquement porte CD5 + lymphocytes comme le montre la figure 1D. Acquisition du reste de l&#39;échantillon de pré-enrichissement sur le cytomètre de flux. </li&gt; </ol></li><li> Ajuster le nombre de cellules à 10 7 cellules par 90 ul dans du tampon FACS et 10 pl ajouter des microbilles anti-CD5. </li><li> Incuber à 4 ° C pendant 15 min, laver une fois avec 4 ml tampon FACS et remettre en suspension dans 500 pi de tampon FACS froid. Enrichissez pour CD5 + lymphocytes en utilisant séparation magnétique de cellules (MACS) colonnes selon les recommandations du fabricant. </li><li> Facultatif) Stain 10 5 cellules avec FITC conjugué anti-CD5 (4 pi de dilution 1h30 / 10 6 cellules) et DAPI comme en 2.1.1. Acquérir 10 4 lymphocytes enrichis sur le cytomètre de flux et de vérifier que les portes électroniques créés en 2.1.1. sélectionnez CD5 + lymphocytes dans la fraction cellulaire enrichie. Acquérir 10 5 cellules sur le cytomètre de flux et d&#39;analyser le pourcentage CD5 + lymphocytes présents dans la fraction enrichie comme représenté sur la figure 1E - H. </li></ol></li><li> La détection et l&#39;isolement des souris iNKT thisLLS par FACS. <ol><li> Remettre en suspension les lymphocytes CD5 + enrichis à une concentration d&#39;environ 10 6 cellules par 20 ul de tampon pour FACS contenant anti-CD16 / CD32 (4 pi d&#39;une dilution 1:50 / 10 6 cellules) et aGalCer PE-conjugué / tétramère CD1d (1 pg / 10 6 cellules). </li><li> Incuber pendant 30 min à température ambiante dans l&#39;obscurité. </li><li> Diluer anticorps de souris (mAb) anti-CD3e V500 (4 pi d&#39;une dilution 1:20 / 10 6 cellules), anti-CD19 E450 (4 ul de dilution 01:50 / 10 6 cellules) et anti-CD8a E450 (4 ul de dilution 1:50 / 10 6 cellules) dans un tampon FACS, ajouter à CD5 + lymphocytes et incuber pendant 30 min à 4 ° C dans l&#39;obscurité. </li><li> Laver les cellules avec 1 ml de tampon FACS par centrifugation à 300 xg pendant 10 min à 4 ° C et remettre en suspension dans 4 ml de tampon FACS à une concentration finale de 10 7 cellules / ml. </li><li> Filtrer les cellules à travers un tamis cellulaire de 30 um et de placer les cellules sur de la glace. </li><li> Calibrer le cytomètre en flux pour le tri selon les recommandations du fabricant 14,15 cellule. Remarque: L&#39;étalonnage d&#39;un cytomètre de flux pour le tri de cellules nécessite une formation et peut être effectuée par un opérateur expérimenté, ou un technicien qualifié. </li><li> Ajouter 10 ul d&#39;une solution de travail de 20 pM de DAPI par 10 6 cellules et incuber à température ambiante pendant 5 min. L&#39;acquisition d&#39;environ 10 4 enrichis CD5 + lymphocytes sur le cytomètre en flux. Électronique porte sur les cellules lo FSC-W à exclure doublets de cellules et d&#39;agrégats (figure 2A). Puis électronique porte sur DAPI + CD8 + CD19 + cellules (canal de décharge) dans le FSC-W lo population (figure 2B), et utiliser SSC-A et FCS-A pour sélectionner électroniquement lymphocytes (figure 2C) comme décrit dans 2.1.1.1 . <ol><li> Terrain aGalCer / CD1d tétramère par CD3e et électroniquement porte aGalCer / CD1dcellules tétramère + CD3e + iNKT comme indiqué sur la Figure 2D. Cellules présentes dans la fraction cellulaire enrichie acquérir plus de 2 x 10 5 CD5 + lymphocytes enrichis pour déterminer le pourcentage aGalCer / CD1d tétramères + CD3e + iNKT. </li></ol></li><li> Utilisation des portes électroniques créés en 2.2.7., Les cellules FACS sorte aGalCer / tétramère CD1d CD3e + + iNKT sur le cytomètre de flux à 70 psi en utilisant une buse de 70 um à une vitesse de &#39;1,5&#39; d&#39;écoulement. Recueillir des cellules iNKT triés dans un tube FACS contenant 1 ml de 100% de FCS. Ensuite rincer cellules iNKT trié du côté du tube FACS utilisant 10 ml IPMB 1640 et centrifuger les cellules à 300 g pendant 10 min à 4 ° C. </li><li> Remettre en suspension triée cellules iNKT dans 1 ml de milieu RPMI 1640 et d&#39;acquérir 40 pi de celui-ci sur un cytomètre de flux pour évaluer la pureté des cellules iNKT triés. Sélectionnez FSC-W lo DAPI - lymphocytes comme décrit dans 2.2.7. (Figure 2E - G) et électroniquement porte aGalCer / CD1d cellules tétramère + CD3e + iNKT comme indiqué dans la figure 2H. </li></ol></li></ol> 3. Culture et l&#39;expansion des cellules de souris iNKT Jour 0 <ol><li> Enduire une plaque à 96 puits à fond plat avec les anticorps anti-CD3e (3 pg / ml) par puits pendant 1 heure à température ambiante. Laver deux fois avec 200 pi de PBS 1x et une fois avec 200 pi milieu RPMI 1640 complet (10% vol / vol de FCS, 100 U / ml de pénicilline-streptomycine, 5,5 uM β-mercaptoéthanol). </li><li> Comptez le nombre de cellules viables triées iNKT (étape 2.2.8) en utilisant 0,4% d&#39;un hémocytomètre comme dans 1.11., Centrifuger à 300 g pendant 10 min à 4 ° C et remettre en suspension à 5 x 10 5 cellules / ml dans du milieu complet RPMI 1640 contenant l&#39;IL-2 murine (10 ng / ml) recombinant, recombinant murine IL-12 (1 ng / ml) et soluble CD28 anti-souris (5 ug / ml). Plate ssignalés auparavant cellules iNKT à 10 5 cellules / puits dans des plaques revêtues anti-CD3e et incuber à 37 ° C dans un incubateur à CO 2 pendant 2 jours. </li></ol> Jour 2 <ol start="3"><li> Transférer les cellules à une plaque de 96 à fond plat non couché frais bien et la culture des cellules dans des conditions de repos en milieu RPMI 1640 complet à 37 ° C dans un incubateur à CO 2 pendant 2 jours. </li></ol> Jour 4 <ol start="4"><li> Déloger les cellules de la plaque par pipetage doux et transférer les cellules dans un nouveau tube de 15 ml. Centrifuger à 300 g pendant 10 min à 4 ° C, remise en suspension à 5 x 10 5 cellules / ml dans du milieu RPMI 1640 complet contenant recombinant IL-7 murine (5 ng / ml), et la plaque 10 5 cellules / puits dans un forfaitaire fond plaque à 96 puits pendant 4 jours à 37 ° C dans un incubateur à CO 2. </li></ol> Jour 8 <ol start="5"><li> Recueillir les cellules dans un tube frais, centrifugeuse à 300 xg pendant 10 min à 4 ° C et remettre en suspension à 5 x 10 5 cellules / ml dans du milieu RPMI 1640 complet contenant anti-CD28 soluble (5 ug / ml). </li><li> Enduire une plaque à 96 puits à fond plat avec les anticorps anti-CD3e (3 pg / ml) par puits et laver comme en 3.1. La plaque 10 5 cellules / puits dans des plaques anti-CD3e revêtues et incuber à 37 ° C dans un incubateur à CO 2 jusqu&#39;à 11 jours. </li></ol> Jour 11-18 <ol start="7"><li> Répétez les étapes 3.4 à 3.6. </li></ol> Jour 19-20 <ol start="8"><li> Transférer les cellules à un tube de 15 ml frais, centrifugeuse à 300 g et remettre en suspension à 5 x 10 5 cellules / ml dans milieu RPMI 1640 complet. </li><li> Cellules de la plaque à 10 5 cellules / puits dans une plaque de 96 puits à fond plat et permettre aux cellules de se reposer pendant 2 jours à 37 ° C dans un incubateur à CO2 avant de l&#39;utiliser dans une expérience. </li></ol> 4. Production de cytokines par les souris des cellules iNKT l&#39;aide d&#39;un CD1d Plate-lié Assay <ol><li> Enduire une plaque à 96 puits à fond plat avec 100 pi de murin recombinant CD1d (10 pg / ml dans du PBS 1X) par puits et incuber pendant 1 heure à 37 ° C. </li><li> Laver quatre fois avec 200 pi de PBS 1x, ajouter 200 pi de 2% de FCS en 1x PBS (solution de blocage) par puits et incuber pendant 2 heures à 37 ° C. </li><li> Retirer la solution de blocage, ajouter 200 pi de aGalCer (5 ng / ul) par puits et incuber la plaque à 37 ° C pendant 16 heures. [Pour la préparation de la solution aGalCer, ajouter 1,085 μ 1 x PBS à 55 pg d&#39;aGalCer dissous dans un véhicule contenant 96 mg / ml de saccharose, 10 mg / ml de désoxycholate de sodium et 5 mg / ml entre 20]. </li><li> Après incubation, jeter la solution aGalCer et laver le puits deux fois avec 200 pi de PBS 1x suivie par 200 pi complète moyennes RPMI 1640. </li><li> Recueillir les cellules iNKT élargi in vitro au jour 21, centrifuger les cellules à 300 g pendant 10 min à 4 ° C et remettre en suspension dans complète RPMI 1640 à une concentration de 0,5 x 10 6 cellules / ml. </li><li> Ajouter 200 ul de la remise en suspension de cellules iNKT par puits et incuber pendant 72 heures à 37 ° C dans un incubateur à CO 2. </li><li> Récupérer le surnageant et mesurer les cytokines d&#39;intérêt par immuno-essai enzymatique (ELISA) selon le protocole du fabricant. </li></ol>

<ol>
	<li>Bendelac, A., Rivera, M. N., Park, S. H., Roark, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+CD1-specific+NK1+T+cells:+development,+specificity,+and+function.">Mouse CD1-specific NK1 T cells: development, specificity, and function.</a> Annu Rev Immunol. 15, 535-562 (1997).</li><li>Kronenberg, M., Gapin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unconventional+lifestyle+of+NKT+cells.">The unconventional lifestyle of NKT cells.</a> Nat Rev Immuno. 2, 557-568 (2002).</li><li>Park, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+CD1d-restricted+repertoire+is+dominated+by+a+few+autoreactive+T+cell+receptor+families.">The mouse CD1d-restricted repertoire is dominated by a few autoreactive T cell receptor families.</a> J Exp Med. 193, 893-904 (2001).</li><li>Zhou, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+glycosphingolipid+recognition+by+NKT+cells.">Lysosomal glycosphingolipid recognition by NKT cells.</a> Science. 306, 1786-1789 (2004).</li><li>Cardell, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD1-restricted+CD4++T+cells+in+major+histocompatibility+complex+class+II-deficient+mice.">CD1-restricted CD4+ T cells in major histocompatibility complex class II-deficient mice.</a> J Exp Med. 182, 993-1004 (1995).</li><li>Kawano, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD1d-restricted+and+TCR-mediated+activation+of+valpha14+NKT+cells+by+glycosylceramides.">CD1d-restricted and TCR-mediated activation of valpha14 NKT cells by glycosylceramides.</a> Science. 278, 1626-1629 (1997).</li><li>Drennan, M. B., Aspeslagh, S., Elewaut, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invariant+natural+killer+T+cells+in+rheumatic+disease:+a+joint+dilemma.">Invariant natural killer T cells in rheumatic disease: a joint dilemma.</a> Nat Rev Rheumatol. 6, 90-98 (2010).</li><li>Terabe, M., Berzofsky, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKT+cells+in+immunoregulation+of+tumor+immunity:+a+new+immunoregulatory+axis.">NKT cells in immunoregulation of tumor immunity: a new immunoregulatory axis.</a> Trends Immunol. 28, 491-496 (2007).</li><li>Molling, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+sustained+expansion+of+mouse+spleen+invariant+NKT+cell+lines+with+preserved+cytokine+releasing+capacity.">Generation and sustained expansion of mouse spleen invariant NKT cell lines with preserved cytokine releasing capacity.</a> J Immunol Methods. 322, 70-81 (2007).</li><li>Chiba, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+reliable+generation+of+invariant+natural+killer+T-cell+lines+in+vitro.">Rapid and reliable generation of invariant natural killer T-cell lines in vitro.</a> Immunology. 128, 324-333 (2009).</li><li>Gumperz, J. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+TCRs+recognize+murine+CD1.">Diverse TCRs recognize murine CD1.</a> J Immunol. 162, 161-167 (1999).</li><li>Watarai, H., Nakagawa, R., Omori-Miyake, M., Dashtsoodol, N., Taniguchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+detection,+isolation+and+culture+of+mouse+and+human+invariant+NKT+cells.">Methods for detection, isolation and culture of mouse and human invariant NKT cells.</a> Nature protocols. 3, 70-78 (2008).</li><li>Houtz, B., Trotter, J., Sasaki, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> BD FACService TECHNOTES. 9 (4), (2004).</li><li>Osborne, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+quantifying+cell+sorting+yield+in+'real+time'.">A method of quantifying cell sorting yield in 'real time'.</a> Cytometry. Part A : the journal of the International Society for Analytical Cytology. 77, 983-989 (2010).</li><li>Drennan, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thymic+microenvironment+differentially+regulates+development+and+trafficking+of+invariant+NKT+cell+sublineages.">The thymic microenvironment differentially regulates development and trafficking of invariant NKT cell sublineages.</a> J Immunol. 193, 5960-5972 (2014).</li></ol>

The authors have no competing interests to disclose.

Les étapes critiques dans le protocole actuel comprennent l&#39;isolement et l&#39;enrichissement ultérieur de CD5 + lymphocytes (Section 1 &amp; 2), tri FACS (section 3) et l&#39;étalement initial de cellules iNKT (section 4). Parmi les étapes effectuées dans la section 1, rappelez-vous à la couche attentivement la suspension cellulaire splénique sur le milieu de gradient de densité telle que une interphase cellulaire distincte est générée après la centrifugation. L&#39;enrichissement ultérieur ...

Murine T (iNKT) les cellules tueuses naturelles invariant constituent une population distincte de lymphocytes T innées choisis dans le thymus thymocytes corticaux par CD1d exprimant 1,2. cellules iNKT expriment un récepteur de lymphocyte T (TCR) comprenant une chaîne Vα14-Jα18 TCR invariant associé à chaque Vß8, Vβ7 ou Vβ2 TCR 3, qui est capable de reconnaître endogène ainsi que des antigènes lipidiques étrangers dans le cadre de CD1d. Par exemple, des cellules murines reconnaissent iNKT et sont activées par un antigène lipide endogène appelé isoglobotrihexosylceramide (iGb3) 4, ainsi que les α-galactosylcéramide (aGalCer) 5,6, un glycolipide isolé à partir d&#39;éponges marines. TCR-dépendante de l&#39;activation des cellules iNKT favorise l&#39;amorçage de réponses immunitaires adaptatives, et, par conséquent, les cellules iNKT se sont révélés être fonctionnellement impliquée dans le développement ou l&#39;amélioration d&#39;une gamme de pathologies, y compris les maladies rhumatismales 7 et le cancer &lt;sup&gt; 8. Actuellement, les ligands de cellules iNKT synthétiques constituent de nouveaux prometteurs adjuvants de vaccins qui peuvent être capables de réguler un nombre de conditions immunopathologiques. Il a précédemment été démontré que les cellules iNKT peuvent être générés in vitro après isolement à partir de tissus de souris toutefois; bon nombre de ces études emploient l&#39;utilisation de cellules primaires présentatrices d&#39;antigène (CPA) et / ou des lignées cellulaires 9, Vα14 TCR transgénique (Tg) de 10 souris, les thymomes ou pour la génération d&#39;hybridomes dérivés de cellules iNKT-11,12. En outre, un grand nombre de souris, des volumes élevés de réactifs tels que les dimères de CD1d aGalCer-chargés, et de longs temps de culture font certains protocoles publiés moins éthiquement et économiquement attrayante 9,13. Dans ce rapport, nous décrivons une méthode adaptée pour l&#39;isolement et dans l&#39;expansion in vitro de cellules iNKT de rate de souris. Plus précisément, le protocole décrit un procédépour enrichir les cellules iNKT de rate de souris qui réduit les souris, les réactifs et le temps requis pour FACS tri cellulaire, et propose une approche optimisé pour l&#39;expansion des cellules iNKT spléniques triées in vitro.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-2</td>
			<td>eBiosciences</td>
			<td>14-8021</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-12</td>
			<td>eBiosciences</td>
			<td>39-8122-65</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-7</td>
			<td>eBiosciences</td>
			<td>14-8071</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD3e (145-2C11)</td>
			<td>eBiosciences</td>
			<td>16-0032-86</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD28 (37.51)</td>
			<td>eBiosciences</td>
			<td>16-0281-85</td>
			<td></td>
		</tr>
		<tr>
			<td>e450-conjugated anti-mouse CD19 (eBio1D3)</td>
			<td>eBiosciences</td>
			<td>48-0193-82</td>
			<td></td>
		</tr>
		<tr>
			<td>e450-conjugated anti-mouse CD8&#945; (53-6.7)</td>
			<td>eBiosciences</td>
			<td>48-0081-82</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-conjugated anti-mouse CD5 (53-7.3)</td>
			<td>eBiosciences</td>
			<td>11-0051-81</td>
			<td></td>
		</tr>
		<tr>
			<td>V500-conjugated anti-mouse CD3&#949; (500A2)</td>
			<td>BD biosciences</td>
			<td>560771</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD5 (Ly-1) microbeads</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-049-301</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD16/32 (2.4G2)</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-092-575</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MS Columns</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue, 0.4% (wt/vol)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Gibco</td>
			<td>12633-020</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Gibco</td>
			<td>10010-056</td>
			<td>[Ca2+/Mg2+ - free]</td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Gibco</td>
			<td>10270</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-123</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4458-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>EDS-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque plus</td>
			<td>GE Healthcare</td>
			<td>71-7167-00 AF</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate F-bottom</td>
			<td>Greiner-bio one</td>
			<td>657-160</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Greiner-bio one</td>
			<td>665-180</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Greiner-bio one</td>
			<td>655-180</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml falcon tube</td>
			<td>Greiner-bio one</td>
			<td>188-271</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml falcon tube</td>
			<td>Greiner-bio one</td>
			<td>227-261</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m filter</td>
			<td>Greiner-bio one</td>
			<td>542-070</td>
			<td></td>
		</tr>
		<tr>
			<td>30 &#181;m filter</td>
			<td>Millipore</td>
			<td>SVGP01050</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS separator</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Water-jacketed CO2 incubator</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Kit</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSAria III</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an optimized method for isolating and expanding invariant natural killer t cells from mouse spleen

La capacité à sécréter des cytokines rapidement lors d&#39;une stimulation est une caractéristique fonctionnelle de l&#39;invariant T tueuses naturelles (iNKT) lignée cellulaire. iNKT cellules sont donc caractérisées par une population de cellules T capables d&#39;activer innée et adaptative direction réponses immunitaires. Le développement des techniques améliorées pour la culture et l&#39;expansion des cellules iNKT murins facilite l&#39;étude de la biologie des cellules iNKT in vitro et dans des modèles in vivo. Nous décrivons ici un procédé optimisé pour l&#39;isolement et l&#39;expansion de cellules spléniques murines iNKT. Les rates de souris C57BL / 6 sont supprimés, disséqués et tendues et la suspension cellulaire obtenue est en couches sur les médias de gradient de densité. Après centrifugation, les cellules mononucléaires spléniques (EMN) sont recueillies et (CD5 +) des lymphocytes CD5-positifs sont enrichies en utilisant des billes magnétiques. iNKT cellules au sein de la fraction CD5 + sont ensuite colorées avec du ^5; GalCer tétramère CD1d-chargé et purifié par tri cellulaire activé par fluorescence (FACS). Triées par FACS des cellules iNKT sont initialement puis cultivées in vitro en utilisant une combinaison de cytokines murines recombinantes et des récepteurs des cellules T de plaque lié (TCR) des stimuli avant d&#39;être détendu dans la présence de murine IL-7 recombinante. En utilisant cette technique, environ 10 8 cellules iNKT peuvent être générés à l&#39;intérieur de 18-20 jours de culture, après quoi ils peuvent être utilisés pour des tests fonctionnels in vitro, ou in vivo pour les expériences de transfert chez la souris.

Isolement des cellules mononucléaires spléniques en utilisant d&#39;un gradient de densité prend environ une heure et élimine l&#39;utilisation de réactifs nécessaires pour lyser les globules rouges (RBC). Un rendement élevé de cellules viables est obtenue en utilisant cette méthode et les débris générés pendant l&#39;organe de déformation est supprimée. Typiquement, la fréquence des cellules iNKT au sein du pool de lymphocytes de la rate est comprise entre 1 et 5% des lymphocytes T totaux Cependant, cel...

Watch this Scientific Journal Video about An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen at JoVE.com

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

Un procédé optimisé pour isoler et expansion Invariant Cellules T Tueuses Naturelles de Spleen souris

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

an-optimized-method-for-isolating-expanding-invariant-natural-killer

Research

JoVE Journal

Immunology and Infection

11.9K vues.  Ghent University Hospital. Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents. 

Vidéo: An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Transduction rétrovirale des récepteurs de cellules T dans les lymphocytes T de souris

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Recherche

Immunologie et infection

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

Préparation et utilisation de VIH-1 primaires CD4 + T-cellules comme cellules cibles dans des cellules tueuses naturelles Tests cytotoxiques

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

L&#39;expansion des lymphocytes T cytotoxiques à partir de sang de cordon ombilical qui cytomégalovirus cible, le virus Epstein-Barr Virus et Adénovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Présentatrice d&#39;antigène artificielle d&#39;activation à médiation cellulaire (AAPC) et l&#39;expansion des cellules T tueuses naturelles

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Tueur antigène artificielle cellules présentatrices (KaAPC) d&#39;utilisation efficace<em&gt; In Vitro</em&gt; L&#39;épuisement des lymphocytes T humains spécifiques de l&#39;antigène

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

Dérivation des cellules T<em&gt; In Vitro</em&gt; De la souris les cellules souches embryonnaires

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

Cytométrie en flux Analyse des cellules tueuses naturelles lytique Activité dans le sang humain

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Un nouveau système exempt d’alimentation pour la Production de masse des cellules tueuses naturelles murins In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Isoler des Lymphocytes du système immunitaire Intestinal souris petit

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

Multicolor Flow Cytometry-basé Quantification des mitochondries et des Lysosomes dans les cellules T

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...