This work was supported by the Swedish Cancer Society (11 0253, 13 0469), the Swedish Research Council (K2012-99X-21959-01-3), Marcus Borgstr&#246;ms Foundation and the Swedish Research Council through a grant to the Uppsala RNA Research Centre (2006-5038-36531-16). We are indebted to the BioVis core facility at Uppsala University for much help with the FACS analysis.

Le protocole décrit l&#39;isolement de cellules lymphoïdes à partir de matériau patient humain et nécessite donc une approbation éthique. Le travail effectué dans la présente étude a été accordée par le Conseil d&#39;examen éthique Uppsala (DNR. 2013/387). 1. Isolement des cellules mononucléaires (MNC) de amygdales palatines humaines ATTENTION: Tout le matériel blindé d&#39;origine humaine comme le sang, les tissus ou les fluides corporels doit être considéré comme du matériel potentiellement infecté. Par conséquent, les pratiques de biosécurité recommandées pour la manipulation des tissus humains doivent être suivies. Remarque: Pour éviter la contamination, toutes les solutions et équipements de culture cellulaire doivent être stériles. Tous les tampons et les solutions doivent être pré-refroidis et conservés dans la glace. Tissus de l&#39;amygdale et cellules isolées doivent être conservées et traitées sur la glace. <ol><li> Obtenir amygdales fraîches provenant de patients ayant subi une amygdalectomie ou amygdalotomie (Figure 2). Plonger la clum de tissusps dans un tube centrifuge de 50 ml rempli d&#39;une solution saline glacée stérile de Hanks équilibrée (HBSS) complétée avec 5% de sérum bovin fœtal (FBS), mM glutamine 10, 0,05 mg / ml de gentamicine et 1% d&#39;antibiotique-antimycotique mélange (Penicillin, streptomycine et amphotéricine B). </li><li> Gardez les tubes avec les échantillons de tissus des amygdales submergées en tout temps sur la glace. Essayez de traiter les amygdales dès que possible, et au plus tard 3 heures après l&#39;ablation chirurgicale. </li><li> Placez l&#39;amygdale sur un 60 mm cellule en plastique plaque de culture sur de la glace et de garder le tissu humidifié avec HBSS. Retirer caillots de sang, les tissus conjonctifs et gras avec une pince de nettoyage de la surface des amygdales. </li><li> Utilisez un nouveau 60 mm plaque de culture cellulaire contenant 5 ml de HBSS. Couper la touffe de tissu d&#39;amygdale en fragments 3-10 mm en utilisant une paire de ciseaux stérile et / ou d&#39;un scalpel. </li><li> Préparer une nouvelle plaque de 60 mm culture cellulaire contenant 10 ml de HBSS et placer un tamis cellulaire en plastique 100 pm dans la solution HBSS. </li><li> Transférer les dissfragments d&#39;amygdale échies dans le tamis cellulaire avec un forceps stériles. En utilisant l&#39;extrémité du piston d&#39;une seringue en plastique, presser doucement les fragments de tissus à travers le tamis de la cellule. Assurez-vous que les fragments de l&#39;amygdale sont totalement immergés dans le HBSS. </li><li> Jeter le tamis cellulaire avec les restes de tissu et transférer la suspension cellulaire obtenue à partir de la plaque de culture cellulaire de 60 mm dans un tube de centrifugation en plastique de 50 ml. Laisser le tube sur la glace tout en procédant à des mesures 1.8 et 1.9. </li><li> Dans le cas de grandes amygdales, de plus de 2 cm de diamètre (Figure 2), serrer les fragments de l&#39;amygdale dans deux crépines de cellules pour éviter le colmatage de la maille dans la passoire. </li><li> Pour obtenir le volume final de 35 ml de la suspension cellulaire. Remarque: À cette étape, il est possible de préparer la suspension de cellules pour la cryoconservation (voir la section 2). </li><li> Ajouter 10 ml d&#39;une solution à gradient de densité de Ficoll comme dans un nouveau tube de centrifugation de 50 ml. Superposer délicatement le suspens cellulairesion (étape 1.9) au-dessus de la solution à gradient de densité. Eviter le mélange de la suspension cellulaire et la solution de gradient de densité. Remarque: La solution à gradient de densité doit être réchauffé à la température ambiante. </li><li> Centrifuger la suspension cellulaire dans un rotor swing-out à 700 g pendant 20 min à température ambiante. Ne pas activer la fonction de frein sur la centrifugeuse que le freinage rapide peut perturber le gradient. Aussi, utilisez la fonction d&#39;accélération le plus bas disponible sur la centrifugeuse. Note: Après cette étape de centrifugation, d&#39;observer que les MNC une couche duveteuse blanche à l&#39;interface, et les globules rouges (RBC), les fibroblastes et les débris cellulaires comme le sédiment au fond du tube. </li><li> Recueillir soigneusement la couche de multinationales en utilisant une pipette 10 ml. Placer la suspension de cellules dans un nouveau 50 ml tube de centrifugation. </li><li> Ajouter 25 ml de PBS glacé à la suspension cellulaire et tourner le tube à 300 g pendant 5 min dans un rotor swing-out à 4 ° C. </li><li> Eliminer le surnageant en utilisant une pipette 25 mlet répéter culot cellulaire étape de lavage deux fois de plus. Enfin remettre en suspension le culot cellulaire dans 15 ml de PBS. Remarque: Après l&#39;étape de lavage final, il est possible de cryoconservation de la suspension cellulaire (voir la section 2). </li><li> Appliquer 10 pi de suspension de cellules dans la cellule de hémocytomètre chambre de comptage et de compter le nombre de cellules sous un microscope. Distribuer la quantité appropriée de cellules (par exemple, 3.2 x 10 7) dans un tube de centrifugeuse de 15 ml pour la séparation par billes magnétiques ultérieure. Gardez cette aliquote sur la glace jusqu&#39;à l&#39;étape 3.2. </li></ol> 2. La cryoconservation des cellules des amygdales <ol><li> Préparer le milieu de congélation (90% de FBS et 10% de DMSO) et le garder sur la glace. Pour le stockage à long terme tenir les médias de congélation à -20 ° C. </li><li> Déterminer le nombre total de cellules en utilisant une chambre de comptage de cellules hémocytomètre (étape 1.15). Calculer la quantité nécessaire de milieu de congélation selon la densité cellulaire congelé souhaité (par exemple, 10 7 celluless / ml de milieu de congélation). </li><li> Centrifuger la suspension de cellules à 300 g pendant 5 min. Décanter le surnageant sans perturber le culot cellulaire et remettre en suspension le culot cellulaire dans du milieu de congélation refroidi à la glace (de l&#39;étape 2.1). </li><li> Distribuer des aliquotes de 1 ml de la suspension cellulaire dans des flacons stériles destinés à l&#39;entreposage à long terme dans de l&#39;azote liquide. Congeler les flacons dans une chambre de l&#39;isopropanol et de les stocker à -80 ° CO / N. Pour la conservation à long terme de transférer les flacons dans un contenant de l&#39;azote liquide réservoir de stockage ou d&#39;une cellule C congélateur -140 °. </li><li> Pour décongeler flacons de cellules congelées, réchauffez-les rapidement dans un bain d&#39;eau à 37 C °. Immédiatement lors de la décongélation, disperser la suspension de cellules dans 10 ml de HBSS préchauffé (avec des suppléments, l&#39;étape 1.1) dans un tube de centrifugeuse de 15 ml. Spin le tube à 300 g pendant 5 min, retirez HBSS et remettre le culot cellulaire à la densité cellulaire souhaitée dans HBSS (avec des suppléments, l&#39;étape 1.1). Remarque: La viabilité du multinationales after dégel est d&#39;importance, car la présence de cellules mortes diminuera le rendement final de B et les lymphocytes T purifiés. Remarque: Selon notre expérience, la viabilité des cellules moins de 80% permettra de réduire l&#39;efficacité de l&#39;isolement cellulaire. </li></ol> 3. positive Sélection des lymphocytes T de la population à partir des amygdales multinationales Note 1: Ce protocole est basé sur la sélection positive des lymphocytes humains T CD3 + de multinationales amygdales utilisant des billes magnétiques couplées à l&#39;anticorps CD3. Il est possible de commencer cette section de frais (Section 1) ou les (section 2) multinationales congelés. Note 2: Commencez avec 3 x 10 7 multinationales. Ne pas dépasser ce nombre de cellules, depuis les colonnes de séparation peuvent obstruer et cela permettra de réduire l&#39;efficacité de l&#39;isolement. Utilisez grandes colonnes si plusieurs cellules seront traitées. Les volumes utilisés dans ce protocole ont été expérimentalement optimisé pour le nombre de cellules de noused dans notre dispositif expérimental. <ol><li> Préparer le tampon de séparation [PBS (pH 7,2), 0,5% d&#39;albumine de sérum bovin (BSA) et de 2 mM d&#39;EDTA]. Filtrez le tampon de séparation à travers un filtre de 0,45 um et stocker à 4 ° C. </li><li> Spin la suspension des multinationales (étape 1.14) à 300 g pendant 5 min à 4 ° C. Remettre en suspension le culot résultant dans des cellules 240 pi (80 pi pour 10 7 cellules) de séparation de tampon glacé. Transférer la suspension cellulaire dans un tube stérile de 2 ml. </li><li> Ajouter 20 ul d&#39;anticorps CD3 magnétique à la solution de cellules. Incuber les tubes pendant 1 heure à 4 ° C avec agitation douce continue pour maintenir les cellules en suspension. </li><li> Transférer la totalité de la suspension cellulaire dans un tube de 15 ml contenant 5 ml séparation tampon de glace froide pour laver les cellules. </li><li> Centrifuger le tube à 300 g pendant 10 min à 4 ° C dans un rotor swing-out. </li><li> Pendant ce temps, mettre en place le séparateur magnétique et de la colonne. Fixez le séparateur magnétique à la barre et placez la colonne dans la separteur. Laver la colonne en appliquant 500 ul de tampon de séparation glacée. Jeter l&#39;écoulement à travers. </li><li> Placer un nouveau tube de collecte de 15 ml au-dessous de la colonne. Garder le tube de collecte sur la glace. </li><li> Jeter le surnageant (étape 3.5) par pipette et remettre en suspension doucement le culot cellulaire dans 500 pi de tampon de séparation. Appliquer la suspension cellulaire sur le dessus de la colonne de pré-lavés et laisser courir à travers. Remarque: amas de cellules peuvent obstruer la colonne et donc de réduire le débit. Pour éviter ce problème, placer un filtre en matière plastique 40 um de la cellule au-dessus de la colonne. Passer les cellules à travers ce maillage sera fortement améliorer l&#39;efficacité de cette méthode. </li><li> Laver la colonne avec 1,5 fois 4 ml de tampon de séparation (500 ul pour 10 7 cellules). Attendre que le réservoir de la colonne à vide (pas d&#39;liquide doit être observée dans la colonne) avant d&#39;appliquer l&#39;étape de lavage suivante. Remarque: Obtenir les cellules non marquées CD3, considérés comme fraction des lymphocytes B, comme elles sontélue dans le tube de collecte. </li><li> Retirer la colonne du séparateur et la placer dans un nouveau tube de 15 ml de collection. Introduire à la pipette 2 ml de tampon de séparation sur la colonne. Utilisation du plongeur fourni avec la colonne éluer les lymphocytes T sélectionnées positivement, qui sont considérés comme la fraction des lymphocytes T. </li><li> Déterminer le nombre de cellules purifiées (étape 1.15), si nécessaire. Les suspensions cellulaires sont maintenant prêts pour des expériences en aval. </li></ol> 4. cytométrie de flux de isolé amygdales B et les lymphocytes T ATTENTION: solution Paraformaldéhyde est irritant et cancérogène suspecté. Porter un vêtement de protection approprié, des gants et une protection des yeux / du visage. Remarque 1: Ce protocole décrit la méthode de coloration directe de la B et les lymphocytes T isolés par tri cellulaire activé par fluorescence (FACS). Le but principal de cette étape est d&#39;évaluer la pureté de la cellule isolée populations after CD3 de séparation d&#39;anticorps magnétique. A cet effet B établi et des marqueurs de cellules T, les anticorps monoclonaux fluorophores conjugué CD20-FITC et CD2-APC, sont utilisés. Aussi l&#39;inclusion d&#39;anticorps de contrôle d&#39;isotype est fortement recommandé de distinguer le &quot;fond&quot; liaison non spécifique des anticorps CD2 et CD20 (voir l&#39;étape 4.8). Remarque 2: Il est possible de conserver les fractions de cellules purifiées dans un paraformaldehyde à 1% (PFA) solution à l&#39;obscurité à 4 ° C jusqu&#39;à ce que le temps de coloration (par exemple, le jour suivant.). De plus, le même procédé est applicable si il existe un écart de temps entre la coloration et une analyse FACS. Rappelez-vous que dans les deux cas, les cellules doivent être lavés correctement avec LNPP (PBS contenant 0,2% de BSA) tampon, avant la coloration ou l&#39;analyse FACS. <ol><li> Sortez 6 x 10 6 cellules des multinationales de l&#39;étape 1.15 (avant d&#39;appliquer à la colonne), fraction des lymphocytes B de l&#39;étape 3.9 et de la fraction des lymphocytes T de l&#39;étape 3.10. </li><li> Spin la cellulesuspensions à 300 g pendant 5 min à 4 ° C en utilisant un rotor swing-out. </li><li> Retirer le surnageant avec pipette et laver le culot cellulaire une fois avec 1 ml de glace-froid LNPP. Faites tourner les suspensions de cellules à 300 g pendant 5 min à 4 ° C. Retirer le surnageant avec pipette et remettre la cellules dans 600 pi de tampon LNPP glacée. </li><li> Ajouter 100 ul de la suspension de cellules au fond d&#39;un tube FACS. </li><li> Ajouter 100 pi de PBS contenant du sérum humain inactivé 10% de la chaleur à chaque tube, bien mélanger et laisser incuber pendant ~ 1 min à température ambiante ou 20 min sur la glace. Remarque: les lymphocytes B portent des récepteurs Fc. Afin de bloquer les récepteurs Fc des fractions de cellules purifiées sont incubées avec du sérum humain inactivé à la chaleur à 10%. Le sérum humain inactivé à la chaleur est d&#39;une incubation à 56 ° C pendant 1 heure. Diviser le sérum inactivé à la chaleur en petites portions et les conserver congelés à -20 ° C. </li><li> Centrifuger la suspension de cellules à 300 xg pendant 5 min à 4 ° C dans un swing Rotor. </li><li> Retirer le surnageant avec pipette et laver le culot de cellules avec 1 ml LNPP. Répéter la centrifugation (300 g pendant 5 min à 4 ° C). </li><li> Ajouter 100 ul LNPP dans chaque tube sur la glace. Ajouter la quantité appropriée de l&#39;anticorps monoclonal fluorophore conjugué, selon la recommandation du fabricant (par exemple, 20 pi d&#39;anti-CD20 et / ou 5 pi d&#39;anticorps anti-CD2 dans 100 pi de LNPP). Note: Ne pas oublier d&#39;attribuer suffisamment de tubes de contrôle pour l&#39;analyse FACS. Dans ce protocole, les contrôles suivants doivent être inclus: 1) cellules colorées avec les anticorps anti-CD2 et anti-CD20 individuellement, 2) des cellules colorées avec les anticorps anti-CD2 et anti-CD20 contrôle isotypique individuellement et, 3) les cellules sans addition d&#39;anticorps. </li><li> Brièvement vortex le tube et incuber pendant 30 min à 4 ° C dans l&#39;obscurité. </li><li> Laver 2 fois avec LNPP. Ajouter 2 ml de solution de PFA à 1% pour les échantillons. Laissez les cellules restent dans la solution PFA à 476; C dans l&#39;obscurité jusqu&#39;à ce que le moment de l&#39;analyse FACS. </li><li> Immédiatement avant d&#39;exécuter les échantillons dans la machine FACS, laver les cellules 2 fois avec LNPP (300 g pendant 5 min à 4 ° C). Échantillons de remettre en suspension dans 1 ml de PBS et de garder à 4 ° C (ou sur glace), protégé de la lumière, avant la séparation sur le cytomètre de flux. </li><li> Faites le tri FACS suivant le protocole du cytomètre en flux fabricant. </li></ol> Détection PCR 5. des adénovirus ADN isolé amygdales B et les lymphocytes T Note 1: Les amorces de gènes d&#39;hexon d&#39;adenovirus sont AdRJC1 (5&#39;-GACATGACTTTCGAGGTCGATCCCATGGA-3 &#39;) et AdRJC2 (5&#39;-CCGGCTGAGAAGGGTGTGCGCAGGTA-3&#39;) 11. Ces amorces génèrent un amplicon de 139 pb. L&#39;hôte gène ARNr 18S peut être détectée avec des amorces tp206 (5&#39;-CCCCTCGATGCTCTTAGCTG-3 &#39;) et TP207 (5&#39;-TCGTCTTCGAACCTCCGACT-3&#39;). La taille de l&#39;amplimère attendu est de 300 pb. Note 2: Chaque PCRterme comprend un contrôle négatif (distillée H 2 O) et un contrôle positif de l&#39;ADN obtenu à partir de la lignée cellulaire B humaine (BJAB) infectés par l&#39;adénovirus de type 5 humain 12. <ol><li> Extraire l&#39;ADN (d&#39;au moins 1 x 10 6 cellules, étape 3.11) en utilisant une membrane de silice procédé de purification sur la base de la colonne. Extraire l&#39;ARN en utilisant du phénol et de l&#39;isothiocyanate de guanidine 13 solution. </li><li> Ajouter 100 ng d&#39;ADN de lymphocytes B et T à un mélange réactionnel contenant un tampon 1x HF, 0,2 mM de désoxynucléotide triphosphate mélange (dNTP), 0,25 uM de chaque amorce et 1 U de haute fidélité de l&#39;ADN polymerase dans un volume total de 20 ul. </li><li> Effectuer une amplification par PCR dans les conditions de cyclage suivantes: 30 s de dénaturation à 98 ° C, suivi par 30 cycles de 98 ° C pendant 10 sec, 67 ° C (hexon) ou 58 ° C (ARNr 18S) pendant 30 s et 72 ° C pendant 30 sec. La réaction PCR a été terminé avec une étape d&#39;extension finale de 7 min à 72 ° C. </li><li> Séparer l&#39;amplif PCR résultantication produits sur un gel d&#39;agarose à 1,5% dans du tampon TBE 1x et visualiser les bandes attendues par coloration avec du colorant d&#39;acide nucléique. </li></ol>

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The authors have nothing to disclose.

Un des facteurs les plus importants qui affectent le résultat de ce protocole est l&#39;utilisation de matériel amygdales frais comme le matériau de départ. Par conséquent, les échantillons d&#39;amygdales doivent être traitées dans un délai de 3 heures après la chirurgie. Amygdales peuvent être obtenus à partir de deux adultes et les enfants. Le matériau des amygdales des enfants est généralement plus petit en raison de l&#39;ablation chirurgicale partielle des amygdales (amygdalotomie). Par conséquent,...

Tonsils are collections of incompletely encapsulated lymphoid tissues that lie under, and in contact with, the epithelium in the upper aero-digestive tract. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. The paired palatine tonsils together with the nasopharyngeal tonsil (adenoid), paired tubal tonsils and lingual tonsils constitute the so-called &#8220;Waldeyer&#180;s ring&#8221;. The latter is responsible for the initial contact between inhaled or ingested pathogens and the lymphoid tissues of the aerodigestive tract1,2. Indeed, numerous reports have shown that both bacterial and viral antigens can be detected in palatine tonsil tissue samples2-6.
The palatine tonsils are composed of dense lymphoid tissue covered by a stratified squamous non-keratinising epithelium. The tonsils have numerous crypts, epithelial invaginations, which penetrate the parenchyma increasing the surface area. Histologically, the palatine tonsils contain numerous lymphoid follicles with germinal centers, which are the sites for B cell maturation and differentiation (B-cell areas). Likewise, the palatine tonsils encompass T cells, which are mainly located in the extrafollicular regions (T-cell areas). In addition to the B and T cells, also various follicular dendritic cells can be detected in palatine tonsils1,2. 
Due to their anatomic location, the palatine tonsils are easily accessible by surgical interventions. For example, surgical removal of tonsils, known as tonsillectomy, is routinely carried out worldwide7. In children with tonsillar hyperplasia, a partial surgical removal of the tonsils (tonsillotomy) is sometimes used, causing less postoperative pain to the patients. Considering the accumulation of various pathogens in tonsils, surgically removed tonsils provide a unique opportunity to study the influence of viral and bacterial agents on tonsillar lymphocyte functions2,8. Furthermore it is possible to study if some pathogens prefer to reside in specific cell subpopulations9. In addition, as the tonsils are rich source of B lymphocytes, isolated tonsillar B lymphocytes can be efficiently used to study the activity of different B cell subpopulations10. However, as the palatine tonsils contain a mixture of cell types an efficient method to separate the different cell subpopulations is needed. 
Here, we describe a simple method for efficient and rapid isolation of tonsillar B and T cell populations from human palatine tonsils by using a magnetic-activated cell separation technique (Figure 1). The method described here is useful for scientists who want to assess the role of different infectious agents in human lymphoid organs such as palatine tonsils.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Hanks balanced salt solution (HBSS)&#160;</td>
			<td>Gibco</td>
			<td>14175-053</td>
			<td>Contains 5% fetal bovine serum (FBS), 10 mM Glutamine,&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>&#160;0.05 mg/ml Gentamicin and 1% Antibiotic-Antimycotic mix</td>
		</tr>
		<tr>
			<td>Freezing medium</td>
			<td></td>
			<td></td>
			<td>90% FBS and 10% DMSO</td>
		</tr>
		<tr>
			<td>MACS buffer</td>
			<td></td>
			<td></td>
			<td>PBS (pH 7.2), 0.5% BSA and 2mM EDTA</td>
		</tr>
		<tr>
			<td>PBSA&#160;</td>
			<td></td>
			<td></td>
			<td>PBS containing 0.2% BSA</td>
		</tr>
		<tr>
			<td>PFA</td>
			<td></td>
			<td></td>
			<td>PBS containing 1% paraformaldehyde (PFA). Make fresh.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>PFA is suspected carcinogen. Wear gloves and goggles.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic mix</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>100-mm petridish</td>
			<td>Nunc</td>
			<td>172958</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting foreceps</td>
			<td>Fisher Scientific</td>
			<td>1381241</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight iris scissors&#160;</td>
			<td>Fisher Scientific</td>
			<td>12912055</td>
			<td></td>
		</tr>
		<tr>
			<td>disposable scalpels</td>
			<td>Swann-Morton</td>
			<td>REF 0501</td>
			<td></td>
		</tr>
		<tr>
			<td>100&#956;m plastic cell strainer</td>
			<td>Corning Life Sciences</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>40&#956;m plastic cell strainers&#160;</td>
			<td>Corning Life Sciences</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>2-ml plastic syringe</td>
			<td>BD Biosciences&#160;</td>
			<td>300185</td>
			<td></td>
		</tr>
		<tr>
			<td>15-ml conical centrifuge tubes</td>
			<td>SARSTEDT</td>
			<td>62554502</td>
			<td></td>
		</tr>
		<tr>
			<td>50-ml conical centrifuge tubes</td>
			<td>SARSTEDT</td>
			<td>62547254</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-speed centrifuge with fixed-angle or swinging-bucket rotor</td>
			<td colspan="2">Thermo Scientific Heraeus Megafuge 16R</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll&#8211;Hypaque</td>
			<td>Sigma-Aldrich</td>
			<td>F5415-50ML</td>
			<td>Ficoll solution</td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Biological industries</td>
			<td>040071A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulphoxide (DMSO)</td>
			<td>SIGMA</td>
			<td colspan="2">D2650-5X5ML</td>
		</tr>
		<tr>
			<td>MACS MS columns&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS human CD3 MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-881</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS separator (Octo MACS)</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-109</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Merck Millipore</td>
			<td>1120180100</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>AnalaR NORMAPUR</td>
			<td>20302.293</td>
			<td></td>
		</tr>
		<tr>
			<td>CD2 conjugated to allophycocyanin (CD2-APC)&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>560642</td>
			<td></td>
		</tr>
		<tr>
			<td>CD20 conjugated to fluorescein isothiocyanate (CD20-FITC)&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>556632</td>
			<td></td>
		</tr>
		<tr>
			<td>Human serum&#160;</td>
			<td>Rockland Immunochemicals</td>
			<td>D119-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>Thermo Scientific</td>
			<td>F-530L</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>BD Falcon</td>
			<td>352003</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotube</td>
			<td>SARSTEDT</td>
			<td>72379</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSRII flowcytometer</td>
			<td>BD Biosciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSDiva 4.1 software</td>
			<td>BD Biosciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleospin Blood&#160;</td>
			<td>Macherey-Nagel</td>
			<td>740951.50</td>
			<td>DNA isolation kit</td>
		</tr>
		<tr>
			<td>TRIzol&#160; reagent</td>
			<td>Life technologies</td>
			<td>15596</td>
			<td>RNA isolation reagent</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>The Paul Marienfeld GmbH &#38; Co. KG</td>
			<td>0610030</td>
			<td>cell counter device</td>
		</tr>
		<tr>
			<td>GelRed</td>
			<td>Biotium</td>
			<td align="right">41003</td>
			<td>Nucleic acid gel stain&#160;</td>
		</tr>
	</tbody>
</table>

efficient isolation protocol for b and t lymphocytes from human palatine tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Une séparation efficace des multinationales amygdales résultats dans les sous-populations hautement purifiées de lymphocytes B et T. Cela a été confirmé par analyse FACS en utilisant des anticorps anti-CD2 et CD20 anti-B et pour détecter des populations de lymphocytes T, respectivement (figure 3A). En revanche, une séparation inefficace des MNC résultats dans les fractions cellulaires qui sont un mélange de cellules provenant des deux sous-populations de lymphocytes B et T <st...

Watch this Scientific Journal Video about Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils at JoVE.com

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Protocole d&#39;isolation efficace pour B et les lymphocytes T à partir des amygdales palatines humaines

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

efficient-isolation-protocol-for-b-t-lymphocytes-from-human-palatine

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15.7K vues.  Uppsala Biomedical Center, Uppsala University. Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Vidéo: Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Human T isolement des lymphocytes, de la Culture et de l&#39;analyse des migrations In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Recherche

Immunologie et infection

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 ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Isolement des lymphocytes de souris muqueuses génitales Tract

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Isolement des précurseurs des cellules B des sous-ensembles à partir de sang de cordon ombilical

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

L&#39;isolement et la différenciation Th17 de Naïve CD4 des lymphocytes T

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Analyse rapide des aberrations chromosomiques dans les lymphocytes B de souris par PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

La transfection très efficace des droits de THP-1 par les macrophages nucléofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

Un protocole simple et rapide aux non enzymatique dissocier frais tissus humains pour l&#39;analyse des lymphocytes infiltrant

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Génération de Recombinant Human IgG anticorps monoclonaux à partir de cellules immortalisées Classé B

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

&lt;em&gt;In Vitro&lt;/em&gt; Differentiation of Human CD4&lt;sup&gt;+&lt;/sup&gt;FOXP3&lt;sup&gt;+&lt;/sup&gt; Induced Regulatory T Cells (iTregs) from Na&amp;#239;ve CD4&lt;sup&gt;+&lt;/sup&gt; T Cells Using a TGF-&amp;#946;-containing Protocol

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

In Vitro Differentiation of Human CD4 + FOXP3 cellules + Induced T régulatrices (de iTregs) de Naïf cellules T CD4 + en utilisant un protocole TGF-β contenant