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.

O protocolo descreve o isolamento de células linfóides a partir de material humano paciente e, por conseguinte, requer uma homologação ética. O trabalho realizado no presente estudo foi concedido pelo Ethical Review Board Uppsala (DNR. 2013/387). 1. Isolamento de células mononucleares (MNC) de tonsilas palatinas Humanos CUIDADO: Todo o material de origem humana sem tela como sangue, tecidos ou fluidos corporais deve ser considerado como material potencialmente infectado. Portanto, as práticas de biossegurança recomendadas para o tratamento dos tecidos humanos devem ser seguidas. Nota: Para evitar a contaminação, todas as soluções e equipamentos de cultura celular deve ser estéril. Todos os buffers e soluções devem ser pré-arrefecido e mantido em gelo. Tecidos das amígdalas e células isoladas devem ser mantidos e manipulados no gelo. <ol><li> Obter amígdalas frescos de pacientes submetidos a amigdalectomia ou tonsilotomia (Figura 2). Mergulhar o tecido ClumPS para um tubo de centrífuga de 50 ml cheio com solução salina arrefecida com gelo estéril de Hanks equilibrada (HBSS), suplementado com 5% de soro fetal de bovino (FBS), 10 mM de glutamina, 0,05 mg / ml de gentamicina e 1% de mistura de antibióticos-antimicótico (penicilina, estreptomicina e Anfotericina B). </li><li> Manter os tubos com as amostras de tecidos das amígdalas submersas em todos os momentos no gelo. Tente processar as amígdalas, logo que possível, eo mais tardar três horas após a remoção cirúrgica. </li><li> Coloque a amígdala em um 60 milímetros placa de cultura de células de plástico no gelo e manter o tecido umedecido com HBSS. Remover coágulos sanguíneos visíveis, gordo e tecidos conjuntivos com uma pinça limpa da superfície da amígdala. </li><li> Usar uma nova placa de 60 mm de cultura de células, contendo 5 ml de HBSS. Cortar o tecido tonsilar moita em fragmentos de 3-10 mm, usando um par de tesouras estéril e / ou um bisturi. </li><li> Prepara-se uma nova placa de 60 mm de cultura de células contendo 10 ml de HBSS e colocar um 100 um filtro de células de plástico na solução de HBSS. </li><li> Transfira os Dissfragmentos da amígdala ected para o filtro de células com pinça estéreis. Usando a extremidade do êmbolo de uma seringa de plástico, sem problemas espremer os fragmentos de tecido através do filtro de células. Certifique-se de que os fragmentos da amígdala está totalmente imerso no HBSS. </li><li> Descartar o filtro celular com os restos de tecido e transferir a suspensão de células resultante a 60 mm de placa de cultura celular num tubo de centrífuga de 50 ml de plástico. Deixe o tubo em gelo enquanto prosseguir com passos 1.8 e 1.9. </li><li> No caso de grandes amígdalas, maior do que 2 cm de tamanho (Figura 2), apertar os fragmentos de amígdalas de dois filtros de células para evitar o entupimento da malha no filtro. </li><li> Obter o volume final de 35 ml da suspensão de células. Nota: Neste passo é possível preparar a suspensão de células para criopreservação (ver secção 2). </li><li> Adicionar 10 ml de solução de gradiente de densidade de Ficoll, tais como para um novo tubo de centrífuga de 50 ml. Delicadamente sobrepor as suspens celularesde iões (passo 1.9) na parte superior da solução de gradiente de densidade. Evitar a mistura da suspensão de células e a solução do gradiente de densidade. Nota: A solução de gradiente de densidade tem de ser aquecida até à temperatura ambiente. </li><li> Centrifugar a suspensão celular num rotor basculante para fora a 700 xg durante 20 min à TA. Não ative a função de frenagem na centrífuga como frenagem rápida pode perturbar o gradiente. Além disso, use a função de aceleração mais baixa disponível na centrífuga. Nota: Após este passo de centrifugação, observar PTM como uma camada fofa branca na interface, e glóbulos vermelhos (RBC), os fibroblastos e os detritos celulares como o sedimento no fundo do tubo. </li><li> Recolher cuidadosamente a camada de multinacionais, usando uma pipeta de 10 ml. Colocar a suspensão de células para um novo tubo de centrífuga de 50 ml. </li><li> Adicionar 25 ml de PBS gelado à suspensão de células e girar o tubo a 300 xg durante 5 min num rotor basculante para fora a 4 ° C. </li><li> Remover o sobrenadante utilizando uma pipeta de 25 mle repita pellet celular lavagem passo mais duas vezes. Finalmente ressuspender o pellet celular em 15 ml de PBS. Nota: Após o passo final de lavagem, é possível criopreservar da suspensão celular (ver secção 2). </li><li> Aplicar 10 ul de suspensão de células para dentro da câmara de contagem de células hemocitómetro e contar o número de células sob um microscópio. Dispensar a quantidade apropriada de células (por exemplo, 2-3 x 10 7) num tubo de centrífuga de 15 ml para subsequente separação esférulas magnéticas. Manter esta alíquota em gelo até o passo 3.2. </li></ol> 2. criopreservação de células tonsilares <ol><li> Prepare meio de congelação (90% de FBS e 10% de DMSO) e mantê-lo em gelo. Para armazenamento a longo prazo manter os meios de congelação a -20 ° C. </li><li> Determinar o número total de células utilizando uma câmara de contagem de hemocitómetro célula (passo 1.15). Calcula-se a quantidade necessária de meio de congelação de acordo com a densidade celular congelado desejado (por exemplo, 10 7 célulasS / ml de meio de congelação). </li><li> Centrifugar a suspensão de células a 300 xg durante 5 min. Decantar o sobrenadante sem perturbar o pelete de células e ressuspender o sedimento de células em meio de congelação arrefecida com gelo (a partir do passo 2.1). </li><li> Distribuir aliquotas de 1 mL da suspensão de células em tubos de ensaio estéreis concebidas para a armazenagem a longo prazo em azoto líquido. Congelar os frascos em uma câmara de isopropanol e armazená-los a -80 ° CO / N. Para a preservação a longo prazo transferir os frascos em azoto líquido que contém um tanque de armazenamento ou uma -140 ° C congelador de células. </li><li> Para descongelar frascos com células congeladas, aquecê-los rapidamente em um banho de água a 37 ° C. Imediatamente quando descongeladas, dispersar a suspensão de células para 10 ml de HBSS pré-aquecido (com suplementos, passo 1.1) em um tubo de centrífuga de 15 ml. Girar o tubo a 300 xg durante 5 minutos, remover HBSS e ressuspender o pellet celular a uma densidade de células desejada em HBSS (com suplementos, passo 1.1). Nota: A viabilidade do af EMNsTer o descongelamento é de importância, uma vez que a presença de células mortas irá diminuir o rendimento final de B e linfócitos T purificados. Nota: De acordo com a nossa experiência a viabilidade celular inferior a 80% irá reduzir a eficiência de isolamento de células. </li></ol> 3. Selecção positiva de Linfócitos T População de tonsilares multinacionais Nota 1: Este protocolo baseia-se na selecção positiva de linfócitos T CD3 + humanos de empresas multinacionais de amígdalas usando pérolas magnéticas acopladas ao anticorpo CD3. É possível começar esta seção de fresco (Seção 1) ou os congelados (Seção 2) as multinacionais. Nota 2: Comece com 3 x 10 7 multinacionais. Não exceder este número de células, uma vez que as colunas de separação podem entupir e isso vai reduzir a eficiência do isolamento. Use colunas maiores se mais células serão tratadas. Os volumes utilizados no presente protocolo tem sido experimentalmente optimizado para o número de células USed em nossa configuração experimental. <ol><li> Preparar o tampão de separação [PBS (pH 7,2), 0,5% de albumina de soro bovino (BSA) e 2 mM de EDTA]. Filtra-se o tampão de separação através de um filtro de 0,45 um e armazenar a 4 ° C. </li><li> Girar a suspensão PTM (passo 1.14) a 300 xg durante 5 min a 4 ° C. Ressuspender o sedimento resultante em 240 uL de células (80 ul por 10 7 células) de tampão arrefecido com gelo de separação. Transferir a suspensão de células para um tubo de 2 mL estéril. </li><li> Adicionar 20 ul de anticorpo CD3 magnética para a solução de células. Incubar os tubos durante 1 hora a 4 ° C com agitação suave contínua para manter as células em suspensão. </li><li> Transferir todo da suspensão de células para um tubo de 15 ml contendo 5 ml de tampão arrefecido com gelo de separação para lavar as células. </li><li> Centrifuga-se o tubo a 300 xg durante 10 min a 4 ° C num rotor basculante para fora. </li><li> Enquanto isso configurar o separador magnético e coluna. Fixar o separador magnético para o suporte e colocar a coluna na SEPARator. Lava-se a coluna por aplicação de 500 ul de tampão de separação gelada. Descartar o fluxo através. </li><li> Coloque um novo tubo de recolha de 15 ml por baixo da coluna. Mantenha o tubo de coleta no gelo. </li><li> Descartar o sobrenadante (passo 3.5), através da pipeta e ressuspender cuidadosamente a pelete de células em 500 ul de tampão de separação. Aplicar a suspensão de células na parte superior da coluna de pré-lavadas e deixá-lo correr através. Nota: aglomerados de células podem obstruir a coluna e, assim, reduzir a taxa de fluxo. Para evitar este problema, coloque um 40 um filtro de células de plástico no topo da coluna. Passando as células através desta malha vai melhorar muito a eficiência deste método. </li><li> Lava-se a coluna 4 vezes com 1,5 ml de tampão de separação (500 ul para 10 7 células). Espere até que o reservatório coluna estar vazio (sem líquido deve ser observado na coluna) antes da aplicação do próximo passo de lavagem. Nota: as células não marcadas obter CD3, consideradas como fracção de linfócitos B, como eles sãoeluiu-se para dentro do tubo de recolha. </li><li> Remova a coluna do separador e coloque em um novo tubo de 15 ml coleção. Pipetar 2 ml de tampão de separação na coluna. Usando o êmbolo fornecidas com a coluna eluir os linfócitos T seleccionados positivamente, os quais são considerados como a fracção de linfócitos T. </li><li> Determinar o número de células purificadas (passo 1.15), se necessário. Suspensões celulares agora está pronto para os experimentos a jusante. </li></ol> 4. Análise de Citometria de Fluxo Isolado tonsilar linfócitos B e T CUIDADO: solução de paraformaldeído é irritante e possivelmente cancerígeno. Usar vestuário de protecção, luvas e proteção para os olhos / face. Nota 1: Este protocolo descreve o método para a coloração directa do isolado B e células T de células activadas por fluorescência Sorting (FACS). O principal objectivo deste passo é o de avaliar a pureza do isolado de células populações afTer anticorpo CD3 separação magnética. Para este efeito, B estabelecida e marcadores de células T, os anticorpos monoclonais a fluoróforos, CD20 e CD2-FITC-APC, são utilizados. Também a inclusão de anticorpos de controlo de isotipo é altamente recomendável para distinguir o &quot;fundo&quot; ligação não específica dos anticorpos CD2 e CD20 (ver passo 4.8). Nota 2: Não é possível manter as fracções de células purificadas numa solução de paraformaldeído a 1% (PFA) no escuro a 4 ° C até o tempo de coloração (por exemplo, no dia seguinte.). Além disso, o mesmo procedimento é aplicável, se houver um intervalo de tempo entre a coloração e análise de FACS. Recordar que em ambos os casos, as células devem ser devidamente lavados com PBSA (PBS contendo BSA a 0,2%) tampão, antes da coloração ou análise de FACS. <ol><li> Retire 6 x 10 6 células de multinacionais do passo 1,15 (antes de aplicar para a coluna), fração de linfócitos B a partir do passo 3.9 e fração de linfócitos T do passo 3.10. </li><li> Gire a célulasuspensões a 300 xg durante 5 min a 4 ° C utilizando um rotor basculante para fora. </li><li> Remover o sobrenadante com uma pipeta e lava-se a pelete celular uma vez com 1 ml de gelo-frio PBSA. Rodar as suspensões celulares a 300 xg durante 5 min a 4 ° C. Remover o sobrenadante e ressuspender com a pipeta células em 600 ul de tampão de PBSA gelada. </li><li> Adicionar 100 ul da suspensão de células para a parte inferior de um tubo de FACS. </li><li> Adicionar 100 ul de PBS contendo soro humano inactivado pelo calor 10% a cada tubo, misturar bem e incubar durante ~ 1 min à temperatura ambiente ou 20 min no gelo. Nota: os linfócitos B transportam receptores de Fc. A fim de bloquear a receptores Fc das fracções purificadas de células são incubadas com soro humano inactivado pelo calor 10%. O soro humano é inactivado pelo calor por incubação a 56 ° C durante 1 h. Divida o soro inactivado calor em pequenas alíquotas e armazenar congelados a -20 ° C. </li><li> Centrifugar a suspensão de células a 300 xg durante 5 min a 4 ° C num SWing-out rotor. </li><li> Remover o sobrenadante com uma pipeta e lava-se a pelete de células com 1 ml de PBSA. Repetir a centrifugação (300 xg durante 5 min a 4 ° C). </li><li> Adicionar 100 ul de PBSA a cada tubo em gelo. Adicionar a quantidade apropriada de anticorpo monoclonal conjugado com fluoróforo, de acordo com a recomendação do fabricante (por exemplo, 20 uL de anti-CD20 e / ou 5 ul de anticorpos anti-CD2 em 100 ul de PBSA). Nota: Não se esqueça de atribuir tubos de controlo suficientes para a análise FACS. Neste protocolo os seguintes controles devem ser incluídos: 1) células coradas com os anticorpos anti-CD2 e anti-CD20 individualmente, 2) células coradas com os anticorpos anti-CD2 e anti CD20 de controle de isótipo individualmente e, 3) células sem adição de anticorpos. </li><li> Resumidamente vortex do tubo e incubar durante 30 min a 4 ° C no escuro. </li><li> Lave 2 vezes com PBSA. Adicionar 2 ml de solução de PFA 1% para as amostras. Permitir que as células permanecem na solução PFA a 476; C no escuro até o momento da análise FACS. </li><li> Imediatamente antes de executar as amostras na máquina de FACS, lavar as células 2 vezes com PBSA (300 xg durante 5 min a 4 ° C). Suspenda as amostras em 1 ml de PBS e manter a 4 ° C (ou em gelo), protegida da luz, antes da separação no citómetro de fluxo. </li><li> Fazer a triagem FACS seguindo o protocolo do citômetro de fluxo fabricante. </li></ol> 5. Detecção de PCR de ADN de adenovirus em Isolado de Tonsillar linfócitos B e T Nota 1: Os iniciadores do gene de adenovirus hexon são AdRJC1 (5&#39;-GACATGACTTTCGAGGTCGATCCCATGGA-3 &#39;) e AdRJC2 (5&#39;-CCGGCTGAGAAGGGTGTGCGCAGGTA-3&#39;) 11. Estes iniciadores gerar um amplicão de 139 pb. O gene de rRNA 18S de acolhimento pode ser detectada com iniciadores tp206 (5&#39;-CCCCTCGATGCTCTTAGCTG-3 &#39;) e tp207 (5&#39;-TCGTCTTCGAACCTCCGACT-3&#39;). O tamanho esperado de 300 pb do amplicon. Nota 2: Cada PCRrun inclui um controle negativo (destilada H 2 O) e um controlo positivo de DNA obtido a partir de linha de células B humanas (BJAB) infectadas com adenovírus humano tipo 5 12. <ol><li> Extracto de ADN (a partir de pelo menos 1 x 10 6 células, etapa 3.11) utilizando membrana de sílica método de purificação com base em coluna. Extrair ARN utilizando uma solução de fenol e isotiocianato de guanidina 13. </li><li> Adicionam-se 100 ng de ADN a partir de linfócitos B e T, a uma mistura de reacção contendo tampão 1x HF, 0,2 mM de mistura de trifosfato de desoxinucleótido (dNTP), 0,25 uM de cada iniciador e 1 U de alta-fidelidade da polimerase de ADN num volume total de 20 ul. </li><li> Efectue a amplificação de PCR sob as seguintes condições de ciclo: 30 s de desnaturação a 98 ° C, seguido de 30 ciclos de 98 ° C durante 10 seg, 67 ° C (hexon) ou 58 ° C (18S rRNA) durante 30 seg e 72 ° C durante 30 seg. A reacção de PCR foi terminada com um passo de extensão final de 7 minutos a 72 ° C. </li><li> Separa-se a amplif PCR resultanteprodutos icação sobre um gel de agarose a 1,5% em tampão 1 x TBE e as bandas esperadas visualização por coloração com mancha de ácido nucleico. </li></ol>

<ol>
	<li>Nave, H., Gebert, A., Pabst, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+and+immunology+of+the+human+palatine+tonsil.">Morphology and immunology of the human palatine tonsil.</a> Anat Embryol (Berl). 204, 367-373 (2001).</li><li>Perry, M., Whyte, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+of+the+tonsils.">Immunology of the tonsils.</a> Immunology today. 19, 414-421 (1998).</li><li>Alkhalaf, M. A., Guiver, M., Cooper, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+quantitation+of+adenovirus+DNA+from+human+tonsil+and+adenoid+tissues.">Prevalence and quantitation of adenovirus DNA from human tonsil and adenoid tissues.</a> J Med Virol. 85, 1947-1954 (2013).</li><li>Brandtzaeg, P., Halstensen, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+and+immunopathology+of+tonsils.">Immunology and immunopathology of tonsils.</a> Adv Otorhinolaryngol. 47, 64-75 (1992).</li><li>Imai, 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=Epstein-Barr+virus+(EBV)-carrying+and+-expressing+T-cell+lines+established+from+severe+chronic+active+EBV+infection.">Epstein-Barr virus (EBV)-carrying and -expressing T-cell lines established from severe chronic active EBV infection.</a> Blood. 87, 1446-1457 (1996).</li><li>Veen, J., Lambriex, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+adenovirus+to+lymphocytes+in+naturally+infected+human+tonsils+and+adenoids.">Relationship of adenovirus to lymphocytes in naturally infected human tonsils and adenoids.</a> Infect Immun. 7, 604-609 (1973).</li><li>Friedman, M., Wilson, M., Lin, H. C., Chang, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Updated+systematic+review+of+tonsillectomy+and+adenoidectomy+for+treatment+of+pediatric+obstructive+sleep+apnea/hypopnea+syndrome.">Updated systematic review of tonsillectomy and adenoidectomy for treatment of pediatric obstructive sleep apnea/hypopnea syndrome.</a> Otolaryngol Head Neck Surg. 140, 800-808 (2009).</li><li>Garnett, C. 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=Latent+species+C+adenoviruses+in+human+tonsil+tissues.">Latent species C adenoviruses in human tonsil tissues.</a> J Virol. 83, 2417-2428 (2009).</li><li>Garnett, C. T., Erdman, D., Xu, W., Gooding, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+quantitation+of+species+C+adenovirus+DNA+in+human+mucosal+lymphocytes.">Prevalence and quantitation of species C adenovirus DNA in human mucosal lymphocytes.</a> J Virol. 76, 10608-10616 (2002).</li><li>Perez, M. E., Billordo, L. A., Baz, P., Fainboim, L., Arana, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+memory+B+cells+isolated+from+blood+and+tonsils+are+functionally+distinctive.">Human memory B cells isolated from blood and tonsils are functionally distinctive.</a> Immunol Cell Biol. 92, 882-887 (2014).</li><li>Cooper, R. J., Yeo, A. C., Bailey, A. S., Tullo, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+polymerase+chain+reaction+assay+for+rapid+diagnosis+of+conjunctivitis.">Adenovirus polymerase chain reaction assay for rapid diagnosis of conjunctivitis.</a> Invest Ophthalmol Vis Sci. 40, 90-95 (1999).</li><li>Zhang, Y., Huang, W., Ornelles, D. A., Gooding, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+adenovirus+latency+in+human+lymphocyte+cell+lines.">Modeling adenovirus latency in human lymphocyte cell lines.</a> J Virol. 84, 8799-8810 (2010).</li><li>Chomczynski, P., Sacchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-step+method+of+RNA+isolation+by+acid+guanidinium+thiocyanate-phenol-chloroform+extraction.">Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.</a> Anal. biochem. 162, 156-159 (1987).</li><li>Watanabe, T., Yoshizaki, K., Yagura, T., Yamamura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+antibody+formation+by+human+tonsil+lymphocytes.">In vitro antibody formation by human tonsil lymphocytes.</a> J Immunol. 113, 608-616 (1974).</li></ol>

The authors have nothing to disclose.

Um dos factores mais importantes que afectam o resultado do presente protocolo é a utilização de material fresco das amígdalas como o material de partida. Portanto, as amostras de amígdalas deve ser processado dentro de 3 horas após a cirurgia. Amígdalas poderiam ser obtidos de adultos e crianças. O material tonsilar das crianças é geralmente menor, devido à remoção cirúrgica parcial das amígdalas (tonsilotomia). Portanto, o número de PTM obtido a partir de amostras tonsilotomia (1 x 10 7 x 10...

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.

Uma separação eficiente de MNCs tonsilares resultados em subpopulações de linfócitos altamente purificados B e T. Isto foi confirmado por análise de FACS utilizando anticorpos anti-CD20 e anticorpos anti-CD2 para a detecção de linfócitos T e B, respectivamente, populações (Figura 3A). Em contraste, uma separação ineficiente das MNCs resulta em fracções de células que são uma mistura de células a partir de ambas as subpopulações de linfócitos B e T (Figura 3B). <...

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.

Protocolo de isolamento eficiente para linfócitos B e T a partir de tonsilas palatinas Humanos

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

Research

Education

JoVE Core

JoVE Journal

Medical-Surgical Nursing

Immunology and Infection

Unit 1

Upper Respiratory Tract Infections

SCIENTISTS IN ACTION

15.7K visualizações.  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.

Vídeo: 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.

Isolamento T de linfócitos humanos, Cultura e Análise da Migração 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 ...

Imunologia e Infecção

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.

Expandindo linfócitos T citotóxicos do sangue do cordão umbilical que citomegalovírus Target, vírus de Epstein-Barr, e Adenovirus

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

O isolamento de linfócitos de rato Genital Tract Mucosa

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.

Isolamento de precursor de células B subconjuntos de Sangue do Cordão Umbilical

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.

Isolamento e Th17 Diferenciação de Linfócitos T CD4 Naïve

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.

Rápida análise de aberrações cromossômicas em linfócitos B mouse por 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.

A transfecção altamente eficiente de THP-1 por macrófagos humanos Nucleofection

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.

Um protocolo simples e rápido para não enzimática Separar frescos Tecidos Humanos para a Análise de infiltrar Linfócitos

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.

Geração de IgG Humana Recombinante de Anticorpos Monoclonais As células seleccionadas a partir de B imortalizadas

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.

Na diferenciação in vitro de células T CD4 + humano FOXP3 + Induzida T reguladoras (iTregs) a partir de células naive T CD4 + usando um protocolo de TGF-β contendo