Name: isolation and flow cytometric analysis of human endocervical gamma delta t cells
Uploaded: 2017-02-06T10:30:00.000+00:00
Duration: 8 min 10 s
Description: Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells at JoVE.com

Agradecemos aos participantes do estudo Miami WIHS para a sua vontade de participar neste trabalho. Os dados apresentados nesta manuscrito foram recolhidos por Interagency HIV Study das Mulheres Miami (WIHS). O conteúdo desta publicação é da exclusiva responsabilidade dos autores e não representam a posição oficial do National Institutes of Health (NIH). Este trabalho foi apoiado pelo Instituto Nacional de Alergia e Doenças Infecciosas e das mulheres Infecção Estudo Interagency HIV (WIHS) [subvenção número U01 AI103397], Instituto Nacional de Alergia e Doenças Infecciosas, Instituto Nacional de Saúde [número de concessão P30AI073961], Centro Nacional para a Promoção Ciências translacionais e do Instituto Nacional sobre Minority Saúde e Saúde disparidades, Instituto Nacional de Saúde [número de concessão UL1TR000460 e 1KL2TR000461], e do Instituto Nacional de Saúde Infantil e Desenvolvimento Humano, Instituto Nacional de Saúde [número de concessão K23HD074489].

atividades de estudo teve lugar na Universidade de Miami Unidade de Pesquisa de HIV em colaboração com Estudo das Mulheres Miami HIV Interagency (WIHS) e do Centro Miami for AIDS Research (CFAR) .Institutional Review Board (Universidade de Miami Miller School of Medicine) aprovação foi obtida antes ao recrutamento e quaisquer procedimentos de avaliação ou de estudos relacionados. Coleta de Amostras 1. Endocervical Escova NOTA: Os participantes foram submetidos a um exame vaginal realizado por MD treinados ou ginecologista e recolha de linfócitos intra-epiteliais foram realizadas em exame especular, inserindo cytobrush. <ol><li> As características dos participantes <ol><li> Recrutar mulheres participantes que estão com idade de 18 a 45 anos de idade, sexualmente ativa, não está grávida e não em medicamentos contraceptivos ou com um dispositivo intra-uterino. </li><li> Antes do exame vaginal, têm participantes passam por um empate de 10 mL de sangue em verdeVACUTAINERS heparina superior para o isolamento periférico de células mononucleares do sangue (PBMC). para servir como um controle para análise intra-epitelial endocervical 9. </li></ol></li><li> Coleta das amostras endocervicais <ol><li> Inserir um espéculo estéril de tamanho apropriado na vagina sem lubrificação. Usar uma água quente para facilitar a inserção do espéculo e gaze estéril para limpar o muco. A posição do espéculo permite a visualização completa do sistema operacional e ectocérvice. </li><li> Insira escova endocervical no canal endocervical até que apenas as cerdas mais próxima da mão são visíveis. Girar a escova para 360 °. Transferir a escova para um tubo de 15 ml contendo 5 ml de meio IMDM. </li><li> Imediatamente após a coleta, colocar o tubo contendo cytobrush no gelo. Para obter uma alta viabilidade das células intra-epitelial recolhidos, entregar o cytobrush para o laboratório e processo dentro de 1 h. </li></ol></li></ol><ol><li> tubo de vórtice contendo cytobrush endocervical aplicando 4 cursos de curta duração (10 seg). </li><li> Centrifuga-se o tubo, contendo cytobrush, durante 10 min a 250 x g. </li><li> Cuidadosamente remover o cytobrush a partir do tubo (não perturbar o sedimento no fundo do tubo) e adicionar 1 mL de meio IMDM e colocar o tubo em gelo. </li><li> Colocar o cytobrush no filtro de células 100 milímetros na parte superior do tubo de 50 ml. </li><li> Lave o cytobrush com 20 ml de IMDM. </li><li> tubo de centrifugação durante 10 min a 250 x g. </li><li> Decantar o sobrenadante e ressuspender as células peletizadas em 1 ml de IMDM e combinam-se com o sedimento ressuspenso já descrito em 2.3. </li><li> Contar as células utilizando um contador de células automatizado 10 ou contar as células utilizando um hemocitómetro, combinando 10 ul da suspensão celular com 10 ul de corante azul de tripano. Suavemente pipeta cima e para baixo dez vezes para misturar as células e corante. Carga de 10 ulda mistura para dentro da abertura de uma das duas câmaras de hemocitómetro. Contar todas as células nas cinco áreas de grade. Manter uma contagem separada de as células viáveis ​​e não viáveis. Determinar a viabilidade celular usando a seguinte fórmula:% de células viáveis ​​= células não viáveis ​​x 100 células / total de número de células. Determinar a concentração de células por ml subsequente (e o número total de células) utilizando os seguintes cálculos. Total de células por ml = x total de células contadas (fator de diluição / número de quadrados contados) x 10.000 células / ml. </li><li> Congelar as células endocervicais isolados utilizando meio de congelamento contendo 10% de DMSO e 90% de FBS. </li></ol> 3. Citometria de Fluxo <ol><li> Re-suspender a 1-3 x 10 5 células endocervicais em 100 ul de tampão de FACS (PBS contendo 1% de albumina de soro bovino </li><li> Adicionar 1 mL / 10 6 Live / Dead fixável kit celular mancha amarela inoperante e anticorpos para marcadores de superfície nas concentrações descritas na Tabela 1. </li><li> Incubam-se as células durante 30 min a 4 ° C, protegidos da luz. </li><li> Adicionar 1 mL de tampão de FACS e as células e tubos de centrifugação durante 5 min a 350 xg </li><li> Re-suspender a pelete de células em 300 uL de paraformaldeído a 1%. </li><li> Preparar controlos de compensação usando grânulos e anticorpos utilizados para a coloração por adição de 100 uL de tampão de SCAF e uma gota de os grânulos e a mesma concentração dos anticorpos utilizados para a coloração da amostra. </li><li> Adquirir pérolas coradas e as amostras num citómetro de fluxo, equipado com 405 nm, 488 nm e 635 nm lasers. </li></ol> 4. Endocervical Gamma delta T Isolamento celular <ol><li> Isolamento esférulas magnéticas <ol><li> Re-suspender a pelete de células endocervicais (≤ 10 7 células totais), descrito em 2.6, em 40 mL de tampão incluídos no kit micropérola gama delta TCR referenciado na Tabela de Materiais e Reagentes. </li><li> Siga as instruções do kit para protocolo de coloração de duas etapas: primeiro, sagacidadeh anti TCR-delta gamma de hapteno-anticorpo e, segundo, com anti-hapteno microbeads-FITC </li><li> Após a incubação de 15 min a 4-8 ° C, lavar as células por adição de 1 ml de tampão e centrifugar a 300 xg durante 10 min. Decantar o sobrenadante por inversão do tubo, e toque em seco com a ajuda de um pedaço de papel. </li><li> Re-suspender o sedimento celular em 500 uL de tampão fornecido e preparar a coluna magnética, colocando-o num separador magnético apropriado de acordo com o protocolo do fornecedor. </li><li> Aplicar a suspensão de células na coluna e recolher células não marcadas que passam através da coluna e lavar 3x com a quantidade apropriada de tampão (500 uL). Executar passos de lavagem através da adição de tampão três vezes, cada vez que uma vez que o reservatório está vazio da coluna. Recolha efluente total. Esta é a fracção de células não marcado, negativo </li><li> Retirar a coluna do separador magnético formar e pipeta de 1 ml de tampão para a coluna e imediatamente expulsar fracção com o rótulo magneticamenteed células, aplicando firmemente o êmbolo fornecidas com a coluna. NOTA: célula isolada T gama delta já estão fluorescente corada para análise de citometria de fluxo desde marcadas magneticamente microesferas anti-hapteno são conjugados com FITC corante fluorescente. </li></ol></li><li> separação de células <ol><li> Para separação de células, as células endocervicais de etiquetas com die viabilidade e painel de anticorpos, como descrito na seção 3. </li><li> Configurar portões eletrônicos na ao vivo, CD45 + CD3 + e delta gamma células positivas e executar separação de células. </li></ol></li></ol>

<ol>
	<li>Kaul, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genital+tract+immune+milieu:+an+important+determinant+of+HIV+susceptibility+and+secondary+transmission.">The genital tract immune milieu: an important determinant of HIV susceptibility and secondary transmission.</a> J Reprod Immunol. 77, 32-40 (2008).</li><li>Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+early+infection+to+prevent+HIV-1+mucosal+transmission.">Targeting early infection to prevent HIV-1 mucosal transmission.</a> Nature. 464, 217-223 (2010).</li><li>Hayday, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma][delta]+cells:+a+right+time+and+a+right+place+for+a+conserved+third+way+of+protection.">gamma][delta] cells: a right time and a right place for a conserved third way of protection.</a> Ann Rev Immunol. 18, 975-1026 (2000).</li><li>Nakasone, C., 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=Accumulation+of+gamma/delta+T+cells+in+the+lungs+and+their+roles+in+neutrophil-mediated+host+defense+against+pneumococcal+infection.">Accumulation of gamma/delta T cells in the lungs and their roles in neutrophil-mediated host defense against pneumococcal infection.</a> Microbes and infection / Institut Pasteur. 9, 251-258 (2007).</li><li>Autran, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+gamma/delta++lymphocyte+subsets+during+HIV+infection.">T cell receptor gamma/delta+ lymphocyte subsets during HIV infection.</a> Clin Exp Immunol. 75, 206-210 (1989).</li><li>Pauza, C. D., Poonia, B., Li, H., Cairo, C., Chaudhry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gammadelta+T+Cells+in+HIV+Disease:+Past,+Present,+and+Future.">gammadelta T Cells in HIV Disease: Past, Present, and Future.</a> Front Immunol. 5, 687 (2014).</li><li>Hayday, A. C., Spencer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barrier+immunity.">Barrier immunity.</a> Sem Immunol. 21, 99-100 (2009).</li><li>Cheroutre, H., Lambolez, F., Mucida, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+light+and+dark+sides+of+intestinal+intraepithelial+lymphocytes.">The light and dark sides of intestinal intraepithelial lymphocytes.</a> Nature Rev Immunol. 11, 445-456 (2011).</li><li>Strbo, N., 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=Loss+of+Intra-Epithelial+Endocervical+Gamma+Delta+(GD)+1+T+Cells+in+HIV-Infected+Women.">Loss of Intra-Epithelial Endocervical Gamma Delta (GD) 1 T Cells in HIV-Infected Women.</a> AJRI. 75, 134-145 (2016).</li><li>Szabo, S. E., Monroe, S. L., Fiorino, S., Bitzan, J., Loper, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+an+automated+instrument+for+viability+and+concentration+measurements+of+cryopreserved+hematopoietic+cells.">Evaluation of an automated instrument for viability and concentration measurements of cryopreserved hematopoietic cells.</a> Lab Hematol. 10, 109-111 (2004).</li><li>Alcaide, M. L., 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=Bacterial+Vaginosis+Is+Associated+with+Loss+of+Gamma+Delta+T+Cells+in+the+Female+Reproductive+Tract+in+Women+in+the+Miami+Women+Interagency+HIV+Study+(WIHS):+A+Cross+Sectional+Study.">Bacterial Vaginosis Is Associated with Loss of Gamma Delta T Cells in the Female Reproductive Tract in Women in the Miami Women Interagency HIV Study (WIHS): A Cross Sectional Study.</a> PloS one. 11, e0153045 (2016).</li><li>Masson, L., 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=Defining+genital+tract+cytokine+signatures+of+sexually+transmitted+infections+and+bacterial+vaginosis+in+women+at+high+risk+of+HIV+infection:+a+cross-sectional+study.">Defining genital tract cytokine signatures of sexually transmitted infections and bacterial vaginosis in women at high risk of HIV infection: a cross-sectional study.</a> Sex Transm Dis. 90, 580-587 (2014).</li><li>Fichorova, R. N., 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=Interleukin+(IL)-1,+IL-6,+and+IL-8+predict+mucosal+toxicity+of+vaginal+microbicidal+contraceptives.">Interleukin (IL)-1, IL-6, and IL-8 predict mucosal toxicity of vaginal microbicidal contraceptives.</a> Biol Reprod. 71, 761-769 (2004).</li><li>Juno, J. A., Boily-Larouche, G., Lajoie, J., Fowke, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection,+isolation,+and+flow+cytometric+analysis+of+human+endocervical+samples.">Collection, isolation, and flow cytometric analysis of human endocervical samples.</a> JoVE. , (2014).</li><li>McKinnon, L. R., 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=Optimizing+viable+leukocyte+sampling+from+the+female+genital+tract+for+clinical+trials:+an+international+multi-site+study.">Optimizing viable leukocyte sampling from the female genital tract for clinical trials: an international multi-site study.</a> PloS one. 9, e85675 (2014).</li><li>Dominguez, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+multiplexed+quantitation+of+gene+expression+on+single+cells.">Highly multiplexed quantitation of gene expression on single cells.</a> J Immunol Methods. 391, 133-145 (2013).</li></ol>

Autores não têm associação comercial ou outra, que podem representar um conflito de interesses (por exemplo, a propriedade de ações farmacêutica, consultoria, a adesão Advisory Board, patentes relevantes, ou o financiamento da investigação).

Avaliação da vulnerabilidade das mucosas do tracto genital inferior feminino é um componente essencial de estudos clínicos que abordam risco de aquisição e transmissão do HIV. Tipicamente FGT vulnerabilidade à infecção do HIV é avaliada através da medição moduladores imunitários solúveis em secreções genitais (citocinas, quimiocinas e péptidos antimicrobianos) 12, 13. No entanto, estes biomarcadores são apenas indicativos da inflamação vagi...

Nós desenvolvemos metodologia de utilização de amostras escova endocervical para avaliar células GD T intra-epitelial. O canal endocervical é recoberto por uma camada única de epitélio colunar. Linfócitos intra-epiteliais representam linfócitos linha de frente que residem na camada epitelial que podem iniciar rapidamente as respostas imunes ao encontrar micróbios patogênicos 7, 8. Compreender os eventos imunológicos do compartimento intra-epitelial endocervical é importante para a concepção de estratégias eficazes para prevenir infecções, incluindo o HIV. O nosso método pode ser aplicado a amostras endocervicais humanos recentemente recolhidos, bem como células endocervicais congelados para explorar ainda mais o papel das células T intra-epiteliais GD em mulheres com infecção por VIH ou em risco de infecção pelo HIV. O principal objectivo deste protocolo é descobrir novos eventos imunológicos no compartimento intra-epitelial endocervical e Avaliado comrespostas de células e GD T como um marcador de vulnerabilidade da mucosa em mulheres com infecção pelo HIV. As lacunas substanciais no conhecimento em torno imunidade FGT são parcialmente devido à dificuldade na recolha e processamento de amostras de mucosa com sucesso. Além disso, o complexo de heterogeneidade das células imunes das mucosas, e a sua interligação uns com os outros, são os principais desafios para a identificação de medições clinicamente relevantes que reflectem o estado e a capacidade do sistema imunitário. Nós desenvolvemos um método para obter células T GD intra-epitelial altamente purificados que podem ser analisados ​​usando tecnologias altamente multiplexados, de uma única célula que pode ser fundamental para identificar os mecanismos subjacentes da imunidade FRT.

<table><tbody><tr><td>Cytobrush Plus GT&amp;nbsp;</td><td>(CareFusion, San Diego, CA, USA</td><td></td><td></td></tr><tr><td>IMDM,Iscove&#039;s Modified Dulbecco&#039;s Medium&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>12440061</td><td></td></tr><tr><td>Beckman Coulter automated cell counter (The Vi-CELL Series Cell Viability Analyzer</td><td>Beckman Coulter</td><td>496178</td><td></td></tr><tr><td>DMSO, Dimethyl sulfoxide&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>D2650</td><td></td></tr><tr><td>FBS, Fetal Bovine Serum</td><td>Invitrogen, Gibco</td><td>26140-079</td><td></td></tr><tr><td>PBS</td><td>Invitrogen, Gibco</td><td>10010023</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A-9647</td><td></td></tr><tr><td>LIVE/DEAD Fixable Yellow Dead Cell Stain kit&amp;nbsp;</td><td>Life Technologies,</td><td>L349S9</td><td></td></tr><tr><td>1% paraformaldehyde</td><td>Sigma Aldrich&amp;nbsp;</td><td>D6148</td><td></td></tr><tr><td>Fortessa flow cytometer&amp;nbsp;</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>UltraComp eBeads</td><td>eBioscience</td><td>01-2222-42</td><td></td></tr><tr><td>ArC Amine Reactive Compensation Beads (Life Technologies, Grand Island, NY, USA)</td><td>Life Technologies</td><td>A10346</td><td></td></tr><tr><td>anti-TCR gamma/delta MicroBead Kit&amp;nbsp;</td><td>Milteny Miltenyi Biotec Inc.</td><td>130-050-701</td><td></td></tr><tr><td>MS column</td><td></td><td>103-042-201</td><td></td></tr><tr><td>MACS separator</td><td></td><td>4124</td><td></td></tr></tbody></table>

isolation and flow cytometric analysis of human endocervical gamma delta t cells

O trato reprodutivo (FRT) da mucosa sistema imunológico feminino serve como a primeira linha de defesa. Melhor conhecimento da mucosa genital é, portanto, essencial para a compreensão da patogenicidade de diferentes patógenos, incluindo o HIV. Gama células delta (GD) T são o protótipo de células T &#39;não convencionais&#39; e representam um subconjunto relativamente pequeno de células T definidos pela sua expressão de receptores de células T heterodiméricos (TCRs) compostas de cadeias gama e delta. Isso os diferencia das células T clássicos e muito melhor conhecidos CD4 + auxiliares e células CD8 + T citotóxicos que são definidos por TCRs alfa-beta. GD células T muitas vezes mostram a localização específica do tecido e são enriquecidos em epitélio. GD células T orquestrar respostas imunes em inflamação, vigilância de tumores, doenças infecciosas e auto-imunidade. Aqui, apresentamos um método para isolar reprodutível e analisar linfócitos intra-epiteliais endocervicais humanos GD T. que have amostras cytobrush endocervicais usados ​​mulheres participantes Infecção Estudo Interagency HIV das Mulheres (WIHS). Conhecimento sobre GD células T interações durante as condições em que não é um insulto à mucosa vaginal poderia ser aplicada a qualquer estudo clínico em que a vulnerabilidade das mucosas são os destinatários, incluindo o desenvolvimento de adição microbicides.In vaginal, o conhecimento sobre as respostas das células da mucosa GD T tem potencial para aplicação de terapia imunitária baseada em células T GD no tratamento de doenças infecciosas.

Recentemente, analisamos células GD T intraepiteliais endocervicais e fomos o primeiro a relatar sobre a perda de células T gama delta endocervical em mulheres infectadas pelo HIV 9, 11. Aqui, descrevemos o protocolo para isolar e analisar o subconjunto de células T gama delta endocervical. Como mostrado na Figura 1, as células T GD pode ser facilmente detectado em amostras de células endocervicais humanos. ...

Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells at JoVE.com

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

Isolamento e Citometria de Fluxo Analysis of Human Endocervical Gama Cells Delta T

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

isolation-flow-cytometric-analysis-human-endocervical-gamma-delta-t

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

8.7K visualizações.  Miller School of Medicine, University of Miami. We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

Vídeo: Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Expansão do sangue periférico humano γδ células T usando zoledronato

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Imunologia e Infecção

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Coleção, isolamento e Citometria de Fluxo de Análise de Amostras endocervix Humano

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

Medicina

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

Isolamento e<em&gt; Ex Vivo</em&gt; Cultura de Vδ1<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; Células T, γδ um Extrathymic αβT de células progenitoras

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Isolamento e Análise de Citometria de Fluxo infiltrantes Glioma Células Mononucleares do Sangue Periférico

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Caracterização de derivados de monócitos células dendríticas humanas por imagem Citometria de Fluxo: Uma Comparação entre dois protocolos de isolamento de monócitos

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and activity of cytotoxic T cells within tumors and their tumor microenvironment (TME) are now well-established prognostic and predictive biomarkers for numerous tumor types. However, it is well-known that various tumor treatment modalities, including radiotherapy, chemotherapy, immunotherapy, and targeted therapy, modulate not only the immunogenicity of the tumor but also the immune system itself. Consequently, the interaction between tumor cells and T cells requires more intensive study in different therapeutic contexts to fully understand the complex role of T cells during tumor therapy. To address this need, a protocol was developed to analyze the activity and proliferative capacity of human cytotoxic (CD8+) T cells in co-culture with pre-treated tumor cells. Specifically, CD8+ T cells from healthy donors are stained with the non-toxic proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated using CD3/CD28-coated plates. Subsequently, T cells are co-cultured with pre-treated tumor cells. As a readout, T cell proliferation is quantified by measuring CFSE signal distribution and assessing the expression of surface activation markers via flow cytometry. This can be further complemented by quantifying cytokine release using enzyme-linked immunosorbent assay (ELISA). This method facilitates the evaluation of treatment-induced changes in the interaction between tumor cells and T cells, providing a foundation for more detailed analyses of tumor treatment modalities and their immunogenicity in a human ex vivo setting. Additionally, it contributes to the reduction of pre-clinical in vivo analyses.

The present protocol describes an experimental workflow that allows for the ex vivo analysis of human T cell stimulation in an allogenic co-culture system with pre-treated tumor cells.

Análise da atividade de células T humanas em um ambiente de co-cultura alogênica de células tumorais pré-tratadas

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and ...

Pesquisa do câncer

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

Isolamento de células dendríticas do tracto reprodutivo feminino humano para estudos fenotípica e funcionais

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

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

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

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

Discriminação de sete subconjuntos de células imunes por dois-fluorocromo citometria de fluxo

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

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

Isolamento de células linfoides inatas uterinas para análise por citometria de fluxo

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the lymph node microenvironment in B-cell non-Hodgkin&#39;s lymphoma is analyzed. Subsets of circulating T-follicular helper cells (cTfh), the circulating memory compartment of Tfh cells in the blood, are also perturbed in disease and therefore represent potential novel predictive biomarkers. Peripheral blood-based testing is advantageous because it is relatively non-invasive and allows simple serial monitoring.This article describes a method for isolating CD4+ T-cells from human blood, and further analysis by flow-cytometry to enumerate cTfh cells and the proportions of their various subsets (cTfhPD-1-/+/hi, cTfh1,2,17 and cTfh1/17). The level of these subsets was then compared between normal subjects and patients with lymphoma. We found that the method was robust enough to obtain reliable results from routinely collected patient material. The technique we describe for the analysis can be easily adapted to cell sorting and downstream applications such as RT-PCR.

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper cTfh Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Here, a protocol for the isolation and characterization of CD4+ T-cell subsets from human peripheral blood is described. Purified CD4+ T-cells are analyzed by flow cytometry to determine proportions of T-follicular helper cell subsets.

Isolamento de CD4+ T-células e análise de circulação folicular-T Helper (cTfh) célula subconjuntos de citometria de fluxo de 6 cores usando o sangue periférico

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the ...

Isolamento e Citometria de Fluxo Analysis of Human Endocervical Gama Cells Delta T

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