Financiamento deste projeto foi fornecido pelo National Science Foundation concede OCE1029607 (para XM) e MCB0702125 (para MAM) eo Gordon e Betty Moore Foundation (a MAM).

1. Processamento de amostra de água <ol><li> Filtro de água 10L ambiental através de uma filtros mM de poros de tamanho de membrana para remover partículas grandes e bacteriovores. Recolher o filtrado em um garrafão de água. </li><li> Transferência de 36 ml cada filtrado em 3 tubos estéreis Eppendorf (50 ml) contendo 4 ml de solução recém-preparada de paraformaldeído (PFA; 10%). Incubar por 2 horas em temperatura ambiente para preservar as células. Coletar células para 0,22 mícrons de tamanho de poros filtros de membrana branca por filtração a vácuo. Lavar o filtro, passando de 10 ml tampão fosfato (PBS) através do filtro por filtração a vácuo. Rotular os filtros como controles negativos e armazená-los a -20 ° C. </li></ol> 1,3-1,4 passos são opcionais para o estabelecimento de DOC-limitada condições. <ol start="3"><li> Adicionar uma mistura de nitrogênio inorgânico e fósforo (5 mM NH 4 Cl, 5 M NaNO 3 e 1 Hm NaH 2 PO 4 a concentração final) para o garrafão. </li><li> Incubar no escuro, na temperatura local por 48 horas, com agitações ocasionais. </li></ol> 2. Estabelecimento microcosmo e incubação <ol><li> Preencha cada um dos seis frascos de 1 L de vidro com 800 ml de amostra de água do garrafão de 1,4 passo para estabelecer microcosmos. Adicionar BrdU (10 mM, concentração final) para cada um microcosmo. Misture bem. </li><li> Adicionar 1 ml de solução de modelo composto DOC em três dos microcosmos, estes servirão como alterações DOC. Adicionar 1 ml estéril PBS em três microcosmos restantes, estes servirão como sem adição de controles. </li><li> Incubar todos os microcosmos em uma incubadora shaker e incubar no escuro, na temperatura situ agitando a 100 rpm. </li><li> Coletar 36 ml de amostra de água de cada microcosmo e transferir para 50 ml tubos estéreis Eppendorf em momentos de 0, 8, 16 e 24 horas. Imediatamente adicionar 4 ml de PFA fresco (10%) para tubos de coleta e incubar por 2 horas em temperatura ambiente para preservar as células. </li><li> Filtro preservada células através de 0,22 mícrons de tamanho de poros filtros de membrana de policarbonato. Lavar os filtros com 10 ml de PBS. Proceder de imediato para a próxima etapa ou armazenar os filtros a -20 ° C. </li></ol> 3. Na imunodetecção situ de Incorporação BrdU <ol><li> Descongelar os filtros (amostras DOC alterada, sem adição de controles e os controles negativos) em temperatura ambiente. </li><li> Aplicar 1 ml de solução de lisozima [10 mg / ml lisozima clara de ovo em 100 mM Tris, 50 nM EDTA (pH = 8)] 4 para cobrir as células bacterianas no filtro. Incubar à temperatura ambiente por 30 minutos. Lavar o filtro, passando de 10 ml PBS através dela sob sucção. </li><li> Adicionar 1 ml de solução de proteinase K [2 mg / ml proteinase K em 100 mM Tris, 50 nM EDTA (pH = 8)] 4 para cobrir as células bacterianas no filtro. Incubar à temperatura ambiente por 30 minutos. Lavar o filtro, passando de 10 ml PBS através dela sob sucção. </li><li> Adicionar 1 ml solução exonuclease [exonuclease III (50 U / ml) em 5 mM MgCl 2 e 50 mM Tris-HCl] 5 para cobrir as células bacterianas no filtro. Incubar a 37 ° C por 30 minutos. Lavar o filtro, passando de 10 ml PBS através dela sob sucção. </li><li> Montar câmaras quadro selo de incubação (Bio-Rad) de acordo com as instruções do fabricante. </li><li> Fatia do filtro em oitavos utilizando uma lâmina estéril em um álcool limpo superfície seca. </li><li> Utilizando uma pinça, coloque uma oitava seção de um filtro em uma câmara de incubação montadas quadro selo (oito quadros selo câmaras são necessários para cada amostra do filtro). O lado da seção de filtro que contém células deve estar voltado para cima. A umidade na parte de trás do filtro permitirá a vara filtro para o slide. Se o filtro torna-se seca, aplique uma pequena gota (2 L) de dih 2 O no centro da câmara antes de colocar a seção de filtro no slide. </li></ol> Passos 3,8-3,20 use os reagentes da Roche em Kit Situ Proliferação Celular, FLUOS seguintes procedimentos modificados a partir de instruções do fabricante. Exceto para PBS, todos os reagentes são fornecidos dentro do kit. <ol start="8"><li> Aplicar tampão de incubação suficiente (0,5% albumina sérica bovina, 0,1% Tween20 em PBS), para cobrir uniformemente toda a seção de filtro na câmara de incubação, sem causar estouro de uma vez o selo é aplicado. </li><li> Coloque uma capa de quadro de poliéster sobre o quadro câmara de incubação. Pressione para baixo para selar o bem câmara de incubação. Evite bolhas de ar acima do filtro. Incubar as câmaras seladas por 10 min em temperatura ambiente no escuro. </li><li> Retire o selo de poliéster e abrir as câmaras de incubação. (Nota: a abertura das câmaras, por vezes, vai puxar para cima o selo moldura do slide Nesse caso, prepare uma nova câmara para as etapas seguintes..) </li><li> Pipeta a solução de incubação de um canto da câmara. Evitar o contacto com o filtro. </li><li> Lavar o filtro com cuidado pipetando 100 mL PBS efora da câmara de 3 vezes. </li><li> Prepare anti-BrdU-FLUOS solução de trabalho, seguindo os passos recomendados pelo fabricante imediatamente antes da utilização. </li><li> Pipetar 120 mL anti-BrdU-FLUOS solução de trabalho para o filtro, tendo o cuidado de cobrir uniformemente toda a superfície. </li><li> Feche a câmara com uma capa de poliéster novo. Evite bolhas de ar no topo do filtro. Incubar a câmara no escuro a 37 ° C por 3 horas. Esta etapa irá rótulo BrdU incorporado DNA in situ com fluoresceína isotiocianato (FITC). </li><li> Remova a tampa de poliéster e abrir a câmara de incubação. Pipeta com a solução anti-BrdU-FLUOS de trabalho. Lavar o filtro 3 vezes com PBS. </li><li> Transferir o filtro da câmara de incubação a uma superfície estéril. Fatia da seção de filtro em pedaços pequenos usando uma lâmina estéril. </li><li> Transferir os pedaços de filtro em 2 tubos de microcentrífuga ml, cada um contendo 1 ml de PBS. Firmemente a tampa do tubo e vedação com parafilme. Incubar a 37 ° C e 200 rpm por 10 min. </li><li> Segura os tubos em um vortex vortexer e à velocidade máxima por 5 minutos. Pipetar o sobrenadante para um tubo de 15 ml estéril Eppendorf. Repita as etapas de incubação e vortex por 5 vezes mais. Combine o sobrenadante no mesmo tubo de 15 ml Eppendorf para cada amostra. Normalmente, 80% de células podem ser recuperados na suspensão. </li><li> Armazenar o sobrenadante com células ressuspenso a 4 ° C. Ordenar dentro de 2 dias. </li></ol> 4. Análise FACS Um procedimento para um citômetro de fluxo BD FACSAria e software correspondente é descrito aqui. <ol><li> Otimizar a configuração do fluxo passos seguintes citómetro recomendado pelo fabricante. Isto implica: ajustando o controle de amplificação para definir os parâmetros necessários e breakoff o ponto doce; otimizando o atraso laser e fatores de escala da área para a pressão de bainha de experiência e otimizar as configurações para FSC e SSC tensões, limiar FSC, FSC escala de fluorescência, fluorescência tensões PMT , etc </li><li> Executar amostras de controlo negativo no citômetro de fluxo (FCM) com base na intensidade de fluorescência FITC e de dispersão lateral (SSC). Aumentar o limite até que não FITC células nos controles negativos podem ser visualizados pelo visor de aquisição FITC-SSC. </li><li> Executar amostras sem adição de controle e examinar o padrão de distribuição de 10.000 células com base em FITC e aquisição SSC. Definir uma porta para colocar todas as células e designá-los como &quot;células de baixa intensidade&quot; (LIs). </li><li> Run-DOC alterado amostras e analisar o padrão de distribuição de 10.000 células com base em FITC e SSC. Algumas células irão aparecer na porta LI predefinido. Definir um outro portão para colocar as células que têm maior intensidade de fluorescência (HIS) do que LIs. Adquirir as estatísticas para ver a abundância relativa de células fechadas. </li><li> Ordenar HI células em tubos de coleta contendo 500 mL PBS em &quot;1 gota purificar&quot; o modo. Terminar a classificação quando o número dos seus atinge 500.000 contagens. </li></ol> 5. Filtro de amplificação por PCR do 16S rRNA genes Procedimentos filtro de PCR são modificados a partir Kirchman et al 6. <ol><li> Filtro de células classificadas em um branco, 0,22 mM de poros de tamanho, 25 mm de diâmetro, filtro de membrana de policarbonato. Corte a borda do filtro que não contém células bacterianas usando uma lâmina estéril. Fatia do filtro em 8 pedaços iguais de tamanho. </li><li> Coloque um pedaço do filtro único em um tubo de reação de PCR, com as células virado do tubo. Adicionar 45 mL de água grau PCR em tubo de reação de PCR. Submergir o filtro totalmente na água. </li><li> Adicione 2 ROCHE illustra PuReTaq Read-To-Go Beads PCR em tubo de reação de PCR, vortex por alguns instantes. Adicione 2 mL cada um dos frente e verso 16S rRNA gene primers (0,4 mM concentração final para cada primer), como 27F e 1492R 7, em cada um dos tubos de reação de PCR. </li><li> (Opcional) Adicione 1μl solução de soro albumina bovina (BSA, a concentração final é 30μg/100 mL) à mistura de reação de PCR para ajudar a inibidores de amplificação adsorver. Se alguém opta por não adicionar BSA, adicione 1μl de água em seu lugar. </li><li> Realizar a amplificação em um termociclador. Um toque para baixo programa de PCR é recomendado, que tem a temperatura de recozimento em seqüência decrescente 62-52 ° C por 1 ° C por ciclo para 11 ciclos, seguidos de 15 ciclos com temperatura de anelamento de 52 ° C. Todos os ciclos incluem desnaturação (a 95 ° C), anelamento (a 62-52 ° C) e extensão (a 72 ° C) medidas de duração 50s. Uma desnaturação 3 min-inicial e etapa de extensão final de 10 min também está incluída no programa de PCR. </li><li> Confirmar a amplificação PCR por eletroforese em um brometo de etídio manchada de 1% agarose gel. Excise o PCR amplicons do gel e limpa com o kit de extração QIAGEN QIAquick gel. </li><li> Realizar dois adicionais amplificações PCR para cada amostra, cada vez que utilizar uma nova secção do filtro. Após PCR gel purificatina, o pool de amplicons da mesma amostra juntos. Amplicons purificados 16S rDNA está agora pronto para uma série de análises moleculares que permitem a identificação taxonômica, como a construção clone biblioteca e seqüenciamento. </li></ol> 6. Resultados representativos: Resultados representativos são descritos através de um estudo de putrescina de degradação de bactérias como um exemplo. Amostras de água foram coletadas de um site costeira da Geórgia e processadas seguindo os procedimentos descritos acima. Análise FACS revelou que além putrescina induzida desenvolvimento de um grupo de bactérias com intensidade de fluorescência FITC alta, indicando alta taxa de incorporação BrdU (Figura 1). Essas células foram designadas como de alto BrdU incorporação de células (HIS) e era esperado que contêm principalmente putrescina de degradação bactérias. Seu estavam faltando nos controles não-adição, que só continha células com menores níveis de BrdU incorporação (LIs). LIs se esperava que contêm principalmente bacterioplâncton que foram incapazes de usar putrescina acrescentou. Sua foram classificados em tubos separados e depois coletados em filtros de membrana. 16S rRNA gene amplicons foram obtidas para células de alta HI usando filtro de PCR. <img src="/files/ftp_upload/2855/2855fig1.jpg" alt="Figura 1"/> Figura 1. Análises por citometria de fluxo sem adição de controle e modelo composto-alterada (putrescina como um exemplo aqui) amostras coletadas após 24 h de incubação. Análise da distribuição das células foi baseada em (1) intensidade de fluorescência de FITC rotulagem (eixo-x), que é positivamente relacionada com o nível de incorporação de BrdU, e (2) dispersão lateral (SSC, y-eixo), que é positivamente relacionada com a célula tamanho. Notação portão é baseada no nível de BrdU incorporação, (HI, high-BrdU incorporação; LI, baixa incorporação BrdU). A percentagem relativa de HI e células LI são apresentados na portões correspondente.

<ol>
	<li>Pernthaler, A., Pernthaler, J., Schattenhofer, M., Amann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+DNA-synthesizing+bacterial+cells+in+coastal+North+Sea+plankton.">Identification of DNA-synthesizing bacterial cells in coastal North Sea plankton.</a> Appl. Environ. Microbiol. 68, 5728-5728 (2002).</li><li>Urbach, E., Vergin, K. L., Giovannoni, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunochemical+detection+and+isolation+of+DNA+from+metabolically+active+bacteria.">Immunochemical detection and isolation of DNA from metabolically active bacteria.</a> Appl. Environ. Microbiol. 65, 1207-12 (1999).</li><li>Mou, X. Z., Hodson, R. E., Moran, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterioplankton+assemblages+transforming+dissolved+organic+compounds+in+coastal+seawater.">Bacterioplankton assemblages transforming dissolved organic compounds in coastal seawater.</a> Environ. Microbiol. 9, 2025-2025 (2007).</li><li>Hodson, R. E., Dustman, W. A., Garg, R. P., Moran, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+PCR+for+visualization+of+microscale+distribution+of+specific+genes+and+gene+products+in+prokaryotic+communities.">In situ PCR for visualization of microscale distribution of specific genes and gene products in prokaryotic communities.</a> Appl. Environ. Microbiol. 61, 4074-4074 (1995).</li><li>Dinjens, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bromodeoxyuridine+(BrdU)+immunocytochemistry+by+exonuclease+III+(Exo+III)+digestion.">Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion.</a> Histochemistry. 98, 199-199 (1992).</li><li>Kirchman, D. L., Yu, L. Y., Fuchs, B. M., Amann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+bacterial+communities+in+aquatic+systems+as+revealed+by+filter+PCR.">Structure of bacterial communities in aquatic systems as revealed by filter PCR.</a> Aquat. Microb. Ecol. 26, 13-13 (2001).</li><li>Delong, E. F., Wickham, G. S., Pace, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+stains:+ribosomal+RNA-based+probes+for+the+identification+of+single+cells.">Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells.</a> Science. 243, 1360-1360 (1989).</li><li>Artursson, V., Jansson, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+bromodeoxyuridine+immunocapture+to+identify+active+bacteria+associated+with+arbuscular+mycorrhizal+hyphae.">Use of bromodeoxyuridine immunocapture to identify active bacteria associated with arbuscular mycorrhizal hyphae.</a> Appl. Environ. Microbiol. 69, 6208-6208 (2003).</li><li>Mou, X. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+carbon+processing+by+generalist+species+in+the+coastal+ocean.">Bacterial carbon processing by generalist species in the coastal ocean.</a> Nature. 451, 708-708 (2008).</li><li>Mou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-cytometric+cell+sorting+and+subsequent+molecular+analyses+for+culture-independent+identification+of+bacterioplankton+involved+in+dimethylsulfoniopropionate+transformations.">Flow-cytometric cell sorting and subsequent molecular analyses for culture-independent identification of bacterioplankton involved in dimethylsulfoniopropionate transformations.</a> Appl. Environ. Microbiol. 71, 1405-1405 (2005).</li><li>Dean, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+human+genome+amplification+using+multiple+displacement+amplification.">Comprehensive human genome amplification using multiple displacement amplification.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 5261-5261 (2002).</li></ol>

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Casais a nossa abordagem incorporação de BrdU, FACS e 16S rDNA para permitir a análise em nível de espécie de identificação do bacterioplâncton que metabolizam os componentes individuais DOC em ambientes aquáticos. O ensaio de incorporação BrdU rótulos células bacterianas com base em atividades metabólicas, que permite a análise somente em bactérias ativas e, portanto, não inclui as células adormecidas. Em nossa abordagem, a incorporação é BrdU em bactérias in situ immunodetected ...

<table border="1"><tbody><tr><td> Nome </td><td> Tipo </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> BrdU </td><td> Reagente </td><td> Sigma </td><td> B5002-5G </td><td></td></tr><tr><td> Lisozima </td><td> Reagente </td><td> Sigma </td><td> L6876-5G </td><td></td></tr><tr><td> Proteinase K </td><td> Reagente </td><td> Sigma </td><td> P2308-25mg </td><td></td></tr><tr><td> In Situ Proliferação Celular Kit, FLUOS </td><td> Conjunto </td><td> Roche </td><td> 11810740001 </td><td> Consumir mais de Anti-BrdU-FLUOS e tampão de incubação por reação do que o sugerido pelo fabricante. </td></tr><tr><td> Quadro Seal-Chambers Incubação </td><td> Material </td><td> Bio-Rad </td><td> SLF-1201 </td><td></td></tr><tr><td> Filtros de membrana de policarbonato (142 mm de diâmetro, 1,0 mM de poros de tamanho) </td><td> Material </td><td> Millipore </td><td> FALP14250 </td><td></td></tr><tr><td> Filtros de membrana de policarbonato (25 mm de diâmetro, 0,2 m de poros de tamanho) </td><td> Material </td><td> Millipore </td><td> FGLP02500 </td><td></td></tr><tr><td> illustra PuReTaq Ready-To-Go PCR Beads </td><td> Conjunto </td><td> GE Healthcare </td><td> 27-9559-01 </td><td></td></tr><tr><td> QIAquick extração de gel kit </td><td> Conjunto </td><td> QIAGEN </td><td> 28704 </td><td></td></tr><tr><td> FailSafe PCR System </td><td> Conjunto </td><td> Epicentro </td><td> FS99060 </td><td></td></tr></tbody></table>

bromodeoxyuridine (brdu) labeling and subsequent fluorescence activated cell sorting for culture-independent identification of dissolved organic carbon-degrading bacterioplankton

Micróbios são agentes importantes mediando a degradação do carbono dissolvido numerosos orgânico (DOC) substratos em ambientes aquáticos. No entanto, a identificação da taxa de bactérias que transformam a grupos específicos de DOC na natureza representa um desafio técnico. Aqui nós descrevemos uma abordagem que os casais bromodeoxiuridina incorporação (BrdU), separação de células ativadas por fluorescência (FACS), e 16S rRNA gene baseada em análise molecular que permite que a cultura independente de identificação do bacterioplâncton capazes de degradar compostos DOC uma específicos em ambientes aquáticos. Microcosmos bacterioplâncton triplicado são configurados para receber tanto BrdU e um composto modelo DOC (DOC alterações), ou apenas BrdU (sem adição de controle). Substitutos BrdU as posições de timidina no DNA recentemente sintetizado bacteriana e BrdU marcado DNA pode ser prontamente 1,2 immunodetected. Através de uma de 24 horas de incubação bacterioplâncton, que são capazes de usar o composto adicionado DOC são esperados para ser seletivamente ativado, e, portanto, têm níveis mais elevados de incorporação de BrdU (células HI) que os não-responsivos células nas alterações DOC e células em no- Além controles (células de baixa incorporação de BrdU, células LI). Depois de imunodetecção de fluorescência, as células HI são distinguidos e separados fisicamente das células LI por separação de células ativadas por fluorescência (FACS) 3. Ordenado DOC-responsiva (células HI) são extraídos de DNA e identificados taxonomicamente por meio de 16S rRNA gene subseqüentes análises baseadas incluindo PCR, clonagem e sequenciamento de construção da biblioteca.

Watch this Scientific Journal Video about Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degradin

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Bacterioplâncton ambientais são incubados com um modelo de carbono orgânico dissolvido composto (DOC) e um reagente rotulagem DNA, bromodeoxiuridina (BrdU). Depois, DOC-degradante células são separadas da comunidade em massa com base em sua incorporação BrdU elevada usando separação de células ativadas por fluorescência (FACS). Estas células são então identificadas por análises subseqüentes molecular.

Bromodeoxiuridina Labeling (BrdU) e separação de células subseqüentes Fluorescence Activated de Cultura independente de Identificação de carbono orgânico dissolvido de degradação bacterioplâncton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

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Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

20.8K visualizações.  Kent State University. Bacterioplâncton ambientais são incubados com um modelo de carbono orgânico dissolvido composto (DOC) e um reagente rotulagem DNA, bromodeoxiuridina (BrdU). Depois, DOC-degradante células são separadas da comunidade em massa com base em sua incorporação BrdU elevada usando separação de células ativadas por fluorescência (FACS). Estas células são então identificadas por análises subseqüentes molecular.

Vídeo: Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

Geração de humanos CD40-activated células B

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Biologia

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

Separação de células de fluorescência Activated de Protoplastos Usina

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Cromatografia de afinidade lipídica mediada por vesículas com separação de células Magnetic Ativado (LIMACS): um novo método para analisar proteínas lipídios Interação

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

Análise de célula específico<em&gt; Arabidopsis</em&gt; Folhas utilizando ordenação células activadas por fluorescência

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Optimizado coloração e Proliferação Métodos de Modelagem para Monitoramento Divisão Celular usando Corantes Rastreamento celulares

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

Isolamento de fibroblastos normais e associados ao câncer de tecidos frescos, classificando células activadas por fluorescência (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belonging to this network of organelles interconnected by vesicular transport, cause severe pathologies including cancer and neurobiological disorders. It is therefore of prime relevance to understand the mechanisms underlying the biogenesis and organization of the endosomal system. The yeast Saccharomyces cerevisiae has been pivotal in this task. To specifically label and analyze at the ultrastructural level the endosomal system of this model organism, we present here a detailed protocol for the positively charged nanogold uptake by spheroplasts followed by the visualization of these particles through a silver enhancement reaction. This method is also a valuable tool for the morphological examination of mutants with defects in endosomal trafficking. Moreover, it is not only applicable for ultrastructural examinations but it can also be combined with immunogold labelings for protein localization investigations.

Yeast, Saccharomyces cerevisiae, has been a key model organism to identify and study genes regulating the biogenesis and functions of the endosomal system. Here we present a detailed protocol for the specific labeling of the endosomal compartments for ultrastructural studies.

Nanogold Labeling do Sistema de levedura Endossomal de Análises ultraestruturais

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Rotulagem específica de seqüência de ácidos nucléicos e proteínas com Metiltransferases e cofator Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Magnético-ativado da pilha classificação estratégias para isolar e purificar sinovial fluidos derivados mesenquimais células-tronco de um modelo de coelho

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

Micro-aspiração de célula única como uma estratégia alternativa à classificação celular ativada por fluorescência para separação de mistura de vírus gigante

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...