Reconhecemos o fluxo USF Facilidade Núcleo Citometria. Gostaríamos de agradecer ao Dr. Denise Cooper para a partilha de recursos. Gostaríamos também de agradecer a Maya Cohen, Laura Pendleton e Diana Latour por sua assistência na criação e filmagens deste vídeo. NN suportado pela NSF FG-LSAMP Ponte para o Doutorado Fellowship HRD # 0929435. Este trabalho foi financiado pela Sociedade Americana de Câncer Research Grant Institucional # 93-032-13/Moffitt Cancer Center, atribuído ao TG.

Antes da partida, preparar as seguintes soluções: 3% de coloração Media (SM): -3% De soro fetal bovino (FBS) em 1X tampão fosfato salino (PBS) MACS buffer (MB): - 0,5% de albumina de Soro Bovino (BSA) em 1XPBS 1. Os baços de colheita de Ratos <ol><li> Por via subcutânea injectar 6-8 semanas de idade C57BL / 6 machos (Harlan) com 1,5 x 10 5 murinos Panc02 células em suspensão em 100 uL de PBS 1x (tumor de rolamento; TB). Camundongos controle (Naïve) recebem 100 1XPBS ul. </li><li> Cerca de 4 semanas pós-injeção, eutanásia ratos por asfixia dióxido de carbono. </li><li> Colheita baços de camundongos por dissecção romba com pinça e tesoura, em seguida, pesar com uma balança. Baços Coloque em separado, chamado tubos de 50 ml cônicos contendo 1 x PBS. </li></ol> 2. Gerar um suspensio de uma única célulan de leucócitos de baços Todos os procedimentos devem ser realizadas em um ambiente estéril sob uma capa de segurança biológica e as células e os anticorpos mantidos em gelo. <ol><li> Montar a célula de dissociação peneira, inserindo a tela de malha para dentro da abertura da taça para o fundo. Em seguida, insira o anel de retenção para a área de rosca com o lado da fenda e usar o anel de chave para apertar o anel de retenção, mantendo desta forma a tela no lugar. Coloque a peneira montado numa placa de Petri contendo 10 ml de 1 x PBS. </li><li> Baços de piscina e pilão de vidro utilização para moer baços contra a tela de malha de célula de dissociação peneira e para dentro da placa de Petri. Repetir para cada grupo de tratamento de ratinhos. </li><li> Suspensão de células de filtro para um tubo cónico de 50 ml usando um filtro de 70 pm e uma célula pipeta de 5 ml serológico. Centrifugar a 12.000 rpm (250-300 xg) durante 5 min. </li><li> Remover o sobrenadante e ressuspender o pellet em 5 1 x ml de tampão de lise dos glóbulos vermelhos por baço. Pipetar cima e para baixo vigorosamente. Incubar a temperatura ambiente durante 5 min. Parar a reacção por adição de 20 ml de 1 x PBS. Pipetar cima e para baixo vigorosamente. Centrifugar a 12.000 rpm (250-300 xg) durante 5 min. </li><li> Remover o sobrenadante e ressuspender o pellet em 20 ml estéril 1 x PBS. Pipetar cima e para baixo vigorosamente. </li><li> Contar as células utilizando azul de tripano e um hemacitómetro e ressuspender em concentração desejada em SM 3% de modo a que 50 uL é equivalente a 5x10 5-1x10 6 células (1x10 7 células / ml - 2x10 7 células / ml). </li></ol> 3. Superfície celular de coloração / Imunofenotipagem de MDSC por citometria de fluxo <ol><li> Rotular os poços de uma placa de fundo 96-V bem para o controlo e as amostras experimentais e manchas únicas para os controlos de compensação (nenhuma mancha, NS; Mac-1-FITC; Gr-1-APC e DAPI). </li><li> Adicionar 5x10 5-1x10 6 células equivalentes a 50 uL / poço de esplenócitos para os seus respectivos poços na placa de 96 poços de fundo em V. Centrifugarem placas a 12.000 rpm (250-300 xg) durante 5 min. </li><li> Preparar &quot;Mix Master&quot; (MM) do rato BD Fc Block (Rat anti-rato CD16/32 anticorpo monoclonal) diluída em SM 3%, em um tubo de microcentrífuga de 1,5 ml em gelo. Como ponto de partida, usar 1 ug Bloco Fc em SM 3% para um volume final de 50 ul, por poço. </li><li> Cuidadosamente remover o sobrenadante de cada poço da placa de 96 poços de fundo em V por rapidamente invertendo a placa sobre e para trás, ao longo de um recipiente de resíduos ou pia sem interrupção dos peletes celulares. </li><li> Vortex, brevemente centrifugar Fc MM Bloco durante 5 segundos e adicionar 50 uL a todos os peletes na placa de 96 poços de fundo em V. Misture bem cuidado pipetando para cima e para baixo, deixando amostras em seus poços. Incubar placa durante 15 min no escuro em gelo. Centrifugar em placas a 12.000 rpm (250-300 xg) durante 5 min. </li><li> Preparar &quot;Mix Master&quot; (MM) de fluorocromo-anticorpos diluídos em SM 3% em um tubo de microcentrífuga de 1,5 ml em gelo, enquanto as amostras incubar com o Bloco Fc. Anticorpos deve ser tituladapara determinar diluições óptimas para a coloração de procedimentos. Como ponto de partida, combinar uma diluição 1:25 do Mac-1-FITC e diluição 1:20 de Gr-1-APC em SM 3% para um volume final de 50 ul, por poço de amostra. </li><li> Cuidadosamente remover o sobrenadante de cada poço da 96-V bem inferior como descrito anteriormente. </li><li> Vortex, brevemente centrifugar MM de anticorpos fluorescente-conjugados de coloração durante 5 segundos e adicionar 50 uL de controlo e de peletes experimentais. Misture bem cuidado pipetando para cima e para baixo. Para uma única mancha compensações, adicionar uma diluição 1:25 do Mac-1-FITC, diluição 1:20 de Gr-1-APC e 75 ng / ml de DAPI, em SM 3% para um volume final de 50 uL por poço para os seus respectivos poços. Adicionar 50 ul SM 3% a imaculada bem (não mancha). Misturar bem e incubar as células na placa de 96 poços de fundo em V durante 30 min no escuro em gelo. </li><li> Tubos de etiquetas FACS (5 ml, 12 mm x 75 mm de poliestireno tubos de fundo redondo) para corresponder a cada poço na placa de 96 poços de fundo em V. Adicionar 200 uL SM 3% a cada um FACStubo. </li><li> Centrifugar em placas a 12.000 rpm (250-300 xg) durante 5 min e remover os sobrenadantes. Lave pelotas pela adição de 100 ul SM 3% a cada pellet e misture bem cuidado pipetando para cima e para baixo. Centrifugar em placas a 12.000 rpm (250-300 xg) durante 5 min. Repita o passo de lavagem mais uma vez. </li><li> Cuidadosamente remover o sobrenadante de cada poço da 96-V bem inferior como descrito anteriormente. Ressuspender cada sedimento em 100 SM ul 3% e misture bem. </li><li> Transferir 100 uL sedimento ressuspenso a partir de cada poço da placa de 96 poços de fundo em V à sua tubo FACS respectivamente rotulado. </li><li> Antes da análise de fluxo de citometria, adicionar 75 ng / ml DAPI para controlar e as amostras experimentais e de controlo de compensação DAPI única mancha. </li><li> Execute o fluxo de aquisição de dados por citometria de percentagens MDSC. Execute a compensação usando o negativo (nenhum controlo mancha) e os controlos positivos individuais. Configurar um gráfico de pontos que mostra a dispersão frontal (FSC) versus lateral (SSC) em escala logarítmica, para que populações de leucócitos deinteresse pode ser identificada. Desenhe um grande portão em todos os leucócitos, excluindo detritos e aglomerados com menor dispersão para a frente e lateral. A partir deste portão principal, criar um novo gráfico de pontos que exibe SSC contra DAPI e DAPI em porta (ao vivo) células. Seleccionar esta população recém-gated e criar um gráfico de pontos que exibe Mac-1 versus Gr-1 e portão no seu positiva dupla (Mac-1 + Gr-1 +) MDSC. </li></ol> 4. Enriquecimento magnético de GR-1 + Leucócitos <ol><li> Alíquotas 1x10 7 restantes leucócitos não coradas em tubos adequadamente marcados FACS e centrifugar a 12.000 rpm (250-300 xg) durante 5 min. </li><li> Preparar MM de Gr-1-PE anticorpo em um tubo de microcentrífuga de 1,5 ml. Para até 10 7 células, utilizar uma diluição de 1:10 de Gr-1-PE anticorpo em 50 MB ul, por amostra. Para um maior número de células, aumentar os volumes de acordo. Resumidamente centrifugar durante 5 segundos, adicionar aos leucócitos em tubos de FACS e incubar durante 15 min a 4 ° C no escuro. </li&gt; <li> Adicionar 2 ml de MB para tubos de FACS, centrifugar a 12.000 rpm (250-300 xg) durante 5 min e elimine o sobrenadante. </li><li> Preparar MM de Anti-PE micropérolas em um tubo de microcentrífuga de 1,5 ml. Para até 10 7 células, utilizar uma diluição de 1:4 de anti-PE micropérolas em 200 MB ul, por amostra. Para um maior número de células, aumentar os volumes de acordo. Resumidamente centrifugar durante 5 segundos, adicionar aos leucócitos em tubos de FACS e incubar durante 15 min a 4 ° C no escuro. </li><li> Adicionar 2 ml de MB para tubos de FACS, centrifugar a 12.000 rpm (250-300 xg) durante 5 min e elimine o sobrenadante. Ressuspender o granulado em 3 ml de SB. Filtrar através de uma peneira iM 70 para um novo tubo ml, rotulados 50 cónico. </li><li> Prepare-se e prepare Auto MACS Separador Pro. Recarga de todos os frascos com as soluções adequadas e esvaziar o depósito de resíduos, se necessário. Vire instrumento e examinar o status de recipientes de fluido e de coluna (s) após a inicialização. Todos os símbolos devem ser verdes. No menu, selecione &quot;separação&quot; do superiorbarra de menu, seguido de &quot;Lavar Agora&quot; na barra de menu inferior. Selecione &quot;Lavar&quot; a partir da opção pop-up, seguido de &quot;Executar&quot; para iniciar o processo de preparação. Uma vez que o processo de priming é concluída com êxito, o instrumento, então, mostrar que é &quot;Ready for Separation&quot; no menu Status. </li><li> Escolha suporte de tubos refrigerados adequado para tamanhos de tubos e coloque 50 ml de tubo cônico com células marcadas magneticamente na linha A, 50 ml tubo cônico de coleta de fração negativa na linha B e 15 ml tubo cônico para a coleta de fração positiva na linha C. </li><li> Escolha &quot;POSSEL_S&quot; programa de separação de células para seleção positiva de células alvo marcadas no modo sensível a partir de amostras. Células alvo magneticamente marcadas são retidos na coluna automáticos; células não marcadas são libertados para o tubo de recolha negativo fracção em B. Em linha de retracção automática do magneto, as células alvo marcadas irá ser libertado para o tubo de recolha positiva na linha C do tubo rack. </li></ol>Leucócitos + enriquecidos <ol><li> Recontagem Gr-1 + e Gr-1 - frações utilizando Trypan Blue e um hemocitômetro. Ressuspender as células em concentração desejada em meio de coloração com 3% de modo a que 50 uL é equivalente a 5x10 5-1x10 6 células (1x10 7 células / ml - 2x10 7 células / ml) e transferir 50 uL para tubos correspondentemente rotulados FACS. </li><li> Prepara-se uma MM de Mac-1 só, a uma diluição 1:25 em SM 3% para um volume final de 50 uL por amostra. Corar as células e preparar individuais compensações mancha de Gr-PE, Mac-1 FITC e DAPI para análise de citometria de fluxo. Adicionar 200 uL SM 3% e DAPI corante de viabilidade, como descrito anteriormente. </li><li> Execute o fluxo de aquisição de dados por citometria de determinar Gr-1 + e Gr-1 - Percentagens e também para comparar as proporções MDSC enriquecimento pré-e pós-autoMACS. </li></ol> 6. Os resultados representativos Aqui nós mostramos r<html> resultados epresentative para autoMACS enriquecimento de Gr-1 + leucócitos a partir do conjunto, baço ingénuas para FACS subsequentes de triagem de MDSC (Figura 1). 1x10 6 leucócitos naive foram coradas com Mac-1-FITC e Gr-1-APC anticorpos para identificar percentagens MDSC usando um instrumento BD LSRII, antes de triagem autoMACS. 1x10 7 leucócitos naive foram então coradas com anti-Gr-1-PE anticorpos e PE-micropérolas para o enriquecimento de Gr-1 + leucócitos utilizando um autoMACS Separator Pro. Pós-autoMACS enriquecimento, Gr-1 percentagens em Gr-1 + e Gr-1 - fracções recolhidas foram avaliados usando citometria de fluxo. 1x10 6 Gr-1 + leucócitos foram removidas e coradas com Mac-1-FITC de anticorpos para avaliar e comparar percentagens MDSC de enriquecido, pooled leucócitos virgens de não enriquecido, reunidas portadores de tumor leucócitos por citometria de fluxo. fig1.jpg &quot;/&gt; Figura 1. Enriquecimento AutoMACS de Naïve Gr-1 + leucócitos para FACS MDSC Ordenação. Os baços foram colhidas a partir de ratos portadores de tumor pancreático e ingénuos e transformados em suspensões de célula única. Análise de citometria de fluxo de superfície de leucócitos ingénuo coradas com Mac-1 e Gr-1 fluorescente anticorpos conjugados, antes de autoMACS enriquecimento (A). Análise de citometria de fluxo de Gr-1 + (B) e Gr-1 - (C) As fracções de enriquecimento pós-autoMACS de Gr-1 + células a partir do conjunto leuckocytes naive coradas com Gr-1-PE anticorpos e anti-PE micropérolas. Análise por citometria de fluxo de MDSC e Gr-1 + percentagens enriquecimento pós-autoMACS de Gr-1 + células a partir do conjunto de leucócitos ingênuo (D) em comparação aos não enriquecidos leucócitos obtidos a partir de camundongos portadores de tumor (E) (ratos ingênuos, n = 5 ; camundongos portadores de tumor, n = 3). MDSC e Gr-1 percentagens são fechado nos gráficos de contorno representativas e histogramas.

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Este é um método detalhado para o processamento e immunophentyping populações MDSC que é aplicável a diferentes tecidos linfóides a partir de vários modelos animais. Em particular, o enriquecimento autoMACS pode ser usado para o isolamento de várias populações de leucócitos, incluindo Gr-1 depleção de esplenócitos 4, a purificação de subconjuntos mielóides de esplenócitos e gânglios linfáticos 5, o isolamento dos neutrófilos da medula óssea de 14 e purificação de...

<table border="1"><tbody><tr><td> REAGENTE </td><td> EMPRESA </td><td> Catalogo </td><td> COMENTÁRIOS </td></tr><tr><td> Fosfato 1X Tampão Salina </td><td> Thermo Scientific Hyclone </td><td> SH30028.02 </td><td> Ca 2 + / Mg 2 + / Vermelho de Fenol-livre </td></tr><tr><td> Albumina de Soro Bovino (BSA) </td><td> Sigma-Aldrich </td><td> A7906 </td><td> Deixe BSA dissolver imperturbável em PBS; ambiente estéril </td></tr><tr><td> Soro fetal de bovino (FBS) </td><td> Thermo Scientific Hyclone </td><td> SV3001403HI </td><td> Inativados pelo calor; ambiente estéril </td></tr><tr><td> Rat anti-rato CD16/32 anticorpo monoclonal (Fc Block) </td><td> BD Biosciences </td><td> 553142 </td><td> Ambiente estéril </td></tr><tr><td> Anti-ratinho CD11b (Mac-1) FITC </td><td> eBiosciences </td><td> 11-0112 </td><td> EstérilAmbiente </td></tr><tr><td> Anti-ratinho Ly6G (Gr-1) APC </td><td> eBiosciences </td><td> 17-5931 </td><td> Ambiente estéril </td></tr><tr><td> Anti-ratinho Ly6G (Gr-1) PE </td><td> eBiosciences </td><td> 12-5931 </td><td> Ambiente estéril </td></tr><tr><td> DAPI </td><td> Invitrogen </td><td> D1306 </td><td> Ambiente de Diluição Estéril </td></tr><tr><td> A dissociação de células Sieve </td><td> Sigma-Aldrich </td><td> CD1-1KT </td><td> Autoclave antes do uso </td></tr><tr><td> 70 m de filtro </td><td> BD Biosciences </td><td> 352350 </td><td> Ambiente estéril </td></tr><tr><td> 1X Tampão de Lise RBC </td><td> eBiosciences </td><td> 00-4333-57 </td><td> Aquecer até à temperatura ambiente antes da utilização; ambiente estéril </td></tr><tr><td> As placas de Petri </td><td> Fisher Scientific </td><td> 08-757-12 </td><td> Ambiente estéril </td></tr><tr><td> 50ml contubos ical </td><td> Thermo Scientific </td><td> 339652 </td><td> Ambiente estéril </td></tr><tr><td> 5ml 12X75mm de poliestireno de fundo tubos redondos </td><td> BD Biosciences </td><td> 352054 </td><td> Conhecido como tubos FACS; ambiente estéril </td></tr><tr><td> 96 poços de fundo em V placas </td><td> Corning </td><td> 3897 </td><td> Ambiente estéril </td></tr><tr><td> Trypan Blue </td><td> Cellgro </td><td> 25-900-CI </td><td> Ambiente estéril </td></tr><tr><td> PE micro </td><td> Miltenyi Biotec </td><td> 130-048-801 </td><td> Ambiente estéril </td></tr><tr><td> AutoMACS Separador Pro </td><td> Miltenyi Biotec </td><td> 130-092-545 </td><td></td></tr><tr><td> Colunas AutoMACS </td><td> Miltenyi Biotec </td><td> 130-021-101 </td><td></td></tr><tr><td> AutoMACS tampão de corrida </td><td> Miltenyi Biotec </td><td> 130-091-221 </td><td&gt; </td></tr></tbody></table>

preparation of myeloid derived suppressor cells (mdsc) from naive and pancreatic tumor-bearing mice using flow cytometry and automated magnetic activated cell sorting (automacs)

MDSC são uma população heterogénea de macrófagos, células dendríticas imaturas e granulócitos que se acumulam nos órgãos linfóides em condições patológicas, incluindo infecção por parasitas, inflamação, stress traumático, enxerto-versus-hospedeiro da doença, a diabetes eo cancro 1-7. Em ratinhos, MDSC expressa Mac-1 (CD11b) e Gr-1 (Ly6G e Ly6C) antigénios de superfície 7. É importante notar que MDSC são bem estudada em vários hospedeiros portadores de tumor, onde são significativamente expandidas e suprimir anti-tumorais respostas imunes homólogos em comparação com naive 7-10. No entanto, dependendo do estado patológico, existem diferentes subpopulações de MDSC com mecanismos distintos e alvos de supressão 11,12. Assim, os métodos eficazes para isolar populações viáveis ​​MDSC são importantes na elucidação suas diferentes mecanismos moleculares de supressão in vitro e in vivo. Recentemente, o Ghansah grupo relatou a expansão do MDSC num modelo murino de pâncreas cancro. O nosso portadores de tumor MDSC exibir uma perda de homeostase e aumento da função supressora em comparação com MDSC ingénuos 13. Percentagens MDSC são significativamente menos nos compartimentos linfóides de camundongos normais vs tumor-rolamento. Esta é uma ressalva importante, que muitas vezes dificulta precisas análises comparativas destes MDSC. Portanto, enriquecendo Gr-1 + leucócitos de camundongos ingênuos antes de separação de células activadas por fluorescência (FACS) fortalece a viabilidade, pureza e reduz significativamente o tempo de classificação. Todavia, o enriquecimento de Gr-1 + leucócitos de camundongos portadores de tumor é opcional uma vez que estes estão em abundância para FACS rápidas de classificação. Portanto, neste protocolo, nós descrevemos um método altamente eficiente de imunofenotipagem MDSC e enriquecendo Gr-1 + leucócitos de baços de camundongos normais para classificar MDSC em tempo hábil. Camundongos imunocompetentes C57BL / 6 são inoculadas com murino Panc02 cells subcutânea enquanto camundongos ingênuos receber 1XPBS. Aproximadamente 30 dias após a inoculação; baços são removidos e processados ​​em suspensões de célula única, utilizando uma célula de dissociação peneira. Os esplenócitos são então glóbulo vermelho (RBC) lisadas e uma alíquota destes leucócitos são coradas usando fluorocromo-anticorpos contra Mac-1 e Gr-1 a percentagens imunofenotipo MDSC utilizando citometria de fluxo. Numa experiência paralela, os leucócitos inteiras a partir de ratinhos naive são coradas com fluorescência conjugados Gr-1, incubados com anticorpos PE-micropérolas e positivamente seleccionadas utilizando uma separação de células automatizado Magnética Activado (autoMACS) Pro Separator. Em seguida, uma alíquota de Gr-1 + leucócitos são coradas com Mac-1 anticorpos para identificar o aumento em percentagem MDSC utilizando citometria de fluxo. Agora, estas GR1 leucócitos + enriquecidos estão prontos para a selecção de FACS MDSC para ser usado em análises comparativas (ingénuo vs portadores de tumor) em in vivo e in vitro </in&gt; ensaios.

Watch this Scientific Journal Video about Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell S

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

Este é um método rápido e abrangente de imunofenotipagem mielóides derivadas de células supressoras (MDSC) e enriquecendo Gr-1<sup&gt; +</sup&gt; Leucócitos a partir de baços de rato. Este método utiliza a citometria de fluxo e Cell AutoMACS classificação para enriquecer para viável Gr-1<sup&gt; +</sup&gt; Leucócitos anteriores FACS de triagem de MDSC para uso<em&gt; In vivo</em&gt; E<em&gt; In vitro</em&gt; Ensaios.

Preparação de Mielóides derivadas de células supressoras (MDSC) de ingênuos e pâncreas Tumor-rolamento Ratos utilizando citometria de fluxo e separação de células automatizado magnético ativado (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

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Research

JoVE Journal

Medicine

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

26.1K visualizações.  University of South Florida Morsani College of Medicine. Este é um método rápido e abrangente de imunofenotipagem mielóides derivadas de células supressoras (MDSC) e enriquecendo Gr-1 + Leucócitos a partir de baços de rato. Este método utiliza a citometria de fluxo e Cell AutoMACS classificação para enriquecer para viável Gr-1 + Leucócitos anteriores FACS de triagem de MDSC para uso In vivo E In vitro Ensaios.

Vídeo: Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Processamento de tecido primário do tumor cerebral para Ensaios de Células Tronco e Fluxo de Classificando

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Medicina

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Isolamento, caracterização e comparativa Diferenciação de células-tronco da polpa dentária Derivado de dentes permanentes usando dois métodos diferentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Cultura de Células e Isolamento paciente Derivado de CD133<sup&gt; +</sup&gt; Células-tronco de câncer putativos Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

High-throughput Citometria de Fluxo Ensaio Celular para detectar anticorpos em N-metil-D-aspartato ou dopamina-2 Receptor em Soro Humano

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Adaptação do Semi-Automática de circulação Tumor de Células (CTC) Ensaios para aplicações clínicas e pré-clínica de Pesquisa

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

Validação de nanocorpo e Antibody Based<em&gt; In Vivo</em&gt; Experimentos Tumor Xenoenxerto NIRF-imagem em ratos usando<em&gt; Ex Vivo</em&gt; Citometria de fluxo e microscopia

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Isolamento de linfático humano endotelial por multi-parâmetro activadas por fluorescência celular

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Estudar o cancro do pâncreas Stem características celulares para desenvolver estratégias Novo tratamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. Determining the real time state of a microbial cell goes beyond live or dead categories, as microbes can exist in a dormant state, whereby cell division and metabolic activities are reduced. Given the need for detection and quantification of microbes, flow cytometry (FCM) with molecular probes provides a rapid and accurate method to help determine overall population viability. By using SYTOX Green and SYTOX Orange in the model cyanobacteria Microcystis aeruginosa to detect membrane integrity, we develop a transferable method for rapid indication of single cell mortality. The molecular probes used within this journal will be referred to as green or orange nucleic acid probes respectively (although there are other products with similar excitation and emission wavelengths that have a comparable modes of action, we specifically refer to the fore mentioned probes). Protocols using molecular probes vary between species, differing principally in concentration and incubation times. Following this protocol set out on M.aeruginosa the green nucleic acid probe was optimized at concentrations of 0.5 &#181;M after 30 min of incubation and the orange nucleic acid probe at 1 &#181;M after 10 min. In both probes concentrations less than the stated optimal led to an under reporting of cells with membrane damage. Conversely, 5 &#181;M concentrations and higher in both probes exhibited a type of non-specific staining, whereby &#39;live&#39; cells produced a target fluorescence, leading to an over representation of &#39;non-viable&#39; cell numbers. The positive controls (heat-killed) provided testable dead biomass, although the appropriateness of control generation remains subject to debate. By demonstrating a logical sequence of steps for optimizing the green and orange nucleic acid probes we demonstrate how to create a protocol that can be used to analyse cyanobacterial physiological state effectively.

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry

Microbial populations contain substantial cell heterogeneity, which can dictate overall behavior. Molecular probe analysis through flow cytometry can determine physiological states of cells, however its application varies between species. This study provides a protocol to accurately determine cell mortality within a cyanobacterium population, without underestimating or recording false positive results.

Molecular Probe Optimization para determinar mortalidade celular em um fotossintético organismo (<em&gt; Microcystis aeruginosa</em&gt;) Utilizando citometria de fluxo

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. ...

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.