Este trabalho foi apoiado pelo Departamento de Anestesia e Cuidados perioperatórios UCSF.

1. Chapeamento e manutenção das células endoteliais microvasculares <ol><li> Descongelar e crescer suas células endoteliais microvasculares acordo com os fabricantes fornecido instruções. Neste protocolo, as células foram cultivadas em EGM-2 media MV (Lonza), e recomenda-se que todas as experiências são realizadas no prazo de duas semanas de descongelamento para minimizar qualquer variação devido ao número de passagens. As experiências são realizadas com HMVEC-pulmonar entre as passagens 4-9. </li><li> Dia 1: Quando as células atingem 80-90% de confluência, trypsinize e ressuspender em 120 mil células por ml. Placa de 36.000 células (0,3 ml) por poço em 48 poços, placas de cultura de tecidos de poliestireno (es). Incluir poços de HMVEC que não serão incubadas com os neutrófilos a fim de obter uma leitura de fluorescência de fundo. A menos que usando placas de 48 poços projetados especificamente para ensaios de fluorescência, linhas alternadas ou colunas irá minimizar qualquer contaminação luz de poços vizinhos. NOTA: Wells pode ser pré-revestida com poli-Lysine, colagénio ou fibronectina. </li><li> Dia 2: Adicionar novas mídias. </li><li> Dia 3: A microscopia de contraste de fase, confirmar que as células são saudável (isto é, a aparência &quot;Cobblestone&quot; e pouco detritos de células), e eles são 100% confluentes (Figura 1). Se as células não são 100% confluentes, mudar os meios de comunicação a cada dia até que estejam prontos. </li><li> Uma vez que as monocamadas HMVEC está pronto para o tratamento, lave as células uma vez com solução salina equilibrada de Hank (HBSS) e adicionar 0,3 ml de meio EGM-2 MV frescas ou meios contendo agonista inflamatória aos poços apropriados. Neste protocolo HMVEC-pulmão foram tratadas com TNFa [100 ng / ml] (PeproTech, Nova Jersey), durante 3 horas uma vez que este é um activador bem descrito de ECs 21. NOTA: Recomenda-se que o tempo a sua experiência para que suas células HMVEC ativadas (Passo 4.1) e Calcein AM neutrófilos marcados (Passo 3.5) está pronto para usar ao mesmo tempo. Ela nos leva aproximadamente 2,5 horas para isolar e rotular neutrophils. </li></ol> 2. Isolamento de neutrófilos utilizando Polymorphprep <ol><li> Isolar neutrófilos, como descrito anteriormente 22, ou com uma solução de gradiente de densidade pronta para isolar granulócitos polimorfonucleares de sangue total. Empregando Polymorphprep seguindo o protocolo de Nuzzi, et ai. 2,007 resulta em rendimentos de cerca de 2-3 x 10 neutrófilos por seis ml de sangue total 23. NOTA: Para evitar a lise dos eritrócitos e excessivo, uma sedimentação de dextrano para remover os glóbulos vermelhos podem ser efectuadas antes da centrifugação em gradiente de densidade 24. </li><li> Levar a solução do gradiente de densidade Polymorphprep à TA. </li><li> Recolha de 30 ml de sangue total de um voluntário humano saudável, na presença de um anti-coagulante, como a heparina, citrato ou EDTA. O sangue deve ser usado dentro de 2 horas, e mantida entre 18-22 ° C. </li><li> Camada 5 ml de sangue total ao longo de 5 ml da solução de gradiente de densidade num tubo cónico de 15 ml ( <strong&gt; Figura 2A). Evite alterar volumes de sangue ea solução gradiente de densidade, pois isso pode alterar as condições de centrifugação subseqüentes. </li><li> Centrifugar a 450 g durante 30 min a 18-22 ° C. Certifique-se de acelerar lentamente para cima e para baixo da centrífuga (ou seja, desligar o freio centrífuga), e equilibrar os tubos bem para evitar vibrações para ajudar a reduzir a ativação dos neutrófilos e evitar a mistura de camadas. Tempos de centrifugação mais longos ou força centrífuga maior irá resultar na camada inferior de leucócitos, que contém os neutrófilos, que migram mais para baixo em direcção às células vermelhas do sangue peletizadas. </li><li> Aspirar o plasma e leucócitos banda superior contendo PBMC para evitar a contaminação da camada contendo neutrófilos (Figura 2B). </li><li> Usar uma pipeta serológica 1-2 ml para eliminar a camada inferior contendo leucócitos neutrófilos. Combinar as camadas de neutrófilos em 30 ml de PBS (sem Ca 2 + e Mg 2 +), de 50 mltubo cónico. </li><li> Levar o volume até 50 ml com PBS (sem Ca 2 + e Mg 2 +), e centrifugar a 450 xg durante 10 min a 18-22 ° C. </li></ol> Nota: Os passos 2.9 e 2.10 são opcionais, mas altamente recomendado. <ol start="9"><li> Aspirar o sobrenadante. Ressuspender as neutrófilos em 8 ml de água estéril durante 30 segundos para lisar os glóbulos vermelhos, imediatamente adicionar a suspensão de células de 2 ml de 5x em PBS (sem Ca 2 + e Mg 2 +), e misturar suavemente à mão. NÃO incubar células água por mais de 30 segundos desde a morte de neutrófilos significativa pode ocorrer. </li><li> Centrifugar a 250 xg durante 5 min a 18-22 ° C. </li><li> Lave as células uma vez com 10 ml de PBS (sem Ca 2 + e Mg 2 +) e centrifugar novamente a 250 xg durante 5 min a 18-22 ° C. </li><li> Ressuspender as neutrófilos em 10 ml de meio RPMI-1640 (sem vermelho de fenol), e contar as células vivas utilizando o azul de tripanométodo de exclusão de corante e. Evitar períodos prolongados de densidades celulares superiores a 5 x 10 6 por ml uma vez que este pode resultar na activação dos neutrófilos. </li></ol> 3. Neutrófilos Labeling com Calcein AM <ol><li> 6 x 10 5 neutrófilos são utilizados por poço de uma placa de 48 poços. </li><li> Após a contagem, a transferência das células ressuspensas para um tubo de 50 ml e dilui-cónica os neutrófilos a 2-4 x 10 6 culas por ml com vermelho fenol livre de RPMI-1640. </li><li> Adicionar solução de calceína AM de 3 uM (ou seja, 2,98 ul de calceína AM Estoque (1 mg / ml) por ml de meio) e misture bem sacudindo suavemente o tubo. Superior a 5 microns de calceína AM resultará no vazamento dos neutrófilos Nota:. Não-fluorescente calceína AM é clivada no interior das células por esterases endógenas para produzir a molécula altamente fluorescente calceína (isto é, as células mortas não ficam fluorescentes). </li><li> Tapar o tubo com folha de alumínio e incubar fou 30 min a 37 ° C no escuro Nota:. tempos de incubação mais longo pode resultar em mais calceína ser libertado a partir dos neutrófilos ao longo do ensaio de adesão. </li><li> Centrifugar a 450 xg durante 5 min a 18-22 ° C, e lavar os neutrófilos 2x com 10 ml de RPMI-1640 (sem vermelho de fenol; pré-aquecido a 37 ° C). Após a ressuspensão das células para a segunda lavagem, passar primeiro os neutrófilos com um filtro estéril de 40 uM para remover aglomerados, e depois centrifugar mais uma vez a 450 xg durante 5 min a 18-22 ° C. </li><li> Ressuspender as neutrófilos em meio RPMI 1640 (sem vermelho de fenol; pré-aquecido a 37 ° C) a 2 x 10 6 por ml neutrófilos. </li></ol> 4. Ensaio de ligação de neutrófilos <ol><li> Lavar as monocamadas HMVEC 3x com 0,5 ml de filtro esterilizado (0,22 um) meio RPMI 1640 (sem vermelho de fenol), contendo 3% de BSA por poço. </li><li> Aspirar a mídia e adicione 6 x 10 5 neutrófilos calceína-rotulados (0,3 ml de células suspension), ou 0,3 ml de meio RPMI 1640 (sem vermelho de fenol) sem neutrófilos, aos poços apropriados da placa de 48 poços Nota:. Isto é o equivalente de 8 x 10 5 por cm 2 de neutrófilos. </li><li> Incubar durante 20 min no banho a 37 ° C, 5% CO 2 incubadora. </li><li> Fluorescência de neutrófilos totais (pré-lavagem) a medição da intensidade de fluorescência de cada poço com um comprimento de onda de excitação de 485 nm e um comprimento de onda de emissão de 520 nm (conjunto de filtros de fluoresceína) num Fluostar, ou equivalente, com um espectrofotómetro. Executar esta etapa pouco antes do ponto final de incubação Nota: Neste protocolo, calceína excitação e a detecção da luz emitida foram realizadas a partir do topo dos poços a descoberto, no entanto, ambos também pode ser realizada a partir do fundo dos poços.. </li><li> Lavar os poços contendo monocamadas HMVEC e neutrófilos 5x com 0,5 ml por poço de PBS (w / Ca 2 + e Mg 2 +). Remova a mídia e lavagensinvertendo a placa e batendo suavemente sobre toalhas de papel para retirar o líquido restante. </li><li> Adicionar 0,3 ml de volta de RPMI 1640 (sem vermelho de fenol) por poço. </li><li> Aderente a fluorescência de Neutrófilos (lavagem POST): medir a intensidade de fluorescência de cada poço como na etapa 4.4. </li><li> Determinar a aderência e a adesão relativa cento por poço: </li></ol> Nota: Nesta fase, as imagens dos neutrófilos marcados com calceína podem ser obtidas usando um microscópio de fluorescência equipado com capazes filtros de fluoresceína padrão. As imagens na Figura 4 foram obtidas em um microscópio invertido Olympus IX51 equipado com uma câmera 2000R Retiga (Q Imaging) utilizando Q-Captura software Pro7 (Q Imaging).

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U.S.A. 82, 8667-8671 (1985).</li><li>Braut-Boucher, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-isotopic,+highly+sensitive,+fluorimetric,+cell-cell+adhesion+microplate+assay+using+calcein+AM-labeled+lymphocytes.">A non-isotopic, highly sensitive, fluorimetric, cell-cell adhesion microplate assay using calcein AM-labeled lymphocytes.</a> Journal of Immunological Methods. 178, 41-51 (1995).</li><li>Ait-Oufella, H., Maury, E., Lehoux, S., Guidet, B., Offenstadt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelium:+physiological+functions+and+role+in+microcirculatory+failure+during+severe+sepsis.">The endothelium: physiological functions and role in microcirculatory failure during severe sepsis.</a> Intensive Care Medicine. 36, 1286-1298 (2010).</li><li>Andonegui, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelium-derived+Toll-like+receptor-4+is+the+key+molecule+in+LPS-induced+neutrophil+sequestration+into+lungs.">Endothelium-derived Toll-like receptor-4 is the key molecule in LPS-induced neutrophil sequestration into lungs.</a> The Journal of Clinical Investigation. 111, 1011-1020 (2003).</li><li>Sharma, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+endothelial+cell+platelet-activating+factor+production+and+inflammatory+cell+adherence+are+increased+in+response+to+cigarette+smoke+component+exposure.">Lung endothelial cell platelet-activating factor production and inflammatory cell adherence are increased in response to cigarette smoke component exposure.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 302, L47-L55 (2012).</li><li>Deban, L., Correale, C., Vetrano, S., Malesci, A., Danese, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+pathogenic+roles+of+microvasculature+in+inflammatory+bowel+disease:+a+Jack+of+all+trades.">Multiple pathogenic roles of microvasculature in inflammatory bowel disease: a Jack of all trades.</a> The American Journal of Pathology. 172, 1457-1466 (2008).</li><li>Gross, W. L., Trabandt, A., Csernok, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Wegener&amp;#39;s+granulomatosis.">Pathogenesis of Wegener&amp;#39;s granulomatosis.</a> Annales de Medecine Interne. 149, 280-286 (1998).</li><li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+inflammatory+cell+involvement+in+brain+arteriovenous+malformations.">Evidence of inflammatory cell involvement in brain arteriovenous malformations.</a> Neurosurgery. 62, 1340-1349 (2008).</li><li>Martens, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptides+which+bind+to+E-selectin+and+block+neutrophil+adhesion.">Peptides which bind to E-selectin and block neutrophil adhesion.</a> J. Biol. Chem. 270, 21129-21136 (1995).</li></ol>

Os autores não têm nada a revelar.

Os passos mais críticos para uma bem-sucedida de neutrófilos / ensaio de adesão da célula endotelial microvascular são: 1) O uso do baixo número passagem (&lt;9), células endoteliais saudáveis; 2) Manter os neutrófilos isolados com uma densidade baixa (ou seja, &lt;5 x 10 6 células / ml) e usá-los dentro de duas horas de isolamento, e 3) lavagem Fastidious dos neutrófilos calceína AM-rotulados e uso dos neutrófilos calceína AM-rotulados em tempo hábil para minimizar a contaminação C...

Desde o endotélio vascular está em contato direto com o sangue circulante, que tem uma localização única para iniciar uma resposta inflamatória rápida durante a infecção ou lesão. As células endoteliais (ECs) de receptores de reconhecimento de padrões express que reconhecem uma variedade de componentes bacterianos conservadas e as moléculas de perigo, e receptores de mediadores inflamatórios hospedeiras, tais como o TNFa. A activação destes receptores induz a ECs para secretar citocinas (por exemplo, IL-6, IL-8, CXCL1 e CCL2), e para sobrerregular moléculas de adesão (por exemplo, E / P-selectina, VCAM-1 e ICAM-1) na sua superfície celular uma , 2. Estas moléculas de todos facilitar a localização de leucócitos aos locais de infecção e lesão a fim de limpar o exército dos agentes infecciosos e iniciar a reparação tecidual 3,4. A resposta de neutrófilos para a infecção envolve uma interação bem coordenada entre o endotélio vascular ea resposta neutrófilos. Após a ativação CE, IL-8 é segregada e que forma um gradiente intravascular sobre o endotélio, que permite que a casa de neutrófilos para o local da infecção ou lesão 5,6. E / P-selectinas mediata captura de neutrófilos e de rolamento através de associações relativamente fracos com glycomolecules na superfície celular de neutrófilos. Estas interacções, juntamente com a ligação de IL-8 aos seus receptores cognatos, facilitar a robusta ligação integrina-mediada e eventual detenção dos neutrófilos sobre a superfície de células endoteliais 7-10. Depois da prisão, os neutrófilos podem migrar para fora da vasculatura para os locais específicos de infecção para eliminar os patógenos directamente, gerar armadilhas extracelulares de neutrófilos para prevenir a disseminação de agentes patogénicos, e promover a cicatrização de feridas libertam factores adicionais que recrutam leucócitos, tais como monócitos, macrófagos e dendríticas células 11-17. Descritos neste relatório é um método in vitro para quantificar a adesão de neutrófilos para microVascular CEs após a ativação por parte do anfitrião mediador inflamatório TNF. Este ensaio foi concebido para avaliar a ativação de células endoteliais, e não neutrófilos. Neutrófilos humanos primários são primeiro isolados utilizando separação em gradiente de densidade, e depois são marcados com calceína acetoximetilo (AM). Esterases de células vivas no interior da hidrólise calceína AM de calceína a molécula altamente fluorescente com uma excitação de 492-495 nm e emissão de 513-516 nm, 18. Os neutrófilos marcados com fluorescência são então incubadas com monocamadas de EC, e os neutrófilos não aderentes são subsequentemente removidos. A fluorescência das restantes, neutrófilos ligados é então medido usando um espectrofotómetro de fluorescência, e calculada como uma percentagem de neutrófilos de entrada fluorescência total por poço. Este método tem a vantagem adicional de que os neutrófilos ligados marcados com calceína utilizados em espectrofotometria pode ser directamente visualizada utilizando microscopia de fluorescência para dar uma leitura mais qualitativa de ac CEvação. Uma vez que este ensaio é realizado em condições estáticas, apenas os eventos muito iniciais que ocorrem na cascata de adesão dos neutrófilos serão avaliados. Isto é confirmado no presente relatório usando anticorpos bloqueadores de E-selectina para mostrar que a adesão de neutrófilos a TNFa humano tratado com pulmão microvascular CE (HMVEC-pulmão) monocamadas é drasticamente reduzida quando a interacção com a E-selectina é interrompido. Além de TNFa, temos utilizado com sucesso este ensaio para determinar a extensão da activação CE veia umbilical humana (HUVEC) pelo receptor Toll-like 1/2 agonistas lipoproteína associada a peptidoglicano (PAL), mureína lipoproteína (MLP) e e Pam3Cys ativação HMVEC-Lung por Pam3Cys 19,20. Além disso, foram utilizados com êxito este ensaio com os inibidores da quinase e depois knockdown RNAi mediada de superfície e proteínas citoplasmáticas em HMVEC-pulmonar, sugerindo que esta metodologia é compatível com uma variedade de bioquímica e screening ensaios 20. Em resumo, este ensaio proporciona um fácil de usar, forma reprodutível, mais funcional para aceder ao grau de activação CE por mediadores inflamatórios in vitro.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>HMVEC-Lung</td>
 <td>Lonza</td>
 <td>CC-2527</td>
 <td></td>
 </tr>
 <tr>
 <td>EGM-2 MV</td>
 <td>Lonza</td>
 <td>CC-3202</td>
 <td></td>
 </tr>
 <tr>
 <td>HBSS</td>
 <td>Life Technologies</td>
 <td>14175-095</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>48-well Tissue Culture Plates</td>
 <td>BD Falcon</td>
 <td>353078</td>
 <td></td>
 </tr>
 <tr>
 <td>PBS (without phenol red)</td>
 <td>UCSF Cell Culture Facility</td>
 <td>CCFAL001 </td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>D-PBS (without Ca2+ and Mg2+ and phenol red)</td>
 <td>UCSF Cell Culture Facility</td>
 <td>CCFAL003</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>RPMI-1640 (without phenol red)</td>
 <td>Life Technologies</td>
 <td>11835-030</td>
 <td></td>
 </tr>
 <tr>
 <td>Polymorphprep</td>
 <td>Axis-Shield</td>
 <td>1114683</td>
 <td></td>
 </tr>
 <tr>
 <td>calcein AM</td>
 <td>Life Technologies</td>
 <td>C3099</td>
 <td>(0.995 mM) stock soln</td>
 </tr>
 <tr>
 <td>Trypan Blue Solution</td>
 <td>Sigma-Aldrich</td>
 <td>T8154</td>
 <td></td>
 </tr>
 <tr>
 <td>40 &mu;M filters</td>
 <td>VWR</td>
 <td>21008-949</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>FLUOstar OPTIMA Spectrophotometer</td>
 <td>BMG LABTECH</td>
 <td>-</td>
 <td></td>
 </tr>
 </tbody>
</table>

quantitative in vitro assay to measure neutrophil adhesion to activated primary human microvascular endothelial cells under static conditions

O endotélio vascular desempenha um papel fundamental na resposta inflamatória. Durante a fase aguda da inflamação, as células endoteliais (ECs) são activados por mediadores hospedeiras ou directamente por componentes microbianos conservados ou moléculas derivadas do hospedeiro de perigo. Activo CEs citocinas, quimiocinas e express de moléculas de adesão que mobilizam, activar e manter leucócitos no local da lesão ou infecção. Os neutrófilos são os primeiros a chegar leucócitos, e aderem ao endotélio através de uma variedade de moléculas de adesão presentes na superfície de ambas as células. As principais funções dos neutrófilos são para eliminar directamente as ameaças microbianas, promover o recrutamento de outros leucócitos através da liberação de fatores adicionais, e iniciar o reparo de feridas. Portanto, o recrutamento e fixação ao endotélio vascular é um passo crítico na iniciação da resposta inflamatória. Neste relatório, nós descrevemos um ensaio de adesão in vitro de neutrófilos utilizandocalceína AM marcada com neutrófilos humanos primários para quantificar a extensão da activação da célula endotelial microvascular sob condições estáticas. Este método tem a vantagem adicional de que as mesmas amostras quantificada por espectrometria de fluorescência também pode ser visualizada directamente através de microscopia de fluorescência para uma avaliação mais qualitativa da ligação de neutrófilos.

A fim de obter resultados fiáveis ​​e reprodutíveis usando um ensaio de ligação de neutrófilos, é essencial que a saúde e a confluência das células endoteliais microvasculares são óptimas no dia do ensaio, como se ilustra com HMVEC-pulmão na Figura 1. Além disso, é imperativo que o número baixo passagem microvascular ECs são utilizados (ou seja, menos de 9 passagens) e, consequentemente, recomendamos realizar todos os experimentos dentro de duas semanas de descongelamento. A...

Watch this Scientific Journal Video about Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions at JoVE.com

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

A aderência de neutrófilos ao endotélio activado em locais de infecção é um componente integral da resposta inflamatória do hospedeiro. Descritos neste relatório é um ensaio de ligação de neutrófilos, que permite a<em&gt; In vitro</em&gt; Quantificação de neutrófilo humano primário de ligação às células endoteliais activadas por mediadores inflamatórios, sob condições estáticas.

Quantitativo<em&gt; In vitro</em&gt; Ensaio medir a adesão de neutrófilos ativados primárias células humanas endoteliais microvasculares em condições estáticas

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

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JoVE Journal

Immunology and Infection

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

17.0K visualizações.  University of California, San Francisco. A aderência de neutrófilos ao endotélio activado em locais de infecção é um componente integral da resposta inflamatória do hospedeiro. Descritos neste relatório é um ensaio de ligação de neutrófilos, que permite a In vitro Quantificação de neutrófilo humano primário de ligação às células endoteliais activadas por mediadores inflamatórios, sob condições estáticas.

Vídeo: Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

Ensaio in vitro da adesão bacteriana em mamíferos Células Epiteliais

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Imunologia e Infecção

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Isolamento de Células endoteliais da veia umbilical humana e sua utilização no estudo de transmigração de Neutrófilos sob condições de fluxo

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Um<em&gt; In vitro</em&gt; Modelo de Co-infecção para Estudar<em&gt; Plasmodium falciparum</em&gt;-HIV-1 Interacções em monócitos humanos primários derivados de células imunes

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

In vitro Ensaio de cocultura para avaliar migração trans epitelial de neutrófilo induzida por patógenos

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Um fluxo de Adesão Ensaio para o Estudo de Recrutamento de leucócitos para hepáticas humanas senoidais endotélio sob condições de estresse de cisalhamento

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Fluxo de Neutrófilos Humanos Secção Ensaio de Adesão

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

Estática Adesão Ensaio para o Estudo da integrina Ativação em Linfócitos T

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

Imagens em tempo real de endoteliais junções célula-célula durante Neutrophil Transmigração Sob Fisiológica Fluxo

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In Vitro e In Vivo modelo para estudar a adesão bacteriana para o Vaso de parede em condições de escoamento

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Ensaio de Adesão Sob tensão de cisalhamento para o Estudo da Linfócitos T-Molécula de adesão de Interações

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...