Financiamento para esse projeto foi fornecida por um prêmio da Sociedade Americana de Câncer Research Scholar, (06-024-01-CSM para Charles J. Dimitroff), um dos Institutos Nacionais de Saúde / Instituto Nacional do Câncer de subvenção (RO1CA118124 para Charles J. Dimitroff) e um dos Institutos Nacionais de Saúde / Instituto Nacional de Artrite e Doenças músculo-esqueléticas e da pele de subvenção (P30 AR042689 o Dr. Thomas S. Kupper; Hospital Brigham and Women de Boston, MA).

1. Preparação de hBMEC (HBMEC-60 Cell) monocamada em placas de Petri para a Câmara de Fluxo <ol><li> Revestimento 35 x 10mm cultura de tecidos de pratos com 2ml de fibronectina 20μg/ml (em PBS) durante a noite a 4 ° C ou por 3 horas a 37 ° C. </li><li> Aspirar solução fibronectina dos pratos e acrescente 1,5 x10 5 células HBMEC-60 [Meio 199 com HEPES e Glutamina, 10% FBS, 10% de soro humano, 5 unidades / ml de heparina, 1ng/ml fator de crescimento humano recombinante de fibroblastos, 1% Penicilina- estreptomicina] para o prato e permitir-lhes crescer a&gt; confluência de 90%. (Isso leva 2 dias) </li><li> Aspirar meios de crescimento e, para se regular-E-selectina expressão, adicione meio fresco com IL-1β 50ng/mL para 4-6 hrs. Monocamadas de células agora está pronto para rolar ensaio. Para bloquear E-selectina função, neutralizando anti-humano E-selectina Moab (clone BBIG-E4 (5D11)) podem ser adicionados a 20μg/ml para ~ 1h a 37 ° C. </li></ol> 2. Preparação de células progenitoras hematopoéticas KG1a para Câmara de Fluxo <ol><li> Células progenitoras hematopoéticas KG1a são cultivadas a confluência (1x10 6 células / ml) em RPMI-1640 com glutamina e 10% FBS / 1% de penicilina-estreptomicina e são pipetados diretamente do frasco para utilização em ensaio de câmara de fluxo. </li><li> Número de células é calculada usando um hemocitômetro e depois as células são re-suspenso em 1 x 10 6 células / mL em HBSS com 10 mM HEPES (H / H buffer) e CaCl 2 2mM </li><li> Células são armazenadas no gelo até a sua utilização no ensaio. </li></ol> 3. Preparação de Microscópio Câmara de Fluxo, de placas paralelas e bomba de seringa (Figura 1) <img src="/files/ftp_upload/old/1009_2009_1_26_7/1009_figure1.png" alt="Figura 1" width="600" height="409" /> Figura 1. Ilustração de placas paralelas Análise Câmara de Fluxo. Microscópio invertido, Harvard bomba de seringa, computador de câmera / e de placas paralelas câmara de fluxo são colocados em um arranjo ideal para análise de células eficientes e reprodutíveis. <ol><li> Ligue a alimentação e fonte de luz para microscópio invertido e poder de bomba de seringa e selecione a objetiva de 10x no microscópio. </li><li> Coloque 50 mL em tubo cônico titular e encher com H / H e 2mM CaCl 2. </li><li> Lugar de tubos de plástico de 5 ml de teste em tubo de ensaio titular abaixo do tubo de 50 ml cônico </li><li> Coloque 60ml seringa na bomba de seringa (Certifique-se que tanto o êmbolo da seringa e do corpo são ambos garantidos). </li><li> Anexar 3 vias-stop pau-de-final da seringa 60ml e anexar seringa de 5 ml para 3 vias-stop-galo. </li><li> Coloque aparelho Câmara paralela de fluxo (GlycoTech, Inc.) no palco microscópio com tubo de entrada para a direita, tubo de saída para a esquerda e tubo de vácuo para trás. </li><li> Anexar tubos de vácuo para vácuo e válvula de ar livre para que os aparelhos de placas paralelas para aderir ao palco. (Isso testa se a saia de vácuo na câmara de fluxo é hermético.) Desligue a válvula de ar. </li><li> Ligar a linha de saída de tubos (à esquerda de microscópio) para 3-way-stop-galo. Aparelho está agora preparado para a colocação no HBMEC-60 placa monocamada. </li><li> Coloque placa de monocamada HBMEC-60 e colocar no centro do microscópio. </li><li> Ligue vácuo e posição do aparelho câmara de fluxo acima da placa de tal forma que a placa monocamada está no centro. </li><li> Delicadamente mais baixo do aparelho câmara de fluxo na placa monocamada HBMEC-60 e que os aparelhos câmara de fluxo de sucção para baixo. (Não deve haver nenhum som de ar escapando e, neste ponto você pode precisar de aplicar pressão sobre a câmara para comprimir a junta de borracha). </li><li> Uma vez vácuo foi estabelecido, tubo de entrada local (à direita) para dentro do tubo cônico contendo 50mL H / H com 2mM CaCl 2. </li><li> Abrir 3 vias-stop-galo para permitir o fluxo para a seringa 60ml e bomba lugar no &quot;modo de bomba.&quot; </li><li> Para retirar o ar de entrada / saída de linhas sem levantar as células na placa, use a bomba de seringa fixada em 2mL/minute para encher o tubo de entrada, em seguida, definir rapidamente a 0,5 mL / minuto apenas antes de o líquido atinge o aparelho. É imperativo para preparar este jogo com cuidado e evitar bolhas de ar que vai levantar a monocamada da placa. </li><li> Uma vez que o fluido é visto para entrar na seringa 60ml, a câmara de fluxo e bomba de seringa foram preparadas e da câmara de fluxo está pronto para uso. Esta configuração será repetido para cada execução de entrada de célula e para cada novo prato necessário. </li></ol> 4. Captura dos leucócitos Eventos do Rolling usando NIS Elements <ol><li> Elementos aberta NIS no desktop. </li><li> Pressione o botão &quot;play&quot; ícone para imagem ao vivo. Exposição automática permitirá que você ver a placa mais fácil para agora e é importante para focagem. </li><li> Certifique-se que microscópio está focado corretamente na monocamada de células. </li><li> Uma vez focada, a exposição re-set a 1 luz de quadros / segundo e ajustar no microscópio. A imagem deve aparecer no computador e não estar mais visível no visor do microscópio, devido ao nível de pouca luz. Histograma de luz pode então ser adajustada para eliminar a exposição de fundo ou melhorar a clareza da célula. </li><li> Clique em bin 2X2 ou Bin Live para obter imagem que é ideal para captura de filme </li><li> Clique em &quot;Macro&quot; na barra de menu e selecione &quot;gravar na porta&quot;; selecione a porta &quot;COM3&quot; e &quot;aberto&quot;. (Isso abre a porta para coordenar com a bomba de seringa. </li><li> Pipetar 1 ml de suspensão de células KG1a no tubo de ensaio 5ml perto da linha de tubulação de entrada e em seguida, coloque linha de tubulação de entrada para o fundo do tubo de ensaio. </li><li> Ir para barra de menus, clique em &quot;Capture&quot;, &quot;aquisição de lapso de tempo.&quot; </li><li> Um novo menu irá aparecer. Selecione o protocolo com duração 210 segundos, esta é a duração do programa para rolar em HBMEC-60 Cells. (Cada fase é programado para tirar uma foto a cada segundo para a duração do protocolo de bomba. Também é configurado para enviar um comando através da porta COM3 para ligar a bomba.) Se lapso de tempo é interrompida, a bomba não vai parar por conta própria e deve ser reposto. Pressione Cancelar para voltar a viver da imagem. </li><li> Definir a bomba de seringa para &quot;Modo de Programação&quot; de acordo com manual de instruções. (Modo Programa permite a programação manual de taxas de fluxo específico (ou seja, níveis de estresse de cisalhamento), a ser transmitido dentro da câmara e ditar as interações célula KG1a sobre a monocamada de células HBMEC-60.) Configurações para KG1a célula rolando em Células hBMEC foram os seguintes 4,2 dinas / cm 2 por 45 s, 0,3 dinas / cm 2 por 60 s, e aumenta gradualmente a cada 15 s até um máximo de 4,2 dinas / cm 2 </li><li> Parede de tensão de cisalhamento (W st) na câmara foi calculada de acordo com a seguinte equação: W st = 6μQ / a b 2, onde μ é a viscosidade estimada dos meios de comunicação (0,0076 P), Q é a vazão volumétrica em ml / s, a é a altura do canal (espessura da junta) em cm (0,03 cm), e b é a largura do canal em cm (0,5 cm). Vazões foram regulamentados usando o modo de programação da bomba de seringa. </li><li> Microscópio e computador agora está pronto para capturar lapso de tempo fotografia. Pressione &quot;start run&quot; no computador para começar a aquisição de dados. Durante a execução do programa, não se esqueça de adicionar mídia para o tubo de ensaio contendo células KG1a a fim de manter a tubulação de entrada / saída completa do meio (Certifique-se que não há bolhas de ar). </li></ol> 5. Dependência de sialilação Terminal e glicoproteína de superfície de E-selectina-mediada por células KG1a 5. Rolamento (Figura 2; Vídeos Clipes de 5,1-5,5) <ol><li> Dosagem de KG1a célula rolando sobre a não-estimulada HBMEC-60 células. (Controle negativo), células KG1a foram preparados e injetado na câmara de fluxo mais viver não-IL-1β estimulada HBMEC-60 conforme descrito acima. </li><li> Dosagem de célula KG1a rolando no HBMEC-60 células pré-tratadas com IL-1β. (E-selectina-dependência controle) KG1a células foram preparados e injetado na câmara de fluxo mais viver IL-1β estimulada HBMEC-60 conforme descrito acima. </li><li> Dosagem de célula KG1a rolando no HBMEC-60 células pré-tratadas com IL-1β e neutralizantes anti-humano E-selectina Moabe. (E-selectina-dependência controle) KG1a células foram preparados e injetado na câmara de fluxo mais viver IL- 1β estimulada HBMEC-60 pré-tratados com anti-humano neutralizando Moabe E-selectina como descrito acima. </li><li> Dosagem de células-sialidase digerida KG1a rolando no IL-1β estimulada HBMEC-60. KG1a células pré-tratadas com 0.1U/ml Vibrio cholerae neuraminidase de 1hr a 37 ° C foram preparados e injetado na câmara de fluxo mais viver IL-1β estimulada HBMEC-60 conforme descrito acima. </li><li> Dosagem de célula protease-digerida KG1a rolando no IL-1β estimulada HBMEC-60. KG1a células pré-tratadas com protease, 0.2U/ml Bromelain de 1h a 37 ° C, foram preparados e injetado na câmara de fluxo mais viver IL-1β estimulada HBMEC-60 conforme descrito acima. </li></ol> 6. Análise do NIS Elements-Cells KG1a Capturado e gráficos de Cell KG1a Dados do Rolling <ol><li> Após seqüência de tempo é adquirido, salvar os dados. </li><li> Ir para &quot;medir&quot; a guia na barra de menu e selecione &quot;definir limite.&quot; Mover barras de modo que as células desejadas são coloridos em vermelho. Selecione &quot;todas as imagens&quot; e depois &quot;Ok&quot;. A seqüência de lapso de tempo inteiro deve agora ser modificado com vermelho visível células de rolamento. </li><li> Vá para &quot;Medir&quot; e selecione &quot;restrições&quot;. (Restrições permitem ao usuário excluir objetos thresholded que não representam as células de rolamento. Isto é principalmente de fundo criado pela monocamada HBMEC-60.) Selecione &quot;área&quot; e min de entrada e os valores máximo para a área de células de rolamento. Repita este passo para &quot;alongamento&quot; e &quot;circularidade&quot;. </li><li> Selecione &quot;medida&quot; e selecione &quot;criar binários usando restrições&quot;, e definir os valores de restrição para otimizar NIS Elements software de reconhecimento de células rolando em monocamada de células HBMEC-60. </li><li> Voltar para &quot;medir&quot;, selecione &quot;Os dados de campo&quot; e selecione &quot;dados reset&quot;. Fechar este menu. </li><li> Retorno à &quot;Medida&quot; e SEselecione &quot;campo scan&quot;. </li><li> &quot;Medida&quot; aberto &quot;; Os dados de campo&quot;, selecione &quot;número ou Objects&quot; e selecione &quot;todos os campos&quot; e exportar dados para o Microsoft Excel. </li><li> Selecione &quot;dados claros&quot; para preparar-NIS Elements aquisição de células para a coleta de dados seguinte. </li></ol> Resultados: <img src="/files/ftp_upload/old/1009_2009_1_26_8/1009_figure3.png" alt="Figura 3" width="600" height="382" /> Figura 3. Parallel Plate-Análise Câmara Fluxo de E-selectina-Binding Atividade em Células KG1a. KG1a células tratadas com sialidase ou protease foram analisados ​​para rolar a eficiência em monocamadas de HBMEC-60 células pré-tratadas com IL-1β. Controle negativo rolando experimentos também foram realizados por meio da análise de células KG1a rolando sobre a não-IL-1β estimulada HBMEC -60 células ou IL-1β estimulada HBMEC-60 células tratadas com anti-humano neutralizando E-selectina Moab (5D11). Freqüências de rolamento de células foram realizadas em triplicata e analisados ​​com NIS Elements software. (Significância estatística *, p &lt;0,001) Neste relatório, baseado em vídeo, nós fornecemos uma representação visual de como analisar leucócitos - células endoteliais interações fisiológicas sob condições de estresse de cisalhamento. Em particular, analisamos o papel da E-selectina - E-selectina ligantes na mediação tethering leucócitos e rolando, um comportamento adesivo necessário para o compromisso do ensino secundário mais estável atividades de ligação através de receptores, como integrinas e receptores de ácido hialurônico. Usando a câmara de fluxo de placas paralelas adaptada para uso com um microscópio invertido, bomba de Harvard Seringa, câmera de vídeo e computador / celular de aquisição de hardware, realizamos experimentos em que E-selectina ligante + células KG1a foram usados ​​como nosso modelo de leucócitos e células hBMEC (HBMEC- 60) estimuladas com citocinas pró-inflamatórias, IL-1β, foram usados ​​como o nosso E-selectina modelo de células endoteliais humanas + (1-6). Como relatado anteriormente, observamos células KG1a robusta rolando no IL-1β estimulada HBMEC-60 células em um intervalo de níveis de tensão de cisalhamento de uma maneira E-selectina-dependente (Figura 3) (diferença estatisticamente significativa quando comparada com células KG1a rolando no não-IL-1β HBMEC-60 estimulou as células). Controles negativos, que consistia em KG1a célula rolando sobre a não-IL-1β estimulada HBMEC-60 em células ou IL-1β estimulada HBEMC-60 células pré-tratadas com mAb anti-humano E-selectina, foram realizados em paralelo para demonstrar a dependência de estimulação de citocinas e E-selectina expressão para a atividade de rolamento (Figura 3). Além disso, análise de rolamento de células pré-tratadas com KG1a Vibrio cholerae neuraminidase (sialidase) ou com bromelina, uma protease não específica conhecida para digerir superfície E-selectina glicoproteína ligante, destacou a importância dos resíduos de ácido siálico terminais e glicoproteína de superfície de E-selectina ligante atividade (2,3).

<ol>
	<li>Dimitroff, C. J., Kupper, T. S., Sackstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+leukocytic+migration+to+inflamed+skin+with+a+novel+fluorosugar+modifier+of+cutaneous+lymphocyte-associated+antigen.">Prevention of leukocytic migration to inflamed skin with a novel fluorosugar modifier of cutaneous lymphocyte-associated antigen.</a> J. Clin. Invest. 112, 1008-1018 .</li><li>Dimitroff, C. J., Lechpammer, M., Long-Woodward, D., Kutok, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rolling+of+human+bone-metastatic+prostate+tumor+cells+on+human+bone+marrow+endothelium+under+shear+flow+is+mediated+by+E-selectin.">Rolling of human bone-metastatic prostate tumor cells on human bone marrow endothelium under shear flow is mediated by E-selectin.</a> Cancer Res. 64, 5261-5269 (2004).</li><li>Dimitroff, C. J., Descheny, L., Trujillo, N., Kim, R., Nguyen, V., Huang, W., Pienta, K. J., Kutok, J. L., Rubin, 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=Identification+of+leukocyte+E-selectin+ligands,+P-selectin+glycoprotein+ligand-1+and+E-selectin+ligand-1,+on+human+metastatic+prostate+tumor+cells.">Identification of leukocyte E-selectin ligands, P-selectin glycoprotein ligand-1 and E-selectin ligand-1, on human metastatic prostate tumor cells.</a> Cancer Res. 65, 5750-5760 (2005).</li><li>Descheny, L., Gainers, M. E., Walcheck, B., Dimitroff, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ameliorating+Skin-Homing+Receptors+on+Malignant+T+cells+with+a+Fluorosugar+Analog+of+N-acetylglucosamine:+P-selectin+Ligand+is+a+More+Sensitive+Target+than+E-selectin+Ligand.">Ameliorating Skin-Homing Receptors on Malignant T cells with a Fluorosugar Analog of N-acetylglucosamine: P-selectin Ligand is a More Sensitive Target than E-selectin Ligand.</a> J Invest Dermatol. 126 (9), 2065-2073 (2006).</li><li>Gainers, M. E., Descheny, L., Barthel, S. B., Liu, L., Wurbel, M., Dimitroff, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin-homing+receptors+on+effector+leukocytes+are+differentially+sensitive+to+glyco-metabolic+antagonism+in+allergic+contact+dermatitis.">Skin-homing receptors on effector leukocytes are differentially sensitive to glyco-metabolic antagonism in allergic contact dermatitis.</a> J. Immunology. 179 (12), 8509-8518 (2007).</li><li>Rood, P. M., Calafat, J., von dem Borne, A. E., Gerritsen, W. R., van der Schoot, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+human+bone+marrow+endothelial+cells:+characterisation+of+new+cell+lines.">Immortalization of human bone marrow endothelial cells: characterisation of new cell lines.</a> Eur J Clin Invest. 30 (7), 618-629 (2000).</li></ol>

Os autores não têm conflitos de interesse ou divulgações.

De placas paralelas análise câmara de fluxo é uma empresa especializada sistema in vitro utilizado para estudar leucócitos - interações adesivas endoteliais sob condições de estresse de cisalhamento semelhantes aos transmitidos na superfície de uma vênula pós-capilar. Tal como é descrito aqui, em nossa apresentação visual, que demonstram o valor deste sistema para investigar o papel da E-selectina - E-selectina ligantes na mediação tethering leucócitos e rolando sobre a superfície do endotélio microvascular ativado. Nós também revelam a aplicação de glicosidase protease, e os tratamentos de anticorpos bloqueadores para elucidar o papel da E-selectina e seus ligantes neste sistema. Estas análises são altamente reprodutíveis e ajudar a formar uma base experimental para o estudo de E-selectina - E-selectina função de ligante in vivo. Além de estudar a adesão celular com monocamadas de citocinas ativadas células endoteliais microvasculares (veia umbilical hBMEC ou humanos ou células endoteliais microvasculares dérmicas), os ensaios podem ser realizados utilizando humana recombinante purificada E-selectina ou E-selectina-Ig moléculas quiméricas como substrato de adesão celular. Interações outro adesivo mediada por leucócitos (L)-selectina e de plaquetas (P)-selectina também podem ser analisadas utilizando-placa paralela tecnologia de câmara de fluxo. Variando o tipo de substrato celular monocamada / proteína ou de leucócitos de entrada / modelos-selectina transfectant célula, os pesquisadores podem estudar o papel dessas interações sob condições de estresse fisiológico de cisalhamento.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>F6178-500ML</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14190</td>
<td>&nbsp;</td>
<tr>
<td>0.5 M EDTA</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15575-038</td>
<td>&nbsp;</td>
<tr>
<td>1M HEPES</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Parallel-Plate Flow Chamber Kit</td>
<td>&nbsp;</td>
<td>GlycoTech</td>
<td>31-001</td>
<td>&nbsp;</td>
<tr>
<td>PHD2000 Programmable Syringe Pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>PHD 2000</td>
<td>&nbsp;</td>
<tr>
<td>35x10mm Tissue Culture Dish</td>
<td>&nbsp;</td>
<td>BD FALCON</td>
<td>353001</td>
<td>&nbsp;</td>
<tr>
<td>Fibronectin , from human plasma</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>86088-83-7 F2006-2MG</td>
<td>&nbsp;</td>
<tr>
<td>Interleukin-1_, Human; Recombinant</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>I9401-5UG</td>
<td>&nbsp;</td>
<tr>
<td>HBSS (Hank s Buffered Saline Solution)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14170</td>
<td>&nbsp;</td>
<tr>
<td>Medium 199 w/ HEPES & Glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12320-039</td>
<td>&nbsp;</td>
<tr>
<td>Human Serum</td>
<td>&nbsp;</td>
<td>Innovative Research, Inc.</td>
<td>IPLA-SER1</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Abraxis Pharmaceutical Products</td>
<td>504011</td>
<td>&nbsp;</td>
<tr>
<td>Recombinant human fibroblast growth factor</td>
<td>&nbsp;</td>
<td>R&D Systems</td>
<td>233-FB</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin Streptomycin</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Plastic Test tubes</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>352008</td>
<td>&nbsp;</td>
<tr>
<td>Anti-human E-selectin clone BBIG-E4(5D11)</td>
<td>&nbsp;</td>
<td>R&D Systems	</td>
<td>BBA16</td>
<td>&nbsp;</td>
<tr>
<td>Bromelain</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>B-4882</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
				 	
	

		</div>

analysis of physiologic e-selectin-mediated leukocyte rolling on microvascular endothelium

E-selectina é uma proteína de membrana tipo-1 em células endoteliais microvasculares que ajuda a iniciar o recrutamento de leucócitos circulantes para os tecidos ósseos cutânea, e inflamado. E-selectina expressão é constitutiva de microvasos dérmica e óssea e é induzível por citocinas pró-inflamatórias, como IL-1α / TNF-α e, em microvasos em tecidos inflamados. Este receptor medeia a lectina fracas interações de ligação com carboidratos contra receptor-ligantes em leucócitos circulantes, o que resulta em um comportamento característico de rolamento. Porque essas interações precedem eventos adesivo mais estável e atividade diapedese, caracterização da atividade rolling de leucócitos e identificação de leucócitos E-selectina ligantes foram objetivos principais em estudos de tráfico de leucócitos e inflamação e no desenvolvimento de terapêuticas anti-inflamatórios (1-5) . A intenção deste relatório é fornecer uma descrição, visual abrangente da tecnologia mais amplamente utilizada para estudar a E-selectina E-selectina interações ligante sob condições de fluxo fisiológico de sangue. Nosso laboratório em conjunto com o Centro de Pesquisas de Doenças da Pele Harvard usa um state-of-the-art de placas paralelas aparelhos de câmara de fluxo acompanhada através da visualização digital e software de gravação de novo, NIS Elements. Esta tecnologia permite-nos analisar os eventos de adesão em tempo real para visualização na tela assim como a atividade registro rolando em um formato de vídeo. Parâmetros de adesão celular, tais como a resistência ao rolamento, frequência de corte e ligação / tethering eficiência, são calculados com NIS Elements software, exportados para uma planilha do Excel e submetidos à análise estatística. Na demonstração apresentada aqui, foi empregado o câmara de fluxo de placas paralelas para investigar E-selectina-dependente a atividade de leucócitos rolando ao vivo medula óssea humana endotelial (hBMEC). Células progenitoras hematopoéticas KG1a, que expressam um alto nível de E-selectina ligante, foram utilizados como nosso modelo de leucócitos, enquanto que uma linha celular imortalizada hBMEC, HBMEC-60 celas, foi usado como nosso modelo de células endoteliais (6). Para induzir e simular expressão E-selectina nativa na câmara de fluxo, HBMEC-60 células foram ativado pela primeira vez com a IL-1. Nossa apresentação de vídeo mostrou que placas paralelas análise de fluxo é um método adequado para estudar a E-selectina mediada fisiológico de leucócitos atividades rolamento e que a caracterização funcional de ligante E-selectina leucócitos (s) na câmara de fluxo pode ser verificada através da implementação de protease ou glicosidase digestões.

Watch this Scientific Journal Video about Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium at JoVE.com

Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium

Este relatório fornece uma representação visual de placas paralelas análise câmara de fluxo para o estudo de interações leucócitos endoteliais sob tensão de cisalhamento fisiológico. Este método é particularmente útil para investigar o papel do endotélio (E)-selectina e E-selectina leucócitos ligantes que leucócitos gatilho rolando na superfície das células endoteliais.

Análise da Rolling Leucócitos Fisiológica E-Selectina-Mediated no endotélio microvascular

E-selectin is a type-1 membrane protein on microvascular endothelial cells that helps initiate recruitment of circulating leukocytes to cutaneous, bone ...

analysis-physiologic-e-selectin-mediated-leukocyte-rolling-on

Research

JoVE Journal

Biology

19.0K visualizações.  Brigham and Women's Hospital. Este relatório fornece uma representação visual de placas paralelas análise câmara de fluxo para o estudo de interações leucócitos endoteliais sob tensão de cisalhamento fisiológico. Este método é particularmente útil para investigar o papel do endotélio (E)-selectina e E-selectina leucócitos ligantes que leucócitos gatilho rolando na superfície das células endoteliais.

Vídeo: Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A Neuronal e astrocitária Ensaio Co-Cultura de Análise de Conteúdo de Alta de neurotoxicidade

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biologia

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

Bioengenharia Humanos Networks microvascular em camundongos imunodeficientes

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

RNA-seq Análise de transcriptomas de trombina-tratados e controle humano pulmonar células endoteliais microvasculares

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

Isolamento de tubos endoteliais microvasculares de Artérias Rato Resistência

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Medição e Análise de Produção de Ácido Extracelular determinar Glicosidases Classificação

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Quantificação de Estoicometria de Acetilação e Succinilação de Lisinas Proteicas em Sítios Específicos Usando Espectrometria de Massa de Aquisição de Dados Independente

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Expressão de proteínas da fusão fluorescente em murino derivadas da medula óssea as células dendríticas e macrófagos

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Medições simultâneas de cálcio intracelular e potencial de membrana no endotélio Cerebral rato recém isoladas e intacta

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In vitro Análise da Função Ligase E3 Ubiquitin

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.