Agradecemos a equipe do Laboratório de Anatomia Patológica do Príncipe Real Alfred e Hospitais Repatriação Concord para a recolha de amostras frescas de CRC e na mucosa intestinal normal. O trabalho foi financiado por um New South Wales Instituto do Câncer Grant Programa translacional.

<img src="/files/ftp_upload/3322/3322fig1.jpg" alt="Figura 1" /> O fluxo de trabalho figura 1. Para a preparação de uma suspensão de células vivas a partir de uma amostra cirúrgica do CRC. 1. Desagregação amostra clínica Todas as amostras foram coletadas do Príncipe Real Alfred Hospital (Camperdown, NSW, Austrália) e Hospital Repatriação Concord (Concord West, NSW, Austrália) com o consentimento informado nos termos do Protocolo n º X08-164. <ol><li> Coletar câncer colorretal fresco (CRC) ou espécimes adenoma, e mucosa intestinal normal, pelo menos, 10 cm do tumor. Armazene as amostras em pH equilibrado Hank solução salina 7,3 (HBSS) a 4 ° C por até 12 h após a ressecção. </li><li> Siga as normas de segurança para patógenos humanos, processo de todas as amostras clínicas em um gabinete de segurança biológica classe II. Dissecar as amostras em dois cubos mm em uma placa de Petri usando duas lâminas de bisturi. </li><li> Incubar tumor e tecido normal em tubos separados Eppendorf com ocasionais mistura suave por 60 min a 37 ° C com um volume igual de meio RPMI 1640 contendo 2% (v / v) colagenase tipo 4 (Worthington, Lakewood, NJ, EUA) e 0,1 % (w / v) desoxirribonuclease I de pancrease bovina (DNAse I; Sigma-Aldrich). </li><li> Força semi-digerida tecido através de uma peneira fina malha de arame usando um êmbolo de uma seringa de 10 mL, as células através de lavagem com HBSS. </li><li> Passe resultando suspensão celular através de 200 mm e 50 filtros Filcon mM (BD Biosciences) para remover agregados celulares. A maioria dos agregados muco DNA, células e são removidos nesta série de filtrações. </li><li> Centrífuga suspensões de células a 400 xg a 20 ° por 5 min. </li><li> Pellets celulares ressuspender em inativado pelo calor FCS contendo 10% de dimetil sulfóxido (DMSO), congelar lentamente em criotubos e armazenar a -80 °. O processo de congelamento tende a reduzir o muco na amostra e lise de células vermelhas do sangue. </li></ol> 2. Preparação de amostras para a captura de células <ol><li> Descongelar as amostras rapidamente em banho-maria 37 ° e células ressuspender em 10 ml de HBSS para lavar o DMSO. </li><li> Centrífuga suspensões de células a 410 xg a 20 ° por 5 min. </li><li> Decantar o sobrenadante e ressuspender o pellet celular em 500 mL de HBSS. </li><li> Tratar a amostra com 0,1% (w / v) DNAse I por 20 min em temperatura ambiente. </li><li> Misturar 10 mL de cada suspensão de células com um volume igual de azul de tripano e carga de 10 L da mistura em um hemocitômetro. Usando um microscópio de luz em 100 vezes de ampliação, a contagem de células viáveis, que aparecem claros devido à exclusão de tripan azul, enquanto as células mortas assumir o corante. Um mínimo de 4 x 10 6 células viáveis ​​é necessária para a captura de células na microarray. </li><li> Após o tratamento DNAse, ressuspender a suspensão de células em 10 mL HBSS e centrifugar a 410 xg a 20 ° por 5 min. </li><li> Decantar o sobrenadante e ressuspender pellets de células em RPMI 1640 para um volume final de 200 mL. </li></ol> 3. Anticorpo de captura de células microarray <ol><li> Umedecer o microarray de anticorpos DotScan por imersão a seção de nitrocelulose em tampão fosfato salino (PBS) por aproximadamente 20 s. Limpe cuidadosamente as bordas de vidro do microarray com Kimwipes dobrado, evitando tocar a seção de nitrocelulose. </li><li> Adicionar água à bandeja de incubação microarray para fornecer uma câmara úmida. Coloque o microarray para a câmara e pipetar a suspensão de células em RPMI 1640 para a seção de nitrocelulose úmida. Pipeta gotas em cada canto da nitrocelulose para assegurar uma repartição equilibrada das células. </li><li> Incubar os microarrays, a 37 ° C por 1 h. A incubação possibilita que as células para resolver e entrar em contato com os anticorpos no microarray. Células que expressam antígenos de superfície correspondentes aos anticorpos que pousar em será capturada. </li><li> Após a incubação, mergulhe a microarrays delicadamente e verticalmente em três calhas contendo pelo menos 15 mL PBS para lavar as células não ligado (20 s por lavagem). </li><li> Prepare 3,7% (w / v) de formaldeído em PBS para consertar as células e anticorpos pelo cross-linking. Pipeta suavemente cerca de 1 mL para cobrir a seção de nitrocelulose do microarray. Incubar por 20 min em temperatura ambiente. </li><li> Em seguida mergulhe microarrays em 3 trocas de PBS (15 mL; 30 s cada) para lavar o excesso de formol. </li><li> Limpe as bordas e parte de trás da lâmina de vidro com Kimwipes e digitalizar o microarray usando o scanner DotScan enquanto, a seção de nitrocelulose é úmido. A digitalização óptica fornece o padrão de expressão de antígenos de uma mistura de células população células por exemplo, CRC, leucócitos e outras células do estroma do tumor. </li></ol> 4. Fluorescência de multiplexação <ol><li> Remover o microarray do scanner e aplicar 200 mL de tampão de bloqueio (2% w / v BSA, 2% inativado pelo calor humano AB soro, PBS, pH 7.3). Incube-o na bandeja microarray em temperatura ambiente por 20 min. </li><li> Prepare o multiplexing solução em um tubo Eppendorf coberto com folha de alumínio: 20 mL Ficoeritrina-anti-CD3 (Beckman Coulter, Gladesville, NSW, Austrália, # IM12824; 1/7.5 diluição final), 10 mL Alexa Fluor 647-anti-EpCAM (Biolegend, San Diego, CA, EUA; 15/01 diluição), 2 mL da inativado pelo calor humano soro AB (Sigma-Aldrich, Castle Hill, NSW, Austrália) e 118 mL de tampão de bloqueio. </li><li> Drenar o excesso de tampão de bloqueio do microarray e pipetar a solução de multiplexação para a seção de nitrocelulose, espalhando de maneira uniforme. Incubar por 30 min no escuro à temperatura ambiente. </li><li> Mergulhe o microarray verticalmente em três bebedouros de 15 ml PBS fresco; (30 s cada). </li><li> Deixe o microarrays seca no escuro e armazenar a 4 ° C em uma caixa de slides. O microarray pode ser armazenado no escuro por até 3 meses sem perda de fluorescência. </li><li> Digitalizar o microarray seco usando um scanner Typhoon FLA 9000 (GE Healthcare, Rydalmere, NSW, Austrália) com a resolução definida para 50 (532 nm laser, 580 BP30 emissão filtro para PE. Laser de 633 nm e 670 BP30 emissão filtro para Alexa 647). Os microarrays são verificados com o lado de nitrocelulose virada para baixo na bandeja de vidro do scanner. </li><li> Guardar as imagens fluorescentes como arquivos TIFF e usando Photoshop tamanho de imagem ajustado para 17 x 25 cm e resolução de 72 pixels / cm. Importar a imagem para o software de análise DotScan para analisar a intensidade de pontos. </li><li> O DotReader capta uma imagem digital do padrão de pontos de ligação e quantifica a densidade de ligação de célula em cada ponto de anticorpos em escala cinza de 8 bits (1-256 U). Ocasional ligação não específica de controle isotipo foi subtraído dos valores de ligação para os anticorpos com isotipos de imunoglobulina correspondente. Intensidades de fluorescência de pontos para cada microarray foram normalizados contra os mais brilhantes dot fixada em 100% de intensidade. Sinal / spot força foi gravada em um arquivo xml. (Dados brutos) ou representado como um gráfico de barras em um arquivo. Pdf (relatório final) </li><li> Microarray heatsmaps e agrupamento hierárquico foram realizados utilizando MultiExperiment Visualizador de versão (MeV) 4.4 do TM4 Microarray Software Suite ( <a href="http://www.tm4.org/mev.html">http://www.tm4.org/mev.html</a> ). Agrupamento hierárquico foi realizada sobre dados de fundo ajustado usando MeV com análise de ligação completa. Distância euclidiana foi utilizada para a medida de similaridade. O Estudante 2-tailed t-teste com variância igual foi usada para determinar a significância estatística dos resultados. </li></ol> 5. Resultados representativos: Os resultados do microarray DotScan deve mostrar padrões consistentes de células de ligação entre matrizes duplicado. Forte alinhamento de pontos de ligação (CD44/CD29) permite uma grade para ser colocado sobre a área da matriz. A Figura 2 mostra um exemplo de captura de célula ideal e multiplexação. A Figura 3 mostra alguns problemas comuns encontrados durante a captura de células e as possíveis soluções. Os resultados das células microarray ligação pode ser quantificado através da medição intensidades dot expressa numa escala de cinza que varia de 1 a 256. A Figura 4 mostra dados numéricos de 58 peças cirúrgicas CRC, corados com anticorpo Alexa EpCAM-647, como um heatmap com agrupamento hierárquico. Mesmo que o número de amostras é limitada, CRCs do mesmo estágio tendem a se agrupar em um mesmo grupo. <img src="/files/ftp_upload/3322/3322fig2.jpg" alt="Figura 2" /> Figura 2. Padrão de ligação de celular do tumor do câncer colorretal clínicas (estadiamento clínico-patológico da Austrália, ACP estágio B1). (A) chave de anticorpos DotScan mostrando a localização de anticorpos para a metade esquerda do microarray duplicados (delineado). A seção superior contém o original 82 anticorpos do microarray DotScan leucemia. Um adicional de 40 anticorpos, correspondentes a antígenos de superfície específicos encontrados para ser sobre-regulada na literatura, foram adicionados como microarray &#39;satélite&#39; a CRC. A seção inferior consiste de anticorpos controle isotipo (b) imagem óptico de células CRC ligação ao microarray. (C) imagem da fluorescência CD3 mostrando células-T. (D) EpCAM imagem de fluorescência mostrando células CRC. <img src="/files/ftp_upload/3322/3322fig3.jpg" alt="Figura 3" /> Figura 3. Exemplos de maus resultados DotScan e possíveis soluções. (A) célula de baixa taxa de ligação; solução: certifique-se, pelo menos, 4x106 células viáveis ​​sobre a matriz (b) Isotype célula de controle de ligação e ligação não específica; solução: adicionar inativado pelo calor humano AB soro a amostra antes da incubação de microarray para minimizar isotipo controle de ligação. Ocasionalmente, uma pequena quantidade de ligação não específica de células para a nitrocelulose ocorre com amostras de CRC e não afetar significativamente os resultados. (C) nitrocelulose secar durante a incubação; solução: garantir a amostra abrange a seção de nitrocelulose todo e microarray é incubada em uma superfície plana. (D) artefatos de fundo de alta; solução: garantir amicroarray é cuidadosamente lavados de incubação seguinte. <img src="/files/ftp_upload/3322/3322fig4.jpg" alt="Figura 4" /> Figura 4. DotScan software de análise gerados gráficos de barras que representam as densidades de células de ligação em uma escala de cinza que varia de 1 a 256. Números no eixo referem-se aos antígenos CD. Outras abreviações são TCR, receptor de células T; κ, λ, as cadeias de imunoglobulina luz; sig, imunoglobulina de superfície; DCC, apagados em proteínas câncer colorretal; EGFR, receptor do fator de crescimento epidérmico; FAP, proteína de ativação de fibroblastos; HLA-A, B, C HLA-DR, DR antígenos leucocitários humanos e A, B, C, respectivamente; MICA, MHC classe I relacionadas à cadeia de proteína A; MMP-14, matriz metallopeptidase 14; PIGR, receptor de imunoglobulina poliméricos; TSP-1, trombospondina-1; Mabthera, humanizado anti-CD20. <a href="http://www.jove.com/files/ftp_upload/3322/3322fig4large.jpg">Clique aqui para ver a imagem ampliada</a> .

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Não há conflitos de interesse declarados.

Neste vídeo, demonstramos como o microarray de anticorpos DotScan pode ser usado em uma forma simples e semi-quantitativa para estudar perfis de antígeno de superfície para as populações de células do tecido CRC. Obtenção de uma suspensão de células viáveis ​​único a partir do tecido é fundamental para o sucesso do experimento, porque a energia dependente de processos (por exemplo, antígeno de nivelamento e / ou formação de pseudópodos) parecem ser necessárias para a li...

The author's email has been corrected in the publication of <a href="http://www.jove.com/video/3322">Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing</a>. There was an error with the author, Jerry Zhou's, email. The author's email has been updated to:
j.zhou@uws.edu.au
from:
jzho7551@mail.usyd.edu.au

Erratum: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

<table border="1"><tbody><tr><th> Nome do reagente ou equipamento </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> Solução de Hanks salina balanceada </td><td> Sigma-Aldrich </td><td> H6136-10X1L </td><td> Tamponado com Hepes 25 mM (Sigma # H3375) </td></tr><tr><td> Airpure gabinete de segurança biológica classe II </td><td> Westinghouse </td><td> 1687-2340/612 </td><td></td></tr><tr><td> Lâminas cirúrgicas </td><td> Livingstone </td><td> 090609 </td><td> Pacote de 100 </td></tr><tr><td> RPMI 1640 com 2 mM Hepes </td><td> Sigma-Aldrich </td><td> R4130-10X1L </td><td></td></tr><tr><td> Colagenase tipo 4 </td><td> Worthington </td><td> 4188 </td><td></td></tr><tr><td> Desoxirribonuclease 1 </td><td> Sigma-Aldrich </td><td> DN25-1G </td><td></td></tr><tr><td> Terumo Seringa (10 mL) </td><td> Terumo </td><td> SS L 10 </td><td> Caixa de 100 </td></tr><tr><td> Filcon filtro (200 mm) </td><td> BD Biosciences </td><td> 340615 </td><td></td></tr><tr><td> Filcon filtro (50 mm) </td><td> Filcon filtro (50 mm) </td><td> Filcon filtro (50 mm) Filcon filtro (50 mm) 340603 </td><td></td></tr><tr><td> Soro fetal bovino </td><td> Gibco / Invitrogen </td><td> 10099-141 </td><td></td></tr><tr><td> Centrífuga 5810 R </td><td> Eppendorf </td><td> 7017 </td><td></td></tr><tr><td> Dimetilsulfóxido </td><td> Sigma-Aldrich </td><td> D2650 </td><td></td></tr><tr><td> Azul de tripano </td><td> Sigma-Aldrich </td><td> T8154 </td><td></td></tr><tr><td> Hemocymeter Technocolor Neubar </td><td> Hirschmann </td><td> não disponível </td><td></td></tr><tr><td> Microscópio de luz </td><td> Nikon </td><td> Nikon TMS </td><td></td></tr><tr><td> Tubos Cyrovial </td><td> Greiner bio-one </td><td> 121278 </td><td></td></tr><tr><td> Cryo contrainer congelamento </td><td> Nalgene </td><td> 5100-0001 </td><td></td></tr><tr><td> DotScan anticorpos microarray kit </td><td> Medsaic </td><td> não disponível </td><td></td></tr><tr><td> DotScan bandeja de lavar microarray </td><td> Medsaic </td><td> não disponível </td><td></td></tr><tr><td> KimWipes </td><td> Kimberly-Clark </td><td> 4103 </td><td></td></tr><tr><td> Formol 37% </td><td> Sigma-Aldrich </td><td> F1635-500ML </td><td></td></tr><tr><td> DotReaderTM </td><td> Medsaic </td><td> não disponível </td><td></td></tr><tr><td> Albumina de soro bovino </td><td> Sigma-Aldrich </td><td> A9418-10G </td><td></td></tr><tr><td> Inativado pelo calor soro AB 2% </td><td> Invitrogen </td><td> 34005100 </td><td></td></tr><tr><td> Ficoeritrina conjugada CD3 </td><td> Beckman Coulter </td><td> ET386 </td><td></td></tr><tr><td> AlexaFluor647 conjugada EpCAM </td><td> BioLegend </td><td> 324212 </td><td></td></tr><tr><td> Typhoon FLA 9000 </td><td> GE Healthcare </td><td> 28-9558-08 </td><td> 532 nm do laser, 580 BP30 emissão filtro para PE. 633 nm do laser e 670 BP30 emissão filtro para Alexa647 </td></tr><tr><td> MultiExperiment Visualizador v4.4 </td><td> TM4 Microarray Software Suite </td><td> Open - software de código (Ref 11) </td><td></td></tr></tbody></table>

colorectal cancer cell surface protein profiling using an antibody microarray and fluorescence multiplexing

O prognóstico atual e classificação do CRC conta com encenação de sistemas que integram achados histopatológicos e clínicos. No entanto, na maioria dos casos CRC, disfunção das células é o resultado de numerosas mutações que modificam a expressão da proteína e pós-translacionais modificação 1. Um número de antígenos de superfície celular, incluindo cluster de diferenciação (CD) de antígenos, foram identificados como prognósticos ou potenciais biomarcadores metastático em CRC. Estes antígenos fazer biomarcadores ideal como sua expressão muitas vezes muda com a progressão do tumor ou interações com outros tipos de células, como linfócitos tumor infiltrantes (TILs) e tumor associado macrófagos (TAMs). O uso da imuno-histoquímica (IHQ) para o câncer de sub-classificação e prognóstico é bem estabelecida para alguns tipos de tumor 2,3. No entanto, não &#39;marcador&#39; single mostrou significado prognóstico maior do que clínico-patológica de teste ou ganhou ampla aceitação para uso em relatórios de patologia de rotina de todos os casos CRC. Uma abordagem mais recente para a estratificação prognóstica de fenótipos da doença depende de perfis de proteínas de superfície usando vários &quot;marcadores&quot;. Enquanto perfil de expressão de tumores através de técnicas proteômicas como iTRAQ é uma poderosa ferramenta para a descoberta de biomarkers4, não é ideal para uso rotineiro nos laboratórios de diagnóstico e não pode distinguir diferentes tipos de células em uma população mista. Além disso, grandes quantidades de tecido tumoral são necessários para a caracterização de glicoproteínas da membrana plasmática purificada por estes métodos. Neste vídeo nós descrevemos um método simples para criar perfis de superfície proteoma de células viáveis ​​a partir de amostras desagregadas CRC usando um anticorpo DotScan microarray CRC. O microarray 122-anticorpo consiste em uma região de anticorpos 82-standard reconhecendo uma série de marcadores de linhagem específica de leucócitos, moléculas de adesão, receptores e marcadores de inflamação e resposta imune 5, bem como uma região satélite para a detecção de 40 marcadores potencialmente prognóstico para CRC . Células são capturadas apenas em anticorpos para os quais eles expressam o antígeno correspondente. A densidade de células por ponto, determinado pelo escaneamento óptico, reflete a proporção de células que expressam esse antígeno, o nível de expressão do antígeno e afinidade do anticorpo 6. CRC de tecido ou mucosa intestinal normal, scans óptica refletem o imunofenótipo de populações mistas de células. Multiplexação de fluorescência pode então ser usada para perfil selecionado sub-populações de células de interesse capturado no array. Por exemplo, Alexa molécula de adesão 647-anti-células epiteliais (EpCAM; CD326), é um antígeno de diferenciação pan-epiteliais que foi usado para detectar células CRC e também células epiteliais da mucosa intestinal normal, enquanto Ficoeritrina-anti-CD3, foi usado para detectar infiltração de células T-7. O CRC DotScan microarray deve ser o protótipo de uma alternativa de diagnóstico para o sistema de estadiamento baseado anatomicamente CRC.

Watch this Scientific Journal Video about Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing at JoVE.com

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

Nós descrevemos um procedimento para a desagregação de câncer colorretal (CRC) para produzir células viáveis ​​única, que são, então, capturado em microarrays de anticorpos personalizado reconhecer antígenos de superfície (CRC DotScan microarray). Sub-populações de células vinculada ao microarray pode ser perfilado por multiplexação de fluorescência usando anticorpos monoclonais marcados com corantes fluorescentes.

Câncer Colorretal Profiling proteína de superfície celular Usando um Microarray de anticorpos e Multiplexação de fluorescência

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

colorectal-cancer-cell-surface-protein-profiling-using-an-antibody

Research

JoVE Journal

Immunology and Infection

13.9K visualizações.  University of Sydney. Nós descrevemos um procedimento para a desagregação de câncer colorretal (CRC) para produzir células viáveis ​​única, que são, então, capturado em microarrays de anticorpos personalizado reconhecer antígenos de superfície (CRC DotScan microarray). Sub-populações de células vinculada ao microarray pode ser perfilado por multiplexação de fluorescência usando anticorpos monoclonais marcados com corantes fluorescentes.

Vídeo: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

Usando um ensaio Microarray Pan-Viral (Virochip) para Tela de amostras clínicas para patógenos virais

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Imunologia e Infecção

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular localization can provide insights into the functional role of bacterial proteins. Surface localization of bacterial proteins is a key step towards identification of virulence factors involved in mechanisms of pathogenicity. 
Methods for fractionating leptospiral membranes 2-5 may be selective for a certain class of outer-membrane proteins (OMPs), such as lipoproteins vs. transmembrane OMPs, and therefore lead to misclassification. This likely is due to structural differences and how they are associated to the outer membrane. Lipoproteins are associated with membranes via a hydrophobic interaction between the N-terminal lipid moiety (three fatty acids) and the lipid bilayer phospholipids 6, 7. In contrast, transmembrane OMPs are typically integrated into the lipid bilayer by amphipathic &beta;-sheets arranged in a barrel-like structure 8, 9. In addition, presence of a protein in the outer-membrane does not necessarily guarantee that the protein or its domains are exposed on the surface. Spirochetal outer membranes are known to be fragile and therefore necessitate methods involving gentle manipulation of cells and inclusion of sub-surface protein controls to assess the integrity of the outer membrane.
Here, we present an immunofluorescence assay (IFA) method to directly assess surface exposure of proteins on intact leptospires. This method is based on recognition of leptospiral surface proteins by antigen-specific antibodies. Herein, antibodies specific for OmpL5410 are detetcted aftero binding to native, surface exposed epitopes. Comparison of antibody reactivity to intact versus permeabilized cells enables evaluation of cellular distribution and whether or not a protein is selectively present on leptospiral surface. The integrity of outer membrane should be assessed using antibody to one or more subsurface proteins, preferably located in the periplasm.
The surface IFA method can be used to analyze surface exposure of any leptospiral protein to which specific antibodies are available. Both the usefulness and limitation of the method depends on whether the antibodies employed are able to bind to native epitopes. Since antibodies often are raised against recombinant proteins, epitopes of native, surface-exposed proteins may not be recognized. Nevertheless, the surface IFA method is a valuable tool for studying components of intact bacterial surfaces. This method can be applied not only for leptospires but also other spirochetes and gram-negative bacteria. For stronger conclusions regarding surface-exposure of OMPs, a comprehensive approach involving several cell localization methods is recommended 10.

An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction.

Imuno-fluorescência Ensaio de Leptospirose superfície exposta Proteínas

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Análise de uma única célula de Bacillus subtilis Os biofilmes utilizando microscopia de fluorescência e citometria de fluxo

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Visualização das respostas toxina bacteriana induzida por microscopia de fluorescência de células vivas

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Uma abordagem comparativa para caracterizar a paisagem de Patógeno-Hospedeiro interações proteína-proteína

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

A Miniaturized Glycan Microarray Ensaio para a Avaliação de avidez e especificidade do Hemaglutininas vírus da gripe A

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, immunodiagnostics and more recently as therapeutic agents. Traditional methods for labeling antibodies with small molecules require purified antibodies at relatively high concentration, involve multiple dialysis steps and have limited throughput. However, several applications, including the field of Antibody Drug Conjugates (ADCs), will benefit from new methods that will allow labeling of antibodies directly from cell media. Such methods may allow antibodies to be screened in biologically relevant assays, for example, the receptor-mediated antibody internalization assay in the case of ADCs. Here, we describe a method (on-bead method) that enables labeling of small amounts of antibodies directly from cell media. This approach utilizes high capacity magnetic Protein A and Protein G affinity beads to capture antibodies from the cell media followed by labeling with small molecules using either amine or thiol chemistry and subsequent elution of the labeled antibodies. Taking fluorescent dyes as surrogates for small molecules, we demonstrate the on-bead labeling of three different mouse antibodies directly from cell media using both amine and thiol labeling chemistry. The high binding affinity of antibodies to Protein A and Protein G ensures high recoveries as well as high purity of the labeled antibodies. In addition, use of magnetic beads allows multiple samples to be handled manually, thereby significantly improving labeling throughput.

The on-bead method for labeling antibodies with small molecules enables labeling of a small amount of antibodies directly from cell media. This method is compatible with amine and thiol chemistry, and can handle multiple samples in parallel, manually or using automated platforms.

Rotulagem de anticorpos com corantes fluorescentes utilizando proteína Magnetic A e esferas de proteína G

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

Um ensaio de Microarray da proteína para determinação sorológica do antígeno-específicas anticorpo respostas seguintes Clostridium difficile infecção

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

Célula única quantificação de mRNA e expressão de proteínas de superfície em CD4 infectados por vírus de imunodeficiência simiana+ T células isoladas de macacos Rhesus

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.