Os autores reconhecem o financiamento através do Departamento de Defesa pelo Congresso dirigiu programas de Investigação Médica (# W81XWH-10-1-0445), National Institutes of Health (NIH R21 HL110056) e American Heart Association (# 11SDG4890030).

1. Preparação e Esterilização de Buffers e agentes de teste <ol><li> 150 mM de NaCl: Dissolver 4,383 g de cristais de NaCl em 500 ml de água nanopura. </li><li> Os tampões de pH: Preparar tampão fosfato a pH 5,6, 6,2, 6,8, e 7,4 por mistura de quantidades apropriadas de fosfato monobásico e fosfato de sódio dibásico. Se as amostras são testadas a valores de pH mais baixos (isto é, pH &lt;5,6), em seguida, um tampão mais apropriado, tal como tampão de citrato, deve ser usado. Receitas tampão estão facilmente disponíveis, e um exemplo de referência tenha sido fornecido aqui 12. </li><li> Esterilizar todos os buffers acima indicadas através de um aparelho de filtração garrafa-top vácuo e re-check tampão pH. </li><li> 20% de Triton X-100 (testemunha positiva): Misturar 20 ml puro Triton X-100 em 80 ml de água nanopura. Vortex vigorosamente e sonicar a dissolver-se. Deixar a temperatura ambiente durante a noite antes da utilização. </li></ol> 2. Preparação de eritrócitos <ol><li> Obter 25 ml de sangue from um anônimo doador humano, elaborado diretamente em tubos de K 2-EDTA revestidos Vacutainer para impedir a coagulação. </li></ol> NOTA: Todos os procedimentos devem ser pré-aprovados pelo Conselho de Revisão Institucional apropriado (IRB), e punção venosa e coleta de sangue deve ser realizada por um flebotomista treinado, a fim de minimizar o risco para o doador. Os procedimentos padrão de flebotomia ter sido publicado anteriormente 13. <ol start="2"><li> Centrifugar o sangue a 500 xg durante 5 min, e os níveis de hematócrito marca (vermelho, a camada inferior) e de plasma (camada superior amarelada) no tubo. </li><li> Aspirar plasma suavemente através de uma micropipeta, adicionar em água sanitária, e descartar em resíduos com risco biológico. </li><li> Encher o tubo hematócrito a linha marcada (nível original de plasma) com 150 mM de solução de NaCl. Cap e inverter algumas vezes para misturar suavemente. Centrifugar a 500 xg durante 5 min. </li><li> Repita o passo 2,3-2,4 para lavar as células do sangue novamente. Em seguida, aspire supernatformiga e substituir com PBS a pH 7,4. Inverter para misturar. </li><li> Dividir sangue uniformemente em quatro tubos, correspondendo a cada pH que será testado. Rotular os tubos de acordo com cada valor de pH a ser testado (5.6, 6.2, 6.8, 7.4). </li><li> Centrifugar os tubos de sangue a 500 xg durante 5 min. Marcar níveis em tubos, em seguida, aspirar o sobrenadante. </li><li> Encher cada tubo de linha marcada com tampão de pH apropriado (como indicado em 2.6). </li><li> Etiqueta quatro tubos de 50 ml (um por pH de teste), e pipeta de 49 ml de PBS de pH apropriado para cada tubo cónico. </li><li> Adicionar 1 ml de eritrócitos (mesmo pH) para dentro do tubo correspondente para uma diluição de 1:50. Inspecionar visualmente o sangue diluído, que deve ser turva e vai resolver, se deixado em repouso. Se nenhuma forma de pelotas, as células têm lisadas. </li></ol> 3. Lise Ensaio 96 Configuração da Bem e Quantificação <ol><li> Preparar soluções de reserva de todos os agentes de entrega de droga experimental, a 20x a concentração final desejada para ser testado (Ensaioterá 10 ul de agente de entrega de droga + 190 ul diluídas células vermelhas do sangue, conduzindo a uma diluição de 1/20 do original de agente de libertação de fármacos para a mistura de ensaio final). Os estoques de 20, 100, e 800 ug / ml são sugeridas, resultando em concentrações de ensaio finais de 1, 5, e 40 ug / ml, respectivamente. </li><li> Pipetar 10 ul de cada solução de estoque em Fundo em V de 96 poços. Para melhores resultados, coloque cada amostra em triplicado ou quadruplicado. </li></ol> NOTA: Para facilidade, recomenda-se que uma placa de 96 poços separada deve ser preparada para cada pH a ser testado, com cada uma das amostras (a cada concentração) carregado com n = 3-4 por prato. <ol start="3"><li> Para poços de controlo positivo, adicionar 10 ul de Triton X-20% 100. </li><li> Para poços de controlo negativo, adicionam-se 10 ul de tampão de fosfato. Utilize uma solução tampão ao mesmo pH a ser testado. </li><li> Pipete 190 pi de eritrócitos diluídas (ver 2.10) a cada poço, tornando solução stock certeza célula permanecehomogénea durante a transferência. Dica: Use uma pipeta multi-canal para simplificar essa tarefa. </li><li> Incubar as placas a 37 ° C durante uma hora (Opcional: Utilizar um agitador orbital ou oscilante). </li><li> Centrifugar as placas durante 5 minutos a 500 xg para eritrócitos intactos pelota. Nota: durante a remoção da placa da centrífuga e transportá-lo para o passo seguinte, manusear com cuidado e ter a certeza de não perturbar o sedimento celular. </li><li> Utilizando uma pipeta multicanal, transferir 100 ul do sobrenadante de cada poço para uma clara, de fundo chato de placas de 96 poços. Observação: se o sedimento de células foi acidentalmente perturbado por qualquer amostra (s), pode-se re-centrifugar a placa e, em seguida, proceder à transferência de sobrenadante. </li><li> Medir a absorvância do sobrenadante com um leitor de placas. Note-se que uma gama de comprimentos de onda podem ser utilizados (400 - 541 nm). </li></ol> NOTA: leitores de placas diferentes podem ter pontos de saturação e sensibilidades diferentes, para a escolha de um comprimento de onda para measurements depende ou não de dados de hemólise de amostras experimentais podem ser confiavelmente normalizado contra hemólise máximo induzido por tratamento com detergente. Isto requer uma medição precisa dos valores de absorvância das amostras de controlo positivo. <ol start="10"><li> Usando o Microsoft Excel ou um software de análise de dados semelhante, encontre a média das leituras de absorvência de fundo a partir das amostras de controlo negativo criados para cada pH (passo 3.4). Subtrair o valor de absorvância de fundo a partir de todas as outras amostras que foram medidas naquele pH. </li><li> Após subtracção do fundo, encontrar a absorvência média do controlo positivo detergente tratados com amostras (passo 3.3.) Então normalizar todos os pontos de dados experimentais, para este valor de absorvância média, a qual deve representar 100% de hemólise. Finalmente, multiplicar cada valor bem por 100% para calcular% de hemólise, que ocorreu em cada poço individual em relação ao controlo de detergente. </li></ol>

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Aprovação IRB: Procedimentos que envolvem seres humanos tenham sido aprovados pelo Conselho de Revisão Institucional da Universidade Vanderbilt (IRB; Protocolo # 111251). Não há conflitos de interesse declarados.

pH-responsivos polímeros ou outros agentes destinados a função endosomolytic pode ser rápida e eficazmente rastreados com base na lise de células vermelhas do sangue em valores de pH encontrados no endossoma (Figura 1, pH 6,8 - endossomo precoce, a pH 6,2 - endossoma tardio, pH 5,6 - lisossoma). 14-17 hemólise dependente do pH foi usado para analisar a capacidade de mediar a libertação transportadoras endossomal da terapêutica biomacromolecular péptidos (por exemplo, siRNA,...

Embora existam muitos potenciais de alto impacto alvos terapêuticos no interior da célula, a entrega intracelular de agentes representa um desafio significativo. Frequentemente, as drogas, especialmente produtos biológicos, são internalizados pelas células e de tráfico dentro de vesículas que, ou levar a uma degradação do seu conteúdo através da via endo-lisossomal, ou são transportados de volta para fora da célula através de exocitose. 1 No último processo, o pH interno das vesículas é acidificada para cerca de 5-6, que é o pH óptimo para a actividade das enzimas que funcionam neste compartimento, tal como a lisozima 2. Recentemente, um certo número de materiais foram especificamente concebidos para aproveitar a acidificação dos endossomas para facilitar a entrega citosólica da sua carga. Um exemplo desta abordagem utiliza sintéticos, as nanopartículas de polímeros de micelas cujo núcleo é zwitteriónicos e carga neutra a um pH fisiológico (isto é, 7.4). No entanto, a pH 6,0- 6,5, os polímeros tornam-se protonado e adquirem uma carga positiva, que desestabiliza o núcleo micelar, e os segmentos de polímeros expostos interagir com e romper a membrana endossomal. Esta actividade tem sido mostrado para promover a fuga endossomal de péptidos e ácidos nucleicos baseados em terapêutica, o que lhes permite aceder aos seus alvos citosólicas. 3,4 Outros exemplos de métodos desenvolvidos para mediar fuga endossomal que interromper a barreira da membrana incluem as &quot;&quot; péptidos fusogénicos ou proteínas que podem mediar a fusão da membrana, ou a formação de poros transientes na bicamada fosfolipídica. 5 homopolímeros de ácidos acrílicos aniónicos de alquilo, tais como poli (ácido propylacrylic) são outra abordagem bem estudada e, nestes polímeros, o estado de protonação do ácido carboxílico pendente dita transição em um hidrofóbico, estado da membrana disruptiva em gamas de endo-lisossomais pH 6,7. Um sistema modelo útil para a triagem comportamento endosomolytic é o e-x dependente do pH in vivo ensaio de hemólise. 8 Neste sistema modelo, a membrana do eritrócito serve como um substituto para a membrana de camada dupla de lípido que encerram endo-lisossomais vesículas. Este modelo generalizável tem sido utilizado por outros para avaliar o comportamento de células endosomolytic penetrantes péptidos e outros sistemas de entrega de genes poliméricos. 8-11 Nesta experiência, as células vermelhas do sangue e os materiais de teste são co-incubados em tampões a pH definidos que imitam extracelulares (7,4), no início endossomais (6,8), e no final endo-lisossomal (&lt;6,8) ambientes. A quantidade de hemoglobina libertada durante o período de incubação é quantificada como uma medida da lise dos glóbulos vermelhos, que é normalizado para a quantidade de hemoglobina libertada nas amostras de controlo positivo lisadas com detergente. De rastreio de uma pequena biblioteca de materiais de teste potencialmente endosomolytic, pode-se inferir que as amostras que não produzem hemólise, a pH 7,4, mas bainha significativamente elevadosolysis a pH &lt;6,5, serão os candidatos mais eficazes e cytocompatible para a entrega da droga citosólica. Os materiais que se enquadram nestes critérios seria de esperar que permanecem inertes e não indiscriminadamente destruir bicamadas lipídicas (isto é, que poderia causar a citotoxicidade) até serem expostos a uma queda no pH local na sequência de internalização em endo-lisossomais compartimentos. Neste protocolo, os eritrócitos são isoladas a partir de um dador humano e co-incubou-se a pH 5,6, 6,2, 6,8, ou 7,4 com experimentais agentes de entrega de droga endosomolytic. Eritrócitos intactos são sedimentadas e os sobrenadantes (que contêm hemoglobina libertada a partir de eritrócitos lisados) são analisadas para a característica de absorção de hemoglobina através de um leitor de placa (Figura 1).

<table border="1"><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> BD Vacutainer - K2EDTA Vacutainer Tubos </td><td> Fisher Scientific </td><td> 22-253-145 </td><td> Para coleta de sangue </td></tr><tr><td> BD Vacutainer sangue agulhas para coleta, 20,5 calibre- </td><td> Fisher Scientific </td><td> 02-665-31 </td><td> Para coleta de sangue </td></tr><tr><td> BD Vacutainer Tubo Adaptador Titular / Agulha </td><td> Fisher Scientific </td><td> 22-289-953 </td><td> Para coleta de sangue </td></tr><tr><td> BD Marca álcool isopropílico Swabs </td><td> Fisher Scientific </td><td> 13-680-63 </td><td> Para coleta de sangue </td></tr><tr><td> BD Vacutainer Latex-Free Tourniquet </td><td> Fisher Scientific </td><td> 02-657-6 </td><td> Para coleta de sangue </td&gt; </tr><tr><td> Ácido clorídrico (cone.) </td><td> Fisher Scientific </td><td> A144-500 </td><td> Para o ajuste do pH de D-PBS. </td></tr><tr><td> Triton X-100 </td><td> Sigma-Aldrich </td><td> T8787 </td><td> Controle positivo </td></tr><tr><td> PBS Dulbecco </td><td> Invitrogen </td><td> 14190 </td><td></td></tr><tr><td> Nalgene MF75 estéril Unidade de frascos de filtro descartável com SFCA Membrana </td><td> Fisher Scientific </td><td> 09-740 44A- </td><td></td></tr><tr><td> BD placas de 96 poços, de fundo chato de poliestireno tecido, a cultura tratada com </td><td> Fisher Scientific </td><td> 08-772 2C- </td><td> Por placa de leitura no final do ensaio. </td></tr><tr><td> BD placas de 96 poços, de fundo redondo de poliestireno de tecido, a cultura tratada com </td><td> Fisher Scientific </td><td> 08-772-17 </td><td> Por incubação de células vermelhas do sangue com agentes experimentais. </td></tr></table&gt;

ex vivo red blood cell hemolysis assay for the evaluation of ph-responsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs

Bicamadas de fosfolipídios que formam endo-lisossômico vesículas podem representar uma barreira para a entrega de medicamentos biológicos para alvos intracelulares. Para ultrapassar esta barreira, uma série de transportadores de drogas sintéticas têm sido desenvolvidos para a perturbação activa da membrana endossomal e entregar a carga para dentro do citoplasma. Aqui, nós descrevemos o ensaio de hemólise, a qual pode ser usada como uma rápida, tela de alto rendimento para a actividade e cytocompatibility endosomolytic de sistemas de entrega de drogas intracelulares. No ensaio de hemólise, as células vermelhas do sangue e os materiais de teste são co-incubados em tampões a pHs definidos que imitam extracelulares, cedo endossomais e lisossomais tarde endo-ambientes. Após um passo de centrifugação a pelota intactas as células vermelhas do sangue, a quantidade de hemoglobina libertada para o meio é medida por espectrofotometria (405 nm para melhor a gama dinâmica). A interrupção por cento de glóbulos vermelhos é então quantificada em relação ao controle positivo samples lisadas com detergente. Neste sistema modelo da membrana do eritrócito serve como um substituto para a membrana de camada dupla de lípido que encerram endo-lisossomais vesículas. O resultado desejado é a hemólise insignificante a um pH fisiológico (7,4) e hemólise robusto na gama de pH a partir de endo-lisossomal aproximadamente pH 5-6,8.

<html> Tipicamente, os agentes que exibem um comportamento dependente do pH ideal hemolítica têm o maior potencial para a entrega de drogas citosólica, ácidos nucleicos ou outras moléculas bioactivas. Isto é exemplificado pelo agente # 1, como mostrado na Figura 2, o qual exibe a hemólise mínima a um pH de 7,4, mas um aumento significativo no comportamento hemolítica em gamas de pH endossomais (&lt;6,5). Alguns agentes podem exibir níveis significativos de comportamento hemolítica em gamas de ...

Watch this Scientific Journal Video about Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs at JoVE.com

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Um ensaio de hemólise podem ser usados ​​como um meio rápido, tela de alto rendimento de cytocompatibility sistemas de entrega de drogas e atividade endosomolytic para entrega intracelular de carga. O ensaio mede a ruptura das membranas dos eritrócitos em função do pH do meio ambiente.

Ex Vivo Blood Red Ensaio Hemólise celular de Avaliação de pH-responsivos agentes Endosomolytic para entrega citosólico de Drogas Biomacromolecular

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this ...

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JoVE Journal

Immunology and Infection

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

34.0K visualizações.  Vanderbilt University. Um ensaio de hemólise podem ser usados ​​como um meio rápido, tela de alto rendimento de cytocompatibility sistemas de entrega de drogas e atividade endosomolytic para entrega intracelular de carga. O ensaio mede a ruptura das membranas dos eritrócitos em função do pH do meio ambiente.

Vídeo: Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Apresentando tensão de cisalhamento em o Estudo da adesão bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Imunologia e Infecção

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Uma avaliação quantitativa da migração celular pelo ensaio Phagokinetic faixa Motilidade

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Antígenos funcionais protegidos glóbulos vermelhos pela membrana enxerto de compactos Poligliceróis hiper

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Métodos para a detecção quantitativa de anticorpos induzida pela ativação do complemento no Glóbulos Vermelhos

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

De alto rendimento quantitativo em tempo real de RT-PCR Ensaio para Determinar Perfis de Expressão do tipo I e III do interferão subtipos

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Uso de um monócito monocamada Ensaio para Avaliar a fagocitose mediada pelo receptor Fcy

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran aumenta a eficiência de Lentivirus transdução das células NK primário murino e humano

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

Um glóbulo vermelho simples método de lise para o estabelecimento de linhas celulares linfoblastóides B

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Na Vivo Ensaio para detecção de células T antígeno-específicas função citolítica usando um modelo de vacinação

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

Medição de deformabilidade e heterogeneidade de glóbulos vermelhos no sangue por Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...