Os autores gostariam de agradecer financiamento do NIH 3R01AI080289-02S1, e Harvard University Center for AIDS Research Scholar Fellowship sob NIH / NIAID 2P30AI060354-07.

1. As células fagocíticas cultura <ol><li> Cultura de células THP-1 10 em RPMI 1640 suplementado com 10% de soro fetal bovino como uma suspensão estacionária em frascos T. Densidade celular deve ser mantida abaixo de 0.5x10 6 mL / a fim de manter níveis consistentes de expressão FcγR e desempenho do ensaio. </li></ol> 2. Prepare antígeno biotinilado <ol><li> Calcular a quantidade de sulfo-NHS kit reagente LC biotina necessário biotinylate o antígeno alvo de interesse de acordo com as instruções do fabricante. </li><li> Dissolver o reagente Biotinilação em água e imediatamente adicionar o montante calculado ao antígeno em um buffer que não contém aminas primárias. Permitir que a reação continue por 1 hora em temperatura ambiente, mexendo ocasionalmente. </li><li> Remover biotina, o excesso não conjugada por troca de buffer em uma unidade de concentração Amicon centrífuga de um corte de peso molecular apropriada para reter o targeantígeno t. Adicione a amostra para a câmara superior da unidade de concentração e adicione PBS para levar o volume total de até 15 mL linha de enchimento. Centrifugar a 4.000 xg para trazer volume para cerca de 1,5 mls. Repetindo este processo duas vezes irá remover 99% da biotina livre para garantir a cobertura máxima de antígeno para contas. </li></ol> 3. Prepare Beads Antígeno Saturada <ol><li> Lavar 100 l de 1 mm contas neutravidin fluorescentes duas vezes em 1 ml de 0,1% PBS-BSA após girar para baixo em uma microcentrífuga em alta velocidade. Ressuspender lavados contas em 100 l PBS-BSA, e alíquota em 10 tubos. </li><li> Para determinar as condições de antígeno revestimento que saturam as contas, combine 10 ul da suspensão lavados talão com concentrações variando de antígeno biotinilado em duas vezes passos. Incubar overnight a 4 ° C em um tubo de microcentrífuga em um rotador. </li></ol> NOTA: Saturação de contas devem ser determinadas experimentalmente. Isso pode ser feitoidentificando-revestimento talão condições que produzem fagocitose máximo quando contas são posteriormente opsonizadas com um anticorpo monoclonal controle. <ol start="3"><li> Remover antígeno não ligado por lavagem com 1 mL de PBS-BSA e centrifugar em alta velocidade até contas são peletizadas. Remover PBS-BSA e repita. </li><li> Ressuspender antígeno revestido contas em um volume final de 1 ml em PBS-BSA. Contas pode ser armazenado por até uma semana a 4 ° C antes do uso. </li></ol> 4. Prepare amostras de anticorpos <ol><li> Amostras clínicas de anticorpos pode ser purificado a partir do plasma usando um gel Melon kit de purificação de IgG de acordo com as instruções dos fabricantes. IgG purificada pode ser armazenado a 4 ° C até que esteja pronto para uso. </li></ol> NOTA: as devidas precauções ao manusear amostras humanas devem sempre foram tomadas. <ol start="2"><li> Determinar a concentração de anticorpos purificados por absorvência a A280, e diluir as amostras a 1 mg / ml em PBS. </li><li> Prepare positive e negativos anticorpos monoclonais controle por diluição de 1 mg / ml em PBS. </li></ol> 5. Chapeamento do Experimento <ol><li> Ressuspender o lavado, o antígeno solução saturada talão preparada acima em vortex, e transferir 10 ml em cada poço de um de fundo redondo de 96 placa também. Cuidados devem ser tomados para melhorar continuamente a agitar a suspensão de esferas para assegurar igual número de partículas fluorescentes são adicionados a cada poço. </li><li> Adicionar diferentes concentrações dos anticorpos de interesse de cada bem, criando uma curva dose-resposta para cada anticorpo. Concentrações ideais será diferente entre as amostras, dependendo da titulação de anticorpos presente, mas uma gama de 0,01-100 mcg / ml concentração final irá proporcionar uma boa cobertura e permitir a identificação da faixa de concentração de interesse. Garantir anticorpos são adicionados em volumes não maior do que 20 l. </li><li> Contas de incubar e amostras de anticorpos para 2 horas a 37 ° C para permitir que os anticorpos para opsonize as contas. &lt;/ Li&gt; <li> Prepare uma suspensão de células THP-1 a 2,5 x 10 5 células / ml, e adicionar 200 mL da suspensão a cada poço, para um total de 5 x 10 4 células THP-1 em cada poço. </li><li> Incubar durante a 37 ° C, 5% CO 2, em uma incubadora estacionária, permitindo que as células e os grânulos de pellet através da gravidade. </li></ol> NOTA: Sinal de ruído pode ser melhorada através da determinação do talão ideal: anticorpo: THP-1 índices de células para uma fonte de antígeno-anticorpo dado. 6. Análise por citometria de fluxo <ol><li> Remova 100 ul de sobrenadante de cada poço com cuidado para não perturbar o pellet celular. Sobrenadante podem ser salvas se as determinações de citocinas secreção ou outras análises são desejados. </li><li> Para fixação, adicione 100 ml de paraformaldeído 4% a cada poço e pipeta para ressuspender as células e misture. </li><li> Análise de fluxo de alta vazão citometria pode ser realizada utilizando uma BD LSR II equipado com um leitor de placas HTS.</li><li> Software programa para misturar todos os poços três vezes (100 mL de volume mix) e analisar 30 mL, ou pelo menos 2.000 eventos de células, de cada amostra. </li><li> Os dados coletados podem ser analisados ​​no programa FlowJo ou software equivalente. Métricas úteis incluem o percentual de + talão, ou células fluorescentes, que fornece uma medida do número de células fagocíticas presentes, bem como a intensidade de fluorescência das células fagocíticas, que fornece uma medida do número de contas fagocitados. Multiplicando esses valores gera uma intensidade média integrada fluorescentes ou iMFI. </li><li> O número médio de grânulos fagocitados por cada célula fagocítica pode ser calculado dividindo o iMFI pela fluorescência média de um talão. </li></ol> 7. Resultados representante Deve haver uma diferenciação clara de amostras de anticorpos de indivíduos afetados e não afetados. Figura 1A apresenta os histogramas FACS de uma amostra de um anticorpo nega HIVtiva (traço preto) e um sujeito HIV positivo (traço cinza), e demonstra a fagocitose aumentou impulsionada pela presença de antígeno-anticorpos específicos. Ótima sensibilidade do ensaio é dependente de saturação das contas com o antígeno biotinilado. Figura 1B apresenta a fagocitose observado quando grânulos revestidos com diferentes quantidades de antígeno foram opsonizadas com 3 anticorpos monoclonais controle (incluindo um anticorpo não vinculativo, triângulos), e estabelece 2μg antígeno / mL contas como uma concentração de saturação para este antígeno. A curva dose-resposta é apresentada na Figura 2, e demonstra a capacidade diferencial de amostras de anticorpos assunto para induzir fagocitose em uma faixa de concentração de anticorpos de 0,05-5 mcg / ml. Este fagocitose diferencial pode ser movida por qualquer diferença no título ou propriedades de domínio Fc como subclasse IgG e estadual de glicosilação. Quando células THP-1 são imaged por microscopia fluorescente, há clara evidência de fagocitose talão. A Figura 3 apresenta duas imagens estáticas 63x de células THP-1 após a incubação com o anticorpo opsonizadas (verde) e não-opsonizadas (vermelho) contas, demonstrando a falta de absorção fagocíticas na ausência de anticorpos. Quando a microscopia de lapso de tempo é realizada, a absorção de anticorpos específicos fagocítica de partículas fluorescentes é ainda mais impressionante (Movie 1, ampliação de 20x). Trabalho anterior confirmou interiorização das contas associadas com as células, e experimentos com monócitos primários concordaram bem com os índices de fagocitose (valores iMFI) neste ensaio alta taxa de transferência (dados não mostrados). <img src="/files/ftp_upload/3588/3588fig1.jpg" alt="Figura 1"/> Figura 1. Controle de Qualidade de Ensaio. 1A, histogramas citometria de fluxo de fagocitose de uma amostra de anticorpos a partir de um tema HIV negativo (traço preto) e um sujeito HIV positivo (traço cinza).1B: determinação experimental do óptimas condições de revestimento talão para um antígeno da amostra. Antígeno-anticorpos monoclonais específicos (círculo, quadrado) demonstram fagocitose máxima de contas revestidas com&gt; 2 mg de antígeno / mL de contas, enquanto um anticorpo de controle (triângulo) demonstra nenhuma atividade fagocítica. <img src="/files/ftp_upload/3588/3588fig2.jpg" alt="Figura 2"/> Figura 2. Fagocitose curva dose-resposta. Clínica amostras de anticorpos HIV positivo (tratado, tratado, e exibindo controle da replicação viral na ausência de anti-retrovirais) e fagocitose disco HIV negativo assuntos da gp120 (envelope HIV) grânulos revestido de forma diferenciada. <img src="/files/ftp_upload/3588/3588fig3.jpg" alt="Figura 3"/> Figura 3. Internalização eficiente. Microscopia confirma a internalização de anticorpos opsonizadas contas (verde), enquanto os não-opsonizadas contas permanecer no solution (63x ampliação). Movie 1. Time-lapse de microscopia de anticorpos dirigidos a fagocitose foi realizado ao longo de 14 horas e permite a visualização da atividade das células fagocíticas THP-1 utilizados no ensaio de alto rendimento. Verde partículas fluorescentes são o anticorpo opsonizadas, vermelho partículas fluorescentes fornecer um controle negativo. <a href="http://www.jove.com/files/ftp_upload/3588/3588movie1.mov">Clique aqui para assistir o filme.</a>

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O ensaio aqui descrito permite high-throughput análise da atividade fagocítica de amostras clínicas de anticorpos, utilizando uma linha celular monocítica muito utilizada para estudar os processos fagocíticas 10 e placa de citometria de fluxo baseado automatizado. Este ensaio é vantajoso sobre os outros em sua capacidade de caracterizar precisamente subconjuntos antígeno-anticorpo específico, permitindo não só para o estudo das diferenças de fagocitose impulsionado pela doença de anticorpos específicos de diferentes disciplinas, mas também estudar as especificidades de antígenos múltiplos dentro do mesmo assunto. Porque o recrutamento de anticorpos de células efetoras inata, como monócitos e células antígeno apresentando outros impactos fortemente a evolução da doença 11-13, e é biologicamente variável, dependendo da geometria de anticorpos, subclasse IgG, e glicosilação em Asparagine297 do domínio Fc 14-16, este método prevê avaliação de um mecanismo de anticorpos críticos da ação. Além disso, como medi-anticorpofagocitose ated é explorada por alguns patógenos durante o curso da infecção 7,17,18, este ensaio é uma promessa na avaliação do processo de dependente de anticorpos acessório, e destaca a natureza dual da fagocitose como potencialmente protetor, bem como potencialmente prejudiciais atividade de anticorpos. O ensaio descrito pode ser utilizado para analisar os anticorpos a partir de amostras clínicas de populações de pacientes, mas também dos vacinados e podem ser adaptadas para utilizar soro em vez de IgG purificada. Além disso, a secreção de citocinas em resposta a fagocitose pode ser analisado a partir do sobrenadante da cultura, ea influência de FcR específico pode ser determinado usando FcR-anticorpos bloqueadores, permitindo não só as diferenças de potência fagocíticas a ser determinado, mas a jusante de sinalização de eventos, com uma visão em mecanismo, e pode ajudar a resolver e aperfeiçoar a compreensão deste mecanismo imunológico.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> EZ-Link Micro sulfo-NHS-LC-Biotinilação Kit </td><td> Thermo scientfic </td><td> 21935 </td><td></td></tr><tr><td> Amicon Ultra-15 Unidade Centrífuga de filtro com membrana Ultracel-50 </td><td> Millipore </td><td> UFC905024 </td><td> Tamanho da unidade de filtro pode variar de acordo com o tamanho do antígeno </td></tr><tr><td> FluoSpheres NeutrAvidin rotulados microesferas, 1,0 mM, amarelo-verde fluorescente (505/515) </td><td> Invitrogen </td><td> F-8776 </td><td> Fabricantes produzem este produto em várias cores </td></tr><tr><td> Melão Gel Kit de Purificação de IgG </td><td> Thermo </td><td> 45212 </td><td> Cuidados devem ser tomados para verificar a pureza das amostras Ab por SDS-PAGE. </td></tr></tbody></table>

determining the phagocytic activity of clinical antibody samples

Anticorpo-driven fagocitose é induzida através do envolvimento de receptores Fc em fagócitos profissionais, e pode contribuir tanto para a depuração, bem como a patologia da doença. Enquanto as propriedades dos domínios variáveis ​​de anticorpos têm sido considerados cruciais para função in vivo, a capacidade de anticorpos para recrutar células do sistema imunológico inato via seus domínios Fc tornou-se cada vez mais apreciada como um fator importante para sua eficácia, tanto no cenário de recombinante terapia com anticorpos monoclonais, bem como no curso da infecção natural ou vacinação 1-3. Importante, apesar de sua nomenclatura como um domínio constante, o anticorpo de domínio Fc não tem função constante, e é fortemente modulada por subclasse IgG (IgG1-4) e glicosilação de asparagina 297 4-6. Assim, este método para estudar as diferenças funcionais de antígeno-anticorpos específicos em amostras clínicas facilitará correlação do pote fagocíticaspreferencial de anticorpos para a susceptibilidade à doença do Estado, à infecção, a progressão ou evolução clínica. Além disso, esta função efetora é particularmente importante à luz da capacidade documentado de anticorpos para aumentar a infecção através do acesso patógenos em células hospedeiras através de fagocitose receptor Fc-driven 7. Além disso, há algumas evidências de que a captação fagocitária de complexos imunes podem afetar a polarização Th1/Th2 da resposta imune 8. Aqui, descrevemos um teste desenvolvido para detectar diferenças de anticorpos induzida pela fagocitose, que pode ser causada pelo diferencial de subclasse IgG, estrutura de glicano de Asn297, bem como a capacidade de formar complexos imunes de antígeno-anticorpos específicos de uma forma de alto rendimento . Para este fim, 1 mícron partículas fluorescentes são revestidas com antígeno, em seguida, incubadas com amostras clínicas de anticorpos, gerando fluorescente antígeno específico complexos imunes. Estes Antibody-opsonizadas grânulos são então incubadas com uma linha celular monocítica expressar FcγRs múltiplas, incluindo tanto inibitórios e ativar. Saída de ensaio pode incluir atividade fagocítica, secreção de citoquinas e padrões de uso FcγRs, e são determinados de forma padronizada, tornando este um sistema altamente útil para a análise de diferenças nesta função dependente de anticorpos efetoras em ambas as infecções e vacinas mediada protecção 9.

Watch this Scientific Journal Video about Determining the Phagocytic Activity of Clinical Antibody Samples at JoVE.com

Determining the Phagocytic Activity of Clinical Antibody Samples

Nós apresentamos um ensaio de fluxo de alto rendimento por citometria de determinar a atividade fagocítica de antígenos-anticorpos específicos a partir de amostras clínicas, utilizando antígenos fluorescentes revestido contas e uma linha celular monocítica expressar múltiplos receptores Fc-proporcionando o uso do receptor e determinações atividade fagocitária em um padronizados e forma reproduzível para qualquer antígeno de interesse.

Determinação da atividade fagocítica de amostras de anticorpos Clínica

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

determining-the-phagocytic-activity-of-clinical-antibody-samples

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JoVE Journal

Immunology and Infection

23.3K visualizações.  Ragon Institute of MGH, MIT, and Harvard. Nós apresentamos um ensaio de fluxo de alto rendimento por citometria de determinar a atividade fagocítica de antígenos-anticorpos específicos a partir de amostras clínicas, utilizando antígenos fluorescentes revestido contas e uma linha celular monocítica expressar múltiplos receptores Fc-proporcionando o uso do receptor e determinações atividade fagocitária em um padronizados e forma reproduzível para qualquer antígeno de interesse.

Vídeo: Determining the Phagocytic Activity of Clinical Antibody Samples

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37&deg;C for 60 minutes. After this time, the samples can then be easily scored for  ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.

Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

Determinação da Reatividade e Titre de soro usando um ensaio de hemaglutinação

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood ...

Imunologia e Infecção

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 ...

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

Detecção de multiplex de bactérias em amostras clínicas e Complexo Ambiental utilizando Oligonucleotídeos-coupled microesferas fluorescentes

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

Determinando a atividade citotóxica Optimal específicos de células CD8 Her2neu Humanos T, comparando a Cr51 Ensaio de Libertação do Sistema xCELLigence

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

A saliva, glândula salivar, e Coleta de hemolinfa de carrapatos Ixodes scapularis

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Fluorescência métodos de microscopia para determinar a viabilidade de bactérias em associação com células de mamíferos

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

Selecção de alta Scalable Rendimento de bibliotecas de anticorpos em fagos sintética exibida-

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

Uma estratégia rápida para o isolamento de novos Faustoviruses de amostras ambientais Usando<em&gt; vermiformis Vermamoeba</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

Avaliação rápida e específica da atividade da peroxidase de halogenação em amostras de sangue enriquecido por leucócitos

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Avaliando Retinal microglia fagocítica Função<em&gt; In Vivo</em&gt; Usando um ensaio baseado em citometria de fluxo

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...