O autor gostaria de agradecer a senhorita Lora Matthews para executar a técnica eo Sr. Paul Pastor para a cinematografia. Este projeto foi financiado pela Universidade da Austrália do Sul, Laboratório curso de Medicina.

Preparação de Saline Veronal 5x Buffered (VBS) <ol><li> Para preparar a solução salina Veronal Buffered (VBS), três soluções distintas precisa estar preparado. </li><li> Prepare uma solução através da dissolução de NaCl e 21.25gm 0.94gm de Barbitone de sódio em 350ml de água destilada. A concentração final de NaCl e de sódio Barbitone são 1,02 e 13mm, respectivamente. </li><li> Prepare a solução 2, dissolvendo 1.44gm de Barbitone em 125ml de água destilada quente. A concentração final de Barbitone é 62,5 mm. </li><li> Prepare a solução 3 dissolvendo 20.33gm de MgCl 2 e 4.41gm de CaCl 2 em 100ml de água destilada. A concentração final de MgCl 2 e CaCl 2 é 2.18m e 440mm, respectivamente. </li><li> Mix de soluções 1 e 2 e arrefecer à temperatura ambiente. </li><li> Uma vez que a solução combinada tenha arrefecido, adicione 1.25ml de solução 3 e ajustar o pH a 7,3-7,5 usando HCl 1M. </li><li> Ajustar o volume final de 500ml com água destilada para preparar uma solução estoque de 5x. </li><li> Para preparar uma solução 1x de trabalho, diluir a 1:05 estoque com água destilada. </li></ol> Sensibilização de hemácias de carneiro com hemolisina <ol><li> Prepare a hemolisina, em primeiro lugar diluição 1:50 com VBS </li><li> Para adicionar 4ml de SRBC 6ml de VBS e misture delicadamente por inversão </li><li> Centrifugar a 600g x 5 minutos </li><li> Desprezar o sobrenadante e lavar as células mais 2 vezes </li><li> Após a lavagem final, centrifugar as células de 900g x 5 minutos para arrumar as células </li><li> Descartar o sobrenadante e ressuspender as células em VBS suficiente para preparar uma solução de 10% ou seja, 0,5 ml de hemácias são ressuspenso em 5 ml de tampão </li><li> Gotas adicionar igual volume de hemolisina (coelho anti-ovelha anticorpos de glóbulos vermelhos) para as células enquanto agita continuamente </li><li> Incubar a 30 ° C por 30 minutos em banho-maria </li><li> Misture suavemente as células a cada 15 minutos </li><li> SRBC sensibilizados podem ser armazenados durante a noite a 4 ° C </li></ol> CH 50 ensaio <ol><li> Rotular uma série de tubos em duplicata com 1:08, 1:16, 1:32, 1:64 e 1:128. </li><li> Prepare uma série de duas diluições de controle e soro em cada VBS em duplicado </li><li> Início às 01:04 (100ml + 300ml de soro VBS) e transferência de 200ml de amostra dentro do tubo próxima rotulados. </li><li> Misturar bem entre diluições e 200ml de transferência para a diluição seguinte com uma ponteira de pipeta nova. </li><li> Repita até que todos os cinco diluições são feitas. </li><li> Descartar 200ml da diluição 1:128 final. </li><li> Adicione 200 ml de suspensão SRBC sensibilizados para todos os tubos. </li><li> Identifique dois tubos separados como em branco e adicionar 200ml de sensibilizados SRBC + 200ml VBS. Estes tubos medirá lise espontânea do SRBC na VBS. </li><li> Rótulo mais dois tubos separados como lise TOTAL e adicionar 200ml de água + 200ml SRBC sensibilizados destilada. </li><li> Misture suavemente todos os tubos. </li><li> Incubar a 37 ° C por 30 minutos em banho-maria misturando após 15 minutos. </li><li> Centrifugar as amostras a 1.500 g por 5 minutos para sedimentar as hemácias. </li><li> Transferência de 100ml do sobrenadante de cada tubo para um bem em uma placa inferior 96 bem plana. </li><li> Adicione 100 ml de água destilada para cada poço. </li><li> Ler a absorbância das amostras em 540 nm usando um espectrofotômetro de placas. </li></ol> Cálculos <ol><li> Calcular a média de absorbância para cada amostra </li><li> Subtrair a absorbância BLANK (lise espontânea) de todas as amostras </li><li> Calcular a lise% para cada diluição com a seguinte fórmula: <img src="/files/ftp_upload/1923/1923eq1.jpg" alt="Equação" /></li><li> Plotar a porcentagem de lise (eixo vertical) versus a diluição de soro no eixo horizontal. </li><li> Calcular a diluição necessária para a hemólise 50% para o controle e soro de ensaio (Fig. 2). </li></ol> Resultados representativos: O vídeo inclui um exemplo de resultados representativos. Na prática, um soro de controlo também é executado ao mesmo tempo, como o soro de teste e é tratado da mesma maneira. Abaixo (Tabela 1) são os dados da amostra a partir de uma amostra testada soro. Os dados são manipulados de acordo com a equação apresentada em 5.3. <table border="1"><tbody><tr><td> Amostra </td><td> OD 540 </td><td> Média </td><td></td><td></td></tr><tr><td> Em branco </td><td> 0,042, 0,044 </td><td> 0,043 </td><td></td><td></td></tr><tr><td> Lise total </td><td> 0,183, 0,183 </td><td> 0,183 </td><td></td><td></td></tr><tr><td colspan="5"></td></tr><tr><td> Diluição </td><td> OD 540 </td><td> Média </td><td> Dizer-Blank </td><td> Lise% </td></tr><tr><td> 01:08 </td><td> 0,200, 0,219 </td><td> 0,210 </td><td> 0,168 </td><td> 116 </td></tr><tr><td> 01:16 </td><td> 0,179, 0,173 </td><td> 0,176 </td><td> 0,134 </td><td> 93 </td></tr><tr><td> 01:32 </td><td> 0,134, 0,110 </td><td> 0,122 </td><td> 0,08 </td><td> 55 </td></tr><tr><td> 1:64 </td><td> 0,053, 0,066 </td><td> 0,059 </td><td> 0,017 </td><td> 12 </td></tr><tr><td> 1:128 </td><td> 0,044, 0,045 </td><td> 0,045 </td><td> 0,003 </td><td> 2 </td></tr></tbody></table><img src="http://www.jove.com/files/ftp_upload/1923/1923fig1.jpg" alt="Figura 1" /> Figura 1. Ativação da via clássica do complemento. A via clássica é ativada pela ligação imunoglobulina M (IgM) ou imunoglobulina g (IgG) na superfície de uma célula-alvo. A porção Fc da Ab liga a C1q, C1r é ativado e este por sua vez ativa outra molécula de C1r que, juntos, ativar duas moléculas de C1s. C1s agora cliva C4 que expõe o sítio de ligação para C2 que também é clivada. A ligação do C4b e C2a leva à formação de um complexo chamado de C3 convertase. Este complexo cliva C3 formando agora C3a e C3b, alguns dos quais combinam com o C3 convertase formando uma convertase C5. Este complexo age agora em C5, com a resultante C5b ligação com C6 iniciando a formação do complexo de ataque à membrana (MAC). C5b6 atua em C7, que por sua vez funcionam em C8 e C9 em última análise, resultando na formação do MAC final. (Goldsby et al, 2003). <img src="http://www.jove.com/files/ftp_upload/1923/1923fig2.jpg" alt="Figura 2" /> Figura 2. Plotagem de dados de amostra e cálculo do CH50 para um controle e amostra de soro. (A), a percentagem calculada (%) lise de tanto o controle (•) e teste () amostras de soro são plotados contra o fator de diluição. (B) Para calcular a lise de 50%, uma linha é desenhada a partir do valor percentual de 50% até se cruza com as linhas do gráfico e, em seguida, uma linha vertical é traçada até a diluição. Neste exemplo, uma diluição de 35 vezes, do controle e 21,6 vezes diluição do soro de teste são necessários para alcançar a lise de 50%. Estes dados indicam que há menos complemento presente no soro de teste em relação ao controle, uma vez que necessitou de menos de diluição, antes de lise de 50% foi alcançado. A amostra de controlo também mostra lise&gt; 100% na diluição de 1:08. Isto é provavelmente devido a lise incompleta do SRBC pela água destilada e mais eficiente lise pelos componentes do complemento.

<ol>
	<li>Goldsby, R. A., Kindt, T. J., Osborne, B. A., Kuby, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunology. , (2003).</li></ol>

O CH 50 ensaio está sujeito a muitas interferências. Alguns SRBC são mais frágeis do que outros, resultando em hemólise espontânea que não está relacionado para complementar a atividade. A afinidade do anticorpo de coelho varia de lote para lote e de um fabricante para outro, o que afeta a quantidade de anticorpo que se liga ao SRBC. Além disso, o processo de sensibilização SRBC com resultados de anticorpos em células com diferentes quantidades de anticorpos do revestimento SRBC. Coleta e armazena...

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<td>Sheep red blood cells</td>
<td>&nbsp;</td>
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<td>Serum</td>
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<td>&nbsp;</td>
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<td>Human serum</td>
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<td>Rabbit Anti-Sheep Haemolytic serum (RBCs), Unconjugated</td>
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<td>C12HSB</td>
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<td>96 well flat bottom plate</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
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<td>Veronal buffered saline</td>
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measuring the 50% haemolytic complement (ch50) activity of serum

O sistema complemento é um grupo de proteínas que, quando ativado levar a meta a lise das células e facilita a fagocitose através opsonisation. Componentes do complemento individual pode ser quantificado no entanto, este não fornece qualquer informação sobre a atividade da via. O CH<sub&gt; 50</sub&gt; É um ensaio de triagem para a ativação da via clássica do complemento (Fig. 1) e é sensível à redução, ausência e / ou inatividade de qualquer componente da via. O CH<sub&gt; 50</sub&gt; Testa a capacidade funcional de componentes do complemento sérico da via clássica para lisar hemácias de carneiro (SRBC) pré-revestida com anticorpos de coelho anti-células vermelhas do sangue de ovelhas (hemolisina). Quando revestidos com anticorpo SRBC são incubadas com soro de teste, a via clássica do complemento é ativado e os resultados hemólise. Se um componente do complemento está ausente, o CH<sub&gt; 50</subNível&gt; será zero, se um ou mais componentes da via clássica estão diminuídas, o CH<sub&gt; 50</sub&gt; Vai ser diminuída. Um volume fixo de SRBC ideal sensibilizados é adicionado a cada diluição do soro. Após a incubação, a mistura é centrifugada eo grau de hemólise é quantificada pela medida da absorbância da hemoglobina liberada no sobrenadante a 540 nm. A quantidade de atividade complementar é determinada examinando a capacidade de várias diluições do soro de ensaio para lisar SRBC revestidas de anticorpo. Este vídeo descreve os passos envolvidos na análise experimental do nível de atividade complementar da via clássica do complemento.

Watch this Scientific Journal Video about Measuring the 50% Haemolytic Complement CH50 Activity of Serum at JoVE.com

Measuring the 50% Haemolytic Complement CH50 Activity of Serum

A via clássica é ativada por anticorpos e culmina com a lise das células alvo. O CH<sub&gt; 50</subEnsaio&gt; fornece uma medida da atividade de complemento de uma amostra de soro. Este vídeo mostra os passos envolvidos na determinação do CH<sub&gt; 50</sub&gt; De uma amostra de soro, os cálculos e interpretação de resultados.

Medir o Complemento de 50% hemolítica (CH 50) Atividade de soro

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual ...

measuring-the-50-haemolytic-complement-ch50-activity-of-serum

Research

JoVE Journal

Immunology and Infection

Measuring the 50% Haemolytic Complement (CH50) Activity of Serum

37.5K visualizações.  University of South Australia. A via clássica é ativada por anticorpos e culmina com a lise das células alvo. O CH 50 fornece uma medida da atividade de complemento de uma amostra de soro. Este vídeo mostra os passos envolvidos na determinação do CH 50 De uma amostra de soro, os cálculos e interpretação de resultados.

Vídeo: Measuring the 50% Haemolytic Complement CH50 Activity of Serum

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

Esgotamento das populações de células específicas de Esgotamento Complemento

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Imunologia e Infecção

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 ...

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Medir a atividade das calpaínas em células fixas e Vivendo por citometria de fluxo

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Medir a carga bacteriana e resposta imune em camundongos infectados com Listeria monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

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 ...

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 ...

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 ...

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

Medindo Anafilaxia locais em Ratos

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

Medição de granulócitos e monócitos fagocitose e oxidativo Atividade Burst no Sangue

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

Ensaios para estudar o papel do vitronectina na adesão bacteriana e resistência de soro

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...

Methods Article

Medir o Complemento de 50% hemolítica (CH₅₀) Atividade de soro