Gostaria de agradecer Sreemanti Basu pelo fornecimento dos dados para a Figura 1. Este trabalho foi financiado em parte pelo NIH conceder AI069358 ea Fundação de Pesquisas BloodCenter.

A. complemento de coelho <ol><li> Complemento de coelho ações devem ser descongeladas em temperatura ambiente, e depois aliquotado em 0,5 e / ou volumes de 1 ml e congelado a -20-80 ° C para armazenamento de longo prazo. </li><li> Cada lote de complemento deverá ser titulado para determinar a concentração ideal de trabalho. Normalmente, uma diluição de 1:10 - 01:20 é o ideal. </li><li> Complemento de coelho não é sempre esterilizado antes da venda, dependendo do uso final das células, o complemento deve ser filtrado esterilizado quando aliquotado e congelado ou antes disso para as células. </li></ol> B. Seleção de anticorpos <ol><li> Nem todos os anticorpos são eficazes na ativação do complemento, os anticorpos, assim, primeiro deve ser testado para a atividade. </li><li> Ambos os ratos e ratinhos mAb são eficazes na ativação do complemento. </li><li> Anticorpos IgM são geralmente os mais eficientes para uso em complemento induzida pela lise celular seguido de IgG. Os isotipos IgG melhor para ativação do complemento variam entre as espécies. </li><li> Cada anticorpo deve ser ajustada para uso otimizado com uma densidade celular de 10 7 células / ml. </li></ol> C. Básico Protocolo <ol><li> Ajustar a população de células começando a 5 x 10 7 células / ml em meio contendo calor FCS inativado. O volume final das células será o dobro do volume inicial. Lembre-se de salvar algumas células para que o esgotamento pode ser quantificado. </li><li> Cada anticorpo deve ser adicionada à suspensão celular na concentração adequada predeterminado ou diluição. Misture as células bem por inversão. </li><li> Calcular o valor do complemento necessário. Descongelar o estoque complementar à temperatura ambiente. Se o complemento não é estéril, esterilizá-lo usando um filtro de baixa proteína de ligação ponta da seringa. Para a seringa, adicione alguns mililitros de meio antes da adição do complemento e filtro diretamente no tubo que contém as células e anticorpos. </li><li> Ajustar o volume final do tubo de modo que as células estão em 1 x 10 7 células / ml. </li><li> Incubar as células a 37 ° C por uma hora, misturando a cada 15 minutos. </li><li> Centrifugar as células e desprezar o sobrenadante. </li><li> Lavar com média duas vezes. Restos celulares vai ficar para o lado dos tubos de poliestireno. Células mortas e detritos pode ser eliminado usando um coador de celular ou por centrifugação em gradiente de densidade. </li><li> Verificar a pureza de células, comparando a população de células de interesse, antes e após o esgotamento. Citometria de fluxo e imunohistoquímica são dois bons métodos de determinação da pureza celular. </li></ol> Resultados representante Ao usar um mAb que é altamente eficiente em ativar complemento depleção de células, depois de uma rodada é maior do que 95%. Isso é mostrado na Figura 1, onde começamos com esplenócitos do mouse composto de células T αβ 32,2% conforme detectado pela expressão de TCRβ (eBioscience, San Diego, CA) por citometria de fluxo. Nós esgotados células T usando um mAb específicas para Thy1, uma proteína expressa por todas as células T, mas não outros leucócitos. O anticorpo utilizado foi o Y-19 um rato IgG2c 2. Seguindo o procedimento descrito, a população TCRβ foi reduzida para 1,56%, uma redução superior a 95%. <img src="/files/ftp_upload/1487/1487fig1.jpg" alt="Figura 1" /> Figura 1. Depleção de células T + thy1 esplênica pela ativação do complemento. Esplenócitos do mouse totais foram obtidos pela homogeneização do baço e lise de RBC. Anti-thy1 e complementar bebê coelho em concentrações pré-determinados foram adicionados a uma suspensão única célula de esplenócitos tal que a concentração celular final foi de 1 x 10 7 células / ml. Após a incubação por 1 hora a 37 ° C, as células foram lavadas duas vezes e avaliados pela exclusão de tripan azul para a viabilidade celular. A eficiência da depleção foi avaliada por citometria de fluxo comparando o percentual de TCRβ + células antes de depleção (painel à esquerda) e após a depleção (painel direito). A barra horizontal indica coloração positiva eo número acima da barra é o percentual positivo.

<ol>
	<li>Janeway, C., Travers, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Immune+System+in+Health+and+Disease.">The Immune System in Health and Disease.</a> Immunobiology. , (1994).</li><li>Jones, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+activities+of+antibodies+against+brain-associated+T+cell+antigens+II.+Stimulation+of+T+cell-induced+B+cell+proliferation.">Functional activities of antibodies against brain-associated T cell antigens II. Stimulation of T cell-induced B cell proliferation.</a> Eur J Immunol. 12, 30-37 (1982).</li></ol>

A purificação de populações de células é um procedimento comum necessário para o estudo das suas funções. Aqui nós descrevemos um método rápido e eficaz de esgotar populações de células com antígeno de anticorpos específicos e esgotamento complemento. O esgotamento de qualquer população de células pode ser alcançado se um anticorpo ativadores de complemento está disponível para um marcador de linhagem específica. Aqui demonstramos a depleção de células T usando um mAb específicas para Thy1, uma proteína expressa essencialmente por todas as populações de células T. Nós esgotado mais de 95% das células T αβ e os restantes esplenócitos foram aumentados de 68% para 98%. Este nível de pureza das células é semelhante a outras estratégias de purificação e separação incluindo FACS grânulo magnético. Esgotamento complemento é uma técnica rápida que pode ser realizado em 1,5 horas, uma vez a população de células de partida é obtida. Outra vantagem do esgotamento complemento é que qualquer laboratório pode realizar a técnica, pois não requer equipamento caro e reagentes. As únicas peças principais do equipamento necessário é um banho de água e uma centrífuga. Complemento de coelho pode ser comprado de uma variedade de fornecedores a um custo razoável (ver tabela de reagentes) e mAb muitos pode ser comprado de ATTC e cultivado localmente para controlar custos. Em contraste, FACS requer um citômetro de fluxo com capacidade de separação de células que vai custar nos seis números e caro mAb fluorescente etiquetado. Separação grânulo magnético está com preços moderados, mas requer a compra de um ímã e esferas magnéticas, que podem ser caros. Além disso, algumas estratégias de separação magnética talão também exigem a compra de colunas. O principal obstáculo para o uso de complemento para depleção de células é a disponibilidade de complemento ativando mAb com a especificidade de antígeno necessária. Embora os anticorpos do rato humano, e todos os ratos têm a capacidade de fixar o complemento, alguns isotipos são melhores que outros. Apesar de IgM de todas as três espécies é altamente eficaz na fixação complementar o isotipos IgG variar. Assim, cada mAb deve primeiro ser testada em um estudo piloto para determinar a sua eficácia. Para anticorpos que não são altamente eficientes, duas rodadas de esgotamento complementar pode ser realizada sem afetar a viabilidade das populações de células remanescentes. Como uma economia de custos, os anticorpos devem ser ajustadas para uma eficácia óptima. Além disso, os anticorpos utilizados não precisa ser purificado. Sobrenadantes hibridoma e ascite trabalho fluido tão eficazmente, que é particularmente conveniente para anticorpos IgM, que são difíceis de purificar. Tal como acontece com os anticorpos, é imperativo que o complemento ser titulada em um estudo piloto. Um muito alto de concentração resultará na perda da viabilidade das populações não-alvo celular. Também importante é que o tempo de incubação não exceda 60 minutos. Em nosso protocolo, que co-incubação do anticorpo e do complemento juntos. No entanto, as células podem ser rotulados com anticorpos para 15-30 minutos e lavado antes da adição do complemento. Ao usar esta estratégia, certifique-se de realizar a incubação de anticorpos no gelo para evitar a perda do antígeno de células na superfície da célula, como ocorre com algumas proteínas por patching e nivelamento ou internalização do receptor. Apesar de altamente eficiente, a principal desvantagem para complementar o esgotamento, em comparação com outros métodos é que ele não pode ser usado para a seleção positiva. Assim, é difícil obter subpopulações de células. Por exemplo, seria difícil para purificar as células B de zona marginal de células B folicular, o que seria melhor ser feito por FACS. Nesta situação, a ativação do complemento pode ser usado para retirada de células T para reduzir o número de células que estão sendo classificados. Esta estratégia seria reduzir o tempo de classificação, o que economizar dinheiro e seria menos severo nas células. Desde a purificação de populações de células específicas é um requisito comum uma série de estratégias têm sido desenvolvidas. As principais vantagens da estratégia de esgotamento complemento estão a sua acessibilidade, simplicidade técnica e economia de tempo.

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<td>3-4 week baby rabbit complement</td>
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<td>Water bath</td>
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<td>various</td>
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<td>An incubator set at 37 &deg;C can also be used.</td>
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depletion of specific cell populations by complement depletion

A purificação de populações de células imunes é muitas vezes necessária a fim de estudar as suas funções originais. Em particular, as abordagens moleculares, como PCR em tempo real e análise de microarrays requer o isolamento de populações de células com elevado grau de pureza. Estratégias de purificação comumente usados ​​incluem separação de células fluorescentes ativado (FACS), a separação grânulo magnético e esgotamento complemento. Das três estratégias, esgotamento complemento oferece as vantagens de ser rápido, barato, gentil nas células e um rendimento de células de altura. O sistema complemento é composto por um grande número de proteínas plasmáticas que quando ativados iniciam uma cascata proteolítica culminando com a formação de um complexo de ataque à membrana, que forma um poro em uma superfície da célula, resultando em morte celular<sup&gt; 1</sup&gt;. A via clássica é ativada por anticorpos IgM e IgG e foi descrita pela primeira vez como um mecanismo para matar bactérias. Com a geração de anticorpos monoclonais (mAb), a cascata do complemento pode ser usada para lisar qualquer população de células de uma maneira antígeno-específicas. Depleção de células, a cascata do complemento é obtido pela adição de complemento de fixação antígeno-anticorpos específicos e complemento de coelho para a população de células de partida. As células são incubadas por uma hora a 37 ° C e as células lisadas são posteriormente removidas por duas rodadas de lavar roupa. O mAb com uma alta eficiência de fixação de complemento geralmente destroem 95-100% da população de células-alvo. Dependendo da estratégia de purificação para a população de células alvo, esgotamento complemento pode ser utilizada para a purificação de células ou para o enriquecimento de populações de células que, então, pode ainda ser purificada por um método subseqüentes.

Watch this Scientific Journal Video about Depletion of Specific Cell Populations by Complement Depletion at JoVE.com

Depletion of Specific Cell Populations by Complement Depletion

Para efetivamente estudar a função de populações de células imunes sua purificação é muitas vezes necessária. Esgotamento complemento é uma técnica rápida e barata para o isolamento de populações de células imunes, com alta pureza.

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 ...

depletion-of-specific-cell-populations-by-complement-depletion

Research

JoVE Journal

Immunology and Infection

22.1K visualizações.  Blood Research Institute. Para efetivamente estudar a função de populações de células imunes sua purificação é muitas vezes necessária. Esgotamento complemento é uma técnica rápida e barata para o isolamento de populações de células imunes, com alta pureza.

Vídeo: Depletion of Specific Cell Populations by Complement Depletion

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Purificação da população de células específicas de separação de células de fluorescência Ativado (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Imunologia e Infecção

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

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual complement components can be quantified however this does not provide any information as to the activity of the pathway. The CH50 is a screening assay for the activation of the classical complement pathway (Fig 1) and it is sensitive to the reduction, absence and/or inactivity of any component of the pathway. The CH50 tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (haemolysin). When antibody-coated SRBC are incubated with test serum, the classical pathway of complement is activated and haemolysis results. If a complement component is absent, the CH50 level will be zero; if one or more components of the classical pathway are decreased, the CH50 will be decreased. A fixed volume of optimally sensitised SRBC is added to each serum dilution. After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the supernatant at 540nm. The amount of complement activity is determined by examining the capacity of various dilutions of test serum to lyse antibody coated SRBC. This video outlines the experimental steps involved in analysing the level of complement activity of the classical complement pathway.

Measuring the 50% Haemolytic Complement CH50 Activity of Serum

The classical pathway is activated by antibody and culminates in target cell lysis. The CH50 assay provides a measure of the complement activity of a serum sample. This video demonstrates the steps involved in determining the CH50 of a serum sample, the calculations and interpreting of results.

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 ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

Isolamento de variantes de vírus de RNA Fidelity e Caracterização de frequência de mutação de vírus

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

Depleção e reconstituição de macrófagos em Ratos

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

Visualização do imunológica Synapse por Dual Color Time-fechado Emissão Estimulada Exaustão (STED) Nanoscopia

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

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 ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Assassino Artificial células apresentadoras de antígenos (KaAPC) para a eficiente<em&gt; In Vitro</em&gt; Depleção de células T humanas antígeno específico da

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Macrófago Colesterol Exaustão e seu efeito sobre a fagocitose de<em&gt; Cryptococcus neoformans</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

Gerando<em&gt; De Novo</em&gt; Antígeno-específica T Humano receptores celulares por Transdução retroviral de Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion (STED) Microscopy

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure that optimizes B-cell receptor (BCR) signaling and BCR-mediated antigen acquisition. Both the remodeling of the actin cytoskeleton and the reorientation of the microtubule network towards the antigen contact site are essential for immune synapse formation. Remodeling of the actin cytoskeleton into a dense peripheral ring of F-actin is accompanied by polarization of the microtubule-organizing center towards the immune synapse. Microtubule plus-end binding proteins, as well as cortical plus-end capture proteins mediate physical interactions between the actin and microtubule cytoskeletons, which allow them to be reorganized in a coordinated manner. Elucidating the mechanisms that control this cytoskeletal reorganization, as well as understanding how these cytoskeletal structures shape immune synapse formation and BCR signaling, can provide new insights into B cell activation. This has been aided by the development of super-resolution microscopy approaches that reveal new details of cytoskeletal network organization. We describe here a method for using stimulated emission depletion (STED) microscopy to simultaneously image actin structures, microtubules, and transfected GFP-tagged microtubule plus-end binding proteins in B cells. To model the early events in immune synapse formation, we allow B cells to spread on coverslips coated with anti-immunoglobulin (anti-Ig) antibodies, which initiate BCR signaling and cytoskeleton remodeling. We provide step-by-step protocols for expressing GFP fusion proteins in A20 B-lymphoma cells, for anti-Ig-induced cell spreading, and for subsequent cell fixation, Immunostaining, image acquisition, and image deconvolution steps. The high-resolution images obtained using these procedures allow one to simultaneously visualize actin structures, microtubules, and the microtubule plus-end binding proteins that may link these two cytoskeletal networks.

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy

We present a protocol for using STED microscopy to simultaneously image actin structures, microtubules, and microtubule plus-end binding proteins in B cells that have spread on coverslips coated with antibodies to the B-cell receptor, a model for the initial phase of immune synapse formation.

Visualizando a actina e microtúbulos citoesqueleto na célula B sinapse imune usando microscopia de depleção (STED) emissão estimulada

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure ...