Me gustaría dar las gracias a Sreemanti Basu de proporcionar los datos de la Figura 1. Este trabajo fue apoyado en parte por subvención NIH AI069358 y la Fundación de Investigación BloodCenter.

A. Complemento de Conejo <ol><li> Complemento de archivo de conejo se deben descongelar a temperatura ambiente, y luego en alícuotas de 0,5 y / o volúmenes de 1 ml y congelado a -20 a 80 ° C para almacenamiento a largo plazo. </li><li> Cada lote de complemento se titula para determinar la concentración óptima de trabajo. Normalmente, una dilución de 1:10-1:20 es óptima. </li><li> Complemento de conejo no siempre es esterilizado antes de su venta, en función del uso final de las células, el complemento debe ser filtrada esterilizada y congelada en alícuotas o antes de la adición a las células. </li></ol> B. Selección de anticuerpos <ol><li> No todos los anticuerpos son eficaces en la activación del complemento, por lo tanto los primeros anticuerpos debe ser probado para la actividad. </li><li> Ratón y rata MAB son eficaces en la activación del complemento. </li><li> Anticuerpos IgM son generalmente los más eficaces para su uso en la lisis celular inducida por el complemento seguido de IgG. Los isotipos IgG mejor para la activación del complemento varía entre las especies. </li><li> Cada anticuerpo se debe ajustar para un uso óptimo a una densidad celular de 10 7 células / ml. </li></ol> C. Protocolo Básico <ol><li> Ajuste la población de células a partir de 5 x 10 7 células / ml en medio del calor que contiene FCS inactivado. El volumen final de las células será el doble del volumen inicial. Recuerde guardar algunas células, de modo que el agotamiento puede ser cuantificado. </li><li> Cada anticuerpo se debe agregar a la suspensión celular en la concentración adecuada determinado o la dilución. Mezcla de las células mediante la inversión. </li><li> Calcular la cantidad de complemento necesario. Descongele las acciones complementan a temperatura ambiente. Si el complemento no es estéril, esterilizar usando una proteína de baja unión filtro punta de la jeringa. A la jeringa, añadir algunos mililitros de medio antes de la adición del complemento y el filtro directamente en el tubo que contiene las células y anticuerpos. </li><li> Ajustar el volumen final en el tubo de modo que las células son de 1 x 10 7 células / ml. </li><li> Se incuban las células a 37 ° C durante una hora, la mezcla cada 15 minutos. </li><li> Centrifugar las células y descartar el sobrenadante. </li><li> Lave con el medio dos veces. Los restos celulares se adhieren a la parte de los tubos de poliestireno. Las células muertas y los desechos pueden ser eliminados mediante un filtro de celular o por centrifugación en gradiente de densidad. </li><li> Comprobar la pureza de la célula mediante la comparación de la población celular de interés antes y después de agotamiento. La citometría de flujo e inmunohistoquímica son dos buenos métodos para determinar la pureza de la célula. </li></ol> Resultados representante Cuando se utiliza un mAb que es muy eficaz en la activación del complemento, la depleción de células después de una ronda es mayor que el 95%. Esto se muestra en la Figura 1 donde comenzamos con esplenocitos de ratón compuesto de células T αβ 32,2% detectado por la expresión de TCRβ (eBioscience, San Diego, CA) por citometría de flujo. Hemos agotado las células T con un mAb específico para Thy1, una proteína expresada por todas las células T, pero no otros leucocitos. El anticuerpo que se utilizó Y-19 de una rata IgG2c 2. Siguiendo el procedimiento descrito, la población se redujo a TCRβ 1,56%, una reducción superior al 95%. <img src="/files/ftp_upload/1487/1487fig1.jpg" alt="Figura 1" /> Figura 1. El agotamiento de las células del bazo thy1 + T por la activación del complemento. Esplenocitos totales de ratón se obtuvieron mediante la homogeneización del bazo y la lisis de glóbulos rojos. Anti-thy1 y complementar bebé conejos en concentraciones predeterminadas se han añadido a una suspensión de células individuales de los esplenocitos de tal manera que la concentración celular final fue 1 x 10 7 células / ml. Después de la incubación durante 1 hora a 37 ° C, las células fueron lavadas dos veces y evaluados por la exclusión de azul tripan para la viabilidad celular. La eficiencia de agotamiento se evaluó por citometría de flujo se compara el porcentaje de TCRβ + células antes de agotamiento (izquierda) y después de agotamiento (panel derecho). La barra horizontal indica tinción positiva y el número por encima de la barra es el porcentaje de positivos.

<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>

La purificación de las poblaciones de células es un procedimiento común necesarios para el estudio de sus funciones. Aquí se describe un método rápido y eficaz de agotar las poblaciones de células con antígeno de los anticuerpos específicos y el agotamiento del complemento. El agotamiento de cualquier población de células se puede lograr si un anticuerpo complemento de activación está disponible para un marcador específico de linaje. Aquí hemos demostrado el agotamiento de las células T con un mAb específico para Thy1, una proteína expresada por prácticamente todas las poblaciones de células T. Hemos agotado más del 95% de las células T αβ y los esplenocitos restantes se incrementaron del 68% al 98%. Este nivel de pureza de las células es similar a otras estrategias de purificación y separación entre FACS cuentas magnéticas. El agotamiento del complemento es una técnica rápida que se pueden realizar en 1,5 horas una vez que la población de células de partida se obtiene. Otra ventaja del agotamiento del complemento es que cualquier laboratorio puede realizar la técnica, ya que no requieren equipos costosos y reactivos. Las únicas piezas principales del equipo requerido es un baño de agua y una centrífuga. Complemento de conejo se pueden comprar en una variedad de proveedores a un costo razonable (véase la tabla de reactivos) y muchos otros AcM se pueden comprar de ATTC y de origen local para controlar los costos. Por el contrario, FACS requiere un citómetro de flujo con una capacidad de clasificación de células que tendrá un costo de seis figuras y caras marcado con fluorescencia mAb. Separación de esferas magnéticas es de precio moderado, pero requiere la compra de un imán y las bolas magnéticas, que pueden ser costosos. Además, algunas estrategias de separación con bolas magnéticas también requieren la compra de las columnas. El principal obstáculo para el uso del complemento para el agotamiento de las células es la disponibilidad de complementar la activación de AcMo con especificidad de antígeno requerida. Aunque los anticuerpos humanos, ratones y ratas tienen la capacidad de fijar el complemento, algunos isotipos son mejores que otros. A pesar de IgM de las tres especies es muy eficaz para la fijación de complemento de los isotipos IgG varían. Así, cada AcM primero debe ser probado en un estudio piloto para determinar su eficacia. Los anticuerpos que no son muy eficientes, dos rondas de agotamiento de complemento se puede realizar sin afectar la viabilidad de las poblaciones de células restantes. Como un ahorro de costes, los anticuerpos se debe ajustar para una eficacia óptima. Además, los anticuerpos utilizados no necesita ser purificado. Hibridoma sobrenadantes y el trabajo ascitis fluido con la misma eficacia, que es particularmente conveniente para los anticuerpos IgM, que son difíciles de purificar. Al igual que con los anticuerpos, es imprescindible que el complemento de ser valorados en un estudio piloto. Una alta concentración de demasiado resultará en la pérdida de la viabilidad de las poblaciones de células no específicas. También es importante que el tiempo de incubación no exceda de 60 minutos. En nuestro protocolo, que co-incubar el anticuerpo y el complemento del conjunto. Sin embargo, las células pueden ser etiquetados con anticuerpos durante 15-30 minutos y se lava antes de la adición del complemento. Cuando se utiliza esta estrategia, asegúrese de llevar a cabo las incubaciones de anticuerpos en el hielo para evitar la pérdida del antígeno de células en la superficie celular, como ocurre con algunas de las proteínas mediante parches y la limitación o la internalización del receptor. Aunque altamente eficiente, el principal inconveniente para complementar el agotamiento, en comparación con otros métodos es que no se puede utilizar para la selección positiva. Por lo tanto, es difícil obtener las subpoblaciones de células. Por ejemplo, sería difícil de purificar las células B de zona marginal de células B folicular, que se podría realizar por FACS. En esta situación, la activación del complemento se podría utilizar la depleción de células T para reducir el número de células se van a ordenar. Esta estrategia reduciría el tiempo de clase, lo que ahorraría dinero y podría no ser tan duro en las células. Desde la purificación de poblaciones celulares específicas es un requisito común una serie de estrategias se han desarrollado. Las principales ventajas de la estrategia de reducción del complemento son su accesibilidad, simplicidad técnica y ahorro de tiempo.

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<td>3-4 week baby rabbit complement</td>
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<td>Pel-Freez Biologicals</td>
<td>31061-3</td>
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<td>Water bath</td>
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<td>various</td>
<td>various </td>
<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

La purificación de las poblaciones de células inmunes se requiere a menudo con el fin de estudiar sus funciones exclusivas. En determinados enfoques, molecular como PCR en tiempo real y análisis de microarrays de requerir el aislamiento de las poblaciones de células con alta pureza. Comúnmente utilizado estrategias de purificación son fluorescentes clasificación de células activadas (FACS), la separación de esferas magnéticas y el agotamiento del complemento. De las tres estrategias, el agotamiento del complemento ofrece la ventaja de ser rápido, barato, suave con las células y una producción de células de alta. El sistema del complemento se compone de un gran número de proteínas plasmáticas que cuando se activa iniciar una cascada proteolítica que culminó en la formación de un complejo de la membrana de ataque que forma un poro en la superficie de una célula que resulta en la muerte celular<sup&gt; 1</sup&gt;. La vía clásica es activada por los anticuerpos IgM e IgG y fue descrita por primera vez como un mecanismo para matar las bacterias. Con la generación de anticuerpos monoclonales (mAb), la cascada del complemento se puede utilizar para lisar cualquier población de células de una forma antígeno-específica. Agotamiento de las células de la cascada del complemento se logra mediante la adición de antígeno de fijación del complemento específico de los anticuerpos y complemento de conejo a la población de células de partida. Las células se incubaron durante una hora a 37 ° C y las células lisadas se retira posteriormente por dos rondas de lavado. MAb con una alta eficiencia para la fijación del complemento suele agotar el 95-100% de la población de células específicas. Dependiendo de la estrategia de purificación de la población de células específicas, el agotamiento del complemento se puede utilizar para la purificación de células o para el enriquecimiento de las poblaciones de células que luego pueden ser purificados por el método siguiente.

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 estudiar con eficacia la función de las poblaciones de células inmunes a menudo es la purificación necesaria. El agotamiento del complemento es una técnica rápida y barata para el aislamiento de las poblaciones de células inmunes de alta pureza.

El agotamiento de las poblaciones de células específicas por el agotamiento del 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

22.0K vistas.  Blood Research Institute. Para estudiar con eficacia la función de las poblaciones de células inmunes a menudo es la purificación necesaria. El agotamiento del complemento es una técnica rápida y barata para el aislamiento de las poblaciones de células inmunes de alta pureza.

Video: 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.

La purificación de la población de células específicas de clasificación celular activado por fluorescencia (FACS)

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

Investigación

Inmunología e Infección

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.

La medición del complemento hemolítico 50% (CH 50) La actividad de suero

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.

El aislamiento de las variantes de los virus de ARN de fidelidad y caracterización de la frecuencia de mutación de virus

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.

El agotamiento y la reconstitución de los macrófagos en ratones

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.

La visualización de la sinapsis inmunológica mediante gated-Tiempo color dual Emisión Estimulada de Agotamiento (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.

Los métodos para la detección cuantitativa de anticuerpos inducida por la activación del complemento en los glóbulos rojos

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.

Killer Artificial células presentadoras de antígenos (KaAPC) para la utilización eficiente<em&gt; In Vitro</em&gt; El agotamiento de células T humanas específicas de antígeno

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.

El colesterol de los macrófagos agotamiento y su efecto sobre la fagocitosis 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.

Generando<em&gt; De Novo</em&gt; T humanas receptores de células específicas de antígeno por retroviral Transducción 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.

Visualización de la actina y los microtúbulos citoesqueletos en la sinapsis inmune de células B mediante microscopía de emisión (STED) el agotamiento

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 ...