Vorrei ringraziare Sreemanti Basu di fornire i dati di Figura 1. Questo lavoro è stato sostenuto in parte dal NIH concedere AI069358 e la Fondazione di Ricerca BloodCenter.

A. Coniglio Complemento <ol><li> Coniglio complemento di riserva dovrebbe essere scongelato a temperatura ambiente, e poi aliquotati in 0,5 e / o volumi di 1 ml e congelati a -20-80 ° C per conservazione a lungo termine. </li><li> Ogni lotto di complemento dovrà essere titolato per determinare la concentrazione ottimale di lavoro. Tipicamente, una diluizione di 1:10-1:20 è ottimale. </li><li> Complemento di coniglio non è sempre presterilizzati prima della vendita, a seconda dell&#39;uso finale delle celle, il complemento deve essere filtrato sterilizzati quando aliquotati e congelati o prima di essere aggiunti alle cellule. </li></ol> B. anticorpi selezione <ol><li> Non tutti gli anticorpi sono efficaci in fase di attivazione del complemento, in tal modo gli anticorpi devono essere prima testati per l&#39;attività. </li><li> Sia topo e nel ratto mAb sono efficaci in fase di attivazione del complemento. </li><li> Gli anticorpi IgM sono generalmente i più efficienti per l&#39;uso in complemento indotta lisi cellulare seguito da IgG. L&#39;isotipo IgG meglio per l&#39;attivazione del complemento variano tra le specie. </li><li> Ogni anticorpo deve essere titolato per un uso ottimale delle cellule ad una densità di 10 7 cellule / ml. </li></ol> C. Protocollo di base <ol><li> Regolare la popolazione cellulare di partenza per 5 x 10 7 cellule / ml in mezzo contenente calore FCS inattivato. Il volume finale delle celle sarà il doppio del volume di partenza. Ricordati di salvare alcune cellule in modo che la riduzione può essere quantificata. </li><li> Ogni anticorpo deve essere aggiunto alla sospensione cellulare alla concentrazione corretta predeterminato o di diluizione. Mescolare le cellule bene per inversione. </li><li> Calcolare la quantità di complemento necessario. Scongelare lo stock di complemento a temperatura ambiente. Se il complemento non è sterile, sterilizzare utilizzando un basso legame con le proteine ​​filtro siringa. Alla siringa, aggiungere alcuni millilitri di media prima l&#39;aggiunta del complemento e il filtro direttamente nella provetta contenente le cellule e anticorpi. </li><li> Regolare il volume finale del tubo in modo che le cellule sono a 1 x 10 7 cellule / ml. </li><li> Incubare le cellule a 37 ° C per un&#39;ora, mescolando ogni 15 minuti. </li><li> Centrifugare le cellule ed eliminare il surnatante. </li><li> Lavare con media due volte. Detriti cellulari che si attacchi al lato di tubi in polistirolo. Le cellule morte e detriti possono essere eliminate con un colino o cellulare per centrifugazione a gradiente di densità. </li><li> Verificare la purezza delle cellule confrontando la popolazione di cellule prima e dopo l&#39;esaurimento. Citometria a flusso ed immunoistochimica sono due buoni metodi per determinare la purezza delle cellule. </li></ol> Rappresentante Risultati Quando si utilizza un anticorpo monoclonale che è altamente efficiente ad attivare complemento, deplezione delle cellule dopo un round è superiore al 95%. Questo è illustrato nella figura 1, dove abbiamo iniziato con splenociti del mouse composti da cellule T αβ 32,2%, come rilevato da espressione di TCRβ (eBioscience, San Diego, CA) in citometria a flusso. Abbiamo esaurito le cellule T utilizzando un mAb specifici per Thy1, una proteina espressa da tutte le cellule T, ma non altri leucociti. L&#39;anticorpo che abbiamo usato era Y-19 di un ratto IgG2c 2. Seguendo la procedura descritta, la popolazione TCRβ è stata ridotta a 1,56%, una riduzione superiore al 95%. <img src="/files/ftp_upload/1487/1487fig1.jpg" alt="Figura 1" /> Figura 1. L&#39;esaurimento delle Thy1 + cellule T della milza attraverso l&#39;attivazione del complemento. Splenociti del mouse totali sono stati ottenuti dalla omogeneizzazione della milza e lisi dei globuli rossi. Anti-Thy1 e coniglio complemento bambino a concentrazioni predeterminate sono stati aggiunti una sospensione singola cella di splenociti tale che la concentrazione cellulare finale è stato 1 x 10 7 cellule / ml. Dopo l&#39;incubazione per 1 ora a 37 ° C, le cellule sono state lavate due volte e valutate da esclusione trypan blu per la vitalità cellulare. L&#39;efficienza di deplezione è stata valutata mediante citometria di flusso confrontando la percentuale di TCRβ + cellule prima di esaurimento (pannello a sinistra) e dopo l&#39;esaurimento (pannello di destra). La barra orizzontale indica la colorazione positiva e il numero sopra la barra è la percentuale 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>

La purificazione di popolazioni cellulari è una procedura comune richiesto per lo studio delle loro funzioni. Qui si descrive un metodo veloce ed efficace riducono popolazioni di cellule con antigene-anticorpi specifici e l&#39;esaurimento del complemento. L&#39;esaurimento di ogni popolazione di cellule può essere raggiunto se un complemento, l&#39;attivazione di anticorpi è disponibile a un marcatore specifico lignaggio. Qui abbiamo dimostrato l&#39;esaurimento delle cellule T utilizzando un mAb specifici per Thy1, una proteina espressa da tutti essenzialmente popolazioni cellulari T. Noi impoverito oltre il 95% delle cellule T αβ splenociti e le rimanenti sono stati aumentati dal 68% al 98%. Questo livello di purezza cella è simile ad altre strategie di purificazione compresi FACS e magnetici separazione tallone. Esaurimento complemento è una tecnica veloce che può essere eseguita in 1,5 ore una volta che la popolazione cellulare di partenza è ottenuto. Un altro vantaggio di esaurimento complemento è che ogni laboratorio può eseguire la tecnica in quanto non richiede costose attrezzature e reagenti. L&#39;unica elementi principali degli strumenti richiesti sono un bagno d&#39;acqua e una centrifuga. Complemento di coniglio può essere acquistato da una varietà di fornitori a costi ragionevoli (vedi tabella dei reagenti) e molti mAb possono essere acquistati presso ATTC e cresciuto a livello locale per controllare i costi. Al contrario, FACS richiede un citofluorimetro con capacità delle cellule di smistamento che costerà in sei cifre e costoso fluorescente tag mAb. Separazione magnetica tallone è un prezzo abbordabile, ma richiede l&#39;acquisto di un magnete e sfere magnetiche che possono essere costosi. Inoltre, alcune strategie di separazione magnetica tallone richiedono anche l&#39;acquisto di colonne. Il principale ostacolo per l&#39;utilizzo del complemento per deplezione delle cellule è la disponibilità di complemento attivare mAb con la specificità dell&#39;antigene necessario. Sebbene anticorpi umani, topi e ratti hanno tutti la capacità di fissare complemento, alcuni isotipi sono migliori di altri. Anche se IgM da tutte e tre le specie è altamente efficace a fissare il complemento isotipi IgG variare. Così ogni mAb deve prima essere testato in uno studio pilota per determinare la sua efficacia. Per gli anticorpi che non sono molto efficienti, due turni di esaurimento complemento può essere effettuata senza compromettere la vitalità delle popolazioni di cellule rimanenti. Come un risparmio sui costi, gli anticorpi dovrebbero essere titolati per un&#39;efficacia ottimale. Inoltre, gli anticorpi utilizzati non hanno bisogno di essere purificati. Surnatanti di ibridomi e liquido ascitico lavoro in modo altrettanto efficace, il che è particolarmente conveniente per gli anticorpi IgM, che sono difficili da purificare. Come per gli anticorpi, è indispensabile che il complemento di essere titolati in uno studio pilota. Un troppo elevato di concentrazione comporterà la perdita di vitalità di non mirato popolazioni cellulari. Altrettanto importante è che il tempo di incubazione non superi i 60 minuti. Nel nostro protocollo, abbiamo co-incubare l&#39;anticorpo e il complemento insieme. Tuttavia, le cellule possono essere etichettati con anticorpi per 15-30 minuti e lavati prima l&#39;aggiunta del complemento. Quando si usa questa strategia, assicurarsi di eseguire le incubazioni anticorpi sul ghiaccio per prevenire la perdita dell&#39;antigene cellule sulla superficie della cellula, come accade con alcune proteine ​​di patch e tappatura o internalizzazione recettoriale. Anche se ad alta efficienza, l&#39;inconveniente principale per completare l&#39;esaurimento rispetto ad altri metodi è che non può essere utilizzato per la selezione positiva. Così è difficile ottenere le sottopopolazioni di cellule. Per esempio, sarebbe difficile per purificare le cellule B della zona marginale a partire da cellule B follicolari, che sarebbe meglio essere fatto da FACS. In questa situazione, l&#39;attivazione del complemento potrebbero essere utilizzati per deplezione delle cellule T per ridurre il numero di celle da ordinare. Questa strategia riduce il tempo per specie, che permetterebbe di risparmiare denaro e sarebbe meno dura sulle cellule. Dal momento che la purificazione delle popolazioni di cellule specifiche è un requisito comune una serie di strategie sono state sviluppate. I vantaggi principali della strategia di riduzione del complemento sono la sua economicità, semplicità tecnica e risparmio di tempo.

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<td>3-4 week baby rabbit complement</td>
<td>&nbsp;</td>
<td>Pel-Freez Biologicals</td>
<td>31061-3</td>
<td>&nbsp;</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 purificazione di popolazioni di cellule immunitarie è spesso necessaria per poter studiare le loro funzioni uniche. In particolare, gli approcci molecolari come la PCR in tempo reale e analisi microarray richiedono l&#39;isolamento di popolazioni di cellule con elevata purezza. Strategie di purificazione comunemente usati includono fluorescenti separazione delle cellule attivate (FACS), separazione magnetica tallone e la deplezione del complemento. Delle tre strategie, l&#39;esaurimento complemento offre i vantaggi di essere veloce, poco costoso, delicato sulle cellule e una resa cellula alto. Il sistema del complemento è composto da un gran numero di proteine ​​del plasma che, quando attivato avviare una cascata proteolitica si conclude con la formazione di una membrana attacco complesso che forma un poro sulla superficie cellulare con conseguente morte cellulare<sup&gt; 1</sup&gt;. La via classica è attivata da anticorpi IgM ed IgG ed è stato descritto come un meccanismo per uccidere i batteri. Con la generazione di anticorpi monoclonali (mAb), la cascata del complemento può essere usato per lisare qualsiasi popolazione di cellule in un antigene specifico modo. Deplezione delle cellule dalla cascata del complemento si ottiene con l&#39;aggiunta del complemento di fissaggio antigene-anticorpi specifici e complemento di coniglio alla popolazione di cellule di partenza. Le cellule vengono incubate per un&#39;ora a 37 ° C e le cellule lisate vengono successivamente rimossi con due turni di lavaggio. Mab ad alta efficienza per la fissazione del complemento tipicamente esaurire 95-100% della popolazione di cellule bersaglio. A seconda della strategia di depurazione per la popolazione di cellule bersaglio, l&#39;esaurimento complemento può essere usato per la purificazione delle cellule o per l&#39;arricchimento di popolazioni di cellule che poi può essere ulteriormente purificato con un metodo successivo.

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

Per studiare in modo efficace la funzione di popolazioni di cellule immunitarie loro purificazione è spesso richiesto. Esaurimento complemento è una tecnica veloce ed economico per l&#39;isolamento di popolazioni di cellule immunitarie con elevata purezza.

L&#39;esaurimento delle popolazioni di cellule specifiche da deplezione 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 Views.  Blood Research Institute. Per studiare in modo efficace la funzione di popolazioni di cellule immunitarie loro purificazione è spesso richiesto. Esaurimento complemento è una tecnica veloce ed economico per l'isolamento di popolazioni di cellule immunitarie con elevata purezza.

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.

Purificazione della popolazione di cellule specifiche per fluorescenza separazione delle cellule attivate (FACS)

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

Ricerca

Immunologia e infezioni

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.

Misura del Complemento 50% emolitica (CH 50) Attività di siero

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 di varianti di virus RNA Fidelity e caratterizzazione di frequenza Virus Mutation

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.

Esaurimento e Ricostituzione dei macrofagi nei topi

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.

Visualizzazione della immunologica Synapse da Dual Color Time-gated emissione stimolata Depletion (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.

Metodi per quantitativa individuazione di anticorpi indotta da attivazione del complemento sui globuli rossi

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 artificiale cellule presentanti l&#39;antigene (KaAPC) per una efficiente<em&gt; In Vitro</em&gt; L&#39;esaurimento delle Umana antigene-specifiche cellule T

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.

Macrofagi Colesterolo Depletion e il suo effetto sulla fagocitosi di<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.

Generazione<em&gt; De Novo</em&gt; Antigene-specifici T umana recettori cellulari di trasduzione retrovirale di 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.

Visualizzando l'actina e Cytoskeletons dei microtubuli alla sinapsi della B-cellula Immune usando la microscopia di svuotamento (STED) emissione stimolata

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 ...