Questa ricerca è stata finanziata dal NIH AI062732 K08, K12 HD001401 e 1UL1RR024139-02 e una salute di Grant Bambino Ricerca dalla H. Charles Hood Foundation per SB-M. e dalla Fondazione di Ricerca presso l&#39;Università dello Stato di New York a Stony Brook.

1. EBV magazzino preparazione <ol><li> Subculture in crescita esponenziale B95-8 celle (ATCC # CRL 1612, 13) a 3 x 10 5 cellule / ml in un pallone di 75 centimetri 2 di coltura tissutale con tecnica sterile. Le cellule sono coltivate in RPMI 1640 completo contenente il 10% di siero siero fetale bovino (FBS), penicillina / streptomicina a 100U/ml e 100 mcg / ml, rispettivamente, e amfotericina B a 0,5 mcg / ml a 37 ° C in presenza del 5% di CO 2. </li><li> Quarantotto ore dopo, risospendere le cellule in fresco completo RPMI 1640 con 1 x 10 6 cellule / ml. Per indurre la produzione del virus, stimolano le cellule con acetato di 20ng/ml tetradecanoyl forbolo (TPA) per 1 ora in un incubatore standard di CO 2. Lavare le cellule tre volte con RPMI 1640 per rimuovere TPA. </li><li> Risospendere le cellule del volume originale di completo RPMI 1640 (da 1,2) e posizionare il pallone in CO 2 incubatore per 96 ore. Questo metodo ha dimostrato di produrre alti titoli di par virus infettivoticles 6. </li><li> Centrifugare a 600 x g per 10 minuti a 4 ° C per separare EBV contenenti supernatante della coltura dalle cellule. Surnatante filtrare attraverso un filtro di 0,45 micron, aliquota, e conservare a -70 ° C per più di un anno. Un&#39;alternativa è quella di ottenere il supernatante da B95-8 celle contenenti EBV dalla ATCC (VR-1492) e utilizzare alla diluizione raccomandata dalla ATCC. </li></ol> 2. Isolamento delle cellule mononucleate del sangue periferico (PBMC) <ol><li> Draw 10 ml di sangue da donatore in una siringa eparina o un tubo di sangue eparinato. Diluire il sangue con 20 ml di PBS a temperatura ambiente in una provetta da 50 ml. </li><li> Alla base di sangue diluito con 15 ml di mezzo di separazione Ficoll Hypaque linfociti. Centrifugare a 225 x g senza freno a temperatura ambiente per 30 minuti. </li><li> Rimuovere il buffy coat e trasferire in un nuovo tubo da 50 ml. Aumentare il volume a 50 ml con PBS e spin a 600 xg a temperatura ambiente per 10 minuti. </li><li> Pour off surnatante. Lavare le cellule da risospendere pellet in 50 ml di PBS e filatura a 600 x g a temperatura ambiente per 10 minuti. Lavare due volte in un modo simile. </li><li> Risospendere lavato le cellule in 1 ml di RPMI completo. Usare 5 ml di cellule di preparare una diluizione 1 / 10 in tripan blu. Contare le cellule vive usando un emocitometro. </li><li> Regolare il volume con RPMI completo in un centimetro 25 2 fiasco di coltura per ottenere una concentrazione cellulare di 2 x 10 6 / ml. </li></ol> 3. L&#39;infezione da EBV <ol><li> Aggiungi FK506 (AG scientifico) alla sospensione cellulare da 2.6 a una concentrazione finale di 20 nm. Pallone posto di CO 2 incubatore a 37 ° C per un&#39;ora. </li><li> Scongelare rapidamente una aliquota di EBV. Rimuovere beuta da incubatore e aggiungere EBV nelle cellule di diluizione 1 / 10. In genere, questa diluizione fornisce una MOI di 50-100. Swirl pallone per mescolarne e lo mettono diritto di CO 2 incubatore a 37 ° C. </li></ol> Nota °al punto 4, eseguita per predire l&#39;esito di successo, è un passo opzionale. Se Fase 4 deve essere eseguita, è necessario incubare un pallone aggiuntivo di non-infetti PBMC con 2 x 10 6 cellule / ml come controllo. 4. L&#39;identificazione della popolazione di cellule proliferanti (passo opzionale) <ol><li> Preparare FACS buffer: PBS + 5% FBS. Conservare a 4 ° C. </li><li> Effettuare l&#39;anticorpo seguenti mescola in 50 microlitri di volume ciascuno per la colorazione delle cellule nella fase 4.6. Diluizioni degli anticorpi devono essere effettuati in tampone FACS contenente 1mg/ml del mouse IgG (Sigma) per inibire legame non specifico dagli anticorpi. <ol class="lroman"><li> singolo-colorati con PE coniugato anticorpo anti-CD23 (1 / 50 di diluizione) </li><li> singolo-colorati con FITC coniugato anticorpo anti-CD58 (1 / 50 di diluizione) </li><li> singolo-colorati con PE-Cy5 coniugato anticorpo anti-CD19 (1 / 50 di diluizione) </li><li> triplo macchiato per CD23, CD58 e CD19 (1 / 50 ogni diluizione) </li><li> singolo-colorati con controllo isotipico coniugato PEanticorpi abbinato a CD23-PE (alla stessa concentrazione di anticorpi anti-CD23) </li><li> singolo-colorati con FITC coniugato controllo isotipo abbinato a CD58-FITC (alla stessa concentrazione di anticorpi anti-CD58) </li><li> singolo-colorati con PE-Cy5 coniugato anticorpo di controllo isotipo abbinato a CD19-PE-Cy5 (alla stessa concentrazione di anticorpi anti-CD19) </li><li> tre colorate con tutti gli anticorpi isotipo controllo a tre. </li></ol> Memorizzare temporaneamente la mescola a 4 ° C al buio. </li><li> In tre giorni post-esposizione ad EBV, delicatamente fiasco agitare per ottenere una sospensione cellulare più uniforme. Rimuovere da 2ml matraccio in un tubo di 2 ml Eppendorf. Spin tubo in una microcentrifuga a 3000 rpm a temperatura ambiente per 3 minuti. </li><li> Aspirare il surnatante e risospendere le cellule in tampone FACS a 5 x 10 6 a 1 x 10 7 cellule / ml. </li><li> Rimuovere otto aliquote 50μl in singoli tubi Eppendorf o un 96-well V-parte inferiore della piastra. Tubi di rotazione o la piastra a 1500 x gper 3 minuti. </li><li> Eliminare il surnatante e tubi vortex / piastra. Risospendere le cellule in ogni tubo / pozzetto in 50 ml di tampone FACS contenente diluizioni degli anticorpi adeguati preparati in 4.2. </li><li> Incubare le provette / piastre in ghiaccio al buio per 30 minuti. Cellule centrifugare a 3000 rpm per 3 minuti, togliere il surnatante e aggiungere 200 ml di tampone FACS. Centrifugare le cellule di nuovo e rimuovere il surnatante per completare il lavaggio. Ripetere due lavaggi più. </li><li> Risospendere le cellule in 200 microlitri di buffer FACS ed acquisire i dati utilizzando un citofluorimetro, l&#39;acquisizione di 20.000 eventi / tubo. </li><li> Analizzare i dati utilizzando WinMDI (freeware per l&#39;analisi FACS dati su PC). Porta su cellule vive usando i profili scatter in avanti e laterale. Poi, le cellule porta dal vivo per l&#39;espressione di CD19 + (cellule B marker) dopo il confronto con le cellule colorate con anticorpo di controllo isotipo abbinato a degli anticorpi anti-CD19. Plot CD19 + vivere cellule B con intensità di fluorescenza per CD23 su asse x e l&#39;intensità di fluorescenza per CD58 su asse y. Determinare CD23 + CD58 + e cellule confrontando per EBV-esposto le cellule colorate con anticorpi isotipo di controllo abbinati. Presenza di CD58 + CD23 hi cellule EBV-esposti cultura (Figura 2C) predice escrescenza successo di EBV-infettate crescita delle cellule trasformate. Al contrario, le cellule CD23 + CD58 hi non emergono quando le cellule non siano esposti a EBV (Figura 2A) o esposti ad un non-immortalando ceppo EBV (Figura 2B). Hi CD58 delle cellule CD23 + sono stati sperimentalmente dimostrato di subire proliferazione e successivamente stabilire LCL (14). </li></ol> 5. Espansione e crioconservazione di LCL <ol><li> Visualizzazione delle cellule mediante microscopia ottica: per una settimana dopo l&#39;infezione da EBV, gruppi di cellule sono visibili al microscopio ottico. La figura 3 mostra un esempio dei primi gruppi microscopici (Figura 3B) in un pallone. </li><li> Col passare del tempo, diventano più grandi ammassi microscopici grumi in modo che siano visibilimacroscopicamente nel pallone. Figura 3C mostra grandi ammassi di cellule stabilito LCL al microscopio ottico. </li><li> Alimentazione celle: doppio del volume del terreno di coltura nel pallone cultura il giorno 12. Successivamente, espandere la cultura aumentando il suo volume 2-3 volte con RPMI completo. </li><li> Periodicità di cellule di alimentazione deve essere determinato sulla base del tasso di crescita delle cellule. Quando il terreno di coltura diventa giallo, è generalmente il momento di sostituire i mezzi di comunicazione di cui sopra. Tipicamente, questo accade una volta alla settimana. Tuttavia, alcune linee cellulari può avere bisogno di essere alimentato più o meno frequentemente. Espandere la cultura di 75-100 ml nelle settimane prossime. </li><li> Crioconservazione: congelamento Quando le cellule, le cellule centrifugare a 600 x g per 10 minuti a temperatura ambiente. Rimuovere il surnatante e risospendere pellet di cellule in medium freddo gelido contenente 90% FBS e il 10% dimetilsolfossido a 1 x 10 7 cellule / ml. Tubo posto in azoto liquido. </li></ol> 6. Rappresentante dei risultati: Risultato positivo è previsto per la presenza di CD23 hi cellule CD58 +. Circa il 2-3% di cellule vive per 3-4 giorni dopo l&#39;esposizione a EBV dovrebbe avere questo profilo (Figura 2C). Altri sotto-popolazioni di CD23 +, CD23 -, CD58 +, CD58 e - cellule osservata dopo l&#39;esposizione a EBV mostrare proliferazione minimo 14. Un cellule infettate (Figura 2A) e le cellule esposte a EBV da HH514-16 cellule (Figura 2B), l&#39;ospitare un non-immortalando ceppo EBV, non dimostrano hi CD58 + CD23 delle cellule. Si può anche visualizzare le cellule al microscopio ottico per il monitoraggio dell&#39;esito positivo: Come accennato in precedenza, entro una settimana dopo l&#39;infezione da EBV, piccoli gruppi di cellule nel pallone sono visibili al microscopio ottico (Figura 3B). Col passare del tempo e le cellule si sviluppano in LCL, cluster microscopico diventano più grandi (Figura 3C) tale che macchie sono visibili macroscopicamente nel pallone. Figura 1. Flusso di lavoro per la generazione e la crioconservazione di linee cellulari linfoblastoidi. Sangue periferico viene centrifugato da un gradiente di Ficoll. PBMC presenti nel buffy coat di un gradiente stabilito sono esposti a FK506 seguita da aggiunta di EBV. EBV-esposto le cellule sono coltivate a 37 ° C in presenza del 5% di CO 2 a stabilire e successivamente espandere LCL per la crioconservazione. <img alt="Figura 2" src="/files/ftp_upload/3321/3321fig2.jpg" /> Figura 2. Identificazione di una sotto-popolazione di linfociti B dovrebbe subire proliferazione dopo l&#39;esposizione a EBV. Un-infettati PBMC (A) o PBMC esposti a EBV derivati ​​da HH514-16 cellule (B) o da cellule B95-8 (C) in presenza di FK506 sono state raccolte al giorno 3. Le cellule sono state colorate con fluorocromo-coniugati anticorpi diretti contro CD19, CD23 e CD58. Dopo gating su cellule vives, non-infetti (A) e EBV-esposti (B e C) delle cellule B CD19 + sono stati esaminati per l&#39;espressione di CD23 e CD58. Un sub-popolazione di linfociti B che esprimono livelli elevati di CD58 e CD23 (CD23 hi CD58 +), osservata solo nelle cellule esposte alla trasformazione competente EBV (derivati ​​da cellule B95-8), è raffigurato. <img alt="Figura 3" src="/files/ftp_upload/3321/3321fig3.jpg" /> Figura 3. Visualizzazione di gruppi di cellule in cellule infettate da EBV. PBMC sono stati trattati con FK506 e infettati con EBV. Un-infetti (A) e EBV-esposte (B), le cellule sono state esaminate uno settimane dopo l&#39;infezione mediante microscopia a contrasto di fase (ingrandimento 10x). Cinque settimane di vecchia linea di cellule linfoblastoidi è mostrato in C.

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	<li>Thorley-Lawson, D. A., Gross, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistence+of+the+Epstein-Barr+virus+and+the+origins+of+associated+lymphomas.">Persistence of the Epstein-Barr virus and the origins of associated lymphomas.</a> N. Engl. J. Med. 350, 1328-1337 (2004).</li><li>Kubuschok, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+spontaneous+Epstein-Barr+virus+lymphoblastoid+cell+lines+genetically+modified+to+express+tumor+antigen+as+cancer+vaccines:+mutated+p21+ras+oncogene+in+pancreatic+carcinoma+as+a+model.">Use of spontaneous Epstein-Barr virus lymphoblastoid cell lines genetically modified to express tumor antigen as cancer vaccines: mutated p21 ras oncogene in pancreatic carcinoma as a model.</a> Hum. 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Nessun conflitto di interessi dichiarati.

Il metodo descritto in questo articolo genera LCL da donatore di sangue periferico con immortalizzazione rapidi e tempi di crioconservazione. Attraverso l&#39;uso di FK506, una cellula T immunosoppressiva, e alti titoli del virus infettivo, siamo in grado di promuovere la proliferazione di cellule infettate da EBV-B a partire da cellule mononucleate del sangue periferico. Questi interventi rendono il metodo descritto più efficiente, con conseguente rapida espansione delle cellule per gli esperimenti successivi. Tradizionalmente, la trasformazione la crescita è stata monitorata attraverso la visualizzazione di gruppi di cellule mediante microscopia ottica circa una settimana dopo l&#39;esposizione a EBV 6, 15. Tuttavia, il raggruppamento delle celle non è un indicatore specifico di EBV trasformazione mediata della crescita. Abbiamo già dimostrato di identificazione consistente della popolazione di cellule proliferanti mediante citometria di flusso 14, fornendo un metodo accurato e specifico per determinare la buona riuscita già a tre giorni di poppaer l&#39;esposizione delle cellule B di EBV.

<table border="1"><tbody><tr><td></td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Amfotericina B </td><td> Fisher Bioreagents </td><td> BP 928-250 </td></tr><tr><td> Dimetilsolfossido </td><td> Sigma </td><td> D 2650 </td></tr><tr><td> Siero fetale bovino, inattivato al calore </td><td> Sigma </td><td> F 4135 </td></tr><tr><td> Ficoll Hypaque linfociti separazione dei media </td><td> Mediatech </td><td> 25-072 </td></tr><tr><td> FK506 </td><td> AG scientifico </td><td> F 1030 </td></tr><tr><td> IgG dal siero di topo </td><td> Sigma </td><td> I 8765 </td></tr><tr><td> Topo anti-CD23 umano coniugato con PE </td><td> BD Pharmingen </td><td> 555711 </td></tr><tr><td> Topo coniugato con PE isotipo IgG1 </td><td> BD Pharmingen </td><td> 554680 </td></tr><tr><td> Topo anti-CD58 umano coniugato con FITC </td><td> Pierce Thermo Scientific </td><td> MA1-82159 </td></tr><tr><td> Topo isotipo coniugati con FITC IgG1 </td><td> BD Pharmingen </td><td> 550616 </td></tr><tr><td> Topo anti-CD19 umano coniugato con PE-Cy5 </td><td> BD Pharmingen </td><td> 555414 </td></tr><tr><td> Topo isotipo coniugato con PE-Cy5 IgG1 </td><td> Dako </td><td> X 0955 </td></tr><tr><td> Penicillina / streptomicina </td><td> Gibco </td><td> 15140-122 </td></tr><tr><td> RPMI 1640 i media </td><td> Sigma </td><td> R 8758 </td></tr><tr><td> Tetradecanoyl forbolo acetato (TPA) </td><td> Calbiochem </td><td> 407952 </td></tr><tr><td> FACS Buffer (1X Phosphate Buffered Saline + 5% siero fetale bovino) </td><td></td><td></td></tr></tbody></table>

establishment of epstein-barr virus growth-transformed lymphoblastoid cell lines

L&#39;infezione delle cellule B con virus Epstein-Barr (EBV) porta alla proliferazione e immortalizzazione successive, con conseguente creazione di linee cellulari linfoblastoidi (LCL) in vitro. Dal LCL sono latentemente infettate con EBV, forniscono un sistema modello per studiare la latenza del virus EBV e-driven proliferazione delle cellule B e tumorigenesi 1. LCL sono stati utilizzati per presentare antigeni in una varietà di test immunologici 2, 3. Inoltre, LCL può essere utilizzato per generare anticorpi monoclonali umani 4, 5 e fornire una fonte potenzialmente illimitata quando l&#39;accesso al materiale biologico primario è limitata 6, 7. Una varietà di metodi sono stati descritti per generare LCL. Metodi precedenti hanno incluso l&#39;uso di mitogeni come fitoemoagglutinina, lipopolisaccaride 8 e 9 pokeweed mitogeno per aumentare l&#39;efficienza di EBV-mediata immortalizzazione. Più recentemente, altri ricercatori hanno utilizzato immunosuppragenti essive come la ciclosporina A per inibire T cellulo-mediata uccisione di cellule B infettate 7, 10-12. La considerevole periodo di tempo da infezione da EBV allo stabilimento di linee cellulari unità l&#39;esigenza di metodi più veloce ed affidabile per EBV-driven trasformazione crescita delle cellule B. Usando una combinazione di alto titolo EBV e un agente immunosoppressivo, siamo in grado di infettare in modo coerente, trasformare e generare LCL dalle cellule B nel sangue periferico. Questo metodo utilizza una piccola quantità di cellule mononucleari del sangue periferico che sono infettati in gruppi di cellule in vitro può essere dimostrata. La presenza di CD23 con EBV in presenza di FK506, una cellula T immunosoppressori. Tradizionalmente, conseguenza della proliferazione delle cellule B è controllato da gruppi di visualizzazione microscopica di cellule circa una settimana dopo l&#39;infezione da EBV. Ciuffi di LCL può essere visto ad occhio nudo dopo diverse settimane. Descriviamo un test per determinare in anticipo se EBV-mediata gtrasformazione rescita è successo anche prima di ammassi di cellule microscopiche può essere dimostrata. La presenza di cellule CD23 + CD58 hi osserva a partire da tre giorni post-infezione indica un esito positivo.

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Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Descriviamo un metodo per generare linee cellulari B trasformate con virus di Epstein-Barr virus. Abbiamo anche illustrare un nuovo metodo di analisi in grado di identificare le cellule B destinato a subire trasformazioni già tre giorni dopo l&#39;infezione.

Istituzione di Epstein-Barr Virus Growth-trasformata linee linfoblastoidi

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

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Immunology and Infection

39.8K Views.  State University of New York at Stony Brook. Descriviamo un metodo per generare linee cellulari B trasformate con virus di Epstein-Barr virus. Abbiamo anche illustrare un nuovo metodo di analisi in grado di identificare le cellule B destinato a subire trasformazioni già tre giorni dopo l'infezione.

Video: Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/- and p53-/- consistently develop thymic lymphoma early in life 1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/- [FVB/N-Atmtm1Led/J] 2 and p53-/- [129/S6-Trp53tm1Tyj/J] 5 mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.

Derivazione di timica linfoma a cellule T Linee da Atm - / -</sup E P53 - / -</sup Mouse

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

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Immunologia e infezioni

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Espansione linfociti T citotossici da sangue del cordone ombelicale che Target Citomegalovirus, virus di Epstein-Barr virus, e Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

Imaging cellulare dal vivo di Alphaherpes Virus Trasporti anterograda e Diffusione

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrofili Isolamento e analisi per determinare il loro ruolo nel linfoma a cellule sensibilità agli agenti terapeutici

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Valutazione della efficacia e la tossicità degli RNA targeting HIV-1 Produzione per l&#39;uso in gene terapia farmacologica

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

Un cellulare semplice Red Blood Metodo Lysis per l&#39;istituzione di B linfoblastoidi linee cellulari

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

Un metodo semplice ed efficace per stabilire NK e linee a cellula T da pazienti con infezione attiva cronica del Virus di Epstein - Barr

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

Identificazione di Coding e classi di RNA Non codificanti espressa in suina sangue intero

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Istituzione dell'infezione virale e analisi dell'interazione ospite-Virus in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

Test ELISA in vitro per valutare la potenza del vaccino contro la rabbia

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...