Name: analysis of simian immunodeficiency virus-specific cd8+ t-cells in rhesus macaques by peptide-mhc-i tetramer staining
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Description: Watch this Scientific Journal Video about Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining at JoVE.com

Vorremmo ringraziare David Watkins per supportare gli esperimenti che hanno permesso l&#39;ottimizzazione del presente metodologia. La ricerca riportato nella presente pubblicazione è stato sostenuto in parte da una sovvenzione pilota fornito dal Miami Center for AIDS Research del National Institutes of Health con il numero premio P30AI073961. Il contenuto è di esclusiva responsabilità degli autori e non rappresentano necessariamente il punto di vista ufficiale del National Institutes of Health.

I campioni periferico cellule mononucleate del sangue (PBMC) utilizzati in questo manoscritto sono stati ottenuti da macachi Rhesus indiani alloggiati presso la Wisconsin Nazionale Primate Research Center. Questi animali sono stati curati secondo la relazione Weatherall sotto un protocollo approvato dalla University of Wisconsin Graduate School cura degli animali e del Comitato Usa 33. Tutte le procedure sugli animali sono stati eseguiti in anestesia e tutti gli sforzi sono stati fatti per ridurre al minimo il potenziale sofferen...

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A pochi passi in questa discussione di merito procedura in quanto sono cruciali per cedere risultati ottimali. Innanzitutto, poiché la qualità dei campioni biologici è un forte predittore del successo di qualsiasi citometria a flusso 38, tutto occorre prestare attenzione a garantire che le cellule sono vitali e in sospensione durante la procedura di colorazione. Questo è particolarmente importante quando si lavora con campioni crioconservati poiché sono più inclini a formazione di grumi e contengono tip...

CD8 + T-cellule comprendono una componente fondamentale del sistema immunitario adattativo, come partecipano tumore sorveglianza immunitaria e contribuire alla eradicazione di patogeni intracellulari 1. In termini semplici, CD8 + T-cellule esprimono i recettori delle cellule T (TCR), che riconoscono specificamente di istocompatibilità di classe complessi peptide-major I (pMHC-I) molecole presenti sulla membrana plasmatica delle cellule ospiti. Poiché questi peptidi sono derivati ​​dalla proteolisi di proteine ​​sintetizzate endogeno, pMHC-I superficie cellulare complessi forniscono una finestra nell&#39;ambiente intracellulare. Al momento l&#39;infezione da virus, per esempio, le cellule infettate mostrerà molecole MHC-I contenenti peptidi del virus di derivazione che possono servire come leganti per TCR espresse da pattugliamento CD8 + T-cellule. Nel caso in cui un CD8 + cellule T specifiche per il virus incontra una cellula infetta presentando il suo ligando pMHC-I, TCR impegno si tradurrà in attivazione + cellule T CD8 e ultinell&#39;intervallo portare a destinazione la lisi cellulare. Data la natura critica di queste interazioni TCR / pMHC-I, che determina la grandezza, la specificità, e fenotipo di rispondere CD8 + T-cellule possono spesso rivelare importanti indizi circa le malattie umane. Fino ai primi anni 1990, la quantificazione di Ag-specifici CD8 + T-cellule invocato il saggio tecnicamente impegnativo diluizione limite (LDA) 2,3. Non solo la LDA ha richiesto diversi giorni per essere completato, ma anche omesso di rilevare le cellule che mancava potenziale proliferativo. Come risultato, la LDA notevolmente sottovalutato la frequenza effettiva di antigene (Ag) specifico d&#39;CD8 + T-cellule che partecipano a una risposta immunitaria. Sebbene lo sviluppo di ELISPOT e dosaggi di citochine colorazione intracellulare notevolmente migliorato la capacità di misurare l&#39;immunità cellulare, questi metodi ancora necessari a stimolazione in vitro per la quantificazione Ag-specifiche cellule T 4. Non è stato fino al 1996 che Altman, Davis, e Colleagues pubblicato il loro punto di riferimento articolo segnalato la sviluppo della tecnologia tetramero pMHC-I 5. Fondamentale per il successo di questa tecnica è stata la multimerizzazione delle molecole pMHC-I, che si estendevano l&#39;emivita delle interazioni TCR / pMHC-I, riducendo così la probabilità di tetrameri pMHC-I cadono fuori durante le fasi di lavaggio dei dosaggi di citometria a flusso. Il vantaggio principale di tetrameri pMHC-I sopra i saggi suddetti è la capacità di rilevare con precisione Ag-specifica CD8 + T-cellule direttamente ex vivo senza la necessità di in vitro ri-stimolazione. Inoltre, la combinazione di pMHC-I tetramero colorazione con multicolore citometria a flusso ha consentito analisi dettagliate della fase di differenziazione, la memoria fenotipo, e lo stato di attivazione di Ag-specifici CD8 + T-cellule 2-4. Alla luce dei recenti progressi tecnici per la caratterizzazione repertori + CD8 T-cellule da pMHC-I Multimer colorazione 6, l&#39;ampiezza di applicazioni FOR questa metodologia è destinata a continuare in espansione. Poche zone nella ricerca biomedica hanno beneficiato di più dal pMHC-I tetramero colorazione di campo dell&#39;HIV dell&#39;immunologia 7. Anche se CD8 + T-cellule erano state temporalmente associato al controllo iniziale di viremia HIV dal momento della pubblicazione da Altman, Davis, e colleghi hanno 8,9, l&#39;uso di tetrameri pMHC-I, negli anni successivi notevolmente ampliato la nostra comprensione di HIV-specifica CD8 + risposta T-cellulare. Ad esempio, pMHC-I tetramero colorazione aiutato confermare la dimensione robusta di risposte delle cellule T virus-specifici CD8 + nella maggior parte delle persone con infezione da HIV 10-12. Questa metodologia anche facilitato la caratterizzazione di HIV e SIV-specifici + risposte delle cellule T CD8 limitato da molecole MHC-I associate al controllo spontaneo della replicazione virale in assenza di terapia antiretrovirale, un fenomeno noto come &quot;controllo elite&quot; <sup&gt; 13-15. Inoltre, pMHC-I tetrameri hanno contribuito a stabilire l&#39;asse morte programmata 1 (PD-1) / PD ligando 1 (PD-L1) come un percorso reversibile per il fenotipo disfunzionale di + HIV-specifica CD8 T-cellule nel infezione cronica non controllata 16,17. Collettivamente, questi studi sottolineano l&#39;utilità di tetrameri pMHC-I per monitorare le risposte delle cellule T CD8 + contro il virus dell&#39;AIDS. Sperimentale infezione SIV di macachi Rhesus (Macaca mulatta) rimane il migliore modello animale per la valutazione degli interventi del sistema immunitario contro l&#39;HIV / AIDS 18,19. Negli ultimi 25 anni, sono stati compiuti notevoli progressi nella identificazione e caratterizzazione di SIV-specifica CD8 + T-cellule in questa specie di scimmie, tra cui la scoperta di alleli MHC-I e la definizione di binding peptide motivi 13,20-27 . Come risultato, pMHC-I tetrameri sono stati sviluppati per l&#39;analisi di SIV-specifica CD8 + T-cellulerisposte in questo modello animale 28. La maggior parte di questi reagenti sono realizzati i prodotti genici di quattro macaco rhesus alleli MHC-I: Mamu-A1 * 001, Mamu-A1 * 002: 01, Mamu-B * 008: 01, e Mamu-B * 017: 01. Da segnalare, macachi Rhesus non esprimono un MHC-C locus 29. La stragrande maggioranza dei tetrameri pMHC-ho utilizzato nelle attuali esperimenti sono stati prodotti presso l&#39;impianto di NIH Tetramero core alla Emory University. Tuttavia, alcuni di questi reagenti, compresi Mamu-A1 * 001 tetrameri legati alla immunodominante epitopo Gag CM9, può essere ottenuto solo da fonti commerciali a causa di accordi di licenza. Uso tetrameri pMHC-I dei quattro alleli macaco rhesus elencati sopra abbiamo elencato con successo CD8 + T-cellule contro un totale di 21 epitopi SIV (Tabella 1), che sono stati indotta dalla vaccinazione o infezione primaria SIV 30,31 (Martins et al., osservazioni non pubblicate). Il presente manoscritto fornisce unaottimizzato protocollo tetramero colorazione pMHC-I per determinare il fenotipo frequenza e la memoria di SIV-specifici CD8 + cellule T nei macachi rhesus. Il test inizia con un elettivo 30 min di incubazione con un inibitore della proteina chinasi (PKI, qui, Dasatinib è utilizzato) al fine di diminuire TCR internalizzazione e quindi migliorare pMHC-I tetramero colorazione 32. Come descritto di seguito, questo trattamento è particolarmente utile quando si utilizza Mamu-B * 017: 01 tetrameri. Sono inoltre disponibili le istruzioni su come etichettare le cellule con tetrameri pMHC-I coniugato a un fluorocromo e anticorpi monoclonali (MAK, MAB). Questo protocollo include anche un passo permeabilizzazione cella per la rilevazione intracellulare della citolisi associata molecola granzyme B (GZM B). Il mAb contro CD3 viene aggiunto in questa fase anche per migliorare la rilevazione di questa molecola di segnalazione TCR. Come riferimento, vengono elencati tutti i fluorocromi impiegati in questo pannello colorazione.

<table border="0" cellpadding="0" cellspacing="0" width="1449">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Dasatinib</td>
			<td style="width:236px;">Axon medchem</td>
			<td style="width:127px;">Axon&#160;1392</td>
			<td style="width:757px;">Must be resuspended in DMSO and immediately stored at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI w/ Glutamax</td>
			<td>gibco/ Life Technologies</td>
			<td>61870-036</td>
			<td>Must be stored at 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heat Inactivated FBS</td>
			<td>gibco/ Life Technologies</td>
			<td>10082-147</td>
			<td>Must be stored at 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycinp-Amphotericin B</td>
			<td>Lonza</td>
			<td>17-745E</td>
			<td>Must be stored at 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO, Anhydrous</td>
			<td>Life Technologies</td>
			<td>D12345</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-mL Round-Bottom Polypropylene Tubes</td>
			<td>VWR</td>
			<td>60819-728</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorochrome-conjugated pMHC-I tetramers</td>
			<td>NIH Tetramer Core or MBL, Inc.</td>
			<td></td>
			<td>Must be stored and maintained at&#160; 4 &#176;C. Centrifuge at 20,000 x g for 15 min before use. Do not freeze.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorochrome-conjugated mAbs</td>
			<td>Various companies</td>
			<td></td>
			<td>Must be stored and maintained at&#160; 4 &#176;C. Do not freeze.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD&#160;Fixable Aqua&#160;Dead&#160;Cell Stain Kit</td>
			<td>Life Technologies</td>
			<td>l34957</td>
			<td>Must be stored at -20 &#176;C. Resuspend each aliquot in 50 &#956;L of DMSO prior to use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brilliant Stain Buffer</td>
			<td>BD Biosciences</td>
			<td>563794</td>
			<td>Must be stored at&#160; 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline</td>
			<td>VWR</td>
			<td>97064-158</td>
			<td>Store at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumine Bovine</td>
			<td>VWR</td>
			<td>700011-230</td>
			<td>Must be stored at&#160; 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Azide</td>
			<td>VWR</td>
			<td>97064-646</td>
			<td>Store at room temperature. Toxic substance. Do not mix with bleach.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bleach</td>
			<td>VWR</td>
			<td>89501-620</td>
			<td>Corrosive chemical, cannot be mixed with sodium azide. Handle with care</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714-S</td>
			<td>Flammable, corrosive, and toxic reagent. Handle with care</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polysorbate 20 (Tween-20)</td>
			<td>Alfa Aesar</td>
			<td>L15029</td>
			<td>Store at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Permeabilization Solution 2&#160;</td>
			<td>BD Biosciences</td>
			<td>340973</td>
			<td>Toxic and corrosive reagent. Handle with care</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sarsdet Tubes 1.5mL screw top</td>
			<td>VWR</td>
			<td>72.692.005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2.0ml DNA/RNA Low bind Tubes</td>
			<td>Eppendorf</td>
			<td>22431048</td>
			<td>The use of Sterile microtubes is preffered&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex mixer</td>
			<td>To discretion of scientist</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biosafety Cabinet&#160;</td>
			<td>To discretion of scientist</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Milli Q Intergral Water Purification system</td>
			<td>EMD Millipore</td>
			<td>ZRXQ010WW</td>
			<td>Molecular Biology grade water from any provider may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge</td>
			<td>To discretion of scientist</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>To discretion of scientist</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4 &#176;C&#160; refrigerator</td>
			<td>To discretion of scientist</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD LSR II</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometer must contain lasers and filters that are compatible with the staining panel used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deionized water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum Foil</td>
			<td>VWR SCIENTIFIC INC.</td>
			<td>89068-738</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td></td>
			<td></td>
			<td>Must be able to maintain 37 &#176;C&#160; internal temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS Diva software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flowjo software version 9.6</td>
			<td>Flowjo</td>
			<td></td>
			<td>Used to analyze FCS files generated by FACS Diva software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropippette tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of simian immunodeficiency virus-specific cd8+ t-cells in rhesus macaques by peptide-mhc-i tetramer staining

Istocompatibilità di classe complesso Peptide-major I (pMHC-I) tetrameri sono state uno strumento prezioso per studiare le risposte delle cellule T CD8 +. Poiché questi reagenti si legano direttamente ai recettori delle cellule T sulla superficie delle cellule CD8 + linfociti T, tetrameri fluorocromi marcato pMHC-I consentono il rilevamento accurato di antigene (Ag) + SPECIFICI CD8 T-cellule senza la necessità di re in vitro -stimolazione. Inoltre, se combinato con multicolore citometria a flusso, pMHC-I tetramero colorazione può rivelare aspetti chiave di Ag-specifici CD8 + T-cellule, tra cui fase di differenziazione, la memoria fenotipo, e lo stato di attivazione. Questi tipi di analisi sono stati particolarmente utile nel campo della immunologia HIV dove CD8 + T-cellule possono influenzare la progressione in AIDS. infezione sperimentale di macachi rhesus con virus dell&#39;immunodeficienza delle scimmie (SIV) fornisce uno strumento prezioso per studiare l&#39;immunità cellulare contro il virus dell&#39;AIDS. Come risultato, notevole progress è stato fatto nel definire e caratterizzare le risposte delle cellule T in questo modello animale. Qui vi presentiamo un protocollo ottimizzato per l&#39;enumerazione SIV-specifica CD8 + T-cellule in macachi Rhesus di pMHC-I tetramero colorazione. Il nostro test permette la simultanea quantificazione e la memoria fenotipizzazione di due popolazioni CD8 + pMHC-I tetramero + T-cellule per test, che potrebbe essere utile per il monitoraggio SIV-specifiche risposte delle cellule T CD8 + generati dalla vaccinazione o infezione SIV. Considerando la rilevanza di primati non umani nella ricerca biomedica, questa metodologia è applicabile per studiare le risposte delle cellule T CD8 + in più impostazioni di malattia.

Il protocollo qui descritto è stato utilizzato per determinare l&#39;ampiezza e la memoria fenotipo di vaccino-indotta, Gag CM9-specifiche risposte delle cellule T CD8 + in un Mamu-A1 * 001 + macaco rhesus. Per questa analisi, un APC-coniugato tetramero Mamu-A1 * 001 / Gag CM9 è stato utilizzato in un pannello colorazione citometria a flusso a 8 colori. Figura 1A - F mostra la strategia di gating utilizzato per analizzare i dati, ch...

Watch this Scientific Journal Video about Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining at JoVE.com

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Qui, vi presentiamo un protocollo ottimizzato per l&#39;enumerazione e la caratterizzazione di cellule macaco rhesus T CD8 + contro il virus dell&#39;AIDS. Questo articolo è utile non solo al campo della immunologia HIV, ma anche ad altri settori della ricerca biomedica cui sono note le risposte delle cellule T CD8 + influenzare l&#39;esito della malattia.

L&#39;analisi dei Simian Immunodeficiency Virus specifici CD8<sup&gt; +</sup&gt; T-cellule in Rhesus macachi di peptide-MHC-I Tetramero colorazione

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

The overall goal of this assay, is to enumerate and characterize Simian Immunodeficiency Virus-specific CD8 positive T-cell populations generated by vaccination or primary infection. This method can help answer key questions in the T-cell immunology field, such as what is the frequency of antigen-specific CD8 T-cells induced either by vaccination or infection. The main advantage of this technique is that it can quantify antigen-specific CD8 T-cells in biological samples obtained directly ex vivo without the need for in vitro re-stimulation.The implications of this technique extend toward the evaluation of incision specific CD8 T-cell in the rhesus macaque, and for studying CDB T-cell responses in multiple disease settings. Generally, individuals new to this method will struggle because of the incomplete knowledge available for rhesus macaque MHC genetics, and because the flow cytometric staining requires precise antibody application. Begin by re-suspending the rhesus PBMC in R10 medium.Add a 1.6 times 10 to the seventh cells per milliliter concentration. Aliquot 50 microliters of cells to the appropriate number of flow cytometry tubes, and add 50 microliters of protein kinase inhibitor to each tube. Vortex each tube and incubate the samples at 37 degrees Celsius for 30 minutes.The correct amount of titrated peptide MHC tetramer must be diluted in enough master mix to stain all of the experimental tubes. Therefore, it is important to prepare a 15%excess of the master mix, to account for pippetting errors. While the cells are incubating, pellet the protein aggregates in the tetramer solution by centrifugation, to minimize the background staining.Next, dilute the tetramers in enough stain buffer, so that 25 microliters of the tetramer master mix can be added to each flow cytometry tube. Then vortex the sample tubes and incubate the PBMC tetramer solutions in the dark, at room temperature for 45 minutes. At the end of the incubation, add 50 microliters of staining master mix to the appropriate sample tubes.And mix the tubes for vortexing. After 25 minutes in the dark at room temperature, wash the cells with wash buffer, pellet the cells by centrifugation, and careful decant the supernatants without disturbing the pellets. Vortex the cells in the leftover buffer in each tube, and fix the samples with 250 microliters of 2%paraformaldehyde per tube.Immediately vortex and incubate the tubes in the dark, at 4 degrees Celsius. After 20 minutes, wash the cells in fresh wash buffer. Pellet the cells by centrifugation, and decant the supernatant without disturbing the pellet.Re-suspend the cells in 500 microliters of permeabilization buffer, vortex, and incubate at room temperature for 10 minutes in the dark. Then wash and pellet the cells. Then decant the supernatants as described previously.Proceed to adding 50 microliters of intracellular stain master mix to the appropriate tubes. Vortex each sample, and incubate at room temperature in the dark for 30 minutes. After the incubation is done, wash and pellet the cells.The samples are now ready to be acquired in a flow cytometer. Mamu-B017.01 tetramers typically yield a dim staining, which can be substantially improved by pre-treatment of the cells, with a protein kinase inhibitor. To avoid background staining, MHC class one matched cells that do or do not contain the antigen-specific CD8 positive T-cell population of interest, should be labelled with the relevant tetramer and multiple dilutions, to determine the optimum amount that results in the best separation between tetramer positive and negative CD8 positive T-cells.In these graphs, the gating strategy used to analyze the magnitude and memory phenotype of vaccine-induced Gag CM9 specific CD8 positive T-cells responses in a Mamu-A 01 positive rhesus macaque, are shown. Note that the tetramer positive CD8 positive T-cell population, is well-separated with minimal background staining. And with the memory subsets clearly delineated by their CD28, CCR7 and Granzyme B expression within the tetramer gate.Once mastered, this technique can be completed in about three hours if it is performed properly. While attempting this procedure, it is important to remember to add the correct reagents to each antibody or tetramer master mix, and to work with the experimental tubes each time as demonstrated. After its development, this technique paved the way for researchers in the field of immunology to explore the specificity and phenotype of antigen-specific CD8 T-cells.After watching this video, you should have a good understanding of how to set up an MHC class one tetramer staining in the assay of the rhesus macaque model of human HIV-1 AIDS research. Don't forget that working with biological specimens from rhesus macaques requires special precautions such as wearing the appropriate personal protective equipment during the sample processing.

Research

JoVE Journal

Immunology and Infection

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

Multicolor Citometria a Flusso di analisi Cellular risposta immunitaria in macachi Rhesus

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

Ricerca

Immunologia e infezioni

Retroviral Transduction of T-cell Receptors in Mouse T-cells

Trasduzione retrovirale di recettori delle cellule T nel topo le cellule T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Determinazione ottimale attività citotossica di specifiche cellule T umane Her2neu CD8 confrontando il Cr51 saggio di rilascio al Sistema xCELLigence

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

Peptide: MHC Tetramero basata Arricchimento di epitopi specifici per cellule T

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induzione di Murine infiammazione intestinale per trasferimento adottivo di Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alto</sup&gt; T Cells in topi immunodeficienti

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

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The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

In Situ MHC-tetramero macchiatura e analisi quantitativa per determinare la posizione, l'abbondanza e fenotipo di cellule T antigene-specifiche CD8 nei tessuti

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in ...

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Virus di Zika specifico diagnostico dell'epitopo Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Cella singola quantificazione del mRNA e l'espressione di proteine di superficie CD4 infettati da Virus di immunodeficienza delle scimmie+ T cellule isolate da macachi Rhesus

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector

Determinazione quantitativa simultanea delle anti-vettore e Anti-transgene-Specific CD8+ T cellule tramite MHC I tetramero colorazione dopo la vaccinazione con un vettore virale

Upon viral infection, antigen-specific CD8+ cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of ...