Ringraziamo i partecipanti allo studio Miami WIHS per la loro disponibilità a partecipare a questo lavoro. I dati in questo manoscritto sono stati raccolti dalle donne Miami Interagency HIV Study (WIHS). I contenuti di questa pubblicazione sono di esclusiva responsabilità degli autori e non rappresentano le posizioni ufficiali del National Institutes of Health (NIH). Questo lavoro è stato sostenuto dal National Institute of Allergy e Malattie infettive e Interagency HIV Infection Study femminile (WIHS) [concessione numero U01 AI103397], National Institute of Allergy e Malattie infettive, National Institutes of Health [codice di autorizzazione P30AI073961], Centro Nazionale per l&#39;avanzamento traslazionale Scienze e l&#39;Istituto nazionale sulla minoranza Salute e disparità, Istituto nazionale della Salute [codice di autorizzazione UL1TR000460 e 1KL2TR000461], e National Institute of Child Health e lo sviluppo umano, National Institute of Health [codice di autorizzazione K23HD074489].

attività di studio ha avuto luogo presso l&#39;Università di Unità di Ricerca HIV Miami, in collaborazione con l&#39;HIV Interagency Studi delle Donne Miami (WIHS) e il Miami Center for AIDS Research (CFAR) .Institutional Review Board (Università di Miami Miller School of Medicine) l&#39;approvazione è stata ottenuta prima per il reclutamento e le eventuali procedure di valutazione o di studio correlati. Raccolta del campione 1. endocervicale Brush NOTA: I partecipanti sono stati sottoposti a un esame vaginale eseguito da MD addestrato o il ginecologo e la raccolta dei linfociti intraepiteliali sono stati eseguiti in esame con lo speculum inserendo cytobrush. <ol><li> caratteristiche dei partecipanti <ol><li> Reclutare donne partecipanti che sono tra i 18 ei 45 anni di età, sessualmente attivi, non è incinta e non sui farmaci contraccettivi o con un dispositivo intrauterino. </li><li> Prima di un esame vaginale, hanno i partecipanti sono sottoposti a un pareggio 10 ml di sangue in verdevacutainer eparina top per l&#39;isolamento delle cellule periferiche mononucleate del sangue (PBMC). per servire come controllo per endocervicale analisi intraepiteliale 9. </li></ol></li><li> Raccolta dei campioni endocervicali <ol><li> Inserire uno speculum sterile di dimensioni adeguate in vagina senza lubrificazione. Utilizzare acqua calda per facilitare l&#39;inserimento del speculum e garza sterile per pulire il muco. La posizione del speculum consente la completa visualizzazione del sistema operativo e portio. </li><li> Inserire la spazzola endocervicale nel canale endocervicale fino a quando solo le setole più vicini alla mano sono visibili. Ruotare il pennello in senso orario per 360 °. Trasferire la spazzola in un tubo da 15 ml contenente 5 ml di mezzo IMDM. </li><li> Subito dopo la raccolta, posizionare il tubo contenente cytobrush sul ghiaccio. Per ottenere elevata vitalità delle cellule intraepiteliali raccolti, consegnare il cytobrush al laboratorio e processo entro 1 ora. </li></ol></li></ol><ol><li> Tubo Vortex contenente cytobrush endocervicale applicare 4 colpi (10 sec) brevi. </li><li> Centrifugare la provetta contenente cytobrush, per 10 min a 250 x g. </li><li> Rimuovere con attenzione il cytobrush dal tubo (non disturbare il pellet sul fondo della provetta) e aggiungere 1 ml di mezzi IMDM e posizionare il tubo in ghiaccio. </li><li> Posizionare il cytobrush sul setaccio cella 100 mm superiore del tubo 50 ml. </li><li> Lavare il cytobrush con 20 ml IMDM. </li><li> provetta da centrifuga per 10 minuti a 250 x g. </li><li> Decantare il surnatante e le cellule in pellet risospendere in 1 ml IMDM e si combinano con il pellet già ri-sospensione descritto in 2.3. </li><li> Contare le cellule utilizzando un contatore di cellule automatizzato 10 o contare le cellule utilizzando emocitometro combinando 10 ml di sospensione cellulare con 10 ml di colorante blu trypan. Delicatamente pipettare su e giù dieci volte per mescolare le cellule e colorante. Carico di 10 microlitridella miscela nell&#39;apertura di una delle due camere emocitometro. Contare tutte le celle in cinque aree della griglia. Tenere un conteggio separato delle cellule vitali e non vitali. Determinare la vitalità cellulare utilizzando la seguente formula:% di cellule vitali = cellule non vitali x 100 / totale cellule numero delle cellule. Determinare la concentrazione successiva cellule per ml (e il numero totale di cellule) usando i seguenti calcoli. Totale cellule per ml = x cellule totali contate (fattore di diluizione / numero di piazza contate) x 10.000 cellule / ml. </li><li> Congelare isolate cellule endocervicali utilizzando media congelamento contenente il 10% di DMSO e il 90% FBS. </li></ol> 3. Citometria a Flusso <ol><li> Risospendere 1-3 x 10 5 cellule endocervicali in 100 microlitri di buffer FACS (PBS contenente 1% di albumina sierica bovina </li><li> Aggiungere 1 ml / 10 6 Live / Dead fissabile giallo Morto kit cellulare Stain e anticorpi per marcatori di superficie alle concentrazioni descritte nella tabella 1. </li><li> Incubare le cellule per 30 minuti a + 4 ° C, al riparo dalla luce. </li><li> Aggiungere 1 mL FACS tampone e le cellule e provette da centrifuga per 5 min a 350 xg </li><li> Risospendere pellet cellulare in 300 microlitri di 1% paraformaldeide. </li><li> Preparare controlli compensazione utilizzando perline e anticorpi utilizzati per la colorazione aggiungendo 100 ml di tampone FACS e una goccia delle perle e la stessa concentrazione di anticorpi usati per la colorazione del campione. </li><li> Acquisire perline colorate e campioni su un citofluorimetro, dotato di 405 nm, 488 nm e 635 nm laser. </li></ol> L&#39;isolamento delle cellule 4. endocervicale Gamma Delta T <ol><li> Isolamento tallone magnetico <ol><li> Risospendere endocervicale pellet cellulare (≤ 10 7 cellule totali), descritto in 2.6, in 40 ml di tampone incluso nel kit di Gamma Delta microperla TCR fa riferimento la tabella di materiali e reagenti. </li><li> Seguire le istruzioni del kit per il protocollo di colorazione in due fasi: in primo luogo, ingegnoh anti-TCR gamma delta Hapten-anticorpo e la seconda, con l&#39;anti-aptenici microsfere-FITC </li><li> Dopo incubazione di 15 min a 4-8 ° C, lavare le cellule aggiungendo 1 ml di tampone e centrifugare a 300 xg per 10 min. Decantare il supernatante invertendo la provetta, e toccare secco con l&#39;aiuto di un foglio di carta. </li><li> Risospendere il pellet cellulare in 500 ml di condizione di buffer e preparare la colonna magnetica ponendolo in separatore magnetico appropriata secondo il protocollo del fornitore. </li><li> Applicare sospensione cellulare nella colonna e raccogliere le cellule senza etichetta che attraversano e lavare colonna 3x con adeguate quantità di tampone (500 mL). Eseguire fasi di lavaggio aggiungendo tampone tre volte, ogni volta dopo il serbatoio colonna è vuota. Raccogliere dell&#39;effluente totale. Questa è la frazione di cellule senza etichetta, negativo </li><li> Rimuovere la colonna formano il separatore magnetico e pipetta 1 ml di tampone sulla colonna e subito scovare frazione con l&#39;etichetta magneticamentecellule cato applicando saldamente lo stantuffo fornita con la colonna. NOTA: Isolated Gamma Delta cellule T sono già fluorescente macchiato per analisi di flusso-citometria da magneticamente etichettati microsfere anti-aptene sono coniugati con FITC fluorescenti colorante. </li></ol></li><li> cell sorting <ol><li> Per l&#39;ordinamento delle cellule, le cellule endocervicali etichette con Die vitalità e pannello di anticorpi come descritto nel paragrafo 3. </li><li> Impostare varchi elettronici sul vivo, CD45 + CD3 + e delta gamma cellule positive e effettuare una cernita delle cellule. </li></ol></li></ol>

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Gli autori non hanno commerciale o di altra associazione che potrebbe rappresentare un conflitto di interesse (ad esempio, magazzino farmaceutico di proprietà, di consulenza, l&#39;appartenenza Advisory Board, brevetti rilevanti, o finanziamenti per la ricerca).

Valutazione della vulnerabilità femminile della mucosa del tratto genitale inferiore è una componente essenziale di studi clinici che affrontano il rischio di acquisizione e trasmissione del virus HIV. Tipicamente FGT vulnerabilità alle infezioni da HIV è valutata misurando immunomodulatori solubili nelle secrezioni genitali (citochine, chemochine e peptidi antimicrobici) 12, 13. Tuttavia, questi marcatori sono solo indicative di infiammazione vaginale, sono...

Abbiamo sviluppato la metodologia di utilizzo di campioni endocervicali pennello per valutare le cellule T GD intraepiteliali. Il canale endocervicale è rivestito da un singolo strato di epitelio colonnare. Linfociti intraepiteliali rappresentano linfociti di prima linea che risiedono all&#39;interno dello strato epiteliale che può rapidamente avviare risposte immunitarie su di incontrare microbi patogeni 7, 8. Comprendere gli eventi immunologici del vano intraepiteliale endocervicale è importante per la progettazione di strategie efficaci per prevenire le infezioni, compreso l&#39;HIV. Il nostro metodo può essere applicato per i campioni endocervicali umane appena raccolti, nonché le cellule endocervicali congelati per esplorare ulteriormente il ruolo delle cellule T GD intraepiteliale nelle donne con infezione da HIV o a rischio di infezione da HIV. L&#39;obiettivo principale di questo protocollo è quello di scoprire nuovi eventi immunologici nel vano intraepiteliale endocervicale e evaluatle risposte delle cellule T e GD come marker di vulnerabilità della mucosa nelle donne con infezione da HIV. Le lacune sostanziali nella conoscenza intorno FGT immunità sono in parte a causa della difficoltà di raccolta e l&#39;elaborazione dei campioni delle mucose con successo. Inoltre, il complesso eterogeneità delle cellule immunitarie mucosali, e la loro interconnessione tra loro, sono sfide importanti per identificare misurazioni clinicamente rilevanti che riflettono lo stato e la capacità del sistema immunitario. Abbiamo sviluppato un metodo per ottenere cellule altamente purificate intraepiteliali GD T che possono essere ulteriormente analizzate utilizzando altamente multiplex, tecnologie unicellulari che possono essere critiche per l&#39;identificazione dei meccanismi alla base della FRT immunità.

<table border="0" cellpadding="0" cellspacing="0" width="648">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cytobrush Plus GT&#160;</td>
			<td style="width:147px;">(CareFusion, San Diego, CA, USA</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">IMDM,Iscove&#39;s Modified Dulbecco&#39;s Medium&#160;</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td align="right" style="width:119px;">12440061</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Beckman Coulter automated cell counter (The Vi-CELL Series Cell Viability Analyzer</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">496178</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO, Dimethyl sulfoxide&#160;</td>
			<td style="width:147px;">Sigma Aldrich&#160;</td>
			<td style="width:119px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS, Fetal Bovine Serum</td>
			<td style="width:147px;">Invitrogen, Gibco</td>
			<td style="width:119px;">26140-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:147px;">Invitrogen, Gibco</td>
			<td align="right" style="width:119px;">10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BSA</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">A-9647</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LIVE/DEAD Fixable Yellow Dead Cell Stain kit&#160;</td>
			<td style="width:147px;">Life Technologies,</td>
			<td style="width:119px;">L349S9</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1% paraformaldehyde (Sigma-Aldrich</td>
			<td style="width:147px;">Sigma Aldrich&#160;</td>
			<td style="width:119px;">D6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fortessa flow cytometer&#160;</td>
			<td style="width:147px;">Becton Dickinson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UltraComp eBeads</td>
			<td style="width:147px;">eBioscience</td>
			<td style="width:119px;">01-2222-42</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">ArC Amine Reactive Compensation Beads (Life Technologies, Grand Island, NY, USA)</td>
			<td style="width:147px;">Life Technologies</td>
			<td style="width:119px;">A10346</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-TCR gamma/delta MicroBead Kit&#160;</td>
			<td style="width:147px;">Milteny Miltenyi Biotec Inc.</td>
			<td style="width:119px;">130-050-701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MS column</td>
			<td style="width:147px;"></td>
			<td style="width:119px;">103-042-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACS separator</td>
			<td style="width:147px;"></td>
			<td align="right" style="width:119px;">4124</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and flow cytometric analysis of human endocervical gamma delta t cells

La femmina tratto riproduttivo (FRT) mucosa sistema immunitario serve come la prima linea di difesa. Una migliore conoscenza della mucosa genitale è quindi essenziale per comprendere la patogenicità dei diversi agenti patogeni, tra cui l&#39;HIV. Gamma Delta cellule (GD) T sono il prototipo delle cellule T &quot;non convenzionali&quot; e rappresentano una parte relativamente piccola sottogruppo di cellule T definite dalla loro espressione dei recettori delle cellule T eterodimeriche (TCR) composte da catene di gamma e delta. Questo li distingue dalle cellule T classici e molto meglio noti CD4 + helper e le cellule CD8 + T citotossici che sono definiti da TCR alfa-beta. cellule T GD spesso mostrano la localizzazione tessuto-specifica e sono arricchiti in epitelio. le cellule T GD orchestrare le risposte immunitarie a infiammazione, la sorveglianza del tumore, malattie infettive, e autoimmunità. Qui, vi presentiamo un metodo per isolare ed analizzare riproducibile endocervicali intraepiteliale linfociti T GD umani. noi ettarove utilizzati campioni cytobrush endocervicali da donne che partecipano alle donne Interagency HIV Infection Study (WIHS). La conoscenza GD cellule T interazioni durante le condizioni in cui vi è un insulto alla mucosa vaginale potrebbe essere applicata a qualsiasi studio clinico in cui la vulnerabilità della mucosa è rivolta, compreso lo sviluppo di oltre microbicides.In vaginale, la conoscenza di risposte delle cellule della mucosa GD T ha possibilità di applicazione di GD T cell-based terapia immunitaria nel trattamento delle malattie infettive.

Recentemente, abbiamo analizzato intraepiteliale cellule T GD endocervicali e siamo stati il primo a riferire circa la perdita di cellule endocervicale T gamma delta in donne infette da HIV 9, 11. Qui, descriviamo il protocollo per isolare e analizzare il sottogruppo di cellule T gamma delta endocervicale. Come mostrato in figura 1, le cellule T GD possono essere facilmente rilevati nei campioni di cellule endoce...

Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells at JoVE.com

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

L&#39;isolamento e la citometria a flusso Analisi di Human endocervicale Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

isolation-flow-cytometric-analysis-human-endocervical-gamma-delta-t

Research

JoVE Journal

Immunology and Infection

8.7K Views.  Miller School of Medicine, University of Miami. We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

Video: Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Isolamento linfociti T umani, la cultura e l&#39;analisi delle migrazioni In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

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Immunologia e infezioni

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Flusso Isolamento citometrica di primari murini di tipo II cellule alveolari epiteliali per gli studi funzionali e molecolari

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Imaging Whole-animale e di citometria a flusso tecniche di analisi di Antigen-specific CD8 + cellule T Risposte dopo nanoparticelle vaccinazione

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Isolamento e analisi citofluorimetrica delle cellule immunitarie dal cervello ischemica mouse

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Isolamento e analisi citofluorimetrica di-Glioma infiltrazione sangue periferico mononucleari Cellule

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

Isolamento e caratterizzazione citometria a flusso di Murine Piccolo intestinale Linfociti

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Flusso analisi di citometria di Particle-bound Bet v 1 allergene in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

Flusso analisi di citometria di Natural Killer cellulare Lytic attività nel Sangue umano intero

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolamento e coltura In Vitro dei macrofagi alveolari murini ed umani

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

Digestione del fegato murino per un'analisi citofluorimetrica delle cellule endoteliali linfatiche

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...