The authors would like to thank Joshua Kimani, clinical director of the research program at the University of Nairobi, for his assistance with mucosal immunology studies related to this protocol. The authors would like to acknowledge funding from CHVI grant MOP 86721.

Dichiarazione etica: la ricerca etica consigli di amministrazione di entrambe dell&#39;Università di Manitoba e Kenyatta Nazionale Ospedale / Università di Nairobi ha approvato questo studio e il consenso informato scritto è stato ottenuto da tutti i partecipanti allo studio. 1. Preparazione dei Media e CMC Collection Tubes <ol><li> Preparare la soluzione tampone fosfato (PBS) (137,93 mM NaCl, 2,67 mM KCl, 8.1 mM Na 2 HPO 4, 1.47 mM KH 2 PO 4). Autoclave per la sterilità. Questo può essere conservato a 4 ° C per diversi mesi. </li><li> Pre-aliquota 5 ml di soluzione in 50 ml di tubo falcon (uno per campione da raccogliere) e conservare a 4 ° C fino al momento di raccolta del campione. </li><li> Etichettare 2-4 cryovials o provette da 1,5 ml microcentrifuga per campione per raccogliere la lavanda per la conservazione. </li><li> Preparare cellule di coltura: RPMI 1640 + 1% di penicillina / streptomicina / amfotericina B (concentrazione finale 100 unità / ml, 100 pg / ml e 250 ng / ml, respectively, senza FCS / FBS aggiunto. </li></ol> 2. Insieme di Cytobrush Campioni Raccolta di campioni cytobrush è una procedura non invasiva che deve essere eseguita da un MD o ginecologo esperto. <ol><li> Raccogliere le cellule mononucleate del collo dell&#39;utero (CMC) utilizzando sia un cytobrush e un raschietto di legno o di plastica. Raccogliere CMC dal partecipante in esame speculum inserendo il raschietto e ruotando attorno al canale cervicale (Figura 1). Inserire il cytobrush nell&#39;OS endocervicale, ruotare di 360 °, e collocare immediatamente sia il cytobrush e raschietto nel tubo falcon da 50 ml contenente 5 ml di PBS. </li><li> Conservare i campioni in ghiaccio / a 4 ° C fino al trattamento. Per mantenere la vitalità cellulare, campioni di processo entro 2 ore dal prelievo. </li><li> Se possibile, raccogliere campioni di sangue compensate in vacutainer eparina top verde di cellule mononucleate del sangue periferico (PBMC) isolamento per servire come un comparatore / controllo per l&#39;analisi CMC. </li&gt; </ol> 3. Isolamento di CMC Eseguire l&#39;elaborazione del campione in un laboratorio di biosicurezza di livello 2, in un armadio biosicurezza sterile con doppi guanti. <ol><li> Durante la raccolta, i campioni CMC possono diventare contaminati con sangue. Escludere campioni con contaminazione sangue visibile dallo studio per evitare confusione inclusione di PBMC nel campione finale. A causa del basso numero di linfociti isolati da campioni CMC, la contaminazione del sangue minore può facilmente sopraffare il componente linfociti CMC. </li><li> Tubi Vortex falco contenenti sia il cytobrush e raschietto per 45 secondi di dissociare le cellule dalle spazzole. I campioni possono diventare spumosa durante questo processo; questo è normale. </li><li> Utilizzare il cytobrush per raschiare tutto il materiale residuo fuori il raschietto, e scartare il raschietto in una soluzione di candeggina. Per raccogliere le eventuali cellule rimanendo attaccati alla spazzola / raschietto, usare un guanto fresco per spremere il raschietto tra il pollice e l&#39;indice.Scivolare giù il pennello, permettendo alle cellule e PBS da rilevare nella stessa provetta 50 ml. Eliminare il cytobrush in una soluzione di candeggina, e gettare il guanto. IMPORTANTE: utilizzare un nuovo guanto per ogni campione. </li><li> Montare un 100 micron filtro cella nylon a un nuovo 50 ml tubo falcon. Usando una pipetta di trasferimento, raccogliere la sospensione PBS-cella dal tubo campione e filtrare attraverso il filtro nel tubo fresco. </li><li> Aggiungere 5 ml di RPMI 1640 alla provetta campione originale. Utilizzando la pipetta di trasferimento, lavare i lati del tubo campione con il RPMI e le trasferisce attraverso il filtro per il nuovo tubo di raccolta. Lavare la parte inferiore del filtro di nylon con il RPMI pure. </li><li> Centrifugare i campioni per 10 min a 514 x g. E &#39;fondamentale per liberare il freno centrifuga durante questa fase, al fine di evitare stacchi del pellet CMC. </li><li> Rimuovere delicatamente il surnatante dal tubo, facendo attenzione a non disturbare il pellet. Dimensione pellet sarà molto variabile; in modome campioni, il pellet è abbastanza grande per la presenza di un gran numero di cellule epiteliali, mentre in altri casi, il pellet è appena visibile ed altamente trasparente. </li><li> Risospendere il pellet da lieve agitazione del tubo falco. Aggiungere 5 ml di PBS per lavare il campione. </li><li> Centrifuga nuovamente per 10 min a 514 xg senza freno centrifuga. </li><li> Eliminare attentamente il supernatante e risospendere il pellet come sopra. Le cellule sono pronte sia per la crioconservazione (utilizzando un protocollo di crioconservazione PBMC), la stimolazione o citometria a flusso fenotipizzazione. </li></ol> 4. CMC colorazione superficiale e Citometria a Flusso <ol><li> Risospendere il pellet dal punto 3.10 in 100 ml di soluzione bloccante e trasferire sia in una piastra a 96 pozzetti o il tubo FACS. La soluzione bloccante (per bloccare i recettori Fcy) ricetta è la seguente: IgG murine 1,8 ml (concentrazione finale 0,2 mg / mL), 5 microlitri FBS e 93,2 microlitri FACS lavaggio (PBS +2% FBS).La colorazione può essere eseguita in una piastra a 96 se formati pellet sono abbastanza piccole, ma può avere bisogno di essere eseguita in tubi FACS se pellets sono ordinariamente grande. E &#39;importante per titolare anticorpi conseguenza. </li><li> Bloccare i campioni per 10 min in ghiaccio / a 4 ° C. </li><li> Lavare le cellule con 100 ml di FACS lavaggio (PBS 2% FBS) in piastre a 96 pozzetti, o 500 ml in tubi FACS. Centrifugare a 600 xg per 10 min a 4 ° C con il freno a basso (o fuori, se una regolazione del freno basso non è disponibile). </li><li> Rimuovere il surnatante e risospendere il pellet cellulare mediante agitazione. Aggiungi vitalità Stain (Live Morto risolvibile vitalità colorante). Preparare la redditività colorante secondo le istruzioni del produttore. Potrebbe essere necessario titolato per l&#39;uso in ogni impostazione del laboratorio / citometro. Incubare per 30 minuti al buio a 4 ° C. </li><li> Lavare le cellule μl/500 100 ml di PBS (per i tubi targa / FACS 96 pozzetti). Centrifugare a 600 xg per 10 min a basso / senza freno. Rimuovere supernatant e risospendere le cellule. </li><li> Incubare le cellule con il cocktail di anticorpi marcatori di superficie, in un volume totale di 100 microlitri. IMPORTANTE: Ottimizzare e titolare ogni citometria a flusso pannello prima di assaggiare colorazione. Incubare le cellule per 30 minuti al buio a 4 ° C. </li><li> Ripetere passo 4.5. Se la colorazione in una piastra da 96 pozzetti, le cellule di trasferimento ad un tubo FACS e portare a un volume finale di 750 ml di FACS lavaggio contenente 1% paraformaldeide (PFA) (o CytoFix, diluito 1 a 4). Procedere all&#39;acquisizione dati su un citometro a flusso. </li></ol> 5. Raccolta e preparazione del collo dell&#39;utero vaginale del lavaggio (CVL) <ol><li> Prima cytobrush campionamento, utilizzare una siringa per lavare il endocervice con 2 ml di PBS sterile e aspirare il lavaggio del fornice posteriore. </li><li> Raccogliere il campione di lavaggio aspirato in un tubo da 15 ml falco e tenere in ghiaccio / a 4 ° C fino al trattamento. </li><li> Campione centrifugare a 400 xg per 7 minuti per rimuovere detriti cellulari. </li><li> Collect il surnatante, un&#39;aliquota del campione in 1 ml o 500 microlitri aliquote e conservare a -70 ° C. Aliquote possono essere scongelati per l&#39;ELISA o l&#39;analisi di matrice cordone di proteine ​​CVL, ma evitano cicli multipli di congelamento / scongelamento. </li><li> Se lo si desidera, risospendere il pellet di RNA detriti cellulari in seguito a misurare l&#39;espressione di RNA seguendo il protocollo del produttore. </li></ol> 6. Acquisizione Dati <ol><li> Preparare una serie di tubi di compensazione che corrisponde al pannello di anticorpi. Eseguire i tubi di compensazione per regolare sovrapposizione spettrale. Eseguire i campioni su una qualsiasi flusso multicolore citometro che è configurato per gli anticorpi coniugati con fluorocromi utilizzati nel pannello. </li><li> Acquisire un campione PBMC prima di regolare Forward Scatter (FSC) e Side Scatter (SSC) della tensione, al fine di rilevare la popolazione linfocitaria. Cercate di tenerli a 100 su ciascun asse su una scala lineare. Esecuzione di un controllo PBMC renderà molto più facile trovare la popolazione linfocitaria CMC. </li><li> La soglia FSC per campioni CMC può essere necessario aumentare rispetto ai campioni di PBMC causa spalmare fino all&#39;asse SSC a bassi valori FSC. </li><li> Agitare ogni provetta prima di acquistare. Acquisire l&#39;intero tubo per massimizzare il numero di eventi cancello linfociti raccolti. Monitorare attentamente la macchina per evitare intasamenti. </li><li> Esportare i dati in un programma di analisi di citometria a flusso, come FlowJo. </li></ol> 7. Strategia di gating <ol><li> Selezionare Forward scatter lato alto (FSC-H) / Forward Area side scatter (FSC-A) per determinare la popolazione singoletto. </li><li> Selezionare Ora contro marcatore fluorescente (come ad esempio FITC) per controllare la qualità di acquisizione. Variazione della portata può introdurre artefatti nei dati. Escludere qualsiasi area che mostra discrepanza nei portata. </li><li> Identificare la popolazione linfocitaria su SSC-A/FSC-A trama. Utilizzare un controllo PBMC per facilitare l&#39;identificazione. Si noti che la popolazione linfocitaria CMC potrebbe non essere chiaro come il controllo PBMC. &lt;/ Li&gt; <li> Gate SSC-A/viability tintura di escludere le cellule morte da cellule vive. Le cellule morte possono non specificamente incorporare gli anticorpi, che introdurrà artefatti. </li><li> Porta sul SSC-A/CD3 per identificare la popolazione di cellule T. Da questa porta, individuare le popolazioni CD4 + e CD8 +. </li></ol>

<ol>
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The authors have nothing to disclose.

Date le grandi lacune nella conoscenza per quanto riguarda l&#39;immunità al tratto genitale femminile (FGT), analisi fenotipica di CMC in grado di fornire una vasta gamma di intuizioni più popolazioni linfocitarie alla cervice. Accoppiato con l&#39;analisi proteomica e le misurazioni della carica virale di lavanda cervicale, l&#39;immunità alle infezioni sessualmente trasmissibili (IST) s ed altri agenti patogeni può essere sezionato in varie popolazioni. Considerazioni tecniche...

La maggior parte delle nuove infezioni da HIV in tutto il mondo nascono attraverso la trasmissione eterosessuale, con le donne rappresentano il 47% delle nuove infezioni nel 2011 (UNAIDS 1). Comprendere il tratto genitale femminile (FGT), uno dei principali portali di ingresso per l&#39;HIV e di altri agenti patogeni a trasmissione sessuale, è di grande importanza nel cammino per trovare strategie efficaci per prevenire l&#39;infezione. Risposte immunitarie alla mucosa genitale sono chiaramente unico e differiscono da quelli misurati nel sangue periferico 2. Tuttavia, l&#39;attuale conoscenza delle dinamiche immunodeficienza al FGT è limitata al meglio. Ad oggi, gli studi di ambiente immunitario della mucosa hanno in gran parte concentrata sui tessuti linfoidi dell&#39;intestino associato (GALT), dove è diventato chiaro che i primi eventi nei tessuti della mucosa seguenti infezioni hanno un forte impatto sulla successiva progressione della malattia 3,4. Raccolta dei campioni dalla mucosa genitale rappresenta una grande sfida ed è almeno parzialmente responsabile del lack di comprensione della immunologia del FGT. Risolvere il puzzle della dinamica immunitario tra ospite e patogeno nel contesto dell&#39;ambiente distinto che è il FGT richiede metodi efficienti per raccogliere e analizzare campioni di questo locale. Il FGT è divisa in due sezioni: il tratto riproduttivo superiore che comprende le tube di Falloppio, endometrio e endocervice, e il tratto inferiore che contiene la portio e la vagina (recensito da Kaushic et al 5). Non è ancora chiaro quanto il contributo relativo di questi diversi siti è all&#39;HIV, ma si ritiene che entrambi i siti possono contribuire a voce HIV 6. Cellule T rappresentano il 40-50% dei leucociti nei tratti riproduttivi superiori e inferiori, mentre macrofagi costituiscono circa il 10% (recensito in Rodriguez-Garcia et al 2). Cellule T possono essere rilevati nella vagina, cervice e dell&#39;endometrio. I macrofagi sono più fortemente Localized nell&#39;endometrio e tessuto connettivo miometrio rispetto alla cervice, anche se possono essere rilevati in entrambi i tessuti. Infine, le cellule dendritiche plasmacitoidi (PDC) e cellule di Langerhans possono essere rilevati anche nei tessuti FGT. Il fenotipo e proporzioni delle popolazioni immunitarie e la loro suscettibilità alle infezioni da HIV possono variare importante secondo cicli ormonali, l&#39;uso di contraccettivi ormonali, vaginosi batterica o attività sessuali 5,7-9. Diversi metodi sono stati sviluppati per studiare le popolazioni immunitarie e l&#39;ambiente del FGT. Biopsia cervicale, cytobrushes cervicale e lavaggi cervico-vaginale (CVL), 10-12 sono i più comunemente utilizzato in tutto il letteratura. Collezione CVL da PBS lavanda è il metodo più semplice e permette lo studio delle proteine ​​modulatori immunitari, ma i risultati del rendimento delle cellule estremamente basso, e quindi non è adatto per studiare le popolazioni di cellule immunitarie del FGT 13. Campioni CVL sono, invece, very utili per valutare l&#39;ambiente immunitario del FGT misurando l&#39;espressione di diverse citochine, chemochine o fattori antimicrobici usando metodi quali ELISA, citochine branello matrice 14 o spettrometria di massa 15,16. Caratterizzazione delle frequenze di cellule immunitarie, fenotipi e funzioni può essere ottenuto mediante prelievo di cellule mononucleate cervicali (CMC) da cytobrush cervicale o mediante campionamento biopsia cervicale. Campionamento biopsia cervicale è un metodo invasivo che aumenta il disagio e rischio di sanguinamento e richiede 2 a 11 giorni per guarire seguendo la procedura a seconda dello stato immunitario della donna 12. D&#39;altra parte, cytobrushes cervicali, nonostante la minore resa di cellule raccolte, è un metodo meno invasivo e più conveniente per raccogliere le cellule immunitarie dal FGT. Entrambi i metodi possono raggiungere la stessa resa di leucociti CD45 +, ma due cytobrushes cervicali sequenziali sono necessari per avere la stessa quantità di cellule contenute in una biopsia 13. Tuttavia, il campionamento cytobrush fornisce ancora un numero accettabile di cellule (cellule + circa 5.000 CD45 / cytobrush) per ulteriore ex vivo fenotipizzazione mediante citometria di flusso 14. Inoltre, la caratterizzazione funzionale può essere effettuata su questi campioni, la stimolazione e flusso intracellulare citometria o qPCR sono state effettuate utilizzando CMC cytobrush derivate per identificare HIV-specifiche risposte immunitarie 17 o polarizzazione cellulare Th 18. L&#39;espansione della popolazione di cellule T può anche facilitare gli studi funzionali con CMC 19. È importante notare che le biopsie e cytobrushes campionare porzioni distinte della FGT. Biopsie sono derivati ​​dalla porzione superiore del epitelio e stroma della portio 12,13, mentre cytobrushes cervicali campionare della cervice, raccogliendo cellule derivate dall&#39;epitelio di endocervice e presumibilmente la zona di trasformazione. Campioni Cytobrush quindi degustare un regione composto da un unico strato di epitelio colonnare, mentre biopsie, comprendono una regione rivestita da un epitelio squamoso stratificato 5. Di conseguenza, la natura delle popolazioni di leucociti raccolti da biopsia cervicale e cytobrush differisce. Biopsie raccolgono una percentuale maggiore di cellule T CD3 +, mentre cytobrushes comportano la raccolta di una maggiore percentuale di monociti CD14 + / macrofagi 13. Studiare la immunologia della FGT è stato un interesse per molti anni 20-22 e abbiamo accumulato una grande esperienza con lo studio della CMC cytobrush-derivati. I nostri studi si concentrano principalmente sullo studio del virus HIV-infetti, non infetto e HIV-esposti sieronegativi (HESN) i lavoratori di sesso femminile da Nairobi, Kenya. HIV si replica preferenzialmente nelle cellule T attivate 23 e inferiore numero di cellule attivate che possono essere avviati da HIV nel FGT potrebbero contribuire alla protezione contro l&#39;acquisizione HIV. In linea con questa ipotesi, diversi studies hanno descritto attivazione immunitaria più bassa tra i lavoratori di sesso HESN che sono altamente esposti al virus HIV ancora rimanere infetto 24,25, e questo fenotipo quiescente si osserva anche nel FGT 14. Qui, descriviamo la metodologia per l&#39;elaborazione e la valutazione cellule T di attivazione nei campioni CMC derivati ​​da cytobrushes cervicali da ex vivo citometria a flusso.

<table><tbody><tr><td>100 &amp;mu;m cell strainer for 50 ml Falcon tube</td><td>BD</td><td>352360</td><td>CMC processing</td></tr><tr><td>RPMI 1640</td><td>Hyclone</td><td>SH30027.01</td><td>CMC processing</td></tr><tr><td>Fetal bovine serum</td><td>Life technology</td><td>16000044</td><td>CMC processing</td></tr><tr><td>Fungizone</td><td>Life technology</td><td>15290-018</td><td>CMC processing</td></tr><tr><td>Penicillin/streptomycin</td><td>Sigma</td><td>P4333-20ml</td><td>CMC processing</td></tr><tr><td>50 ml Falcon tube</td><td>Fisher</td><td>14-959-49A</td><td>CMC processing</td></tr><tr><td>Blood Bank disposable transfer pipette</td><td>Fisher</td><td>13-711-6M</td><td>CMC processing</td></tr><tr><td>Cytobrush plus</td><td>Cooper surgical</td><td>C0121</td><td>CMC sampling</td></tr><tr><td>Disposable cervical scraper</td><td>Quick medical</td><td>2183</td><td>CMC sampling</td></tr><tr><td>15 ml Falcon tube</td><td>Fisher</td><td>14-959-70c</td><td>CVL processsing</td></tr><tr><td>1.5 ml tube Eppendorf</td><td>Fisher</td><td>05-402-18</td><td>CVL storage</td></tr><tr><td>LIVE/DEAD Fixable Cell Stain Kit</td><td>Invitrogen</td><td>Various</td><td>Flow cytometry reagent</td></tr><tr><td>Fixation buffer (4% PFA)</td><td>BD</td><td>554655</td><td>Flow cytometry regeant</td></tr><tr><td>IgG mouse</td><td>Sigma</td><td>I8765</td><td>Flow cytometry regeant</td></tr></tbody></table>

collection, isolation, and flow cytometric analysis of human endocervical samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

Flusso multiparametrica citometria è un potente strumento per analizzare i fenotipi e le funzioni dei sottoinsiemi di cellule in tessuti precedentemente non caratterizzati. Analisi dei campioni CMC può dare informazioni sia su linfociti e monociti popolazioni con le strategie di gating adeguate. Un rappresentante CMC strategia gating, rispetto ad un profilo PBMC abbinato, è mostrato in Figura 2. L&#39;FSC-A piuttosto FSC-H trama consente l&#39;esclusione di doppietti cell...

Watch this Scientific Journal Video about Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples at JoVE.com

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Raccolta, isolamento e analisi citofluorimetrica di campioni endocervicali umano

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

collection-isolation-flow-cytometric-analysis-human-endocervical

Research

JoVE Journal

Medicine

19.0K Views.  University of Manitoba. The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays. 

Video: Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Isolamento di cellule endoteliali umane da Colon normale e Carcinoma colorettale - un protocollo migliorato

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Ricerca

Ricerca sul cancro

Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, and nucleic acids originating from normal tissues, but some of these substances also originate from the diseased area. The investigation and analysis of these substances in the body fluids play a pivotal role in the diagnosis of various diseases. Therefore, it is important to accurately separate the required substances, and several techniques are developed to be used for this purpose.
We have developed a lab-on-a-disc type of device and platform named CD-PRIME. This device is automated and has good results for sample contamination and sample stability. Moreover, it has advantages of a good acquisition yield, a short operation time, and high reproducibility. In addition, depending on the type of disc to be mounted, plasma containing cell-free DNA, circulating tumor cells, peripheral blood mononuclear cells, or buffy coats can be separated. Thus, the acquisition of a variety of materials present in the body fluids can be done for a variety of downstream applications, including the study of omics.

This paper describes the application of automated equipment to easily and efficiently separate and collect substances, such as cell-free DNA and circulating tumor cells, from whole blood.

Separazione automatica e raccolta di sostanze correlate al cancro da campioni clinici

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

Due metodi per stabilire primarie cellule umane endometriale stromali da isterectomia campioni

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Medicina

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Isolamento di cellule umane endoteliali linfatico da Multi-parametro fluorescenza-attivato cell sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation and Flow Cytometric Analysis of Human Endocervical 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 essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

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 ...

Immunologia e infezioni

An Efficient Method for Extracting Human Fallopian Tube Epithelia for Single-cell Analyses

Human fallopian tubes are intrinsic to reproduction. The purpose of the fallopian tubes is to allow for the transit of sperm, the ovum, and if fertilization is successful, the embryo. The epithelial cells that line the inner surface of fallopian tubes are integral to both normal and abnormal fallopian tube processes, including disease initiation. After menopause, fallopian tubes cease to have a significant role in the body and the intraepithelial cell makeup changes. We describe a method in which these epithelial cells can be isolated from fresh fallopian tubes with minimal stromal cell contamination into a single-cell suspension. These cells can be grown in culture or used for further analysis such as flow cytometry and single-cell RNA sequencing. This isolation protocol can be achieved in 4-6 h and yields viable cells that can be used for immediate downstream analysis. This efficient protocol facilitates the isolation of fallopian tube epithelial cells with an enriched epithelial population.

This protocol describes a one-day method for the isolation of human fallopian tube epithelial cells. Isolated epithelial cells can be plated in 2-dimensional (2D) culture or dissociated into single-cell suspensions and utilized in downstream experiments, including flow cytometry and single-cell RNA sequencing.

Un metodo efficiente per l'estrazione degli epiteli umani delle tube di Falloppio per l'analisi di singole cellule

Human fallopian tubes are intrinsic to reproduction. The purpose of the fallopian tubes is to allow for the transit of sperm, the ovum, and if ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

Ex Vivo Infezione della Mucosa genitale umana del tessuto linfoide e donna con il Virus dell'immunodeficienza umana 1 e Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

Isolamento di cellule dendritiche dal tratto riproduttivo femminile umano per studi fenotipici e funzionali

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

Isolamento delle cellule linfoidi innate uterine per l'analisi mediante citometria a flusso

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...

Flow Cytometric Analysis of Biomarkers for Detecting Human Sperm Functional Defects

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no abnormalities in conventional semen parameters. Additional technologies are needed to explain the idiopathic infertility and detect subtle sperm defects. Currently, biomarkers of sperm function, including sperm apoptosis, mitochondrial membrane potential (MMP), and DNA damage, reveal sperm physiology at the molecular level and are capable of predicting male fertility.
With flow cytometry (FCM) techniques, each of these markers can be rapidly, accurately, and precisely measured in human semen samples, but time costs substantially increase and results could be obstructed if all the biomarkers need to be tested with a single cytometer. In this protocol, after collection and immediate incubation at 37 &#176;C for liquefication, semen samples were further analyzed for sperm apoptosis using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The MMP was labeled with 5,5&#8242;,6,6&#8242;-tetrachloro-1,1&#8242;,3,3&#8242;-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) probe, and DNA damage was assessed using the sperm chromatin structure assay (SCSA) with acridine orange (AO) staining. Thus, flow cytometric analysis of sperm function markers can be a practical and reliable toolkit for the diagnosis of infertility and evaluation of sperm function at both bench and bed.

The present protocol provides a practical solution that allows for the measurement of apoptosis, mitochondrial membrane potential, and DNA damage in human sperm with a single cytometer.

Analisi citofluorimetrica di biomarcatori per la rilevazione di difetti funzionali degli spermatozoi umani

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no ...