Questo progetto è stato sostenuto dal National Institutes of Health Borse HL087088 (MK) e HL18208 (MK).

1. Isolamento dei linfociti T <ol><li> Ottenere sangue umano da un donatore sano. Lasciare che il sangue a temperatura ambiente (~ 30 min) prima di procedere alla fase successiva. </li><li> Delicatamente Pipettare 3 ml di sala multimediale densità Polymorph gradiente di temperatura in un ml 8 a fondo rotondo in polistirene. Delicatamente aggiungere 3 ml di sangue intero in cima ai media Polymorph. E &#39;importante evitare la sovrapposizione dei due reagenti. </li><li> Centrifugare le provette a 500 xg per 45 minuti a temperatura ambiente. </li><li> Dopo la centrifugazione, le cellule mononucleate del sangue periferico (PBMC) sono ora separati da altri componenti del sangue nello strato superiore delle cellule. Lo strato PBMC appare, dall&#39;alto verso il basso, come la prima band nuvoloso. </li><li> Rimuovere con attenzione il chiaro di colore giallo fase superiore del sangue, sopra il livello PBMC, e quindi utilizzare un P1000 micropipetta per trasferire lo strato PBMC a 15 ml o 50 ml di tubo conico. </li><li> Lavare i PBMC due volte con PBS, centrifugazione le cellule a 500 xg per 5 minuti ogni volta. Il supernatante sarà un po &#39;torbida dopo ogni lavaggio. </li></ol> 2. Cultura dei diritti dell&#39;uomo e Linfociti T <ol><li> Utilizzando una pipetta, trasferire il PBMC in un pallone T-75 della cultura in 20 ml RPMI 1640 supporti contenenti il ​​10% FBS, 1% di penicillina / streptomicina, e 1 mg / ml fitoemoagglutinina (PHA). </li><li> Incubare a 37 ° C e 5% di CO 2 per almeno 1 ora, e fino a 24 ore. Questo passaggio consente monociti, che sarà aderente alla superficie del pallone, di essere separati dai linfociti che rimangono in sospensione. Se una breve incubazione (1 ora) viene utilizzata in questa fase, è accettabile utilizzare mezzi RPMI 1640 contenente il 10% FBS e 1% di penicillina / streptomicina senza integrazione con PHA, come specificato al punto 2.1. </li><li> Rimuovere con cura tutti i supporti dal pallone, aggiungerlo ad un tubo da 50 ml conica, e centrifugare a 500 xg per 5 minuti. </li><li> Risospendere il pellet di cellule, che ora contiene principalmente i linfociti, le cellule e il trasferimento a un nuovo T-75 beuta contenente 25 mL RPMI 1640 supporti contenenti il ​​10% FBS, 1% di penicillina / streptomicina, e 1 mg / mL di PHA. </li><li> Incubare a 37 ° C per 3 giorni (2 giorni se l&#39;incubazione iniziale di PBMC era notte). Dopo 24 ore di crescita, può essere necessario aggiungere 15-20 ml di mezzi freschi e il trasferimento ad un T-175 più grandi pallone. </li><li> Dopo 3 giorni, utilizzare una pipetta di rimuovere il supporto e linfociti sospeso il pallone e in una provetta da 50 ml. Centrifugare a 500 xg per 5 minuti. </li><li> Risospendere il pellet cellulare e le cellule trasferire in un pallone nuovo T-75 o T-175 contenente 25 ml (T-75) o 50 ml (T-175) RPMI 1640 con 10% FBS, 1% di penicillina / streptomicina, e 20 ng / mL umano IL-2 o IL-15. </li><li> Crescere linfociti per 4-7 giorni. Se a partire da un T-75 fiasco, la cultura dovrà essere ampliato e trasferito in un T-175 fiasco dopo 1-2 giorni. </li></ol> 3. Test in vitro la migrazione dei linfociti <ol><li> 1 giorno prima del test di migrazione, il cappotto di un fondo di vetro piatto 0,17 mm con 20 mg / ml di proteina A o G in PBS notte a 4 ° C. </li><li> Lavare il piatto ampiamente con PBS. </li><li> Immobilizzare ICAM-1/Fc umano (10 mg / mL) e umano SDF-1 (2 mg / mL) con l&#39;aggiunta di questi reagenti in soluzione PBS al piatto e incubazione 4 ore a temperatura ambiente. </li><li> Preparare i linfociti T determinando prima la densità delle cellule in coltura nel T-175 con un fiasco emocitometro. Circa 2-5 x 10 5 cellule dovrebbero essere utilizzati per piatto per ottenere una densità cellulare appropriato per l&#39;analisi della migrazione. </li><li> Lavare i linfociti T per due volte con PBS e poi risospendere in 1 ml L-15 supporti contenenti 1 mg / ml di D-glucosio. </li><li> Lavare il piatto rivestito con ICAM-1/Fc e SDF-1 estesamente con PBS. </li><li> Trasferimento dei linfociti T in 1 media ml al piatto e mantenerli a 37 ° C. </li></ol> 4. Catturare una sequenza di immagini Utilizzando il software NIS Elements <ol><li> Aperto il software NIS Elements. </li><li> Nel menu delle impostazioni della fotocamera, scegliere &quot;binning 2x2&quot; come modalità per entrambe le immagini dal vivo e la cattura delle immagini. </li><li> Vai al menu Applicazioni e scegliere Definisci / Esegui esperimento. </li><li> Scegli l&#39;intervallo di tempo tra le immagini e la lunghezza totale della sequenza di acquisizione delle immagini. </li><li> Premere il tasto &quot;Run&quot; per iniziare l&#39;acquisizione di immagini. </li><li> Le celle possono essere monitorati e parametri di migrazione (velocità, lunghezza del percorso, spostamento, ecc) quantificato utilizzando uno dei pacchetti software, tra cui ImageJ, AutoQuant o Volocity (Figura 1). </li><li> Per generare un &quot;complotto ragnatela&quot;, le coordinate XY per ogni cella e timepoint sono raccolti (Figura 2) e proiettate su un grafico con un comune punto di partenza per ogni cella all&#39;origine (Figura 3). </li></ol> 5. Rappresentante Risultati Il giorno 6 di coltura in presenza di uno di IL-2 o IL-15, i linfociti Tcostituiscono&gt; 98% delle cellule, come determinato dalla colorazione CD3 positivi e CD4 positivi e / o CD8 colorazione. Per IL-2, abbiamo trovato 83% + cellule CD4, CD8 + 15% e &lt;1% CD4 + CD8 +. Per IL-15, abbiamo trovato 88% + cellule CD4, CD8 + 11% e &lt;1% CD4 + CD8 +. Durante la migrazione dei linfociti T in ICAM-1/SDF-1 substrati, le cellule mostravano una velocità di circa 15 micron / min che può essere sostenuta per un tempo di 1 ora di tempo. La migrazione dei linfociti T sulle ICAM-1/SDF-1 dipende LFA-1-mediata adesione, come un anti-LFA-1-bloccante ligando anticorpi inibisce fortemente la migrazione. <img src="/files/ftp_upload/2017/2017fig1.jpg" alt="Figura 1" /> Figura 1. Monitoraggio della migrazione delle cellule T linfociti. Linfociti T isolati dal sangue intero e coltivate in presenza di IL-2 o IL-15 per 6 giorni è stato permesso di aderire e migrare sul fondo di vetro piatti rivestiti con 10 mg / ml ICAM-1 e 2 mg / mL SDF-1. Immagini sono state acquisite ogni 10 secondi per 30 minuti. Le cellule sono state identificate in ogni immagine e monitorati nel tempo utilizzando un software Volocity. Questo filmato mostra la migrazione dei linfociti T casuale ad una velocità di circa 15 micron / min. <a href="http://www.jove.com/files/ftp_upload/2017/2017_Kim_Movie1.mov">Clicca qui per vedere il video.</a> Movie 1. Inseguimento cella di migrazione dei linfociti T. Linfociti T isolati dal sangue intero e coltivate in presenza di IL-2 o IL-15 per 6 giorni è stato permesso di aderire e migrare sul fondo di vetro piatti rivestiti con 10 mg / ml ICAM-1 e 2 mg / ml SDF-1 . Immagini sono state acquisite ogni 10 secondi per 30 minuti. Le cellule sono state identificate in ogni immagine e monitorati nel tempo utilizzando un software Volocity. Questo filmato mostra la migrazione dei linfociti T casuale ad una velocità di circa 15 micron / min. <img src="/files/ftp_upload/2017/2017fig2.jpg" alt="Figura 2" /> Figura 2. Posizione della cella spazio-temporale. Le coordinate XY di ogni cella sono stati ottenuti per ogni punto temporale utilizzando il software Volocity. <img src="/files/ftp_upload/2017/2017fig3.jpg" alt="Figura 3" /> Figura 3. &quot;Spider complotto web.&quot; Utilizzando il XYT coordinate per ogni cella, 15 cellule sono state scelte a caso e tracciati con un punto di partenza comune all&#39;origine. Confrontando trame ragnatela dà una rappresentazione visiva rapido delle differenze di migrazione tra diverse condizioni sperimentali. Parcelle per la migrazione dei linfociti T in condizioni di controllo (pannello di sinistra) e in presenza di un anti-LFA-1-bloccante ligando anticorpi (pannello di destra).

<ol>
	<li>Hyun, Y. M., Chung, H. L., McGrath, J. L., Waugh, R. E., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+integrin+VLA-4+localizes+to+the+lamellipodia+and+mediates+t+cell+migration+on+VCAM-1.">Activated integrin VLA-4 localizes to the lamellipodia and mediates t cell migration on VCAM-1.</a> J Immunol. 183, 359-369 (2009).</li><li>Morin, N. A., Oakes, P. W., Hyun, Y. M., Lee, D., Chin, Y. E., King, M. R., Springer, T. A., Shimaoka, M., Tang, J. X., Reichner, J. S., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonmuscle+myosin+heavy+chain+IIA+mediates+integrin+LFA-1+de-adhesion+during+T+lymphocyte+migration.">Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration.</a> The Journal of experimental medicine. 205, 195-205 (2008).</li><li>Hyun, Y. M., Lefort, C. T., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+integrins+and+their+ligand+interactions.">Leukocyte integrins and their ligand interactions.</a> Immunologic research. , (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

In questo esperimento, forniamo i dettagli su un sistema semplice per analizzare la motilità del primario linfociti T umani. Saggi di migrazione in vitro sono stati utilizzati per sezionare i ruoli di molte molecole e le vie di segnalazione coinvolte nella locomozione dei vari tipi di cellule. Alcuni controlli critici da tenere a mente quando si progetta l&#39;esperimento proprio dal nostro protocollo sono: 1) non-adesivo o non-integrina rivestimenti substrato adesivo, come sieroalbumina bovina (BSA) ...

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<td>RPMI 1640</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11875</td>
<td>&nbsp;</td>
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<td>FBS</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH300070.03</td>
<td>&nbsp;</td>
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<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
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<td>1-Step Polymorphs</td>
<td>&nbsp;</td>
<td>Accurate Chemical</td>
<td>AN221725</td>
<td>&nbsp;</td>
<tr>
<td>PHA</td>
<td>&nbsp;</td>
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<td>Rec. human IL-2</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
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<td>&nbsp;</td>
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<td>R&amp;D Systems</td>
<td>247-IL</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>R&amp;D Systems</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>R&amp;D Systems</td>
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human t lymphocyte isolation, culture and analysis of migration in vitro

La migrazione dei linfociti T richiede l&#39;interazione adesiva delle integrine superficie cellulare con ligandi espressi su altre cellule o con proteine ​​della matrice extracellulare. L&#39;attivazione delle integrine preciso spazio-temporale da uno stato a bassa affinità ad uno stato ad alta affinità al bordo cellula principale è importante per la migrazione dei linfociti T<sup&gt; 1</sup&gt;. Allo stesso modo, retrazione del bordo d&#39;uscita delle cellule, o uropod, è un passo necessario a mantenere persistente integrina-dipendente motilità dei linfociti T<sup&gt; 2</sup&gt;. Molti approcci terapeutici per autoimmuni o infiammatorie integrine obiettivo malattie come mezzo per inibire l&#39;eccessiva reclutamento e la migrazione dei leucociti<sup&gt; 3</sup&gt;. Per studiare gli eventi molecolari che regolano la migrazione dei linfociti T umani, abbiamo utilizzato un<em&gt; In vitro</emSistema&gt; per analizzare la migrazione delle cellule su un substrato bidimensionale che simula l&#39;ambiente che un linfocita T incontra durante il reclutamento dal sistema vascolare. Linfociti T vengono prima isolati da donatori umani e vengono poi stimolati e in coltura per 7-10 giorni. Durante il test, i linfociti T possono aderire e migrare su un substrato rivestito con molecola di adesione intercellulare-1 (ICAM-1), un ligando per integrina LFA-1 e cellule stromali derivate fattore-1 (SDF-1). I nostri dati mostrano che i linfociti T presentano una velocità migratorio di ~ 15 micron / min. La migrazione dei linfociti T può essere inibita dal blocco delle integrine<sup&gt; 1</sup&gt; O con inibitori della macchina actomiosina cellulare che regola la migrazione delle cellule<sup&gt; 2</sup&gt;.

Watch this Scientific Journal Video about Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro at JoVE.com

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

La migrazione dei linfociti T si verifica durante homing verso gli organi linfoidi, uscire dal sistema vascolare, e di entrare in tessuti periferici. Qui, descriviamo un protocollo che può essere utilizzato per analizzare la migrazione dei linfociti T<em&gt; In vitro</em&gt;.

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 ...

human-t-lymphocyte-isolation-culture-analysis-migration

Research

JoVE Journal

Immunology and Infection

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

46.1K Views.  University of Rochester. La migrazione dei linfociti T si verifica durante homing verso gli organi linfoidi, uscire dal sistema vascolare, e di entrare in tessuti periferici. Qui, descriviamo un protocollo che può essere utilizzato per analizzare la migrazione dei linfociti T In vitro.

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

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac Transposon modifica del sistema di cellule primarie umane T

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Ricerca

Immunologia e infezioni

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Linfociti Isolamento da Pelle umana per l&#39;analisi fenotipica e<em&gt; Ex Vivo</em&gt; Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Mouse Naïve CD4<sup&gt; +</sup&gt; Isolamento T cellulare e<em&gt; In vitro</em&gt; Differenziazione in T sottopopolazioni cellulari

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

Imaging CD4 T cellule interstiziali migrazione nel derma infiammate

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

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 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 ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

L&#39;analisi dei linfociti stravaso utilizzo di un<em&gt; In Vitro</em&gt; Modello del Sangue-Cervello umano Barriera

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Quantificazione del linfocita murino traffico In Vivo e chemiotassi del monocito umano In Vitro

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic 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 ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Analisi di taglio indotta da flusso di migrazione delle cellule di B di zona marginale murino In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...