Name: isolation and flow cytometric analysis of human endocervical gamma delta t cells
Uploaded: 2017-02-06T10:30:00.000+00:00
Duration: 8 min 10 s
Description: Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells at JoVE.com

Wir danken den Studienteilnehmern Miami WIHS für ihre Bereitschaft, an dieser Arbeit teilzunehmen. Die Daten in diesem Manuskript wurden von der Vermittlungs-HIV-Studie Miami gesammelt Frauen (WIHS). Der Inhalt dieser Veröffentlichung sind allein in der Verantwortung der Autoren und nicht die offizielle Position der National Institutes of Health (NIH) darstellen. Diese Arbeit wurde vom Nationalen Institut für Allergie und Infektionskrankheiten unterstützt und Vermittlungs-HIV-Infektion Studie Frauen (WIHS) [Gewährungsnummer U01 AI103397], Nationales Institut für Allergie und Infektionskrankheiten, National Institutes of Health [Grant-Nummer P30AI073961], National Center for Advancing Translational Sciences und das Nationale Institut für Minderheiten Gesundheit und Gesundheit Disparitäten, National Institute of Health [Gewährungsnummer UL1TR000460 und 1KL2TR000461] und National Institute of Child Health und Human Development, National Institute of Health [Gewährungsnummer K23HD074489].

Study Aktivitäten fand an der Universität von Miami HIV Research Unit in Zusammenarbeit mit dem HIV-Vermittlungs-Studie Miami Frauen (WIHS) und dem Miami Zentrum für AIDS-Forschung (CFAR) .Institutional Review Board (University of Miami Miller School of Medicine) Genehmigung wurde vor erhalten zur Rekrutierung und jegliche Beurteilung oder Untersuchung im Zusammenhang mit Verfahren. 1. Endozervikale Pinsel Probenentnahme HINWEIS: Die Teilnehmer unterzog sich einer vaginalen Untersuchung durch geschultes MD oder Gynäkologen und Sammlung von intraepitheliale Lymphozyten wurden durchgeführt durch das Einfügen Zytobürste unter Speculum Prüfung durchgeführt. <ol><li> Teilnehmer Merkmale <ol><li> Rekrutieren Frauen Teilnehmer, die 18 bis 45 Jahre alt sind, sexuell aktiv, nicht schwanger und nicht auf Verhütungsmittel Medikamente oder mit einem Intrauterinpessar. </li><li> Vor vaginale Untersuchung unterziehen haben die Teilnehmer 10 ml Blut ziehen in grünTop-Heparin Vacutainer für Pbmc (PBMC) Isolierung. dienen als Kontrolle für endocervical intraepitheliale Analyse 9. </li></ol></li><li> Sammlung der endozervikale Proben <ol><li> Legen Sie eine sterile Speculum von geeigneter Größe in die Vagina ohne Schmierung. Verwenden Sie ein warmes Wasser Einführen des Spekulums zu erleichtern und steriler Gaze den Schleim zu reinigen. Die Position des Speculum ermöglicht eine vollständige Visualisierung des Os und ectocervix. </li><li> Legen Sie endocervical Pinsel in die Endozervikalkanal, bis nur noch die Borsten am nächsten an der Hand sichtbar sind. Drehen Sie die Bürste im Uhrzeigersinn um 360 °. Übertragen Sie die Bürste in einem 15 ml-Röhrchen, die 5 ml IMDM-Medium. </li><li> Unmittelbar nach der Entnahme wird das Reagenzglas Zytobürste auf Eis enthält. Um eine hohe Lebensfähigkeit der gesammelten intraepitheliale Zellen zu erhalten, liefern die Zytobürste ins Labor und Prozess innerhalb von 1 h. </li></ol></li></ol><ol><li> Vortex-Röhrchen mit endocervical Zytobürste Anwendung 4 kurz (10 sec) Schlägen. </li><li> Zentrifugieren Sie das Röhrchen, mit Zytobürste, 10 min bei 250 x g. </li><li> Vorsichtig die Cytobrush aus dem Rohr zu entfernen (nicht das Pellet am Boden des Röhrchens zu stören) und 1 ml IMDM Medium hinzuzufügen und das Röhrchen auf Eis stellen. </li><li> Legen Sie die Zytobürste auf der 100 mm Zelle Sieb auf dem 50-ml-Tube. </li><li> Waschen Sie die Zytobürste mit 20 ml IMDM. </li><li> Zentrifugenröhrchen für 10 Minuten bei 250 x g. </li><li> Den Überstand abgießen und pelletierten Zellen resuspendieren in 1 ml IMDM und verbinden sich mit dem bereits wieder suspendierten Pellets in 2.3 beschrieben. </li><li> Zählen Sie die Zellen unter Verwendung eines automatisierten Zellzähler 10 oder die Zellen zählen Zählkammer durch die Kombination von 10 &amp; mgr; l der Zellsuspension mit 10 &amp; mgr; l Trypanblaufarbstoff. Pipettieren vorsichtig nach oben und unten zehnmal um die Zellen zu mischen und färben. Last 10 &amp; mgr; ldes Gemisches in der Öffnung der einen der beiden Kammern Hämozytometer. Zählen Sie alle Zellen in den fünf Rasterflächen. Halten Sie eine separate Zählung der lebensfähigen und nicht lebensfähigen Zellen. Bestimmen Sie die Lebensfähigkeit der Zellen unter Verwendung der folgenden Formel:% Lebensfähige Zellen = Nicht lebensfähige Zellen x 100 / Gesamtzellzahl Zellen. Bestimmen die nachfolgenden Zellkonzentration pro ml (und die Gesamtzahl der Zellen) unter Verwendung der folgenden Berechnungen. Insgesamt Zellen pro ml = Gesamt Zellen gezählt x (Verdünnungsfaktor / Anzahl der gezählten Quadrat) x 10.000 Zellen / ml. </li><li> Gefriert isoliert endozervikale Zellen mittels Einfrieren-Medium, enthaltend 10% DMSO und 90% FBS. </li></ol> 3. Durchflusszytometrie <ol><li> Re-suspendieren 1-3 x 10 5 Zellen endocervical in 100 ul FACS - Puffer (PBS , enthaltend 1% Rinderserumalbumin </li><li> 1 &amp; mgr; l / 10 6 , LIVE / DEAD Fixable Yellow Dead Cell Stain Kit und Antikörper für Oberflächenmarker in den in Tabelle 1 beschrieben Konzentrationen. </li><li> Inkubieren Sie die Zellen für 30 Minuten bei 4 ° C, vor Licht geschützt. </li><li> 1 ml FACS-Puffer und die Zellen und Zentrifugenröhrchen für 5 Minuten bei 350 xg </li><li> Re-suspendieren Zellpellet in 300 &amp; mgr; l 1% Paraformaldehyd. </li><li> Bereiten Kompensationssteuerungen für die Färbung verwendet, um Perlen und Antikörper durch Zugabe von 100 &amp; mgr; l FACS-Puffer und ein Tropfen der Perlen und die gleiche Konzentration der für die Probenfärbung verwendeten Antikörper. </li><li> Erwerben gefärbten Perlen und Proben auf einem Durchflusszytometer, ausgestattet mit 405 nm, 488 nm und 635 nm Laser. </li></ol> 4. Endozervikale Gamma-Delta-T-Zellen Isolation <ol><li> Magnetic - Bead - Isolation <ol><li> Re-suspend endocervical Zellpellet (≤10 7 insgesamt Zellen) beschrieben in 2,6, in 40 &amp; mgr; l Puffer, die in der TCR gamma delta Mikrobead Kit in der Tabelle der Materialien und Reagenzien verwiesen. </li><li> Folgen Sie den Anweisungen Kit für zweistufigen Färbeprotokolls: Erstens, Witzh anti-TCR gamma delta Hapten-Antikörper und der zweite, mit anti-Hapten-Mikrobeads-FITC </li><li> Nach einer Inkubation von 15 min bei 4-8 ° C, wasche die Zellen durch Zugabe von 1 ml Puffer und Zentrifugieren bei 300 xg für 10 min. Den Überstand umfüllen durch das Rohr Umkehren und tippen Sie trocken mit Hilfe von einem Stück Papier. </li><li> Re-suspendieren des Zellpellets in 500 &amp; mgr; l Puffer zur Verfügung gestellt und bereiten die magnetische Säule, indem sie in geeigneten Magnetscheider Platzierung gemäß dem Protokoll des Herstellers. </li><li> Bewerben Zellsuspension auf die Säule und sammeln unmarkierten Zellen, die durchlaufen und waschen Säule 3x mit entsprechenden Menge an Puffer (500 ul). Führen Waschschritte durch Zugabe von Puffer dreimal, jedes Mal, wenn die Spalte Behälter leer ist. Sammeln gesamten Ausflusses. Dies ist die unmarkierten, negativen Zellfraktion </li><li> Entfernen Sie die Säule bilden die Magnetscheider und Pipette 1 ml Puffer auf die Säule und sofort spülen Fraktion mit dem magnetisch-Labeled-Zellen, indem Sie den Kolben mit der Säule geliefert Anwendung. HINWEIS: Isolierte gamma delta T-Zellen sind bereits für durchflusszytometrische Analyse fluoreszenz gefärbt, da magnetisch markierten Anti-Hapten-Mikrokügelchen mit Fluoreszenzfarbstoff FITC konjugiert sind. </li></ol></li><li> Zellsortierung <ol><li> Für Zellsortierung, Etikett Endozervikalzellen mit Lebensfähigkeit sterben und Antikörper-Panel, wie in Kapitel 3 beschrieben. </li><li> Richten Sie elektronische Tore auf dem Live - CD45 + CD3 + und Gamma - Delta - positive Zellen und Zellsortierung durchführen. </li></ol></li></ol>

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	<li>Kaul, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genital+tract+immune+milieu:+an+important+determinant+of+HIV+susceptibility+and+secondary+transmission.">The genital tract immune milieu: an important determinant of HIV susceptibility and secondary transmission.</a> J Reprod Immunol. 77, 32-40 (2008).</li><li>Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+early+infection+to+prevent+HIV-1+mucosal+transmission.">Targeting early infection to prevent HIV-1 mucosal transmission.</a> Nature. 464, 217-223 (2010).</li><li>Hayday, A. 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D., Poonia, B., Li, H., Cairo, C., Chaudhry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gammadelta+T+Cells+in+HIV+Disease:+Past,+Present,+and+Future.">gammadelta T Cells in HIV Disease: Past, Present, and Future.</a> Front Immunol. 5, 687 (2014).</li><li>Hayday, A. C., Spencer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barrier+immunity.">Barrier immunity.</a> Sem Immunol. 21, 99-100 (2009).</li><li>Cheroutre, H., Lambolez, F., Mucida, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+light+and+dark+sides+of+intestinal+intraepithelial+lymphocytes.">The light and dark sides of intestinal intraepithelial lymphocytes.</a> Nature Rev Immunol. 11, 445-456 (2011).</li><li>Strbo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+Intra-Epithelial+Endocervical+Gamma+Delta+(GD)+1+T+Cells+in+HIV-Infected+Women.">Loss of Intra-Epithelial Endocervical Gamma Delta (GD) 1 T Cells in HIV-Infected Women.</a> AJRI. 75, 134-145 (2016).</li><li>Szabo, S. 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Autoren haben keine gewerblichen oder anderen Verein, der einen Interessenkonflikt (zB Pharma Aktienbesitz, Beratung, Beirat Mitgliedschaft, relevante Patente oder Forschungsförderung) darstellen könnten.

Bewertung der weiblichen Genitaltrakts unteren Anfälligkeit Schleimhaut ist ein wesentlicher Bestandteil von klinischen Studien Risiko einer HIV-Erfassung und Übertragung Adressierung. Typischerweise wird FGT Anfälligkeit für eine HIV - Infektion durch Messung löslichen Immunmodulatoren in Genitalsekreten (Zytokine, Chemokine und antimikrobielle Peptide) bewertet 12, 13. Allerdings sind diese Biomarker nur indikativ für vaginale Entzündung, werden von phy...

Wir entwickelten Methodik der Verwendung von endozervikalen Bürstenproben intraepitheliale GD-T-Zellen zu bewerten. Der Endozervikalkanal wird durch eine einzige Schicht von Epithel ausgekleidet. Intraepitheliale Lymphozyten repräsentieren Front - Lymphozyten in der Epithelschicht Wohnsitz, die sich schnell Immunreaktionen auslösen können pathogene Mikroben 7 nach der Begegnung, 8. die immunologischen Ereignisse des endozervikalen intraepitheliale Kompartiment zu verstehen, ist für das Design von wirksamen Strategien wichtig zur Verhinderung von Infektionen, einschließlich HIV. Unser Verfahren kann für frisch gesammeltem humanen endozervikalen Proben sowie gefrorene endozervikalen Zellen angewendet werden, um die Rolle von intraepithelialen GD T-Zellen bei Frauen mit HIV-Infektion erforschen oder einem Risiko für eine HIV-Infektion. Das Hauptziel dieses Protokolls ist es, neue immunologische Ereignisse in endocervical intraepitheliale Raum zu entdecken und zu evaluate GD T-Zell-Antworten als Marker mit einer HIV-Infektion bei Frauen Anfälligkeit der Schleimhaut. Die erheblichen Lücken im Wissen um FGT Immunität sind teilweise aufgrund der Schwierigkeit bei der erfolgreichen Erfassung und Verarbeitung Schleimhautproben. Darüber hinaus ist die komplexe Heterogenität der Schleimhautimmunzellen und deren Vernetzung untereinander, sind große Herausforderungen zu klinisch relevanten Messungen zu identifizieren, die den Zustand und die Fähigkeit des Immunsystems widerspiegeln. Wir entwickelten eine Methode, hochgereinigtes intraepitheliale GD-T-Zellen zu erhalten, die weiter analysiert werden können hoch Multiplex, Single-Cell-Technologien, die für die Identifizierung der zugrunde liegenden Mechanismen von FRT Immunität von entscheidender Bedeutung sein kann.

<table><tbody><tr><td>Cytobrush Plus GT&amp;nbsp;</td><td>(CareFusion, San Diego, CA, USA</td><td></td><td></td></tr><tr><td>IMDM,Iscove&#039;s Modified Dulbecco&#039;s Medium&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>12440061</td><td></td></tr><tr><td>Beckman Coulter automated cell counter (The Vi-CELL Series Cell Viability Analyzer</td><td>Beckman Coulter</td><td>496178</td><td></td></tr><tr><td>DMSO, Dimethyl sulfoxide&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>D2650</td><td></td></tr><tr><td>FBS, Fetal Bovine Serum</td><td>Invitrogen, Gibco</td><td>26140-079</td><td></td></tr><tr><td>PBS</td><td>Invitrogen, Gibco</td><td>10010023</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A-9647</td><td></td></tr><tr><td>LIVE/DEAD Fixable Yellow Dead Cell Stain kit&amp;nbsp;</td><td>Life Technologies,</td><td>L349S9</td><td></td></tr><tr><td>1% paraformaldehyde</td><td>Sigma Aldrich&amp;nbsp;</td><td>D6148</td><td></td></tr><tr><td>Fortessa flow cytometer&amp;nbsp;</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>UltraComp eBeads</td><td>eBioscience</td><td>01-2222-42</td><td></td></tr><tr><td>ArC Amine Reactive Compensation Beads (Life Technologies, Grand Island, NY, USA)</td><td>Life Technologies</td><td>A10346</td><td></td></tr><tr><td>anti-TCR gamma/delta MicroBead Kit&amp;nbsp;</td><td>Milteny Miltenyi Biotec Inc.</td><td>130-050-701</td><td></td></tr><tr><td>MS column</td><td></td><td>103-042-201</td><td></td></tr><tr><td>MACS separator</td><td></td><td>4124</td><td></td></tr></tbody></table>

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

Die weiblichen Fortpflanzungstrakt (FRT) Schleimhaut-Immunsystem dient als erste Verteidigungslinie. Bessere Kenntnis der Genitalschleimhaut ist daher wesentlich für das Verständnis der Pathogenität von verschiedenen Krankheitserregern wie HIV. Gamma delta (GD) T-Zellen sind der Prototyp &quot;unkonventionelle&quot; T-Zellen und eine relativ kleine Teilmenge von T-Zellen durch ihre Expression von heterodimeren T-Zell-Rezeptoren (TCRs), die aus gamma und delta-Ketten definiert darstellen. Das unterscheidet sie von den klassischen und viel besser CD4 + T - Helferzellen und CD8 + zytotoxischen T - Zellen bekannt , die durch Alpha-Beta - TZR definiert sind. GD-T-Zellen zeigen häufig gewebespezifische Lokalisation und in Epithel angereichert. GD T-Zellen dirigieren Immunreaktionen bei der Entzündung, Tumorüberwachung, Infektionskrankheiten und Autoimmunerkrankungen. Hier präsentieren wir eine Methode, um reproduzierbar zu isolieren und die menschliche endocervical intraepitheliale GD T-Lymphozyten zu analysieren. Wir have verwendet endocervical Zytobürste Proben von Frauen in der Frauen-Vermittlungs-HIV-Infektion Study (WIHS) teilnehmen. Das Wissen über GD-T-Zellen-Wechselwirkungen bei Bedingungen, in denen es eine Beleidigung für die vaginale Schleimhaut ist, könnte zu einer klinischen Studie angewendet werden, bei denen der Schleimhaut Anfälligkeit gerichtet ist, einschließlich der Entwicklung von vaginalen microbicides.In hinaus sind Kenntnisse über Schleimhaut GD T-Zell-Reaktionen hat Potenzial für die Anwendung der GD T-Zell-basierte Immuntherapie bei der Behandlung von Infektionskrankheiten.

Vor kurzem haben wir analysiert , endocervical intraepitheliale GD - T - Zellen , und wir waren die ersten, über den Verlust von endocervical Gamma - Delta - T - Zellen in HIV - infizierten Frauen 9, 11 zu melden. Hier beschreiben wir das Protokoll zu isolieren und die Teilmenge von endozervikalen gamma delta T-Zellen zu analysieren. Wie in Figur gezeigt ist, kann 1, GD T - Zellen leicht in die ...

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.

Isolierung und Analyse über Durchflusszytometrie der Menschen Endozervikale Gamma-Delta-T-Zellen

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 Aufrufe.  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.

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

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Expansion von humanen peripheren Blut γδ T-Zellen mit Zoledronat

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Forschung

Immunologie und Infektion

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.

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.

Sammlung, Isolierung, und Durchflusszytometrische Analyse der Menschen Endozervikale Proben

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

Medizin

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

Isolation und<em&gt; Ex Vivo</em&gt; Kultur des Vδ1<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; γδ T-Zellen, eine extrathymic αβT-Zell-Vorläufer

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

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.

Isolierung und Analyse über Durchflusszytometrie von Gliom-Infiltration peripheren mononukleären Blutzellen

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

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Charakterisierung von humanen Monozyten-abgeleiteten dendritischen Zellen durch Imaging Durchflusszytometrie: Ein Vergleich zwischen zwei Monozyt Isolation Protokolle

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and activity of cytotoxic T cells within tumors and their tumor microenvironment (TME) are now well-established prognostic and predictive biomarkers for numerous tumor types. However, it is well-known that various tumor treatment modalities, including radiotherapy, chemotherapy, immunotherapy, and targeted therapy, modulate not only the immunogenicity of the tumor but also the immune system itself. Consequently, the interaction between tumor cells and T cells requires more intensive study in different therapeutic contexts to fully understand the complex role of T cells during tumor therapy. To address this need, a protocol was developed to analyze the activity and proliferative capacity of human cytotoxic (CD8+) T cells in co-culture with pre-treated tumor cells. Specifically, CD8+ T cells from healthy donors are stained with the non-toxic proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated using CD3/CD28-coated plates. Subsequently, T cells are co-cultured with pre-treated tumor cells. As a readout, T cell proliferation is quantified by measuring CFSE signal distribution and assessing the expression of surface activation markers via flow cytometry. This can be further complemented by quantifying cytokine release using enzyme-linked immunosorbent assay (ELISA). This method facilitates the evaluation of treatment-induced changes in the interaction between tumor cells and T cells, providing a foundation for more detailed analyses of tumor treatment modalities and their immunogenicity in a human ex vivo setting. Additionally, it contributes to the reduction of pre-clinical in vivo analyses.

The present protocol describes an experimental workflow that allows for the ex vivo analysis of human T cell stimulation in an allogenic co-culture system with pre-treated tumor cells.

Analyse der Aktivität humaner T-Zellen in einer allogenen Co-Kultur von vorbehandelten Tumorzellen

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and ...

Krebsforschung

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.

Isolation von dendritischen Zellen aus menschlichen weiblichen Fortpflanzungsorgane für phänotypische und funktionelle Studien

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

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

Diskriminierung von sieben Immunzelle Teilmengen von zwei-Fluorochrom Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

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.

Isolierung von angeborenen lymphatischen Zellen der Gebärmutter zur Analyse mittels Durchflusszytometrie

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 ...

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the lymph node microenvironment in B-cell non-Hodgkin&#39;s lymphoma is analyzed. Subsets of circulating T-follicular helper cells (cTfh), the circulating memory compartment of Tfh cells in the blood, are also perturbed in disease and therefore represent potential novel predictive biomarkers. Peripheral blood-based testing is advantageous because it is relatively non-invasive and allows simple serial monitoring.This article describes a method for isolating CD4+ T-cells from human blood, and further analysis by flow-cytometry to enumerate cTfh cells and the proportions of their various subsets (cTfhPD-1-/+/hi, cTfh1,2,17 and cTfh1/17). The level of these subsets was then compared between normal subjects and patients with lymphoma. We found that the method was robust enough to obtain reliable results from routinely collected patient material. The technique we describe for the analysis can be easily adapted to cell sorting and downstream applications such as RT-PCR.

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper cTfh Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Here, a protocol for the isolation and characterization of CD4+ T-cell subsets from human peripheral blood is described. Purified CD4+ T-cells are analyzed by flow cytometry to determine proportions of T-follicular helper cell subsets.

Isolierung von CD4+ T-Zellen und Analyse von zirkulierenden T-follikulären Helfer (cTfh) Zelle Teilmengen von peripheren Blut mittels 6-Farb Flow Cytometry

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the ...

Isolierung und Analyse über Durchflusszytometrie der Menschen Endozervikale Gamma-Delta-T-Zellen

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