Wir danken Jessica Jones, Erica Clark, Constance Ducar, Donna Smith, Roy Lewis, Lily Apedaile, Joanne Wiesner, Devin Adams, Corey McBain und Stephen Voght für ihre Unterstützung bei der Entwicklung dieser Methode, Manuskript und Video. Diese Arbeit wurde von der Bill and Melinda Gates Foundation CAVD Zuschuss 38.645 (MJM) und National Institutes of Health Zuschüsse UM1 AI068618 und U01 AI069481 (MJM) unterstützt. EA-N. wird durch NIH Grant T32 AI007140 unterstützt. Wir danken dem James B. Pendleton Charitable Trust für ihre großzügige Ausstattung Spende.

Hinweis: Um die Fluorophor-konjugierten Antikörpern vor Licht zu schützen, führen Sie alle Schritte in einer Bio-Sicherheits-Schrank mit dem Licht aus. Ein. Antikörperfärbung Systemsteuerung Vorbereitung <ol><li> Die Antikörper-Färbung Panel kann in Tabelle 1 zu finden. Antikörperkonzentration sollte durch Titration mit Vollblut definiert werden und unter Verwendung des gleichen Durchflusszytometrie Ausrüstungen und Verfahren, die verwendet werden, um die Proben zu erwerben Phänotypisierung gebeizt wird. </li><li> Sobald geeignete Färbung Titer bestimmt werden, kombinieren alle Antikörper in einen einzigen Mischung in einem Lock-Kapselrohr. Hinzufügen Strömung Waschpuffer (Dulbeccos PBS mit 2% hitzeinaktiviertes fötales Rinderserum), um das Gesamtvolumen auf 100 ul zu bringen. Skalieren der Mischung für die Anzahl von Abtastwerten ist angefärbt. Dieses Gemisch kann bei 4 ° C für bis zu acht Wochen gelagert werden. </li></ol> 2. Färbung <ol><li> Wenn das Blut gesammelt wird, ist für andere Zwecke in ad verwendet werdendition diesem Assay, setzen Sie ein Aliquot beiseite, während mehr Zeit-und Kleinschreibung auf die verbleibende Blut durchgeführt werden. Das Aliquot kann bei Raumtemperatur bis zu 4 Stunden nach der Blutabnahme ohne nennenswerte Zellverlust gespeichert. </li><li> Überprüfen, dass es ein intaktes Wulst Pellet am Boden des Rohrs und TruCOUNT Etikett das Rohr zu identifizieren wobei die Probe gefärbt. In HVTN klinischer Studien, deren Laboratory Data Management System (Frontier Science and Technology Research Foundation; Amherst, NY) wird verwendet, um zu kennzeichnen und zu verfolgen gefärbten Proben. </li><li> Notieren Sie die Chargennummern und Verfallsdaten aller Reagenzien. Notieren Sie sich die TruCOUNT tube bead Zählnummer durch den Hersteller auf der Tasche der Rohre vorgesehen, stellen Sie sicher, dass die Chargennummer auf dem Beutel die Lotnummer auf dem Schlauch. </li><li> Verwenden Reverse Pipettieren genau Je 100 ul Vollblut in die TruCOUNT Rohr, knapp oberhalb der Metall-Halterung. Vermeiden Verschmieren Blut an der Seite des Rohres. </li><li&gt; Verwenden von regulären (vorwärts) Pipettiertechnik, Pipette 100 ul des gemischten Antikörperfärbung Panel (siehe Tabelle 1) in die TruCOUNT Röhre. Das Röhrchen und vortexen bei niedriger Drehzahl für ca. 15 Sek. mischen. Sichtprüfen Rohr zu gewährleisten, dass der Wulst Pellet vollständig gelöst ist. </li><li> Inkubieren TruCOUNT Röhrchen für 15 Minuten bei Raumtemperatur (15-30 ° C) im Dunkeln. </li><li> Wenn nötig, Einen aliquoten von 10 × FACS Lyse Lösung 1X mit diH 2 O. Zusatz von 900 ul 1 × FACS Lysing Lösung des Rohres. </li><li> Das Röhrchen verschließen und gründlich vortexen bei niedriger Drehzahl für ca. 15 Sek. mischen. Schieben Sie den Deckel fest in Sperrstellung auf dem Rohr und Dichtung mit Labor-Film. </li><li> Bewahren Sie das Rohr bei -65 ° C bis -95 ° C, bis die Probe für den Versand an die zentrale Analyse Labor oder für die Analyse im Haus bereit. Proben sind in dieser Phase stabil für mindestens vier Wochen. Wenn Versand oder zur quantitativen Bestimmung sofortdiately, kann dieser Schritt weggelassen werden. </li></ol> 3. Versand Hinweis: Die folgenden Anweisungen verwenden einen isolierten Versandsystem von Saf-T-Pak, Inc. speziell für Transportzwecke Kategorie B ausgenommen biologische Stoffe nach International Air and Transport Association (IATA) entworfen. Bei der Analyse der Proben in der gleichen Position wie die Färbung auftrat, Abschnitt 4 gehen. <ol><li> Proben können sofort nach dem Färben oder versendet werden, sobald sie bei -65 ° C eingefroren bis -95 ° C. Wickeln Sie jedes Rohr komplett in Folie und in die gefärbte Probe Box. Legen Sie die gefärbte Probe Box innerhalb einer dichten Poly-Beutel mit einem saugfähigen Material. </li><li> Legen Sie die Dichtheit Poly-Beutel und Inhalt in einen Tyvek Beutel und Dichtung mit so wenig Luft wie möglich in der Tasche. </li><li> Legen Sie die Probe-Paket (Probe innerhalb sekundäre Verpackung) innerhalb des inneren braunen Kasten. </li><li> Setzen Sie den inneren braun box im Styropor Brust, Befestigung in die Vertiefung der Verschiebung zu verhindern. </li><li> Füllen Sie das Styropor Brust mit Trockeneis (ca. 8 kg) und legen Sie den Deckel fest auf der Brust. </li><li> Sicher Band der Versandkarton und Schiff als biologische Substanz, Kategorie B (UN3373) mit der richtigen Trockeneis (UN 1845) Markierungen, folgen IATA PI-650 Anweisungen. </li><li> Bei Empfang werden die Proben bei -65 ° C gelagert bis -95 ° C, bis sie analysiert werden. </li></ol> 4. Auftauen und Durchflusszytometrie-Analyse <ol><li> Entfernen der gefärbten Probe aus dem Gefrierschrank bei Raumtemperatur in der Dunkelheit vor dem Sammeln auf dem Durchflusszytometer aufzutauen. Wenn Daten über mehr als eine Probe, standardisieren den Vorgang für alle Rohre durch Staffelung Auftauen, so dass die Rohre nicht sitzen bei Raumtemperatur für mehr als 1 Stunde lang. </li><li> Proben sollten die unter Verwendung eines Durchflusszytometers vier Laser mit geeigneten Filtern, wie dem BD LSR ausgestattet werdenII. Verwenden Sie Standard-Zytometer Kalibrierung und Fluoreszenzkompensation Methoden zur Datenerfassung 4. </li></ol> Hinweis: Nicht nach vorne gesetzt streuen oder Seitwärtsstreuung Schwellenwerte während collection 5. TruCOUNT Kügelchen können unterhalb der niedrigsten möglichen Threshold-Einstellung für diese Parameter bewirken, dass ein Teil der Perlen nicht bei der Analyse berücksichtigt werden fallen. Wenn durch das Gerät erforderlich, um eine Schwelle, die niedrigste mögliche Am Cyan-Kanal Schwelle. Da CD45 + Leukozyten mit dem Panel gefärbt wird Cyan positive Am, und TruCOUNT Perlen auch fluoreszieren im Am Cyan-Kanal, sollte dies für alle relevanten Daten entsprechend gesammelt werden können. <ol start="3"><li> Vortex das Probenröhrchen für 5 Sekunden vor dem Laden auf dem Durchflusszytometer. TruCOUNT Perlen fluoreszieren stark in viele Kanäle. Tor die Perlen während der Sammlung durch Auffinden der Bevölkerung, die stark doppelt positiv für PE Cy5 und APC (die beiden colors, dass die meisten der Perlen leicht unterscheiden von den Zellen können je nach Instrumentierung variieren). Wählen Sie den Wulst Tor als Stoppen Tor, und Daten aufzeichnen, bis mindestens 20.000 Perlen erworben werden. </li><li> Analysieren Sie die Daten mit Hilfe geeigneter Software wie FlowJo (Treestar; Ashland, OR). Abbildung 1 zeigt die Gating-Schema für die Analyse der verschiedenen Leukozyten-Populationen aus einer repräsentativen Kontrolle Blutentnahme verwendet. </li></ol> 5. TruCOUNT Berechnungen <ol><li> Jeder TruCOUNT Röhre enthält eine lyophilisierte Pellet fluoreszierende Kügelchen. Nach Zugabe von Flüssigkeit zu dem Rohr und Vortexen sollten die Perlen sich gleichmäßig über der Probe verteilt. Die Zahl der Raupen in der Pellet variiert leicht durch Losnummer und kann auf der Aufbewahrungstasche für die Rohre zu entnehmen. </li><li> Tor die TruCOUNT Perlen und Zellpopulationen wie in Abbildung 1 gezeigt, Veranstaltung zählt für jede Population zu bestimmen. Vergleicht man die Anzahl der Ereignisse in die Wulst Gate zur Gesamtzahl von Kügelchen ursprünglich in der Röhre erlauben, das Verhältnis der Probe gesammelt, die dann verwendet werden, um die absolute Konzentration (dh Zellen / ul) für jede Population zu bestimmen bestimmen. Zellkonzentration (# Zellen / Mikroliter Vollblut) = [# Bevölkerung events / (# bead events / # total Perlen in Pellet)] / 100 ul: Die folgende Gleichung kann für diesen Zweck verwendet werden. </li></ol> 6. Repräsentative Ergebnisse <img src="/files/ftp_upload/4302/4302fig1.jpg" alt="Abbildung 1" fo:content-width="5.5in" fo:src="/files/ftp_upload/4302/4302fig1highres.jpg" /> Abbildung 1. Gating-Schema für die Analyse der wichtige Leukozytenpopulationen zeigt repräsentative Daten von einem gesunden Freiwilligen verwendet. A) TruCOUNT Perlen (i) sind gated und ausgeschlossen von Zellen. Granulozyten (ii) werden abgegrenzt und lympohcytes und Monozyten sind in 3 popluations unterteilt: CD14Lymphozyten negativ (iii), alle CD14-negativen Zellen (iv), und nicht-Lymphozyten (v). B) CD14 negativen Lymphozyten sind gated an CD4 + T-Zellen (vi), CD8 + T-Zellen (vii), B-Zellen (viii), CD56 hell NK-Zellen (ix), CD56 dim NK-Zellen (x), und CD56 negativen NK-Zellen zu unterscheiden (xi). C) Alle CD14 negative Zellen sind gated um myeloide (xii) und plasmazytoiden (xiii) dendritische Zellen zu unterscheiden. D) Non-lympocytes sind gated nicht-klassischen (xiv), Mittelstufe (xv) und klassische (xvi) Monozyten zu unterscheiden. <a href="http://www.jove.com/files/ftp_upload/4302/4302fig1large.jpg" target="_blank">Klicken Sie hier für eine größere Abbildung zu sehen</a> . Spezifischere Zelluntergruppen die nicht in 1 (zB NKT-Zellen oder Neutrophile) gezeigt können auch unter Verwendung der Platte unterschieden präsentieren wir werden, und die Gating-Schema kann erweitert oder modifiziert werden, um spezifische Studie Bedürfnisse zu erfüllen. Bestimmte Gating gezeigten Schritte sind einzigartig für diese Methode. Besonders erwähnenswert ist, einer Einbeziehung Tor undAusschluss Gate sind um die Kügelchen TruCOUNT gezogen und auf die Oberseite voneinander ein bis Gate die Perlen zum Zählen und ein um die Kügelchen aus dem zellulären Analyse (Abbildung 1A) auszuschließen. Auch weil Lymphozyten, Monozyten und Granulozyten nicht so leicht in Vollblut unterscheiden, wie sie in peripheren mononukleären Zellen durch Vorwärtsstreuung und seitliche Streuung, Gating diesen Zellen mit CD45-Expression und Seitenstreuung ist oft notwendig (1A) sind. Kontaminierende Granulozyten (eingekreist), die nicht von Lymphozyten und Monozyten mit CD45 und side scatter getrennt werden konnten unterscheidbar sind in einigen Parzellen durch ihre hohe CD16 Expression (Abbildung 1C und 1D). Die Anzahl der kontaminierenden Granulozyten ist üblicherweise klein, und sie nicht mit Monozyten und NK-Zell-Gating stören.

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	<li>Taylor, M. J., London, N. J., Thirdborough, S. M., Lake, S. P., James, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cryobiology+of+rat+and+human+dendritic+cells:+preservation+and+destruction+of+membrane+integrity+by+freezing.">The cryobiology of rat and human dendritic cells: preservation and destruction of membrane integrity by freezing.</a> Cryobiology. 27, 269-278 (1990).</li><li>Reimann, K. A., Chernoff, M., Wilkening, C. L., Nickerson, C. E., Landay, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+lymphocyte+immunophenotype+and+proliferative+responses+in+cryopreserved+peripheral+blood+mononuclear+cells+from+human+immunodeficiency+virus+type+1-infected+donors:+implications+for+multicenter+clinical+trials.+The+ACTG+Immunology+Advanced+Technology+Laboratories.">Preservation of lymphocyte immunophenotype and proliferative responses in cryopreserved peripheral blood mononuclear cells from human immunodeficiency virus type 1-infected donors: implications for multicenter clinical trials. The ACTG Immunology Advanced Technology Laboratories.</a> Clin Diagn Lab Immunol. 7, 352-359 (2000).</li><li>Boonlayangoor, P., Telischi, M., Boonlayangoor, S., Sinclair, T. F., Millhouse, E. 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B., Roederer, M., Herzenberg, L., Blackwell, C., Weir, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The handbook of Experimental Immunology. 43, 1-49 (1996).</li><li>Hulspas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+and+the+stability+of+phycoerythrin-tandem+dye+conjugates.">Flow cytometry and the stability of phycoerythrin-tandem dye conjugates.</a> Cytometry A. 75, 966-972 (2009).</li><li>Le Roy, C., Varin-Blank, N., Ajchenbaum-Cymbalista, F., Letestu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+APC-tandem+dyes+are+degraded+through+a+cell-dependent+mechanism.">Flow cytometry APC-tandem dyes are degraded through a cell-dependent mechanism.</a> Cytometry A. 75, 882-890 (2009).</li><li>Mandy, F., Brando, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+absolute+cell+counts+using+immunophenotypic+techniques.">Enumeration of absolute cell counts using immunophenotypic techniques.</a> Curr. 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Keine Interessenskonflikte erklärt.

In diesem Bericht stellen wir eine Bead-basierte Verfahren zum Aufzählen Leukozytenpopulationen in frischem Vollblut mittels Durchflusszytometrie und decken die notwendigen Parameter für den Einsatz in einer multizentrischen klinischen Studie mit zentraler Probenanalyse. Diese Methode baut auf und optimiert die BD TruCOUNT Protokoll und ermöglicht seinen zuverlässigen Einsatz in einer multizentrischen klinischen Studie. Die Färbung Test ist einfach und dauert etwa 45 Minuten in Anspruch, so dass es möglich ist fü...

<table border="1"><tbody><tr><td> Reagent Name </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> TruCOUNT Absolute Counting Tubes </td><td> BD Biosciences </td><td> 340334 </td></tr><tr><td> 10X FACS Lyselösung </td><td> BD Biosciences </td><td> 349202 </td></tr><tr><td> Kategorie B &amp; Exempt Shipping System, isoliert </td><td> Saf-T-Pak </td><td> STP-320 </td></tr><tr><td> CD45 monoklonalen Antikörpers AmCyan </td><td> BD Biosciences </td><td> 339192 </td></tr><tr><td> CD3 monoklonaler Antikörper FITC </td><td> BD Biosciences </td><td> 349201 </td></tr><tr><td> CD8-PerCP Cy 5,5 monoklonalen Antikörpers </td><td> BD Biosciences </td><td> 341051 </td></tr><tr><td> Alexa Fluor 700 CD4 monoklonalen Antikörperndy </td><td> BD Biosciences </td><td> 557922 </td></tr><tr><td> HLA-DR-ECD monoklonalen Antikörpers </td><td> Beckman Coulter </td><td> IM3636 </td></tr><tr><td> CD14 monoklonalen Antikörper V450 </td><td> BD Biosciences </td><td> 560349 </td></tr><tr><td> CD19 PE monoklonalen Antikörpers </td><td> BD Biosciences </td><td> 555413 </td></tr><tr><td> APC-CD16 monoklonaler Antikörper H7 </td><td> BD Biosciences </td><td> 560195 </td></tr><tr><td> CD56 PE-Cy7 monoklonalen Antikörpers </td><td> BD Biosciences </td><td> 335791 </td></tr><tr><td> CD11c APC monoklonalen Antikörpers </td><td> BD Biosciences </td><td> 559877 </td></tr><tr><td> CD123 PE-Cy5 monoklonalen Antikörpers </td><td> BD Biosciences </td><td> 551065 </td></tr></tbody></table>

enumeration of major peripheral blood leukocyte populations for multicenter clinical trials using a whole blood phenotyping assay

Kryokonservierung von Leukozyten des peripheren Blutes wird häufig verwendet, um Zellen für die Immunantwort Auswertungen in klinischen Studien erhalten und bietet viele Vorteile für eine einfache und Standardisierung von immunologischen Einschätzungen, aber nachteilige Auswirkungen dieses Prozesses haben auf einigen Zellpopulationen, wie Granulozyten, B-Zellen beobachtet und dendritischen Zellen 1-3. Untersuchung frischen Leukozyten gibt ein genaueres Bild der in vivo Zustand der Zellen, sondern ist oft schwierig, im Rahmen der großen klinischen Studien durchzuführen. Frische Zell-Assays sind abhängig von freiwilligen Verpflichtungen und Fristen und wenn zeitaufwendig, ihre Anwendung kann unpraktisch aufgrund der Arbeitszeiten von Laborpersonal erforderlich. Zusätzlich, wenn Prüfungen in mehreren Zentren durchgeführt, können Labors die Mittel und Ausbildung erforderlich, die Assays durchzuführen, die nicht in ausreichender Nähe zu klinischen Standorten befinden. Um diese Probleme anzugehen, haben wir develwickelt einen 11-farbe Antikörperfärbung Panel, das mit TruCOUNT Röhrchen (Becton Dickinson, San Jose, CA) verwendet werden, um Phänotyp und Auflisten der großen Leukozytenpopulationen im peripheren Blut, wodurch stabilere zelltypspezifisch Informationen als Assays, wie ein komplettes Blutbild (CBC) oder Assays mit kommerziell erhältlichen Platten für TruCOUNT Rohre, die nur für ein paar Zelltypen Flecken entwickelt. Die Färbung ist einfach, erfordert nur 100 ml frisches Vollblut, und dauert etwa 45 Minuten, so dass es möglich ist für Standard-Blut-Verarbeitung Labors durchzuführen. Es ist aus dem BD TruCOUNT Rohr technisches Datenblatt (angepasst <a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">Version 8/2010</a> ). Die Färbung Antikörper-Cocktail können im Voraus in der Masse an einem zentralen Assay Labor hergestellt und versendet die Verarbeitung vor Ort Labors. Stained Rohren befestigt werden kannund eingefroren für den Versand an die zentrale Assay Labor für multicolor Durchflusszytometrie. Die Daten aus dieser Färbung Panel generiert werden verwendet, um Veränderungen in Leukozytenkonzentrationen im Laufe der Zeit in Bezug auf Intervention verfolgen und könnte leicht weiter entwickelt werden, um die Aktivierung Staaten von spezifischen Zelltypen des Interesses zu beurteilen. In diesem Bericht zeigen wir, das Verfahren durch den Blut-Verarbeitung Laboranten zur Färbung von frischem Vollblut und die Schritte, um diese gefärbten Proben an einem zentralen Test-Labor Unterstützung einer multizentrischen klinischen Studie zu analysieren durchzuführen. Die Video-Informationen das Verfahren, wie es im Rahmen einer klinischen Studie Blutentnahme in der HIV Vaccine Trials Network (HVTN) durchgeführt wird.

Watch this Scientific Journal Video about Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay at JoVE.com

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

In diesem Bericht zeigen wir, die Färbung und Analyse Schritte eines Phänotypisierung Assay auf frischem Vollblut durchgeführt, um wichtige angeborenen und adaptiven Leukozytenpopulationen aufzuzählen. Wir betonen, Überlegungen für die Durchführung dieser Verfahren im Rahmen einer multizentrischen klinischen Studie.

Aufzählung der wichtigsten peripheren Blut Leukozyten Populationen für Multicenter Clinical Trials mit einem Vollblut Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

enumeration-major-peripheral-blood-leukocyte-populations-for

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Immunology and Infection

14.9K Aufrufe.  Fred Hutchinson Cancer Research Center. In diesem Bericht zeigen wir, die Färbung und Analyse Schritte eines Phänotypisierung Assay auf frischem Vollblut durchgeführt, um wichtige angeborenen und adaptiven Leukozytenpopulationen aufzuzählen. Wir betonen, Überlegungen für die Durchführung dieser Verfahren im Rahmen einer multizentrischen klinischen Studie.

Serie: Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

Depletion spezifischer Zellpopulationen durch Ergänzung Depletion

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

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Immunologie und Infektion

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Reinigung von spezifischen Zellpopulation durch Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Expansion, Reinigung und Functional Assessment of Human peripheren Blut NK-Zellen

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

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 ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Introducing Schubspannung in der Studie der bakteriellen Adhäsion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

A Functional Whole Blood Assay zur Rentabilität von Mykobakterien Measure, mit Reporter-Gene BCG oder M. Tb (BCG Tagged Lux / M. Tb Lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

Schnelle und spezifische Bewertung der Halogenierungsagentien Peroxidaseaktivität in Leukozyten angereicherte Blutproben

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

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.

Durchflusszytometrische Analyse von natürlichen Killerzellen lytische Aktivität in menschlichem Vollblut

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

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Die Verwendung eines Monozyten einschichtiges Assay Fcy rezeptorvermittelte Phagozytose zur Bewertung

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran erhöht die Effizienz der Lentivirale Transduktion von murinen und menschliche primäre NK-Zellen

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...