This research was supported by a grant from the NSERC Discovery program grant number RGPIN 418522-2013. We thank R. Kilgour for helpful discussions, and Katelin Gresty and Dr. A. Berghdal for providing the mouse muscle sections.

Forschung mit menschlichen Blutproben wurde von Concordia University Ethics Review Board, Zertifikat-Nummer 10000618. die Arbeit an dem Maus-Muskel wurde von Concordia University Ethics Review Board, Zertifikat-Nummer 2010BERG zugelassen. 1. PBMC Isolierung aus Vollblut HINWEIS: Führen Sie diesen Vorgang in einer Biosicherheitswerkbank mit steriler Technik und hersteller sterilisiert Ausrüstung. <ol><li> Gießen Sie 10-15 ml Vollblut aus dem heparinisierten (Antikoagulans) Blutentnahmeröhrchen in ein steriles 50 ml konischen Röhrchen. Für kleinere Blutverschmutzungen, wischen Sie mit ddH 2 O und 70% EtOH mit Reinigungstuch. </li><li> Verdünne das Blut in dem konischen Rohr an einen 1: 1-Verhältnis mit Phosphatpuffer-Saline (PBS 1x), pH 7,4. Stellen Sie sicher, dass die maximale Gesamtmenge ist 30 ml nicht überschreiten. </li><li> Mischen Sie vorsichtig mit einem serologische Pipettenpistole blasenfrei. HINWEIS: durch Umdrehen des Röhrchens, um Blut zu vermeiden eine nicht mischenccumulation in der Kappe und nachfolgende Einsickern während der Zentrifugation. </li><li> Hinzufügen 13 ml Ficoll-Paque (siehe Materialien und Geräte-Tabelle), in ein neues konisches Rohr 50 ml Kapazität. Halten Sie das Rohr aufrecht im Rack. </li><li> Mit Hilfe einer Transferpipette, nehmen verdünnte Blut, berühren Sie die Transferpipettenspitze an der Innenwand der Röhre in der Nähe der Spitze. Mit langsamen und stetigen Druck, übertragen das Blut entlang der Innenwand des Rohres bildet eine Blutschicht, die auf der Oberseite des Saccharose-Schicht. Führen Sie diesen Schritt so oft, bis alle Blut übertragen wurde. </li><li> Das Röhrchen verschließen und Zentrifugieren Sie die Röhrchen bei Raumtemperatur für 30 Minuten bei 700 · g in einer Schaukel Rotor mit mittleren Beschleunigungssatz bis 5, und Verzögerung auf Null gesetzt. </li><li> Langsam nehmen Sie das Rohr und ohne die Schichten zu stören, nehmen Sie es zurück zu der Biosicherheitswerkbank. </li><li> Die Buffy-Coat (dünne weiße Schicht bedeckt), wobei PBMCs werden, zwischen dem PBS / Plasma ein gelegt sorgfältig sammelnnd ficoll Schichten mit einer Transferpipette. Vermeiden Sammeln Saccharoseschicht und nicht die Schicht roter Blutkörperchen stören. </li><li> Übertragen Sie die Buffy-Coat in eine neue 50 ml sterilen konischen Rohr. Es kann mehrmals der Wiederholung von Schritt 1.8, alle PBMC sammeln zu nehmen. </li><li> In PBS pH 7,4 in das Röhrchen mit der PBMC und füllen Sie bis zur 45 ml-Marke. Schütteln Sie Rohr gründlich. Nicht mit dem Vortex. </li><li> Waschen Sie 1: Zentrifuge für 15 Minuten bei 480 xg sowohl mit maximaler Beschleunigung und Verzögerung bis 9 eingestellt Bildung eines Pellets aus PBMCs Beachten Sie am unteren Rand der konischen Rohr. </li><li> Entsorgen Sie die PBS (Überstand) in einem Kunststoffbecher mit 10 ml Bleichmittel. </li><li> Lösen Zellpellet vorsichtig &quot;Abstich&quot; gegen einen gewellten Oberfläche (dh leere Rack). Nicht mit dem Vortex. </li><li> Zugeben von 25 ml frischer PBS pH 7.4 mit dem Rohr. Wenn mehr als ein Rohr bearbeitet wird, bündeln die Pellets zusammen in diesem Schritt. </li><li> Waschen 2: zentrifugieren Sie das Röhrchen 12 min bei 480 · g with sowohl maximale Beschleunigung und Verzögerung auf 9 gesetzt. </li><li> Entsorgen Sie die PBS (Überstand) in den Kunststoffbecher mit einem Schuss Bleichmittel. </li><li> Gently &quot;Rack&quot; gegen einen gewellten Oberfläche (dh leere Rack). Nicht mit dem Vortex. </li><li> Zugeben von 25 ml frischer PBS zu dem Rohr. <ol><li> Nehmen Sie 50 ul der Zellen und Überführung in ein Mikrozentrifugenröhrchen für die Lebensfähigkeit Zahl. </li><li> In die gleiche Menge (50 ul) von Trypanblau (a Lebensfähigkeit Färbung) und Pipette auf und ab, um sanft zu mischen. </li></ol></li><li> Nehmen Sie 10 ul und überträgt es auf einem Hämocytometer, um die Lebensfähigkeit der Zellen pro ml zu überprüfen. Nehmen die Anzahl der Zellen / ml. </li><li> Nehmen Sie die gewünschte Menge an Zellen und zentrifugieren Sie das Röhrchen 12 min bei 480 xg sowohl mit maximaler Beschleunigung und Verzögerung auf 9 gesetzt. </li><li> Entsorgen Sie die PBS (Überstand) in den Becher mit Bleichmittel. </li><li> Gently &quot;Rack&quot; gegen einen gewellten Oberfläche (dh leere Rack). In 80 ul PBS in die Röhre. </li></ol> 2. Machen Sie die PBMC Slide <ol><li> Platzieren 80 ul der Zellen auf einen Objektträger. Schmieren Sie das Drop mit Hilfe von einer anderen Folie oder stellen Sie 2 Tropfen von 40 ul jeweils an beiden Enden des Schlittens. </li><li> Lassen Sie Folie in der biologischen Sicherheitswerkbank, um zu trocknen. Beschriften Sie die Folie mit einem Bleistift auf der mattierten Seite. </li></ol> 3. Befestigung der Proben auf den Objektträger <ol><li> Bereiten Sie die Fixierungslösung durch Mischen von 0,5 ml 37% Formaldehyd und 4,5 ml 99% Ethanol. </li><li> Nachdem die Folien getrocknet sind, nehmen Sie 2 ml der frisch zubereiteten Fixierlösung und gießen Sie sie auf der Folie, so dass die gesamte Oberfläche der Folie bedeckt ist. </li><li> Verlassen die Lösung auf den Objektträger für 1 min. Spülen Sie den Schieber für 1 Minute mit Leitungswasser und lassen Sie es an der Luft trocknen. </li></ol> 4. Erstellen Sie die Amylase-Lösung für Negativkontrolle <ol><li> Nehmen 0,25 g Amylase Pulver und in 50 ml Di auflösengestillt Wasser. Füllen Sie die Lösung in einem sauberen 100 ml-Becher. </li><li> Tauchen Sie die Folie in den Becher, so dass die Hälfte der Folie erhält die Behandlung und die andere Hälfte bleibt unbehandelt und dann 15 Minuten bei Raumtemperatur (Abbildung 1 C) inkubieren. </li><li> Notieren Sie sich auf der Seite des Schlittens erhält den Amylase-Behandlung. Ziehen Sie eine Linie auf der Rückseite der Folie, die die Grenze zwischen der Behandlung und Kontrolle. </li><li> Waschen Sie die Objektträger mit ddH 2 O, um die Amylase-Lösung zu entfernen und die Folie an der Luft trocknen. </li></ol> 5. Führen Sie Periodic Acid Schiff (PAS) Färbung und Imaging HINWEIS: PAS Reagenzien sind giftig beim Einatmen und korrosiv sind, so dass die Schritte müssen in einem Laborabzug durchgeführt werden, und die Abfallprodukte müssen fachgerecht gemäß gültiger Richtlinien entsorgt werden. <ol><li> Stellen Sie den Schieber auf eine flache Oberfläche und gießen 1,50-2,00 ml Perjodsäure-Lösung auf die Probe. Incubate für 5 min bei Raumtemperatur. </li><li> Spülen Sie die Folien in mehreren Ladungen destilliertem Wasser. </li><li> Gießen 1,50-2,00 ml Schiffs-Reagenz auf den Objektträger, Inkubation bei Raumtemperatur für 15 min. </li><li> Waschen Sie die Objektträger mit destilliertem Wasser für 5 Minuten und lassen Sie es an der Luft trocknen. </li><li> Übernehmen 10 ul Eindeckmittel auf dem Objektträger geben und mit zwei kleinen Deckgläser oder gelten 50 ul und Nutzung einer großen Deckglas. </li><li> Bewerben klaren Nagellack auf den Kanten des Deckglases, lassen Sie über Nacht trocknen. </li></ol> 6. Rufen Sie Bilder mit dem Fernglas Lichtmikroskop Mit dem 100X Ziel

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+glycogen+content+in+various+disease.">Lymphocyte glycogen content in various disease.</a> J. Clin. Pathol. 15 (1), 36-39 (1962).</li><li>Scott, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+in+human+peripheral+blood+leukocytes.+I.+characteristics+of+the+synthesis+and+turnover+of+glycogen+in+vitro.">Glycogen in human peripheral blood leukocytes. I. characteristics of the synthesis and turnover of glycogen in vitro.</a> J. Clin. Invest. 47 (2), 344-352 (1968).</li><li>Fedele, D., 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=positive+index+of+lymphocytes+and+metabolic+control+in+insulin-treated+and+type+II+diabetes+mellitus.">positive index of lymphocytes and metabolic control in insulin-treated and type II diabetes mellitus.</a> Diabete Metab. 9 (3), 188-192 (1983).</li><li>Breli&#324;ska-Peczalska, R., Mackiewicz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochemical+studies+of+peripheral+blood+granulocytes+and+lymphocytes+in+patients+with+systemic+lupus+erythematosus.">Cytochemical studies of peripheral blood granulocytes and lymphocytes in patients with systemic lupus erythematosus.</a> Pol.Med.Sci.Hist.Bull. 15 (2), 231-234 (1976).</li><li>Yunis, A. A., Arimura, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymes+of+glycogen+metabolism+in+white+blood+cells.+I.+glycogen+phosphorylase+in+normal+and+leukemic+human+leukocytes.">Enzymes of glycogen metabolism in white blood cells. I. glycogen phosphorylase in normal and leukemic human leukocytes.</a> Cancer, Res. 24, 489-492 (1964).</li><li>Hagemans, M. L., 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=PAS-positive+lymphocyte+vacuoles+can+be+used+as+diagnostic+screening+test+for+pompe+disease.">PAS-positive lymphocyte vacuoles can be used as diagnostic screening test for pompe disease.</a> J. Inherit. Metab. Dis. 33 (2), 133-139 (2010).</li><li>Totsuka, Y., 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=Physical+performance+and+soleus+muscle+fiber+composition+in+wild-derived+and+laboratory+inbred+mouse+strains.">Physical performance and soleus muscle fiber composition in wild-derived and laboratory inbred mouse strains.</a> 95 (2), 720-727 (2003).</li><li>Murat, J. C., Serfaty, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+enzymatic+determination+of+polysaccharide+(glycogen)+content+of+animal+tissues.">Simple enzymatic determination of polysaccharide (glycogen) content of animal tissues.</a> Clin. Chem. 20 (12), 1576-1577 (1974).</li><li>Arrizabalaga, O., Lacerda, H. M., Zubiaga, A. M., Zugaza, J. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tagliabracci+V.S.+Glycogen+and+its+metabolism:+Some+new+developments+and+old+themes.">Tagliabracci V.S. Glycogen and its metabolism: Some new developments and old themes.</a> Biochem.J. 441 (3), 763-787 (2012).</li><li>Salmoral, E. M., Tolmasky, D. S., Krisman, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+presence+of+glycogen+in+rat+thymus.">Evidence for the presence of glycogen in rat thymus.</a> Cell Mol.Biol. 36 (2), 163-174 (1990).</li><li>Darlington, P. 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The authors have nothing to disclose.

Die kritischen Schritte dieses Video Artikel waren beim Waschen und Amylase-Behandlung der Zellen. Beim Waschen der Objektträger wurde der entscheidende Schritt mit einem Kunststoff squeezable Waschflasche und ließ das Wasser sanft durch die Probe auf dem Objektträger laufen und nicht direkt mit dem Ziel auf die Proben. Selbst die geringste direkte Wasserdruck würde die Zellen veranlassen, kommen aus der Folie. Ein weiterer wichtiger Schritt war, um dieselbe Folie für ± Amylase Bedingungen. Nachdem die PBMCs mit d...

Perjodsäure Schiff (PAS) -Färbung eine immunohistochemische Technik, die weit verbreitet in der Muskel Forschung und Diagnostik eingesetzt wird. Es wird auch als ein diagnostisches Werkzeug für Blutproben verwendet. Diese Technik funktioniert, indem Periodsäure-Lösung zu der Probe, die innerhalb der Polysaccharid Schaffung Aldehydgruppen, die mit dem farblosen Schiffs-Reagenz, wodurch eine tiefmagenta Produkt reagieren Einheiten oxidiert. Die Schritte dieses Verfahrens sind in Abbildung 1 dargestellt. Der Fleck macht nichts mit Polysacchariden magenta, einschließlich Glykogen, Glycoproteine, Glycolipide, Mucinen, oder andere Moleküle mit Polysaccharid-Gruppierungen. PAS-Färbung wird oft verwendet, um Glykogenspiegel in Muskelfasern messen. Muskeln Gewebeschnitte sind für die Technik, wenn sie fest an dem Schieber aushalten mehrere Wasch- und Färbeschritte. Glykogen ist den meisten in schnell kontra vorhanden Typ-II-Muskelfasern, die einen hohen Bedarf habenfür die schnelle Produktion von ATP Glykogen für maximale Leistung 1,2 erfordern. Glycogen ist ein verzweigtes Polymer von Glucose, die in freie Glucose durch die Wirkung von Glycogenphosphorylase-Enzyme abgebaut werden können. In Zeiten der Ruhe und Nahrungsversorgung, wird Glykogen durch den Prozess der Glykogenese aufgefüllt, während in Zeiten der Mangelernährung oder Hochenergiebedarf; Glykogen in Glukose durch Glykogenolyse gebrochen. Bereits ab den 1950er Jahren Kliniker Wissenschaftler haben erforscht PAS-Färbung auf Blutproben Glykogengehalt bei verschiedenen Erkrankungen 3-7 analysieren. Zum Beispiel bei Morbus Pompe-einer bonafide Glykogenspeicher krankheits- weißen Blutkörperchen sammeln sich große Mengen an Glykogen, die wesentlich von gesunden Kontrollen 8 unterscheidet. Dieses Video-Artikel beschreibt eine angepasste Version von PAS-Färbung für den Einsatz in peripheren mononukleären Blutzellen (PBMC) Proben von venösem Blut von gesunden Probanden. PBMCs enthalten hauptsächlich Lymphozyten T-Lymphozyten und B-Lymphozyten-Familien, wie auch andere Immunzellen, wie NK-Zellen und Monozyten. Der erste Reinigungsschritt entfernt Erythrozyten, Neutrophilen, Granulozyten und andere. Diese Technik liefert Daten zu einer konzentrierten Anteil der Lymphozyten sich die Robustheit Aufzählung von PAS-positiven Zellen im Vergleich zur Verwendung von Vollblutabstrichen. <img alt="Figur 1" src="/files/ftp_upload/52199/52199fig1highres.jpg" /> Abb. 1: Schritt für Schritt Methodik der PAS-Färbung auf PBMC (A) Zunächst wird Isolierung von PBMC durch Ficoll Gradienten erreicht, zeigt der linken Seite die Vorbereitung vor dem Zentrifugieren, zeigt sie nach der Zentrifugation im rechten Feld, wo die Speckhaut, die den PBMC ist in der Mitte des Rohres beobachtet. (B) Isolierte PBMC werden auf den Objektträger unter Verwendung von Formalin-ethanol Fixierungslösungen festtion. Der Schieber wird sanft mit destilliertem Wasser aus einer Spritzflasche gespült. (C) Der Objektträger wird dann in einem 100 ml-Becherglas zur Hälfte mit Amylase-Lösung gefüllt, die Glykogen auflöst platziert. Der Objektträger wird vorsichtig gespült. (D) Der Objektträger wird mit Perjodsäure-Lösung, wobei die Oxidation von Sacchariden erfolgt behandelt. Die Objektträger werden schonend gespült; Dies wird das überschüssige Periodsäure zu entfernen und Beenden des Oxidationsschrittes. (E) Wenn der Schiff&#39;schen Reagenz auf den Objektträgern zu, wird es mit Aldehyden während der Oxidationsstufe erstellt reagieren. Dieser farblose Reagenz wird dann in einem tiefrote magenta Produkt führen. Die Objektträger werden schonend gespült, das überschüssige Schiff-Reagenz zu entfernen.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Periodic Acid Shiff Kit</td>
			<td>Sigma-Aldrich</td>
			<td>395B</td>
			<td>Bring to room temperature prior to use. Materials in this kit are toxic and harmful. Use caution&#160; 
			<a href="http://www.sigmaaldrich.com/catalog/product/sigma/395b?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/395b?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>&#945;-Amylase from porcine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>A3176</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a3176?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/a3176?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>Binocular Microscope&#160;</td>
			<td>Carl Zeiss Microscopy</td>
			<td>Axio Lab A0</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen Assay Kit</td>
			<td>Sigma-Aldrich</td>
			<td>MAK016</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/mak016?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/mak016?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>VWR, GE Healthcare</td>
			<td>17-1440-02</td>
			<td>Nonionic synthetic polymer of sucrose 
			<a href="https://us.vwr.com/store/catalog/product.jsp?product_id=4779441">https://us.vwr.com/store/catalog/product.jsp?product_id=4779441</a></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td>For PBMC isolation, swing buckets were used&#160;</td>
		</tr>
	</tbody>
</table>

detecting glycogen in peripheral blood mononuclear cells with periodic acid schiff staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Um die Reagenzien und grundlegende Technik zu validieren, wurde PAS-Färbung nach Angaben des Herstellers auf der Maus Soleusmuskel Abschnitten durchgeführt. Die Färbung wurde am gleichen Tag wie die Opfer gemacht und im letzten Schritt der Färbung wurden die Schnitte mit Xylol fixiert. Der M. soleus ist bekannt, enthalten ~ 35% Glykogen-positiven Zellen 9. Die gefärbten Muskelzellen angezeigt zwei verschiedene PAS-positiven Features-punktförmige Granulat in der Zelle, und eine durchgehende Linie Demarki...

Watch this Scientific Journal Video about Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining at JoVE.com

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Erkennen von Glykogen in peripheren mononukleären Blutzellen mit Perjodsäure-Schiff-Färbung

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

detecting-glycogen-peripheral-blood-mononuclear-cells-with-periodic

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Immunology and Infection

21.0K Aufrufe.  Centre for Structural and Functional Genomics, PERFORM Centre, Concordia University. Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Serie: Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

Induktion von Alloantigen-spezifische Anergie in humanen peripheren mononukleären Zellen durch Alloantigen Stimulation mit Co-stimulatorische Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

Forschung

Immunologie und Infektion

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  &amp;#947;&amp;#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 ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Generation of Human Alloantigen-spezifischen T-Zellen aus peripherem Blut

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

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 ...

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.

Analyse von Lymphozyten-Extravasation einer Verwendung<em&gt; In Vitro</em&gt; Modell des Barrier Menschliches Blut-Hirn

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

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

Trennung von Immunzellsubpopulationen in peripheren Blutproben von Kindern mit infektiöser Mononukleose

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

Flotationsbasierte T-Zell-Isolierung, -Aktivierung und -Expansion aus mononukleären Blutproben des menschlichen peripheren Blutes unter Verwendung von Mikrobläschen

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

PBMC-Sammlung und Kryolagerung für die mitochondriale Bioenergetikanalyse

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.

Autor im Rampenlicht: Konservierung bioenergetischer Parameter in mononukleären Zellen des peripheren Blutes nach Kryokonservierung 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

Kryokonservierung und bioenergetische Bewertung von mononukleären Zellen des menschlichen peripheren Blutes

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...