Diese Arbeit wurde zum Teil durch die NIH (N01 HHSN272201100019C, U19 AI 089992, und die NCRR / GCRC Programm M01-RR00125) unterstützt. Die Autoren erklären, keine konkurrierenden finanziellen Interessen und danken Herrn Dr. Mark Shlomchik, Direktor des Yale Cell Sorter Core Facility.

1. Isolierung und Stimulation von Immunzellen <ol><li> Erhalten 10-50 ml heparinisierten Blut von gesunden Freiwilligen nach schriftlicher Einwilligung unter den Richtlinien der lokalen Institutional Review Board. Bei Studien von Alterung oder Pathogenese der Erkrankung, Exklusion und Inklusion Kriterien für jeden Freiwilligen oder Patienten müssen explizit bestimmt werden. </li><li> Isolieren menschlichen peripheren mononukleären Blutzellen (PBMCs) mit CPT-Röhrchen oder Ficoll-Paque Plus nach den Anweisungen des Herstellers (GE Healthcare, NJ) und diese durch Experimente am Tag der Isolierung 7 (Reagenzien, Tabelle 1). </li><li> Inkubieren PBMCs (3 × 10 6 / Röhrchen) in 1,5 ml Röhrchen in 0,6 ml Medium allein oder stimuliert mit TLR-Liganden oder andere aktivierende Agenzien, zB TLR7 / 8 Liganden R848 (0,6 ml Medium plus 0,6 ul von 10 mM TLR7 / 8 Liganden R848, Endkonzentration 10 uM) bei 37 ° C für 10-60 min (InvivoGen, CA) 9. </li></ol> 2. LaborEling von Cells <ol><li> Label-Zelllinie und andere Oberflächenmarker auf Live-Zellsuspensionen mit Fluoreszenz-konjugierte Antikörper. Label 3 x 10 6 PBMCs für 20 min bei 4 ° C in 100 l PBS / 2% FBS in einem 1,5 ml Eppendorf-Röhrchen mit einem Cocktail von Antikörpern, die für Oberfläche Monozyt oder DC Linien (Tabelle 2). Optimale Antikörper-Verdünnungen können in Vorversuchen und Proben bestimmt werden sollte in Chargen verarbeitet werden, um viel zu viel Variation zu minimieren. Für Anbieter Antikörper Lager von 0,1 mg / ml verwenden 5 ul Antikörper (1:20 Verdünnung). Verwenden Sie einen separaten Schlauch von Zellen mit nur einer einzigen Farbe Konjugat zum Einrichten der Fluoreszenz-Kanäle auf dem Gerät markiert. </li><li> Nach Oberflächenmarkierung, zu fixieren Zellen in 4% PFA / PBS für 10 min bei RT. Für die Lagerung bis zum Zeitpunkt der Analyse-, Zentrifugen-Zellen bei 500 xg und Resuspendieren Sie die Zellen in dem Gefrierpunkt Puffer (90% FBS mit 10% DMSO) bei -80 ° C bis zum Tag des Assays. </li><li> Für die Beurteilung, Prozess-Chargen of unbehandelten und stimulierten Zellen aus einer Gruppe von Gebern zusammen, um die Variabilität zu minimieren. Am Tag der Analyse, Auftau-Zellen schnell in 37 ° C warmes Wasserbad, Zentrifuge bei 500 xg, um ein Einfrieren Puffer zu entfernen. </li><li> Um Zellen für den Nachweis von Zytokinen oder anderen intrazellulären Marker, resuspendieren Zellsuspensionen in 100 ul BD Perm / Waschpuffer (BD Biosciences, NJ) zu permeabilisieren. Gütesiegel für den intrazellulären Signalkomponenten mit zB 1:20 Verdünnung des Kaninchen-Anti-NF-kB (p65)-Antikörper (Endkonzentration 10 ug / ml, Santacruz Biotechnology, CA) für 20 min bei RT, Zentrifuge bei 500 xg, Überstand absaugen und Die Zellen in 100 ul BD Perm / Wash-Puffer, der 1:250 Verdünnung von Ziege-Anti-Kaninchen-IgG-Alexa647 (Invitrogen, CA) und Inkubation für 20 min bei RT. </li><li> Unmittelbar vor der Bildgebung, Gegenfärbung Zellkerne mit DAPI (0,2 ug / ml, Invitrogen, CA) oder Propidiumiodid (PI; 20 ng / ml, Invitrogen, CA). </li></ol> 3. ImageStream Analyse <ol><li>Durchführung von bildgebenden ImageStream auf batched Proben Variabilität zwischen den humanen Proben zu reduzieren. Geräteeinstellungen für die Leistung der Laser waren wie folgt: 200 mW für 488 nm, 10 mW für 658 nm und 250 mW bei 405 nm gemessen. </li><li> (: Höhe-Verhältnis Breite), um einzelne Zellen (R1) aus Schutt (niedrige Intensität PI) oder Multi-zellulären Ereignissen zu unterscheiden (Um Dateien (Abb. 1), erstens, Tor auf Ereignisse mit normaler und hoher Intensität PI PI Seitenverhältnis analysieren hoher Intensität und geringer PI-Bildformat). Monozyten sind aus dem Gate für CD14-positive Zellen. mDCs innerhalb eines Gate-Zellen, die für hoch sind für CD11c und niedrig für CD4 (R2) als auch niedrigen für Lin-1-Marker (R3). </li><li> Verwendung IDEAS-Software (Amnis, WA), um eine wirksame quantitatives Maß für den Grad der Aktivierung von jeder Zelle bereitzustellen. Bestimmen Ähnlichkeit (oder Co-Lokalisierung) der zytoplasmatischen Transkriptionsfaktor NF-kappaB mit nuklearen Farbstoff PI bis Translokation in den Zellkern (R4) angeben. Eine hohe Korrelation von NF-κB/PI localisierung in einem hohen Ähnlichkeitsbewertung reflektiert und gibt den Grad der Aktivierung 10. </li><li> Vergleichen Sie die Ähnlichkeit Verhältnisse zwischen den verschiedenen Behandlungsgruppen oder Patientengruppen zu relativen Funktionstüchtigkeit der Zellen zeigen. Quantifizieren Sie Daten zwischen den Proben durch statistischen Vergleich der absoluten Werte oder falten Unterschiede. Verwenden Sie IDEAS-Software, um Bilder von Zellen, die aus wichtigen Segmenten der Bevölkerung Histogramme (Abb. 2) anzuzeigen. </li></ol> 4. Repräsentative Ergebnisse Wir haben Signalwege in Reaktion auf ein Modell viralen Liganden quantifiziert, indem es PBMCs mit dem TLR7 / 8 Liganden, R848 (Abb. 1). Wir haben sowohl zelluläre Lokalisation Bilder und Bevölkerungsstatistik in unserer Stichprobe Gruppen (Abb. 2). ImageStream ermöglicht es uns, Tor Zelluntergruppen direkt aus PBMCs und Bild-Zell-Antworten von gated, aber unsortierten Zellpopulationen. Hier haben wir den Effekt der TLR s quantifiziertignaling auf nukleäre Lokalisation von NF-kB (p65) in Lin1-, CD4-dim, CD11c + Dendritischen Zellen (mDCs). Zu Beginn der Studie beobachteten wir einen hohen Prozentsatz an unstimulierten Zellen mit dem Transkriptionsfaktor NF-kB (p65) im Zytoplasma. Nach Stimulation, haben behandelten Zellen eine deutlich höhere Ähnlichkeits-Score von NF-B (p65) mit Kernfärbung PI, was darauf hinweist, dass NF-kB (p65) in den Zellkern transloziert nach Stimulation (Median Ähnlichkeit Partituren sind 1,52 und 3,01 bzw. Abb. 2). Ähnliche Ergebnisse sind in TLR5-stimulierten Monocyten 9 angegeben. <img alt="1" src="/files/ftp_upload/3741/3741fig1.jpg" /> Abbildung 1. Färbung und Gating-Strategie, um Effekte der Stimulation auf die menschliche mDCs quantifizieren. Der NF-kappaB-Transkriptionsfaktor reguliert die Expression von zahlreichen Genen des Immunsystems. Diese Studie misst die nukleäre Lokalisation von NF-kB (p65) in mDCs aus je einem Vertreter unterliegen nach TLR7 / 8 Liga<html> ND Stimulation. In-Fokus einzelne Zellen wurden durch Gating auf PI positive Ereignisse mit hohen nuklearen Seitenverhältnissen (R1) identifiziert. mDCs (Lin1-, CD4dim, CD11c +) waren in gated R3. Nukleäre Lokalisation von NF-kB (p65) wird in R4 dargestellt. <a href="http://www.jove.com/files/ftp_upload/3741/3741fig1large.jpg" target="_blank">Klicken Sie hier für eine größere Abbildung anzuzeigen</a> . <img alt="2" src="/files/ftp_upload/3741/3741fig2.jpg" /> Abbildung 2. . Translokation von NF-kappaB in mDCs nach Stimulation der Translokation von NF-kB (p65) in den Zellkern nach Stimulation (R4) in Populationen von unbehandelten und stimulierten mDCs abgebildet, wobei die mediane Ähnlichkeits-Score ist 1.52 in der unbehandelten Probe und 3,01 in die angeregte Probe (A). Digitale Bilder gleichzeitig der unbehandelten oder R848-stimulierte Zellpopulationen zeigen repräsentative Zellen gesammelt und die Intensität der NF-B in den Zellkern (B) transloziert._upload/3741/3741fig2large.jpg &quot;target =&quot; _blank &quot;&gt; Klicken Sie hier für eine größere Abbildung zu sehen.

<ol>
	<li>Linton, P. J., Dorshkind, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+lymphocyte+development+and+function.">Age-related changes in lymphocyte development and function.</a> Nat. Immunol. 5, 133-139 (2004).</li><li>Shaw, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+Human+Toll-like+Receptor+Function+in+Aging.">Dysregulation of Human Toll-like Receptor Function in Aging.</a> Ageing Research Reviews. 10, 346-353 (2011).</li><li>Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptors+and+innate+immunity.">Toll-like receptors and innate immunity.</a> Nature Rev. Immunol. 1, 135-145 (2001).</li><li>van Duin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+Defect+in+Human+TLR1+Function+and+Expression.">Age-associated Defect in Human TLR1 Function and Expression.</a> J. Immunol. 178, 970-975 (2007).</li><li>Panda, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+decrease+in+TLR+function+in+primary+human+dendritic+cells+predicts+influenza+vaccine+response.">Age-associated decrease in TLR function in primary human dendritic cells predicts influenza vaccine response.</a> J. Immunol. 184, 2518-2527 (2010).</li><li>Kong, K. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+TLR3+impairs+the+innate+immune+response+to+West+Nile+virus+in+the+elderly.">Dysregulation of TLR3 impairs the innate immune response to West Nile virus in the elderly.</a> J. Virol. 82, 7613-7623 (2008).</li><li>Qian, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+interferon+signaling+in+dendritic+cells+from+older+donors+infected+in+vitro+with+West+Nile+virus.">Impaired interferon signaling in dendritic cells from older donors infected in vitro with West Nile virus.</a> J. Infect. Dis. 203, 1415-1424 (2011).</li><li>Hayden, M. S., Ghosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF-kappaB+in+immunobiology.">NF-kappaB in immunobiology.</a> Cell research. 21, 223-244 (2011).</li><li>Qian, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+elevation+in+TLR5+leads+to+increased+inflammatory+responses+in+the+elderly.">Age-associated elevation in TLR5 leads to increased inflammatory responses in the elderly.</a> Aging Cell. 11, 104-110 (2011).</li><li>George, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+nuclear+translocation+events+using+similarity+analysis+of+multispectral+cellular+images+obtained+in+flow.">Quantitative measurement of nuclear translocation events using similarity analysis of multispectral cellular images obtained in flow.</a> Journal of immunological. 311, 117-129 (2006).</li></ol>

Keine Interessenskonflikte erklärt.

Die entscheidenden Schritte bei der Verwendung von ImageStream für translationale Studien sind die Auswahl der relevanten Vergleichsgruppen und Optimierung und Validierung von Antikörper-Spezifität. Unterschiede zwischen Fächergruppen in dem markierten Ziel wird leicht ersichtlich sein durch Histogramme und Zellen geben. In Kombination mit einer gründlichen Analyse der Veränderungen in relevanten Rezeptoren und Signalwege, werden diese Daten wertvolle Einblicke in die Mechanismen, die in spezialisierten Immundysre...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Ficoll-Paque Plus- </td><td> GE Healthcare </td><td> 17-1440-03 </td></tr><tr><td> R848 </td><td> Invivogen </td><td> tlrl-r848 </td></tr><tr><td> Paraformaldehyd (16%) </td><td> Electron Microscopy Sciences </td><td> 15710 </td></tr><tr><td> BD Perm / Wash </td><td> BD Biosciences </td><td> 554723 </td></tr><tr><td> CD14-PE </td><td> eBioscience </td><td> 12-0149-41 </td></tr><tr><td> CD4-PacBlue </td><td> BD Pharmingen </td><td> 68436 </td></tr><tr><td> Lineage-Cocktail 1 (lin 1)-FITC </td><td> BD Biosciences </td><td> 340546 </td></tr><tr><td> CD11c-PE </td><td> BD Biosciences </td><td> 347637 </td></tr><tr><td> CD123-PE </td><td> BD Biosciences </td><td> 340545 </td></tr><tr><td> Kaninchen-Anti-NF-kB (p65) </td><td> SantaCruz </td><td> SC-372 </td></tr><tr><td> Alexa647 F (ab &#39;) 2 Ziege-anti-Kaninchen-IgG </td><td> Invitrogen </td><td> A21246 </td></tr><tr><td> DAPI </td><td> Invitrogen </td><td> D21490 </td></tr><tr><td> ImageStream X </td><td> Amnis </td><td></td></tr></tbody></table> Tabelle 1. Spezifischen Reagenzien und Geräte. <table border="1"><tbody><tr><td> Zelltyp </td><td> FITC </td><td> PE </td><td> Alexa 647 </td><td> PacBlue </td></tr><tr><td> Monozyten </td><td></td><td> CD14 </td><td> NF-kB (p65) </td><td></td></tr><tr><td> mDC </td><td> Lin1 </td><td> CD11c </td><td> NF-kB (p65) </td><td> CD4 </td></tr><tr><td> pDC </td><td> Lin1</td><td> CD123 </td><td> NF-kB (p65) </td><td> CD4 </td></tr></tbody></table> Tabelle 2. Antikörper Platte für Zellreihe Färbung von Signalwegen Monozyten: CD14 + MDC: Lin1-, CD4dim, CD11c + PDC: Lin1-, CD4dim, CD123 + Lin1 (Abstammung) Marker enthält CD3, CD14, CD19, CD20, CD56 Marker für Monozyten, NK-Zellen, T &amp; B-Zellen.

quantitative imaging of lineage-specific toll-like receptor-mediated signaling in monocytes and dendritic cells from small samples of human blood

Individuelle Abweichungen in Immunstatus bestimmen Reaktionen auf Infektionen und zur Schwere der Erkrankung und das Ergebnis. Das Altern ist mit einer erhöhten Anfälligkeit für virale und bakterielle Infektionen assoziiert und verringerte Ansprechbarkeit auf Impfstoffe mit einer gut dokumentierten Rückgang in humorale als auch zellvermittelte Immunreaktionen 1,2. Wir haben vor kurzem die Auswirkungen der Alterung auf die Toll-like Rezeptoren (TLR), wichtige Bestandteile des angeborenen Immunsystems, die mikrobielle Infektion und Trigger mikrobiellen Abwehr Antworten 3 erkennen beurteilt. In einer großen Kohorte von gesunden menschlichen Spendern, haben wir gezeigt, dass die Monozyten im peripheren Blut von älteren Menschen haben die Expression und Funktion bestimmter TLR 4 und ähnlich reduzierte TLR Ebenen und Signalantwort in dendritische Zellen (DCs) verringerte, Antigen-präsentierende Zellen, die entscheidend sind in die Verknüpfung zwischen angeborenen und erworbenen Immunität 5. Wir haben eine Fehlregulation von TLR3 in Makrophagen gezeigt, eind niedrigere Produktion von IFN durch DCs von älteren Spendern in Reaktion auf eine Infektion mit West-Nil-Virus 6,7. Paramount zu unserem Verständnis der Immunoseneszenz und therapeutische Intervention ist ein detailliertes Verständnis der spezifischen Zelltypen reagieren und der Mechanismus (s) der Signaltransduktion. Traditionelle Studien von Immunantworten durch bildgebende von primären Zellen und Vermessung Zellmarker durch FACS oder Immunoblot haben unser Verständnis deutlich, rückte aber diese Studien in der Regel mit technischen Mitteln durch die kleine Stichprobe zur Verfügung stehende Volumen von Patienten und die Unfähigkeit, komplexe Laborverfahren auf mehreren befragen begrenzt humanen Proben. ImageStream kombiniert quantitative Durchflusszytometrie bei gleichzeitiger hoher Auflösung digital imaging und erleichtert somit die Untersuchung in mehreren Zellpopulationen gleichzeitig für eine effiziente Erfassung von Patienten-Anfälligkeit. Hier zeigen wir die Verwendung von ImageStream in DCs zu beurteilen, TLR7 / 8Aktivierung-vermittelten Anstieg der Phosphorylierung und die nukleäre Translokation von einem Schlüssel Transkriptionsfaktors NF-kB, die die Transkription zahlreicher Gene, die für Immunantworten 8 initiiert. Mit dieser Technologie haben wir vor kurzem auch eine bisher unbekannte Veränderung TLR5 Signalisierung und der NF-kappaB Signalweg in Monozyten von älteren Spendern, die zu einer veränderten Immunantwort in alternden 9 beitragen können, unter Beweis gestellt.

Watch this Scientific Journal Video about Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood at JoVE.com

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Wir beschreiben den Einsatz von ImageStream Technologie (<a href="http://www.amnis.com/" target="_blank"&gt; Www.amnis.com</a&gt;), Verbindet die quantitative Durchflusszytometrie bei gleichzeitiger hoher Auflösung digital imaging, um zelluläre Mechanismen der primären Immunzellen aus gut definierten Patientengruppen zu quantifizieren. Unsere Studien liefern eine Blaupause für translationale Untersuchungen zur Abstammung spezifische zelluläre Reaktionen in kleinen Proben von Kohorten Thema zu quantifizieren.

Quantitative Bildgebung von Lineage-spezifischen Toll-like Rezeptor-vermittelten Signaltransduktion in Monozyten und dendritischen Zellen aus kleinen Proben des menschlichen Blutes

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

quantitative-imaging-lineage-specific-toll-like-receptor-mediated

Research

JoVE Journal

Immunology and Infection

35.0K Aufrufe.  Yale University School of Medicine. Wir beschreiben den Einsatz von ImageStream Technologie ( Www.amnis.com), Verbindet die quantitative Durchflusszytometrie bei gleichzeitiger hoher Auflösung digital imaging, um zelluläre Mechanismen der primären Immunzellen aus gut definierten Patientengruppen zu quantifizieren. Unsere Studien liefern eine Blaupause für translationale Untersuchungen zur Abstammung spezifische zelluläre Reaktionen in kleinen Pr...

Serie: Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR G&alpha;i proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl &alpha;-2,3-linked &beta;-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.

The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.

Erkennung von Neu1 Sialidase-Aktivität bei der Regulierung der Toll-like-Rezeptor-Aktivierung

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

Forschung

Immunologie und Infektion

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

Isolierung von myeloider dendritischer Zellen und Epithelzellen Menschliche Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

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 of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Isolierung von Murine Peritonealmakrophagen zur Durchführung Genexpressionsanalyse Nach Toll-like Rezeptoren Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Herstellung von menschlichen Monozyten abgeleiteten Dendritischen Zellen aus Vollblut

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

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.

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

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 ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Live-Imaging von Anti-Pilz-Aktivität von Human Primary Neutrophilen und Monozyten in Reaktion auf<em&gt; A. fumigatus</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

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 ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

Entwicklung und Validierung eines empfindsamen Einzelmolekül Array digitale Enzym-linked Immunosorbentprobe Assay für menschliche Interferon-α

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...