Die Autoren bedanken sich bei Bing Zhang für seine technische Unterstützung danken. Diese Arbeit wurde von der kanadischen Institute for Health Research (MOP-86595) finanziert. TAB ist eine CIHR New Investigator und AHFMR Scholar. Doctoral und einem AIHS Vollzeit Studentship - QH wird durch eine CIHR Canada Graduate Scholarship unterstützt. SAN wird von einer Königin Elizabeth II Graduate Scholarship unterstützt. Doctoral - AYWS wird durch ein NSERC Postgraduate-Stipendium unterstützt.

Siehe Abbildung 1 für eine allgemeine Regelung des experimentellen Protokolls Abbildung. Ein. Präparation <ol><li> Ort sterile Stahlgitter Bildschirm in 60 x 15 mm Petrischale. Eine Einheit wird pro Gewebeprobe nötig. </li><li> 5 ml balanced salt Hank-Lösung (HBSS) zu jedem Gericht. Halten Sie Speisen auf dem Eis. </li><li> Einschläfern Mäuse mit CO 2. </li><li> Sichere Maus Dissektion Oberfläche Bauchseite nach oben. Spray-Maus mit 70% Ethanol für die Sterilisation und sicherzustellen, dass das Fell verfilzt wird unten. </li><li> Verwendung von chirurgischen Schere, beginnen Dissektion indem ein Einschnitt in der ventralen Bauchhaut knapp oberhalb der Genitalien. Verlängern Sie die Inzision nach oben bis zum Kinn. </li><li> Von der Mittellinie, verlängern die Inzision entlang aller Glieder. Ziehen Sie die lose Haut und stecken Sie es bis zur Dissektion Oberfläche. </li><li> Ernten Sie die Thymus: <ol><li> Heben Sie die untere Spitze des Brustbeins, um einen Schnitt zu machen.</li><li> Die Vermeidung der Leber, senken die Membran an den Brustkorb zu lösen und dann geschnitten den Brustkorb zu jeder Seite. Schneiden Sie die Brustkorb nach oben auf jeder Seite, kümmert sich um die Lunge und des Herzens zu vermeiden. </li><li> Ziehen der Brustkorb wieder mit einer Pinzette. Der Thymus ist ein weißes, bilobed Organ oberhalb des Herzens. Mit dem flachen Rand der Zange, um die Unterseite der Flügel zu erfassen, ziehen Sie den Thymus und legen Sie es auf dem Sieb. </li></ol></li><li> Ernten die Milz: <ol><li> Die Milz ist ein roter, Surfboard-förmige Organ auf der linken Seite der Maus der Bauchhöhle, unterhalb der Leber entfernt. </li><li> Einen Einschnitt in die Bauchhöhle. Ziehen Sie vorsichtig die Milz mit einer Pinzette, mit dem zweiten Paar zu necken sich Bindegewebe. Legen Sie die Milz auf einem separaten Sieb. </li></ol></li></ol> 2. Cell Preparation <ol><li> Verwendung eines Kolbens aus einer 3-ml-Spritze, schleifenOrgane in den Sieb, bis nur Bindegewebe und Fett bleibt. Spülen Mesh mit HBSS aus der Schale dreimal. Verwenden Sie einen neuen Kolben für jede Gewebeprobe. <ol><li> Andere Möglichkeiten zur Homogenisierung Gewebe kann anstelle dieses Verfahren verwendet werden. </li></ol></li><li> Zählen von Zellen mit einer Zählkammer. </li><li> Pellet Zellen durch Zentrifugation bei 335 xg für 5 Minuten bei 4 ° C. </li><li> Splenozyten resuspendieren in 500 ul ACK-Lysepuffer (0,15 M NH 4 Cl, 10 mM KHC0 3, 0,1 mM Na 2 EDTA, pH 7,2) für 10 min bei Raumtemperatur. Resuspendieren Thymozyten bei 20 x 10 6 Zellen / ml in FACS-Puffer (PBS, 1% FCS, 0,02% Natriumazid) und beiseite stellen auf Eis. </li><li> Splenozyten zurückzukehren, um Isotonie durch Zugabe von 5 ml HBSS. Pellet Splenozyten durch Zentrifugieren und Resuspendieren bei 20 x 10 6 Zellen / ml in FACS-Puffer. Wenn Zellen steril bleiben müssen, können Sie sterilem RPMI + 10% FCS. </li></ol> 3.Färbung Cells für die Durchflusszytometrie Der Zweck dieses Protokolls ist es, Strategien für die Analyse der positiven und negativen Selektion mit Wildtyp (WT) und HY cd4 Mäusen zu skizzieren. Für allgemeine Überlegungen zur Durchflusszytometrie experimentelles Design, Beschaffung und Analyse verweisen wir die Leserschaft auf eine ausgezeichnete Bewertung von Tung et al. 11 <ol><li> Aliquot 4 x 10 6 pro Thymozyten Durchflußcytometrieprobe zu jeder Vertiefung einer 96-Well-Platte, sowie 1 x 10 6 Splenozyten WT pro Kompensationssteuerung. Bei der Verwendung von transgenen Mäusen, sollten Sie einen WT-Maus als Kontrolle in den Test einbezogen werden. </li><li> Fc-Rezeptoren blockieren, durch Inkubieren von Zellen mit anti-CD16/32 (Klon 2.4G2) für 10 min auf Eis. </li><li> Drallplatte bei 335 × g bei 4 ° C für 5 min. Deckel entfernen und zu zerstreuen Flüssigkeit sorgfältig durch Ausklopfen der Platte einmal nach unten in einer Senke. Resuspendieren jedes Well in 200 ul FACS Puffer. Wiederholen Sie die Wäsche. </li><li> Bereiten Antikörper Cocktails wie unten aufgeführt, über die optimale Konzentration jedes Antikörpers zur Verdünnung in 200 ul FACS-Puffer pro well basiert. Die optimale Konzentration der einzelnen Antikörper wird als die niedrigste Konzentration benötigt wird, um die größte Trennung von positiven und negativen Populationen geben definiert. Wenn es keinen negativen Population für einen gegebenen Antikörper, sollte ein Isotyp-Antikörper enthalten sein. Das Gesamtvolumen pro Vertiefung können verkleinert werden (zB zu 100 ul pro Well), falls gewünscht. <ol><li> WT Thymus: anti-TCRβ, anti-CD4, anti-CD8-, Anti-CD69 oder anti-CD5-, Anti-CD24 </li><li> HY cd4 Thymus: anti-TCR HY (T3.70), anti-CD4, anti-CD8-, Anti-CD69 oder anti-CD5-, Anti-CD24 Um eine bessere Trennung von CD69 und CD69 lo hallo Populationen in Thymocyten zu erhalten, wird empfohlen, dass ein biotinylierter anti-CD69 primärer Antikörper verwendet werden, gefolgt von einem sekundären Fleck enthält Fluorochrom-konjugiertem Streptavidin. Wir use die gleiche Strategie für CD5 Färbung. </li></ol></li><li> Inkubieren von Zellen mit 200 ul Antikörper-Cocktail in FACS-Puffer für 30 min auf Eis im Dunkeln. </li><li> Zeitgleich mit Antikörper-Cocktail Färbung, färben jede Entschädigung Steuerung mit einer einzigen Fluorochrom konjugierte Antikörper. Im Idealfall beinhaltet Entschädigung Färbung die gleichen Antikörper wie in der Antikörper-Cocktail verwendet. Allerdings kann Färbung für jedes einzelne Antigen nicht machbar sein für größere Experimente, wenn viele Antigene untersucht wird. Daher wird Anfärben für entweder Anti-CD4-oder Anti-CD8 empfohlen aufgrund hoher Expression dieser Antigene, oder die Verwendung von Kompensation Kügelchen. Färben in FACS-Puffer für 30 min auf Eis im Dunkeln. Lassen Sie eine Ausgleichsregelung ungefärbt. </li><li> Waschen der Zellen zweimal mit FACS-Puffer. </li><li> Die Zellen in FACS-Puffer und Transfer zum FACS-Röhrchen. </li><li> Erwerben Proben auf Durchflusszytometer. Beinhalten den Erwerb von FSC-A-und FSC-W einllow für Dublett Diskriminierung. </li></ol> 4. Analyse der Durchflusszytometrie Data - Non-TCR transgenen Mäusen Wir verwenden FlowJo für die Durchflusszytometrie Datenanalyse. Siehe 2A für die Gating-Strategie für nicht-TCR transgenen Mäusen Abbildung. <ol><li> Mit FSC-A von SSC, elektronisch Tor auf der &quot;Lymphozyten&quot; Bevölkerung. </li><li> Im Rahmen der &quot;Lymphozyten&quot; Bevölkerung, verwenden FSC-A von FSC-W elektronisch Tor des FSC-W lo Bevölkerung zu Zelle Dubletten und aggregrates auszuschließen. Dies ist die &quot;Singulett&quot; Tor. </li><li> Mit den Veranstaltungen in der &quot;Singulett&quot; Tor, Grundstück CD8 von CD4. Zeichnen Tore für DN, DP langweilig, DP hell, CD4 + CD8 lo, CD4SP und CD8SP Populationen wie in 2B dargestellt. </li><li> Unter dem CD4SP und CD8SP Tore, erstellen TCRβ von CD24 Plots. Verwenden Sie den Quadranten Gating-Tool zu Toren zu ziehen wie in 2C dargestellt. </li><li> Erstellen Sie eine new Plot aus der &quot;Singulett&quot; Tor: TCRβ von CD69. Zeichnen Gatter A, B, C, D wie in 2D dargestellt. </li><li> Erstellen Sie ein weiteres Grundstück von der &quot;Singulett&quot; Tor: TCRβ von CD5. Zeichnen Gatter i, ii, iii, iv, wie in 2E dargestellt ist. Dies ist eine weitere Strategie zur Untersuchung positive Selektion. </li><li> Bewerben DN, DP langweilig, DP hell, CD4 int, CD4SP und CD8SP Toren unter &quot;Singulett&quot; zu Toren i, ii, iii, iv, A, B, C, D (2D, E). </li><li> Berechnen Sie die Anzahl der Zellen in einer bestimmten Untergruppe durch Multiplikation Orgel Zellularität durch die Häufigkeit der einzelnen nachfolgenden Tor, bis Sie Ihre Zielgruppe erreichen. Zum Beispiel kann die Anzahl der TCRβ hallo CD69-CD8SP Thymozyten würde durch Thymus Zellularität x% TCR bhi bestimmt werden CD69-(Fraktion D) x% CD8SP. <ol><li> Zählen berücksichtigt bereits die lebenden und toten Zellen also nicht auch die &quot;lymphocYTE &quot;Tor in Ihren Berechnungen. </li></ol></li></ol> 5. Analyse der Durchflusszytometrie Data - TCR transgenen Mäusen Siehe 3A für die Gating-Strategie für TCR transgenen Mäusen Abbildung. <ol><li> Folgen Sie den Schritten 4,1 und 4,2, wie im vorherigen Abschnitt. </li><li> Unter dem &quot;Singulett&quot; Tor, eine T3.70 von SSC Plot und Tor auf der T3.70 + Zellpopulation Antigen-spezifischen T-Zellen (3B) zu analysieren. In manchen Fällen kann es von Interesse sein, diese Bevölkerung zu dem nicht antigenspezifischen (T3.70-) T-Zell-Population zu vergleichen. </li><li> Innerhalb der T3.70 + Bevölkerung, draw DN, DP, CD4SP und CD8SP Tore (3C). <ol><li> Für WT Kontrollproben, zeichnen diese Tore unter dem &quot;Singulett&quot; Tor oder eine T3.70-Gate, da nur sehr wenige T3.70 +-Zellen sein wird. </li></ol></li><li> Unter dem CD8SP Tor, erstellen Sie eineT3.70 von CD24 Grundstück. Verwenden des Quadranten Gating Werkzeug, um Zellen in vier Populationen (3F) zu unterteilen. </li><li> Neu überlagerte Histogramm, welches die CD69-Expression in den DP-Kompartiment von WT-Mäusen und T3.70 + DP Abteil HY cd4 Mäusen (4A). Wiederholen Sie den Vorgang CD5 Expression (4B) zu untersuchen. </li><li> Berechnen der absoluten Anzahl von Zellen in jeder Untergruppe von Interesse, wie in 4.8. </li></ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+analysis+of+clonal+deletion+in+vivo.">Transcriptional analysis of clonal deletion in vivo.</a> J. Immunol. 179, 837-844 (2007).</li></ol>

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Die hier vorgestellten Protokoll kann verwendet werden, um positive und negative Selektion in nicht-transgenen und TCR TCR-transgenen Mäusen zu untersuchen. Dieses Protokoll beschreibt die Färbung von Oberflächen-Antigenen. Für die weitere Analyse der molekularen Mechanismen, ist es oft notwendig, intrazelluläre Färbung führen. Wir verwenden die BD Biosciences Cytofix / Cytoperm Kit für die meisten intrazellulären Proteinen und der BD Biosciences Foxp3 Staining Kit für Transkriptionsfaktoren. Wir in der Regel ...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> HyClone Hanks balanced salt solution </td><td> Thermo Scientific </td><td> SH30030.02 </td><td></td></tr><tr><td> Drahtgewebeelementen </td><td> Cedarlane </td><td> CX-0080-E-01 </td><td></td></tr><tr><td> Petrischalen (60 x 15 mm) </td><td> Fisher Scientific </td><td> 877221 </td><td></td></tr><tr><td> Spritzen (3 ml) </td><td> BD Biosciences </td><td> 309657 </td><td></td></tr><tr><td> Konischen Röhren (15 ml) </td><td> Sarstedt </td><td> 62.554.205 </td><td></td></tr><tr><td> Mikroskop </td><td> Zeiss - Primo Star </td><td> 415.500-00XX-000 </td><td></td></tr><tr><td> Hemocytometer </td><td> HausserWissenschaftlich </td><td> 3110 </td><td></td></tr><tr><td> 96-Well-Platte </td><td> Sarstedt </td><td> 82.1582.001 </td><td></td></tr><tr><td> Mehrkanalpipette </td><td> Fisherbrand </td><td> 21-377-829 </td><td></td></tr><tr><td> Fötales Kälberserum </td><td> PAA </td><td> A15-701 </td><td></td></tr><tr><td> Phosphat-gepufferter Kochsalzlösung </td><td> Fisher Scientific </td><td> SH3025802 </td><td></td></tr><tr><td> Natriumazid </td><td> IT Baker Chemical Co. </td><td> V015-05 </td><td></td></tr><tr><td> FcR Blockierungsreagenz </td><td></td><td></td><td> Clone 2.4G2 </td></tr><tr><td> Anti-Maus-HY TCR </td><td> eBioscience </td><td> XX-9930-YY * </td><td> Clone T3.70 </td></tr><tr><td> Anti-Maus-CD4 </td><td> eBioscience </td><td> XX-0042-YY * </td><td> Clone RM4-5 </td></tr><tr><td> Anti-Maus CD8a </td><td> eBioscience </td><td> XX-0081-YY * </td><td> Clone 53-6,7 </td></tr><tr><td> Anti-Maus-CD24 </td><td> eBioscience </td><td> XX-0242-YY * </td><td> Clone M1/69 </td></tr><tr><td> Anti-Maus-TCRβ </td><td> eBioscience </td><td> XX-5961-YY * </td><td> Clone H57-597 </td></tr><tr><td> Anti-Maus-CD69 Biotinylierte </td><td> eBioscience </td><td> 13-0691-YY * </td><td> Clone H1.2F3 </td></tr><tr><td> Anti-Maus-CD5 Biotinylierte </td><td> eBioscience </td><td> 13-0051-YY * </td><td> Clone 53-7,3 </td></tr><tr><td> Streptavidin </td><td> eBioscience </td><td> XX-4217-YY * </td><td></td></tr><tr><td> Durchflusszytometer </td><td> BD Biosciences - FACS Canto </td><td> 338962 </td><td></td></tr><tr><td> FACS-Röhrchen </td><td> BD Biosciences </td><td> 352052 </td><td></td></tr><tr><td> Durchflusszytometrie-Analyse-Software </td><td> TreeStar - FlowJo </td><td> FlowJo v7 / 9 </td><td></td></tr><tr><td> HyClone RPMI - 1640 Medium </td><td> Thermo Scientific </td><td> SH30027.01 </td><td></td></tr><tr><td></td><td></td><td></td><td> * XX variiert je nach Fluorochrom und YY variiert je nach Gefäß Größe. </td></tr></tbody></table>

examination of thymic positive and negative selection by flow cytometry

Ein gesundes Immunsystem verlangt, dass T-Zellen fremde Antigene reagieren, während verbleibenden tolerant gegenüber Selbst-Antigenen. Random Umlagerung der T-Zell-Rezeptor (TCR) α und β loci erzeugt eine T-Zell-Repertoire mit riesigen Vielfalt in Antigenspezifität, sowohl sich selbst als auch ausländische. Auswahl des Repertoires während der Entwicklung im Thymus ist kritisch für die Erzeugung und sichere nützlich T-Zellen. Defekte im Thymus Auswahl zur Entwicklung von Autoimmun-und Immunschwäche-Erkrankungen 1-4. T-Zell-Vorläufer geben den Thymus als doppelt negative (DN) Thymozyten, die nicht exprimieren CD4 oder CD8 Co-Rezeptoren. Expression des αβTCR und beide Co-Rezeptoren tritt bei der doppelt positiven (DP) Bühne. Interaktion des αβTCR mit Selbst-Peptid-MHC (pMHC) durch Thymus-Zellen präsentiert bestimmt das Schicksal der DP Thymozyten. Hochaffine Wechselwirkungen zu negativen Selektion und Eliminierung führention von selbst-reaktiven Thymozyten. Low Affinitätswechselwirkungen Ergebnis in positiver Selektion und Entwicklung von CD4 oder CD8 einzigen positiven (SP) T-Zellen in der Lage zu erkennen fremde Antigene durch Selbst-MHC 5 dargestellt. Positive Selektion kann in Mäusen mit einem polyklonalen (Wildtyp) TCR Repertoires werden durch Beobachten der Erzeugung von reifen T-Zellen untersucht. Sie sind jedoch nicht ideal für das Studium der negativen Selektion, die Deletion von antigenspezifischen kleinen Populationen umfasst. Viele Modellsysteme verwendet worden, um negative Selektion zu studieren, aber variieren in ihrer Fähigkeit zu rekapitulieren physiologische Ereignisse 6. Zum Beispiel in-vitro-Stimulation von Thymozyten fehlt die Thymus-Umgebung, die innig in Auswahl beteiligt ist, während Verabreichung von exogenen Antigens an nicht-spezifische Deletion von Thymozyten 7-9 führen kann. Derzeit sind die besten Werkzeuge für die Untersuchung in vivo negative Selektion Mäuse, die eine transg ausdrückenenic TCR spezifisch für endogene Selbst-Antigen. Allerdings sind viele klassischen TCR-transgene Modelle von vorzeitigem Expression des transgenen TCRα Kette am DN Stufe dadurch, was zu einem vorzeitigen negative Selektion. Unser Labor hat den HY cd4 Modell, bei dem der transgene HY TCRα bedingt am DP Stufe exprimiert wird entwickelt, wodurch negative Selektion während der DP bis SP Übergang auftreten wie dies bei Wildtyp-Mäusen 10. Hier beschreiben wir eine Durchflusszytometrie-basiertes Protokoll zum Thymus positive und negative Selektion in der HY cd4 Mausmodell zu untersuchen. Während negative Auswahl HY cd4 Mäusen ist höchst physiologischen können diese Verfahren auch auf andere TCR-transgene Modelle angewendet werden. Wir werden außerdem allgemeine Strategien zur Analyse positive Selektion in einem polyklonalen Repertoire für alle genetisch manipulierten Mäusen.

In physiologischen TCR transgene Modelle und WT Mäusen, beginnt positive Selektion an der DP hellen Bühne, bevor er in den DP langweilig Stufe nach Antigen Begegnung. DP langweilig Thymozyten und geben Sie dann eine Übergangsregelung CD4 + CD8 lo Bühne, bevor er CD4SP oder CD8SP Thymozyten (Abbildung 2B). Mature SP Thymozyten zeichnen sich durch hohe TCR-Expression und den Verlust von CD24 (2C) gekennzeichnet. Während die CD8 d...

Watch this Scientific Journal Video about Examination of Thymic Positive and Negative Selection by Flow Cytometry at JoVE.com

Examination of Thymic Positive and Negative Selection by Flow Cytometry

Wir präsentieren eine Durchflusszytometrie-basierte Methode zur T-Zell-Entwicklung zu untersuchen<em&gt; In vivo</em&gt; Mit genetisch manipulierten Mäusen auf einer Wildtyp-oder T-Zell-Rezeptor transgenen Hintergrund.

Prüfung Thymic positive und negative Selektion mittels Durchflusszytometrie

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

examination-thymic-positive-negative-selection-flow

Research

JoVE Journal

Immunology and Infection

21.6K Aufrufe.  University of Alberta. Wir präsentieren eine Durchflusszytometrie-basierte Methode zur T-Zell-Entwicklung zu untersuchen In vivo Mit genetisch manipulierten Mäusen auf einer Wildtyp-oder T-Zell-Rezeptor transgenen Hintergrund.

Serie: Examination of Thymic Positive and Negative Selection by Flow Cytometry

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Mess-Calpain Aktivität in fixierten und lebenden Zellen mittels Durchflusszytometrie

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Forschung

Immunologie und Infektion

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Mensch In Vitro Suppression als Screening-Tool für die Anerkennung von Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Detektion von fluoreszierenden Nanopartikel Wechselwirkungen mit primären Immun-Zell-Subpopulationen durch Durchflusszytometrie

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria.

In-vivo-Bewertung der Nager Plasmodium Parasitämie und Merozoite Invasion durch Durchflusszytometrie

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Phänotypische Charakterisierung von Makrophagen aus Rattenniere mittels Durchflusszytometrie

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluoreszenz-aktivierte Zellsortierung zur Reinigung von Plasmazytoide dendritischer Zellen aus dem Knochenmark der Maus

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolation und In-vitro- Kultur von murinen und menschlichen alveoläre Makrophagen

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Charakterisierung der menschliche Monocyte Teilmengen von Vollblut Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

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

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

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

Diskriminierung von sieben Immunzelle Teilmengen von zwei-Fluorochrom Flow Cytometry

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

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

Vorbereitung des ganzen Knochenmarks für die Mass Cytometry Analyse von Neutrophilen-Linienzellen

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...