Die Arbeit in diesem Artikel berichtet, wurde von einem National Health and Medical Research Council (NHMRC) of Australia Programm Grant (CRP) und eine NHMRC Project Grant (CRP und BJCQ) unterstützt. Auch BJCQ ist ein NHMRC Peter Doherty Postdoctoral Fellow.

1) Reagenz-Setup 1,1) CFSE-Stammlösung Die CFSE Farbstoff als Carboxyfluoresceindiacetat Succinimidylester (CFDA, SE) (MW 557,47 g / mol) gekauft, in der Regel in 25-mg-Durchstechflaschen. Lösen Sie die 25 mg in 8,96 ml DMSO für eine endgültige Stammlösung von 5 mM (speichern als 50-100 &amp;mgr; l Aliquots bei -20 ° C für mehrere Monate). CFDA, SE wird mit wässriger Lösung reagieren so dass es wichtig ist, dass eine solche Exposition während der Lagerung vermieden werden. 1,2) Lymphozyten Isolieren Lymphozyten aus Milz und Lymphknoten oder von Mäusen und resuspendieren in 1 ml Kulturmedium (z. B. RPMI) mit 5% hitzeinaktiviertem (HALLO) fötalem Kälberserum (FCS), mit einer Zellkonzentration von 0,5 x 06 bis 10 Oktober x 10 7 / ml. 2) CFSE Labeling 2,1) Routine-Methode: <ol class="lalpha"><li> Gründlich die Zellen in der 1 ml Volumen Medium und Ort sorgfältig in den Boden einer frisch (nicht medienberührt) 10 ml konischen Rohr. </li><li> Legen Sie das Rohr horizontal (mit einem nicht benetzte Rohr wird 1 ml-Zellsuspension aus bewegten und vorzeitig Vermischung mit dem CFDA, SE-Lösungen zu verhindern). </li><li> Vorsichtig 110 ul PBS, die nicht benetzten Teil der Kunststoff an der Spitze des Rohres gewährleistet es keinen Kontakt mit der Zelle Lösung. </li><li> Resuspendieren 1,1 ul der 5 mM Bestand CFDA, SE in den 110 ul PBS. </li><li> Schnell Kappe der Röhre und invertieren und Vortex auch einen schnellen gleichmäßigen Durchmischung der Lösungen zu erhalten. Beachten Sie, dass die CFSE Farbstoff in einer Endkonzentration von 5 uM ist, jedoch kann die optimale Konzentration müssen für jede Charge vorbereitet bestimmt werden, und dann alle 6 Monate geprüft, um sicherzustellen, dass es wirksam ist. </li><li> Inkubieren Zellen für 5 min bei Raumtemperatur. Beachten Sie, dass bei der Umwandlung von CFDA, SE CFSE (siehe Diskussion) der Farbstoff Fluoreszenz und somit anfällig für Bleichen von übermäßiger Licht wird. So ist es ratsam, das Rohr aus Licht von diesem Punkt zu schützen auf, z. B. durch Abdecken mit Alufolie. </li><li> Wash-Zellen durch Verdünnung in 10 Bänden von 20 ° C PBS mit 5% FCS HALLO, Sedimentieren durch Zentrifugation bei 300 xg für 5 min bei 20 ° C und der Überstand verworfen. Wiederholen Sie waschen zweimal. </li></ol> 2,2) Alternative Methode, die auch geeignet ist für höhere Zellzahlen (10 bis 30 x 10 7): <ol class="lalpha"><li> Bereiten Sie eine 2 x (10 uM) CFDA, SE-Lösung durch Zugabe von 2 ul der 5 mM Lager zu 1 ml PBS und schnell fügen Sie diese zu 1 mL gründlich resuspendierten Zellen, dann schnell Kappe der Röhre und invertieren und Vortex gut zu bekommen schnelle gleichmäßige Durchmischung. </li><li> Inkubieren Sie die Zellen für 5 min bei Raumtemperatur und waschen wie in den Schritten 2,1 (f) und 2,1 (g). </li></ol> 2,3) Die Zellen können dann in einem Protokoll von Interesse für die Induktion der Zellteilung angewendet werden. Markierten Zellen können sowohl in in vitro und in vivo-Untersuchungen verwendet werden. 2,4) Nach Beendigung der Proliferation Assay werden Zellen geerntet und mittels Durchflusszytometrie analysiert (siehe unten für repräsentative Beispiele). 3) repräsentative Ergebnisse Um zu beurteilen, Lymphozyten-Proliferation durch CFSE Verdünnung, ist es sinnvoll, die Fluoreszenz der ungeteilten Zellen (dh maximale Fluoreszenz) und nicht-markierte Zellen (dh minimale Fluoreszenz) kennen. Daher sollten Sie Ihre experimentellen Design dieser Kontrollen zu berücksichtigen. Nachfolgend sind Beispiele für in vitro und in vivo-Tests mit CFSE zu CD8 + T Zellteilung mittels Durchflusszytometrie gemessen. 3,1) In-vitro-Stimulation Abbildung 1 zeigt die CFSE Profile von CFSE-markierten Ovalbumin (OVA)-spezifischen T-Zell-Rezeptor transgenen CD8 + OT-I T-Zellen nach 3 Tagen Kultur mit dendritischen Zellen mit verschiedenen Mengen von OVA gepulst. CD8 + T-Zellen aus der Milz und Lymphknoten von OT-I-Mäusen aufgereinigt wurden mit CFSE und 1 x 10 5 Zellen kultiviert mit 3,3 x 10 4 DC beschriftet. DC, wo mit OVA für 1 Stunde bei 37 ° C gepulst und zweimal vor dem Kultur. Nach 3 Tagen, Zellen, wo und mit anti-CD8-APC-Antikörper und Hoechst 33258 geerntet markiert und dann mittels Durchflusszytometrie analysiert (50.000 Veranstaltungen gesammelt). Live (Hoechst 33258 -) CD8 +-Zellen gezeigt werden. Als Kontrollen CD8 + T-Zellen kultiviert allein, zeigt die CFSE Intensität der nicht-geteilten Zellen, während die nicht-markierten Zellen zeigt die Autofluoreszenz der Zellen und die Grenzen der nachweisbaren Zellteilungen. Beachten Sie, dass 4 CFSE Gipfel in jede der Tafeln in diesem Beispiel zu sehen, was darauf hinweist, dass die Zellen bis zu 3 Divisionen unterzogen. 3,2) In-vivo-Stimulation Abbildung 2 zeigt die CFSE Profile von CFSE-markierten OVA-spezifischen T-Zell-Rezeptor transgenen CD8 + OT-I T-Zellen nach 3 Tagen in vivo in die Anwesenheit von 20 ug OVA. CD8 + T-Zellen aus der Milz und Lymphknoten von CD45.1 kongene OT-I-Mäusen aufgereinigt wurden mit CFSE und 5 x 10 6 Zellen iv injiziert in die seitliche Schwanzvene C56BL/6J (CD45.2 +)-Mäuse bezeichnet. Nach 2 Stunden Mäusen injiziert wurden iv mit 20 ug OVA. Nach 3 Tagen wurden Milzen der Mäuse gesammelt, machte in einem einzigen Zellsuspension, beschriftet mit anti-B220-PerCP, anti-CD8-APC-Cy7 und anti-CD45.1-PE-Cy7 Antikörper und Hoechst 33258 und anschließend analysiert mittels Durchflusszytometrie (1.000.000 Ereignisse gesammelt). Live (Hoechst 33258 -) B220-Zellen sind in linken und gated CD8 + CD45.1 + Zellen im rechten Panel angezeigt gezeigt. Als Kontrollen unstimulierten CFSE-markierten CD8 + T-Zellen, zeigt die CFSE Intensität der nicht-geteilten Zellen, während die nicht-markierten Zellen zeigt die Autofluoreszenz der Zellen und die Grenzen der nachweisbaren Zellteilungen. Beachten Sie, dass die Verwendung des CD45 allotypische Unterschied entscheidend bei der Lösung der übertragenen CD8 + T-Zellen aus Host, da sie eine kleine Teilmenge der gesamten CD8 + T-Zellen (links) sind. Beachten Sie, dass die meisten Zellen innerhalb von 7 CFSE Gipfel in diesem Beispiel (rechts) fallen, was darauf hinweist, dass die Zellen bis zu 6 Divisionen unterzogen. <img src="/files/ftp_upload/2259/2259fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Die Fähigkeit von CFSE-markierten Ovalbumin (OVA)-spezifischen T-Zell-Rezeptor transgenen CD8 + OT-I T-Zellen in vitro zu DC mit verschiedenen Konzentrationen von OVA gepulsten reagieren, Daten gezeigt, dass nach 3 Tagen Kultur. Beachten Sie, nicht geteilt Bevölkerung von CD8 + T Zellen in Abwesenheit von Antigen (hellgrau Histogramm) und Auto-Fluoreszenz von nicht-markierten Bevölkerung (dunkelgrau Histogramm). Die Abbildung zeigt CD8 + T-Zellen, die 1-3 mal aufgeteilt wurden auf der Grundlage CFSE Verdünnung Gipfel mit mehr T-Zellen teilen bei den höheren Antigen-Konzentrationen. <img src="/files/ftp_upload/2259/2259fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Die Fähigkeit von CFSE-markierten Ovalbumin (OVA)-spezifischen T-Zell-Rezeptor transgenen CD8 + OT-I T-Zellen, wenn adoptiv in C57BL / 6 Mäuse übertragen, um OVA reagieren, Daten gezeigt, 3 Tage nach dem adoptiven Transfer Linke Tafel:. CD8 +, CD45. 1 + OT-1 T-Zellen in Empfängermäuse. Rechts: CFSE Verbreitung Profil. Hinweis nicht geteilt Bevölkerung von CD8 + T-Zellen in Empfängermäuse nicht erhalten Antigen (hellgrau Histogramm) und Auto-Fluoreszenz von nicht-markierten Lymphozyten (dunkelgrau Histogramm). Die Abbildung zeigt CD8 + T-Zellen, 1-6 mal aufgeteilt wurden auf der Grundlage CFSE Verdünnung Gipfel. <img src="/files/ftp_upload/2259/2259fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Eine Darstellung der verschiedenen molekularen Ereignisse, die während der Markierung von Zellen mit Carboxyfluoresceindiacetat Succinimidylester (CFDA, SE) auftreten. Für Einzelheiten des Verfahrens siehe Diskussion Text. Hinweis: Die generische Abkürzung &quot;CFSE&quot;, nicht CFDA, SE, wird verwendet, um die Markierungsreagens und die Kennzeichnung Verfahren im Allgemeinen zu beschreiben. Abbildung basierend auf Parish 4.

<ol>
	<li>Lyons, A. B., Parish, 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=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> J. Immunol. Methods. 171, 131-137 (1994).</li><li>Parish, C. R., Glidden, M. H., Quah, B. J., Warren, H. S., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+intracellular+fluorescent+dye+CFSE+to+monitor+lymphocyte+migration+and+proliferation.">Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation.</a> Current protocols in immunology. , (2009).</li><li>Quah, B. J., Warren, H. S., Parish, 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=Monitoring+lymphocyte+proliferation+in+vitro+and+in+vivo+with+the+intracellular+fluorescent+dye+carboxyfluorescein+diacetate+succinimidyl+ester.">Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester.</a> Nature Protocols. 2, 2049-2056 (2007).</li><li>Parish, 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=Fluorescent+dyes+for+lymphocyte+migration+and+proliferation+studies.">Fluorescent dyes for lymphocyte migration and proliferation studies.</a> Immunol Cell Biol. 77, 499-508 (1999).</li></ol>

Um zu schätzen, wie CFSE funktioniert und warum schnelle einheitliche Kennzeichnung ist für den Einsatz in Proliferation Analyse erforderlich ist, ist es sinnvoll, die molekularen Grundlagen der CFSE Zellmarkierung verstehen. Dies kann in zwei Phasen, 1) Eindringen in die Zelle und 2) Protein-Labelling (Abbildung 3) eingeteilt werden. 1) Handy: Eintragung des Farbstoffes in der Zelle wird durch die diacetylierte Form des Farbstoffes, Carboxyfluoresceindiacetat Succinimidylester (CFDA, SE) vermittelt. Die Acetate machen den Farbstoff sehr Membran Permeationsmittel ermöglicht die rasche Wandel in der Plasmamembran. Esterasen, Gegenwart innerhalb der Zelle, spaltet die Acetate von CFDA, SE und damit Anlass zu der CFSE Form des Farbstoffes, die viel weniger Membran Permeationsmittel ist, so konzentriert sich der Farbstoff in der Zelle. 2) Protein Etikettierung: Während beide CFDA, SE und CFSE aminoreaktiven Succinimidyl Seitenketten haben, ist nur die CFSE Fluoreszenz. Die hohe intrazelluläre Konzentration von CFSE ermöglicht eine schnelle und hohe intrazelluläre Markierung von Proteinen. CFSE Markierung von Zellen müssen schnell durchgeführt werden, um eine homogen bezeichnet Population von Zellen, die entscheidend für die Lösung Zellen, die mehrere Abteilungen durchlaufen haben, wie in der repräsentativen Ergebnisse (Abb. 1 und 2) gezeigt, zu erhalten.

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>CFDA,SE</td>
<td>&nbsp;</td>
<td>Molecular Probes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Cambridge Isotope Laboratories Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Lymphocytes</td>
<td>&nbsp;</td>
<td>eg mouse or human</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>RPMI</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>FCS</td>
<td>&nbsp;</td>
<td>SAFC Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>10-15ml conical tubes</td>
<td>&nbsp;</td>
<td>Falcon, Becton Dickinson</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>PBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Aluminium foil</td>
<td>&nbsp;</td>
<td>Capral Aluminium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Vortex</td>
<td>&nbsp;</td>
<td>Scientific Industries</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf (5810 R)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Flow cytometer</td>
<td>&nbsp;</td>
<td>LSR-II (BD Biosciences)</td>
<td>&nbsp;</td>
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the use of carboxyfluorescein diacetate succinimidyl ester (cfse) to monitor lymphocyte proliferation

Carboxyfluorescein Succinimidylester (CFSE) ist ein effektives und beliebtes Mittel, um Lymphozyten Division 1-3 überwachen. CFSE kovalent Etiketten langlebig intrazellulären Molekülen mit dem Fluoreszenzfarbstoff, Carboxyfluorescein. Wenn also ein CFSE-markierten Zelle teilt, werden seine Nachkommen mit der Hälfte der Anzahl der Carboxyfluorescein-markierten Molekülen dotiert und damit jeder Zellteilung kann durch Messung der entsprechenden Abnahme der Zell-Fluoreszenz über Durchflusszytometrie beurteilt werden. Die Kapazität der CFSE auf Lymphozyten-Populationen mit hoher Fluoreszenzintensität von außergewöhnlich niedrigen Varianz, mit seinem niedrigen Zelltoxizität gekoppelt label, machen ihn zum idealen Farbstoff Zellteilung zu messen. Da es ein Fluorescein-basierte Farbstoff ist es ist auch kompatibel mit einer breiten Palette anderer Fluorochrome so dass es für Multi-Color-Durchflusszytometrie. Dieser Artikel beschreibt die Verfahren, die üblicherweise zur Kennzeichnung Maus-Lymphozyten für die Zwecke der Überwachung von bis zu 8 Zellteilungen verwendet. Diese markierten Zellen können sowohl für in vitro und in vivo-Studien verwendet werden.

Watch this Scientific Journal Video about The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation at JoVE.com

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

CFSE kovalent Etiketten langlebig intrazellulären Molekülen mit dem Fluoreszenzfarbstoff, Carboxyfluorescein. Als solche, als eine CFSE-markierten Zelle teilt, haben seine Nachkommen, die Hälfte der Menge der Fluoreszenz, die damit verwendet werden, um die Zellteilung zu beurteilen. Dieser Artikel beschreibt die Verfahren, die üblicherweise zur Kennzeichnung Maus-Lymphozyten mit CFSE verwendet.

Die Verwendung von Carboxyfluoresceindiacetat Succinimidylester (CFSE) auf Lymphozyten-Proliferation-Monitor

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

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Research

JoVE Journal

Immunology and Infection

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

91.3K Aufrufe.  John Curtin School of Medical Research, Australian National University. CFSE kovalent Etiketten langlebig intrazellulären Molekülen mit dem Fluoreszenzfarbstoff, Carboxyfluorescein. Als solche, als eine CFSE-markierten Zelle teilt, haben seine Nachkommen, die Hälfte der Menge der Fluoreszenz, die damit verwendet werden, um die Zellteilung zu beurteilen. Dieser Artikel beschreibt die Verfahren, die üblicherweise zur Kennzeichnung Maus-Lymphozyten mit CFSE verwendet.

Serie: The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

Finger-Stick Blutprobenentnahme Methodik zur Bestimmung der Übung-induzierte Lymphozyten Apoptose

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

Forschung

Immunologie und Infektion

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Ein Visual Assay zur T6SS-vermittelte bakterielle Wettbewerb überwachen

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

Verwenden Sie von<em&gt; In vivo</em&gt; Imaging zur Überwachung der Progression of Experimental Maus Cytomegalovirus-Infektion bei Neugeborenen

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Mit fluoreszierenden Proteinen zu Glycosome Dynamics in der afrikanischen Trypanosomen auf Monitor

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

Immunfluoreszenz, um die zelluläre Aufnahme von menschlichem Lactoferrin und die damit verbundenen antivirale Aktivität gegen das Hepatitis C Virus-Monitor

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

Die Verwendung von Durchflusszytometrie, um den Zustand von Chromatin in T-Zellen bewerten

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Test der Haftung unter Scherspannung für das Studium der T-Lymphozyten-Adhäsions-Molekül-Wechselwirkungen

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

Nachweis von<em&gt; Trypanosoma brucei</em&gt; Variante Oberflächen-Glykoprotein Switching von Magnetic Activated Cell Sorting und Durchflusszytometrie

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Entwicklung eines Systems Hepatitis-B-Virus Reporter den frühen Stadien des Replikationszyklus zu überwachen

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

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 ...