Le travail présenté dans cet article a été soutenu par une Santé nationale et du Medical Research Council (NHMRC) de l&#39;Australie du Programme de subventions (CRP) et une subvention de projet NHMRC (CRP et BJCQ). Aussi BJCQ est un NHMRC Peter Doherty boursier postdoctoral.

Configuration Réactif 1) 1.1) solution stock CFSE Le colorant CFSE est acheté comme carboxyfluorescéine diacétate succinimidyl ester (CFDA, SE) (MW 557.47 g / mol), généralement dans flacons de 25 mg. Dissoudre le 25 mg de 8,96 ml de DMSO pour une solution stock de finale de 5 mM (magasin que 50-100 aliquotes à -20 ° C pendant plusieurs mois). CFDA, SE va réagir avec une solution aqueuse il est donc essentiel que cette exposition soit évitée lors du stockage. 1.2) Lymphocytes Isoler les lymphocytes de la rate et des ganglions lymphatiques de souris et de remettre en suspension dans 1 ml de milieu de culture (par exemple RPMI) avec 5% inactivé à la chaleur (HI) sérum de veau foetal (FCS), avec une concentration cellulaire de 0,5 x 6 au 10 oct x 10 7 / mL. Étiquetage CFSE 2) 2.1 Méthode de routine): <ol class="lalpha"><li> Bien resuspendre les cellules dans le volume 1 ml de milieu et la place soigneusement dans le fond d&#39;une fraîche (non mouillées) tube conique de 10 ml. </li><li> Posez le tube horizontalement (à l&#39;aide d&#39;un tube non mouillé empêchera l&#39;1 ml de suspension de cellules de se déplacer et prématurément le mélange avec le CFDA, SE solutions). </li><li> Ajouter avec précaution 110 ul de PBS pour la partie non mouillée de la matière plastique au sommet du tube de s&#39;assurer qu&#39;il ne fait pas de contact avec la solution cellulaire. </li><li> Resuspendre 1,1 uL du stock de 5 mM de CFDA, SE dans le PBS 110 pi. </li><li> Vite hermétiquement le tube et inverser et le vortex ainsi d&#39;obtenir rapidement uniformes mélange des solutions. Notez que le colorant est CFSE à une concentration finale de 5 uM, cependant, la concentration optimale peut être déterminée pour chaque lot préparé, puis vérifié tous les 6 mois pour s&#39;assurer qu&#39;il est efficace. </li><li> Incuber les cellules pendant 5 min à température ambiante. Notez que lors de la conversion du CFDA, SE CFSE (voir la discussion) le colorant devient fluorescent et donc sujettes à la décoloration par l&#39;exposition à une lumière excessive. Ainsi, il est conseillé de protéger le tube de la lumière à partir de ce moment, par exemple en le couvrant avec du papier aluminium. </li><li> Laver les cellules en diluant dans 10 volumes de 20 ° C PBS contenant 5% de FCS HI, sédimentation par centrifugation à 300 g pendant 5 min à 20 ° C et jeter le surnageant. Répétez laver deux fois plus. </li></ol> 2.2 Méthode alternative), qui est également approprié pour le nombre de cellules plus élevée (10 - 30 x 10 7): <ol class="lalpha"><li> Préparer un 2 x (10 uM) CFDA, la solution de SE en ajoutant 2 ul du stock 5 mm à 1 mL de PBS et rapidement ajouter à 1 ml de cellules soigneusement remis en suspension, puis rapidement hermétiquement le tube et inverser et le vortex ainsi d&#39;obtenir uniformes rapidement le mélange. </li><li> Incuber les cellules pendant 5 min à température ambiante et laver comme dans les étapes 2.1 (f) et 2.1 (g). </li></ol> 2.3) Les cellules peuvent ensuite être appliqués à un protocole d&#39;intérêt pour l&#39;induction de la division cellulaire. Les cellules marquées peuvent être utilisées dans les deux in vitro et in vivo. 2.4) À la fin de l&#39;essai de prolifération, les cellules sont récoltées et analysées par cytométrie en flux (voir ci-dessous pour des exemples représentatifs). Résultats Représentant 3) Afin d&#39;évaluer la prolifération des lymphocytes par dilution CFSE, il est utile de connaître la fluorescence des cellules indivise (soit un maximum de fluorescence) et non-étiquetées cellules (fluorescence soit un minimum). Par conséquent, votre design expérimental devrait prendre en compte ces contrôles. Voici des exemples d&#39;études in vitro et in vivo utilisant CFSE pour mesurer CD8 + T division cellulaire par cytométrie en flux. 3.1) La stimulation in vitro La figure 1 montre les profils de CFSE CFSE marqué l&#39;ovalbumine (OVA) T spécifiques des récepteurs des cellules transgéniques CD8 + T OT-je cellules après 3 jours de culture avec des cellules dendritiques pulsées avec différentes quantités d&#39;OVA. Lymphocytes T CD8 + purifiées à partir des ganglions lymphatiques de la rate et le OT-je souris ont été marquées avec CFSE et 1 x 10 5 cellules en culture avec 3,3 x 10 4 DC. DC, où pulsées à l&#39;OVA pendant 1 heure à 37 ° C et lavées deux fois avant de la culture. Après 3 jours, les cellules ont été récoltés et étiquetés avec des anti-CD8-APC anticorps et Hoechst 33258, puis analysées par cytométrie en flux (50 000 événements collectés). Live (Hoechst 33258 -) CD8 + sont présentés. Comme contrôles, les cellules CD8 + T cultivés seuls, montre l&#39;intensité de la non-CFSE divisée cellules, tandis que les cellules non marquées montre l&#39;auto-fluorescence des cellules et les limites de divisions cellulaires détectables. Notez que quatre sommets CFSE peut être vu dans chacun des panneaux, dans cet exemple, ce qui indique que les cellules ont subi jusqu&#39;à 3 divisions. 3.2) stimulation in vivo La figure 2 montre les profils de CFSE CFSE marqués spécifiques de l&#39;OVA récepteur des cellules T CD8 + transgéniques OT-I des cellules T après 3 jours in vivo in présence de 20 mg d&#39;OVA. Lymphocytes T CD8 + purifiées à partir des ganglions lymphatiques de la rate et CD45.1 congéniques OT-je souris ont été marquées avec CFSE et 5 x 10 6 cellules injectées iv dans la veine latérale de la queue du C56BL/6J (CD45.2 +) les souris. Après les souris ont été injectés 2 h iv avec 20 ug d&#39;OVA. Après 3 jours, les rates des souris ont été recueillies, faite dans une suspension cellulaire unique, étiqueté avec des anti-B220-PerCP, anti-CD8-APC-Cy7 et anti-CD45.1-PE-Cy7 anticorps et Hoechst 33258 et ensuite analysées par cytométrie en flux (1.000.000 événements collectés). Live (Hoechst 33258 -) B220-cellules sont affichés dans le panneau de gauche et fermée CD8 + CD45.1 + cellules montré dans le panneau de droite. Comme contrôles, non stimulées CFSE marqué cellules T CD8 +, montre l&#39;intensité de la non-CFSE divisée cellules, tandis que les non-étiquetées cellules montre l&#39;auto-fluorescence des cellules et les limites de divisions cellulaires détectables. Notez que l&#39;utilisation de la différence CD45 allotypiques est essentiel pour résoudre les CD8 + transférées cellules T de l&#39;hôte, car ils sont un sous-ensemble mineur de la totalité des cellules CD8 T + (panneau de gauche). Notez que la plupart des cellules tombent dans les 7 sommets CFSE dans cet exemple (à droite), indiquant que les cellules ont subi jusqu&#39;à 6 divisions. <img src="/files/ftp_upload/2259/2259fig1.jpg" alt="Figure 1" /> Figure 1. Possibilité de CFSE marqué l&#39;ovalbumine (OVA) T spécifiques des récepteurs des cellules transgéniques CD8 + OT-I des cellules T à réagir in vitro à la DC pulsée avec différentes concentrations de l&#39;OVA, les données présentées sont après 3 jours de culture. Remarque non divisée population de T CD8 + cellules en l&#39;absence d&#39;antigène (lumière histogramme gris) et l&#39;auto-fluorescence de la population non-étiquetées (histogramme gris foncé). La figure illustre cellules T CD8 + qui ont divisé 1-3 fois basée sur des pics de dilution CFSE, avec plus de cellules T à la division concentrations d&#39;antigène plus élevée. <img src="/files/ftp_upload/2259/2259fig2.jpg" alt="Figure 2" /> Figure 2. Possibilité de CFSE marqué l&#39;ovalbumine (OVA) T spécifiques des récepteurs des cellules transgéniques CD8 + OT-I des cellules T, quand adoptive transférées dans des souris C57BL / 6 pour répondre à l&#39;OVA, les données présentées 3 jours après le transfert adoptif de gauche:. CD8 +, CD45. 1 + cellules T OT-1 dans les souris receveuses. Panneau de droite: profil de prolifération CFSE. Remarque non divisée population de cellules T CD8 + dans les souris receveuses ne recevant pas d&#39;antigène (lumière histogramme gris) et l&#39;auto-fluorescence des lymphocytes non-étiquetées (histogramme gris foncé). La figure illustre les cellules CD8 + T qui ont divisé 1-6 fois basée sur des pics de dilution CFSE. <img src="/files/ftp_upload/2259/2259fig3.jpg" alt="Figure 3" /> Figure 3. Une représentation des différents événements moléculaires qui se produisent pendant le marquage des cellules avec des carboxyfluorescéine diacétate succinimidyl ester (CFDA, SE). Pour plus de détails sur le processus de voir le texte de discussion. Note: «CFSE« Le sigle générique, et non CFDA, SE, est utilisé pour décrire le réactif de marquage et de la procédure de labellisation en général. Chiffre basé sur Paroisse 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>

Afin de comprendre comment fonctionne et pourquoi CFSE étiquetage uniforme rapide est nécessaire pour son utilisation dans l&#39;analyse de la prolifération, il est utile de comprendre les bases moléculaires de marquage des cellules CFSE. Ceci peut être divisé en deux phases, 1) entrée dans la cellule et l&#39;étiquetage des protéines 2) (figure 3). 1) entrée dans la cellule: entrée du colorant dans la cellule est médiée par la forme diacétylés de la teinture, le diacétate de carboxyfluorescéine succinimidyl ester (CFDA, SE). Les acétates rendre la membrane perméants colorant fortement en permettant à ses flux rapide à travers la membrane plasmique. Estérases, présentes dans la cellule, cliver les acétates du CFDA, SE et ainsi donner naissance à la forme CFSE du colorant qui est beaucoup moins pénétrants membrane, concentrant ainsi le colorant dans la cellule. Marquage des protéines 2): Alors que les deux CFDA, SE et CFSE ont aminés des chaînes latérales réactives succinimidyle, seul le CFSE est fluorescent. La forte concentration intracellulaire de CFSE facilite l&#39;étiquetage rapide et de haute niveau intracellulaire des protéines. L&#39;étiquetage des cellules CFSE doit être effectuée rapidement afin d&#39;obtenir une population homogène de cellules étiquetées, ce qui est essentiel pour résoudre les cellules qui ont subi plusieurs divisions, comme indiqué dans les résultats représentatifs (figure 1 et 2).

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>CFDA,SE</td>
<td>&nbsp;</td>
<td>Molecular Probes</td>
<td>&nbsp;</td>
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<td>DMSO</td>
<td>&nbsp;</td>
<td>Cambridge Isotope Laboratories Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Lymphocytes</td>
<td>&nbsp;</td>
<td>eg mouse or human</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>RPMI</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<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>&nbsp;</td>
<td>Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Centrifuge</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Flow cytometer</td>
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the use of carboxyfluorescein diacetate succinimidyl ester (cfse) to monitor lymphocyte proliferation

Carboxyfluorescéine succinimidyl ester (CFSE) est un moyen efficace et populaire pour surveiller la division des lymphocytes 1-3. CFSE étiquettes de façon covalente à long terme avec les molécules intracellulaires colorant fluorescent, carboxyfluorescéine. Ainsi, quand une cellule se divise CFSE-étiquetés, sa descendance sont doués de moitié le nombre de carboxyfluorescéine molécules étiquetées et donc à chaque division cellulaire peut être évaluée en mesurant la diminution correspondante de la fluorescence des cellules par cytométrie en flux. La capacité de CFSE pour étiqueter des populations de lymphocytes avec une haute intensité de fluorescence de la variance exceptionnellement faible, couplé à sa toxicité cellulaire faible, en font un colorant idéal pour mesurer la division cellulaire. Comme il est un colorant à la fluorescéine base, il est également compatible avec une large gamme d&#39;autres fluorochromes rendant applicable à la cytométrie de flux multi-couleurs. Cet article décrit les procédures généralement utilisées pour le marquage des lymphocytes de souris dans le but de surveiller jusqu&#39;à 8 divisions cellulaires. Ces cellules marquées peuvent être utilisées à la fois in vitro et in vivo.

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 étiquettes de façon covalente à long terme avec les molécules intracellulaires colorant fluorescent, carboxyfluorescéine. En tant que tel, quand une cellule se divise CFSE-étiquetés, sa descendance ont la moitié de la quantité de fluorescence, qui peut ainsi être utilisée pour évaluer la division cellulaire. Cet article décrit les procédures généralement utilisées pour le marquage des lymphocytes de souris avec CFSE.

L&#39;utilisation de carboxyfluorescéine diacétate succinimidyl ester (CFSE) pour surveiller la prolifération des lymphocytes

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 vues.  John Curtin School of Medical Research, Australian National University. CFSE étiquettes de façon covalente à long terme avec les molécules intracellulaires colorant fluorescent, carboxyfluorescéine. En tant que tel, quand une cellule se divise CFSE-étiquetés, sa descendance ont la moitié de la quantité de fluorescence, qui peut ainsi être utilisée pour évaluer la division cellulaire. Cet article décrit les procédures généralement utilisées pour le marquage des lymphocytes de souris avec CFSE.

Vidéo: 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 Méthode d&#39;échantillonnage de sang pour la détermination des induite par l&#39;exercice apoptose des lymphocytes

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

Recherche

Immunologie et infection

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.

Un test visuel pour surveiller T6SS à médiation bactérienne concurrence

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.

Utilisation d&#39;<em&gt; In vivo</em&gt; Imagerie à suivre la progression de cytomégalovirus souris une infection expérimentale chez les prématurés

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.

Utilisation de protéines fluorescentes pour surveiller glycosome Dynamics dans le trypanosome africain

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.

Immunofluorescence de suivi de l&#39;absorption cellulaire de la lactoferrine humaine et de son activité associée antivirale contre le virus de l&#39;hépatite C

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

L&#39;utilisation de la cytométrie de flux pour évaluer l&#39;état de la chromatine dans les cellules T

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.

Dosage de Adhérence Sous contrainte de cisaillement pour l&#39;étude des lymphocytes T-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 ...

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.

détection de la<em&gt; Trypanosoma brucei</em&gt; Switching Variant de glycoprotéine de surface par magnétique tri cellulaire activé et cytométrie de flux

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.

Développement d&#39;un système de virus de l&#39;hépatite B Reporter pour surveiller les premiers stades du cycle de réplication

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.

Dextrane augmente l’efficacité de la Transduction des gènes des cellules NK primaires murines et humaines

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