K. Wong, Wong STC sont soutenus par le financement de l&#39;imagerie fonctionnelle et moléculaire Centre, Hôpital Brigham and Women de K. Wong, Wong STC sont soutenus par le Centre de biotechnologie et d&#39;informatique, le Methodist Hospital Research Institute, Weill Cornell Medical College. KK Kwong, I. Chen, JQ Ren sont pris en charge par le financement du Centre Athinoula A. Martinos d&#39;Imagerie Biomédicale, Massachusetts General Hospital. Lenuta Kloetzer, Braden Kuo sont soutenus par un financement du Centre de recherche neurentérique, Massachusetts General Hospital Les auteurs remercient les Drs. Q. Zeng et X. Xu imagerie optique Lab, Hôpital Brigham and Women pour l&#39;aide technique.

<ol><li> Femme HO-1-luciférase souris transgéniques (âge de 4-6 semaines) peuvent être achetés auprès de Taconic Farms, Inc À leur arrivée, les souris sont placées dans un établissement de logement des animaux pendant au moins 4 jours pour permettre l&#39;hébergement. </li><li> Préparation pour l&#39;imagerie par bioluminescence: <ol style="list-style-type:lower-alpha;"><li> Peser chaque souris HO-1. Poids de l&#39;animal est nécessaire pour calculer le dosage de la luciférine. </li><li> Épilation: Utiliser un coton pour appliquer les cheveux remover Nair ® sur l&#39;abdomen et / ou au dos de l&#39;animal. Attendre 5 à 10 secondes. Utilisez un coton propre pour essuyer les cheveux. Tremper un morceau de tissu dans de l&#39;eau distillée pour nettoyer les cheveux restants. </li><li> Placez un papier non fluorescent noir (Strathmore Artagain ® papier noir) sur la plate-forme d&#39;imagerie IVIS d&#39;un poste de 100 pour réduire le bruit de fond. </li><li> Préparer luciférine (de Xenogen Corporation) solution à la concentration de 7,5 mg / ml (dissous dans H 2 O stérile). La dose de luciférine est de 65,5 mg / kg. Ainsi, une souris de 20 grammes nécessite une injection intrapéritonéale de 0,175 ml luciférine solution. Pour y parvenir, un volume simple injection de luciférine solution (0.175ml) est préchargé d&#39;une seringue avec une aiguille de calibre 26 </li></ol></li><li> Gua Sha procédure: Gua Sha est appliqué une seule fois avant l&#39;exécution du protocole d&#39;imagerie par bioluminescence. Depuis Gua Sha n&#39;est pas douloureuse, la souris a seulement besoin d&#39;être brièvement anesthésiés par l&#39;isoflurane à rester calme. Gua Sha procédure comprend les étapes suivantes: <ol style="list-style-type:lower-alpha;"><li> Appliquer l&#39;huile de cuisson ou de l&#39;eau distillée pour lubrifier les zones ciblées par la peau Gua Sha à quelques reprises durant la procédure de Gua Sha. </li><li> Répétitive grattez la région sans poils du dos de la souris en vigueur douce mais ferme à l&#39;aide d&#39;une cuillère à soupe en céramique ou une cuillère en plastique. </li><li> Le raclage se poursuit jusqu&#39;à ce que la peau du dos devient rouge, qui est un signe d&#39;extravasation du sang sous-cutané, habituellement obtenue dans les 2 à 3 minutes. </li></ol></li><li> L&#39;imagerie par bioluminescence peut être lancé immédiatement ou plusieurs heures après que Gua Sha HO-1 régulation à la hausse se construit lentement au fil de quelques heures. La procédure pour l&#39;imagerie par bioluminescence est la suivante: <ol style="list-style-type:lower-alpha;"><li> Anesthésier la souris dans une chambre de l&#39;induction anesthésique rempli d&#39;un mélange d&#39;air de grade isoflurane (1,5%) et médicales. </li><li> Une fois que la souris est anesthésiée, déplacez la souris à la chambre d&#39;imagerie IVIS sur la station de 100 imagerie optique. Placez la souris en position couchée (abdomen vers le haut). La chambre de l&#39;imagerie est continuellement infusé avec 1,5% d&#39;isoflurane. La plate-forme d&#39;imagerie est chauffé à 37 ° C pour garder la souris au chaud. </li><li> Régler l&#39;acquisition d&#39;imagerie à «binning moyen» et le temps d&#39;exposition mis à 30 secondes. Commencez à acquérir des images. Régler la machine à répéter l&#39;acquisition d&#39;imagerie toutes les trois minutes, soit manuellement ou automatiquement. Après l&#39;acquisition de la première image, une région d&#39;intérêt (ROI) est établi pour couvrir les abdominaux, la poitrine, et de la tête. Cette ROI est ensuite copié et collé à l&#39;aide d&#39;images suivantes &quot;image vivante ®&quot;, le logiciel qui accompagne le IVIS 100 stations. </li><li> L&#39;intensité du signal est mesurée en photons par seconde. Temps de Mark pour l&#39;intensité du signal pour atteindre sa valeur maximale et de garder à l&#39;imagerie de la souris pendant environ 5 à 10 minutes après l&#39;heure de pointe. </li><li> Lorsque l&#39;acquisition d&#39;imagerie à la position couchée est fait, retournez la souris et déposez-le dans la position couchée (sauvegarder). Continuer d&#39;imagerie de l&#39;animal pendant 5 à 10 minutes avec le ROI désormais établi pour couvrir le dos et la zone de tête dans l&#39;utilisation de &quot;l&#39;image vivante ®&quot; du logiciel. Le choix de l&#39;imagerie de la position couchée après la position couchée est arbitraire. On peut choisir de commencer par la position couchée, selon les principaux organes de l&#39;intérêt. Une autre option est à l&#39;image de la position couchée et sujettes à des expériences distinctes. </li><li> Lorsque l&#39;acquisition d&#39;imagerie à la position couchée est terminée, éteignez l&#39;isoflurane et déplacer la souris de la chambre de l&#39;imagerie de la chambre de l&#39;induction de récupérer. La chambre de l&#39;induction est désormais imprégné de l&#39;air médical uniquement et la souris se réveille souvent en moins d&#39;une minute. </li><li> Sauvegarder les données d&#39;imagerie pour le post-traitement. </li><li> Après l&#39;étude d&#39;imagerie initiale, des acquisitions d&#39;imagerie complémentaires sont effectués pendant plusieurs jours après Gua Sha. Un exemple d&#39;horaire est répété imagerie à l&#39;heure 12 ème, 24 ème heure, 36 ème heure, 48 ème heure, 72 heures ème et 120 ème heure, mais le moment exact de l&#39;horaire d&#39;imagerie est flexible. Séances d&#39;imagerie supplémentaires peuvent être ajoutés si un suivi plus long est nécessaire. Avant d&#39;imagerie, répétez la procédure d&#39;épiler si les cheveux ont repoussé. </li></ol></li><li> Pour le groupe de contrôle, répéter toute la procédure mais sauter Gua Sha (étape 3). Si le même groupe de souris est utilisé à la fois pour le contrôle et Gua Sha, l&#39;exp de contrôleeriment devrait être effectuée de préférence avant la procédure de Gua Sha ou au moins un mois après. </li></ol> Les résultats représentatifs: Les images de bioluminescence dans la figure 1 montre de régulation à la hausse in vivo de HO-1 en réponse à Gua Sha. Le graphique de la figure 2 montre l&#39;évolution temporelle quantitative de plus de 120 heures dans le flux optique (photons / sec) de tout le corps de la même souris liées à Gua Sha ... <img src="/files/ftp_upload/1385/figure1tmb.jpg" alt="Figure 1" border="0" /> Figure 1. De gauche à droite, des images représentatives de la vue de face (décubitus dorsal) de la même souris, avant Gua Sha, à 18 heures, 36 heures et 120 heures après Gua Sha, respectivement. Après Gua Sha, on observe les progrès de l&#39;augmentation de l&#39;intensité du signal significatif dans plusieurs organes qui englobent les régions du tractus gastro-intestinal, le tractus génital, le foie, les reins (de la vue arrière, non représenté), et d&#39;autres. S&#39;il vous plaît <a href="https://www.jove.com/files/ftp_upload/1385/figure1.jpg">cliquez ici</a> pour une version agrandie de la figure 1. <img src="/files/ftp_upload/1385/figure2tmb.jpg" alt="Figure 2" border="0" /> Figure 2. Changement quantitatif des flux (photons / sec) de l&#39;ensemble du corps suivi de plus de 120 heures suivant Gua Sha dans la même souris de la figure 1. S&#39;il vous plaît <a href="https://www.jove.com/files/ftp_upload/1385/figure2.jpg">cliquez ici</a> pour voir une version agrandie de la figure 2.

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor-1+mediates+transcriptional+activation+of+the+heme+oxygenase-1+gene+in+response+to+hypoxia.">Hypoxia-inducible factor-1 mediates transcriptional activation of the heme oxygenase-1 gene in response to hypoxia.</a> J Biol Chem. 272, 5375-5381 (1997).</li><li>Maines, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heme+oxygenase+system:+a+regulator+of+second+messenger+gases.">The heme oxygenase system: a regulator of second messenger gases.</a> Annu Rev Pharmacol Toxicol. 37, 517-554 (1997).</li><li>Wunder, C., Potter, R. 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The authors have nothing to disclose.

La transcription de HO-1, une forme inductible de l&#39;hème oxygénase, est stimulée par de nombreux facteurs, y compris l&#39;hème, le peroxyde d&#39;hydrogène, irradiation UV, l&#39;hypoxie, et contraintes physiques. L&#39;imagerie du corps entier comme le rapport HO-1 du protocole bioluminescence fournit un aperçu rapide de HO-1 systémique d&#39;expression dans de multiples organes. Dans les petits animaux, l&#39;imagerie par bioluminescence permet à haute sensibilité in vivo en temps réel d&#39;évaluation...

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<td>Luciferin</td>
<td>&nbsp;</td>
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<td>IVIS 100</td>
<td>&nbsp;</td>
<td>Caliper </td>
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bioluminescence imaging of heme oxygenase-1 upregulation in the gua sha procedure

Gua Sha est une thérapie folklorique traditionnelle chinoise qui emploie la peau gratter pour provoquer extravasation sous-cutanée de sang microvasculaires et des contusions. Le protocole d&#39;imagerie optique bioluminescents de HO-1-<em&gt; Luciférase</emSouris&gt; transgéniques rapportés dans ce manuscrit fournit une rapide<em&gt; In vivo</emTest&gt; de la régulation positive de l&#39;hème oxygénase-1 (HO-1) l&#39;expression des gènes en réponse à la procédure de Gua Sha. HO-1 est connue depuis longtemps pour fournir cytoprotection contre le stress oxydatif. La régulation positive de HO-1, évaluée par la sortie de bioluminescence, est pensé pour représenter une réponse antioxydante aux produits d&#39;hémoglobine circulant publié par Gua Sha. Gua Sha a été administré par coups répétés d&#39;un bord de cuillère à lisser la peau lubrifiés sur le dos ou d&#39;autres partie du corps ciblée de la souris transgénique jusqu&#39;à ce pétéchies (hémorragies dissident) ou ecchymoses (bleus) indicatif de l&#39;extravasation du sang des capillaires sous-cutanés a été observée. Après Gua Sha, des séances d&#39;imagerie par bioluminescence ont été effectuées quotidiennement pendant plusieurs jours pour suivre la dynamique de HO-1 expression dans plusieurs organes internes.

Watch this Scientific Journal Video about Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure at JoVE.com

Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

Gua Sha, traditionnelle chinoise grattant la peau thérapeutiques, les causes extravasation sous-cutanée de sang microvasculaires. Nous rapportons un protocole d&#39;imagerie par bioluminescence de HO-1-<em&gt; Luciférase</em&gt; Souris transgéniques pour démontrer que Gua Sha régule l&#39;hème oxygénase-1 (HO-1) dans de multiples organes.

Imagerie par bioluminescence de l&#39;hème oxygénase-1 Régulation à la hausse dans la procédure de Gua Sha

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol ...

bioluminescence-imaging-heme-oxygenase-1-upregulation-gua-sha

Research

JoVE Journal

Biology

14.0K vues.  Massachusetts General Hospital, Harvard Medical School. Gua Sha, traditionnelle chinoise grattant la peau thérapeutiques, les causes extravasation sous-cutanée de sang microvasculaires. Nous rapportons un protocole d'imagerie par bioluminescence de HO-1- Luciférase Souris transgéniques pour démontrer que Gua Sha régule l'hème oxygénase-1 (HO-1) dans de multiples organes.

Vidéo: Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

A Craniotomy Surgery Procedure for Chronic Brain Imaging

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an extremely useful method, because it allows the study of the live intact brain. With appropriate preparations, this technique allows the observation of the same cortical area chronically, from minutes to months. In this video, we show a preparation for chronic in vivo imaging of the brain using two-photon microscopy. This technique was initially pioneered by Dr. Karel Svoboda, who is now a Howard Hughes Medical Institute Investigator at Janelia Farm. Preparations like the one shown here can be used for imaging of neocortical structure (e.g., dendritic and axonal dynamics),  to record neuronal activity using calcium-sensitive dyes, to image cortical blood flow dynamics, or for intrinsic optical imaging studies. Deep imaging of the neocortex is possible with optimal cranial window surgeries. Operating under the most sterile conditions possible to avoid infections, together with using extreme care to do not damage the dura mater during the surgery, will result in successful and long-lasting glass-covered cranial windows.


This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.

Une procédure de chirurgie Craniotomie pour l&#39;imagerie cérébrale chronique

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an ...

Recherche

Biologie

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

In vitro et in vivo en bioluminescence gène rapporteur imagerie des cellules souches embryonnaires humaines

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Murin procédure transplantation rénale

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Imagerie fonctionnelle de la graisse brune chez les souris avec FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

Surveillance de cellules autonomes Rythmes horloge circadienne de l&#39;expression génique par Reporters Bioluminescence luciférase

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Vivre imagerie de dédouanement cellulaire par apoptose au cours<em&gt; Drosophila</em&gt; Embryogenèse

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Étudier les interactions protéine-protéine dans les cellules vivantes en utilisant le transfert d&#39;énergie de résonance de bioluminescence

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Mesure des niveaux Heme synthèse dans des cellules de mammifères

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Haute résolution Time-lapse d&#39;imagerie et d&#39;analyse automatisée des microtubules Dynamics Living ombilicale humaine Cellules endothéliales de veine

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Imagerie du peroxyde mitochondrial dans des cellules de levure vivantes à l’aide de mtHyper7

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, cancer, and normal aging. Here, an approach is described to assess mitochondrial function in living yeast cells at cellular and subcellular resolutions using a genetically encoded, minimally invasive, ratiometric biosensor. The biosensor, mitochondria-targeted HyPer7 (mtHyPer7), detects hydrogen peroxide (H2O2) in mitochondria. It consists of a mitochondrial signal sequence fused to a circularly permuted fluorescent protein and the H2O2-responsive domain of a bacterial OxyR protein. The biosensor is generated and integrated into the yeast genome using a CRISPR-Cas9 marker-free system, for&#160;more consistent expression compared to plasmid-borne constructs.
mtHyPer7 is quantitatively targeted to mitochondria, has no detectable effect on yeast growth rate or mitochondrial morphology, and provides a quantitative readout for mitochondrial H2O2 under normal growth conditions and upon exposure to oxidative stress. This protocol explains how to optimize imaging conditions using a spinning-disk confocal microscope system and perform quantitative analysis using freely available software. These tools make it possible to collect rich spatiotemporal information on mitochondria both within cells and among cells in a population. Moreover, the workflow described here can be used to validate other biosensors.

Pleins feux sur l’auteur : Faire progresser la recherche mitochondriale - Biocapteur mtHyper7 pour l’analyse subcellulaire 

Hydrogen peroxide (H2O2) is both a source of oxidative damage and a signaling molecule. This protocol describes how to measure mitochondrial H2O2 using mitochondria-targeted HyPer7 (mtHyPer7), a genetically encoded ratiometric biosensor, in living yeast. It details how to optimize imaging conditions and perform quantitative cellular and subcellular analysis using freely available software.

Imagerie de mtHyPer7, un biocapteur ratiométrique pour le peroxyde mitochondrial, dans des cellules de levure vivantes

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...