Nous remercions les produits ReadiSorb pour leur généreux soutien de ces études.

1. La culture cellulaire d&#39;astrocytes féline et le traitement par H 2 O 2 <ol><li> Lignée cellulaire Culture G355-5 dans un flacon de 75 cm2 avec les médias DMEM plus nutriments à 37 ° C et 5% de CO 2 jusqu&#39;à confluence de 60% ​​(~ 5 jours). Remplacer les médias tous les 1-2 jours ou au besoin pour maintenir une culture saine. </li><li> Retirez le support et ajouter 2 ml de trypsine à 0,25%. Soulevez les cellules par pipetage pour 45 s - 1 min à température ambiante (RT). </li><li> Transférez rapidement la suspension d&#39;un tube de 15 ml conique contenant 10 ml de médias. Mélanger délicatement. </li><li> Centrifuger à 300 xg, 23 ° C pendant 3 min pour obtenir un culot. Jeter le surnageant sans perturber le culot. </li><li> Ajouter ml 6-7 de milieux frais et de remettre en suspension les cellules. </li><li> Ajouter environ 1 ml de cellules dans chaque puits d&#39;une plaque de 6 puits. Ajouter 2-3 ml de milieu dans chaque puits et incuber jusqu&#39;au confluent de 80-90%. </li><li> Préparer une solution de peroxyde 100 microns (H 2 O 2) dans les médias. </li><li> Retirez le support de chaque puits et les remplacer avec soit 2 ml de milieu frais (contrôle), 2 ml de H 2 O 2 (traitement) ou 2 ml de DPBS. Incuber à 37 ° C CO, 5% 2 pour 3 h. </li><li> Retirer des solutions et ajouter 1 ml de trypsine à lever les cellules. Transfert des cellules dans un tube de 1,7 ml Eppendorf contenant 0,5 ml DPBS. Cette étape doit être effectuée sur chaque bien individuellement afin de prévenir la mort cellulaire à partir d&#39;incubation prolongée trypsine. </li><li> Centrifuger à 300 xg pendant 3 minutes, jetez la. Surnageant et remettre en suspension dans 1 ml de DPBS </li></ol> 2. Coloration pour ROS <ol><li> Toutes les étapes pour la coloration doit être effectuée avec exposition à la lumière minimale afin de prévenir le blanchiment. </li><li> Ajouter 1 ul de 50 mM à CCCP le contrôle positif. Incuber à 37 ° C, 5% de CO 2 pendant 5 min. </li><li> Ajouter 5 pi d&#39;un 10 mm CM-H 2 solution DCFDA à l&#39;H 2 O 2 du groupe. Incuber à 37 ° C, 5% de CO 2 pendant 15-30 min. Aucune tache est ajouté à l&#39;échantillon une solution saline. </li><li> Lavez échantillons avec 2 ml de DPBS pour enlever taches résiduelles. </li><li> Centrifuger à 300 xg pendant 3 minutes pour obtenir un culot. </li><li> Resuspendre les cellules dans 0,5 ml de DPBS. Ajouter une PI ul à chaque échantillon, à l&#39;exception de la saline. </li><li> Transfert des échantillons à un tube de 5ml cytomètre en flux. </li><li> Exécuter sur le cytomètre en flux. </li></ol> 3. La cytométrie en flux <ol><li> Exécuter des échantillons sur une FC500 Beckman Coulter (ou équivalent) équipé d&#39;un laser à 488 nm argon et la bande de passe suivants: 525, 775 et 620 nm, tous les 20 ± nm. </li><li> Préparer les contrôles appropriés: cellules non colorées, CM-H 2 DCFDA taché seulement, seul IP, teinté double (témoin négatif). </li><li> Faire un protocole qui a le histoplots suivantes: FS vs SS, PI vs CM-H 2 DCFDA. Il devrait également avoir les histogrammes suivants: le nombre de cellules vs FL1, FL2 et FL3, ce qui correspond à la tache. </li><li> Avant d&#39;exécuter les échantillons, vérifier l&#39;équipement, des fluides et conteneur à déchets. Warm up de l&#39;équipement pendant au moins 20 min. </li><li> Nettoyer l&#39;équipement et effectuer un calibrage avec les perles appropriée selon les normes des fabricants. </li><li> Sélectionnez votre protocole et d&#39;exécuter les échantillons. </li><li> Nettoyer le matériel comme précédemment. </li><li> Nettoyer la ligne de vide selon les normes des fabricants. </li><li> Eteignez le flux et nettoyer la ligne de tête / vide. </li><li> Eteignez le logiciel et l&#39;ordinateur. </li></ol> 4. Les résultats représentatifs: Résultats de cytométrie en flux ont été analysées à l&#39;aide FlowJo 7.6 et les contrôles ont été utilisés pour tache de définir objectivement le fenêtrage électronique. Sur cette base, les cellules saines apparaissent sur la gauche de l&#39;histogramme et les ROS (stress oxydatif) a été détecté comme un changement de cellules vers la droite. La figure 1 montre les résultats de l&#39;effet du peroxyde d&#39;hydrogène sur la santé des astrocytes félin. Les données de FL1 a été utilisé pour mesurer l&#39;intensité de CM-H 2 DCFDA, qui indique la présence de ROS. Comme prévu, un montant plus élevé de ROS a été détectée dans l&#39;échantillon traité avec H 2 O 2. Ceci est affichée sur l&#39;histogramme comme un changement dans l&#39;intensité de fluorescence de gauche à droite. Lorsque l&#39;on compare les cellules saines traitées avec du DMEM par rapport DPBS, il n&#39;y avait aucun changement significatif dans la quantité de ROS (Fig 2). <img src="/files/ftp_upload/2841/2841fig1.jpg" alt="Figure 1"/> Figure 1. Les résultats de cytométrie en flux des niveaux de stress oxydatif dans les astrocytes sains féline et l&#39;effet de H 2 O 2 sur les cellules saines. Les résultats indiquent que H 2 O 2 augmente les niveaux de ROS dans les cellules saines par rapport aux cellules sans traitement (DMEM). <img src="/files/ftp_upload/2841/2841fig2.jpg" alt="Figure 2"/> Figure 2. Résultats cytométrie de flux de niveaux de ROS détecté dans les cellules saines incubés pendant 3h avec du DPBS.

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Un mécanisme suggère souvent de virus induit des lésions neuronales est le stress oxydatif 1, 4, 7, 9, 13, 15, 16, 18-22, 27, 31. Fondamentalement, il est proposé que l&#39;exposition virale de la glie (astrocytes et microglie) radicaux communiqué réactives de l&#39;oxygène tels que l&#39;hydroxyde, l&#39;anion superoxyde, l&#39;oxyde nitrique, et / ou le peroxyde d&#39;hydrogène, qui sont toxiques pour les neurones. De même, le stress oxydatif a également été suggéré comme un mécanisme majeur ...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> DMEM </td><td> Cellgro </td><td> 15 à 013-CV </td><td></td></tr><tr><td> MEM Vitamines </td><td> Cellgro </td><td> 25 à 020 cl </td><td> 1% (v / v) </td></tr><tr><td> L-glutamine </td><td> Hyclone </td><td> 17-605E </td><td> 1% (v / v) </td></tr><tr><td> Acides aminés non essentiels </td><td> Hyclone </td><td> 25 à 025 cl </td><td> 1% (v / v) </td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30071.03 </td><td> 20% (v / v) </td></tr><tr><td> Acides aminés essentiels </td><td> Cellgro </td><td> 25 à 030 cl </td><td> 0,2% (v / v) </td></tr><tr><td> 500ml système de filtration sous vide </td><td> VWR </td><td> 87006-076 </td><td></td></tr><tr><td> 15 ml tubes coniques </td><td> Falcon </td><td> 352097 </td><td></td></tr><tr><td> 75 culture de tissus cm 2 flacons </td><td> Falcon </td><td> 353136 </td><td></td></tr><tr><td> 6 plaque ainsi la culture de tissus </td><td> Falcon </td><td> 353224 </td><td></td></tr><tr><td> CM-H 2 DCFDA </td><td> Invitrogen </td><td> C6827 </td><td></td></tr><tr><td> Iodure de propidium (PI) </td><td> Sigma </td><td> P4170-10mg </td><td></td></tr><tr><td> Trypsine </td><td> Lonza </td><td> 17-160F </td><td></td></tr><tr><td> H 2 O 2 </td><td> Sigma </td><td> H6520 </td><td></td></tr><tr><td> Antibiotique HyQ </td><td> Hyclone </td><td> SV30079.01 </td><td> 0,1% (v / v) </td></tr><tr><td> G355-5 cellules </td><td> ATCC </td><td> LCR-2033 </td><td> Normal du cerveau féline </td></tr></tbody></table>

screening assay for oxidative stress in a feline astrocyte cell line, g355-5

Un mécanisme souvent suggéré de virus induit des lésions neuronales est le stress oxydatif. Les astrocytes ont un rôle important dans le contrôle du stress oxydatif du Système Nerveux Central (SNC). Les astrocytes aider à maintenir un environnement homéostatique des neurones ainsi que des neurones à partir de protéger les espèces réactives de l&#39;oxygène (ERO). CM-H2DCFDA est un indicateur de cellules perméables à la présence des ROS. CM-H 2 DCFDA pénètre dans la cellule comme un composé non fluorescent, et devient fluorescent après estérases cellulaires éliminer les groupes acétate, et le composé est oxydé. Le nombre de cellules, mesurée par cytométrie en flux, qui sont jugés fluorescent vert est une indication du nombre de cellules qui sont dans un état d&#39;oxydation. CM-H 2 DCFDA est sensible à l&#39;oxydation par un grand nombre de différents ROS. Ce manque de spécificité, au sujet desquels les ROS peuvent s&#39;oxyder CM-H 2 DCFDA, rend ce composé un régent précieux pour l&#39;utilisation dans les premiers stades d&#39;une enquête pathogenèse, comme ce test peut être utilisé pour dépister un environnement oxydatif cellulaire indépendamment de laquelle l&#39;oxygène radicale ou une combinaison de ROS sont responsables des conditions cellulaires. Une fois qu&#39;il a été établi que les ROS sont présents par oxydation du CM-H 2 DCFDA alors des expériences supplémentaires peuvent être effectuées pour déterminer les ROS ou combinaison de Ross sont impliqués dans le processus de pathogénie particulière. Les résultats de cette étude démontrent que grâce à l&#39;ajout de peroxyde d&#39;hydrogène une augmentation de la CM-H 2 DCFDA fluorescence n&#39;a été détectée par rapport aux contrôles saline, ce qui indique que ce test est un test utile pour détecter un environnement oxydatif dans les cellules G355-5, un féline lignée d&#39;astrocytes.

Watch this Scientific Journal Video about Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5 at JoVE.com

Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5

Une méthode de dépistage pour détecter des environnements cellulaires oxydatif est de mesurer l&#39;oxydation de la CM-H2DCFDA. Une fois oxydé dans une cellule, le CM-H2DCFDA changements de la non-fluorescente en un composé fluorescent. Ce changement de fluorescence est mesurée par cytométrie en flux et indique le nombre de cellules dans un milieu oxydant.

Test de dépistage du stress oxydatif dans une lignée d&#39;astrocytes féline, G355-5

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of ...

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Research

JoVE Journal

Neuroscience

14.3K vues.  Western University of Health Sciences. Une méthode de dépistage pour détecter des environnements cellulaires oxydatif est de mesurer l'oxydation de la CM-H2DCFDA. Une fois oxydé dans une cellule, le CM-H2DCFDA changements de la non-fluorescente en un composé fluorescent. Ce changement de fluorescence est mesurée par cytométrie en flux et indique le nombre de cellules dans un milieu oxydant.

Vidéo: Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Le test neuroblaste: Un test pour la génération et l&#39;enrichissement des cellules souches neuronales à partir de différenciation des cellules souches neurales Progeny Par cytométrie en flux

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Recherche

Neurosciences

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

Un test migration cellulaire quantitative pour murins de neurones entériques progéniteurs

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Analyseur portable à base d&#39;impédance en temps réel comme un outil pour délimiter voies moléculaires impliquées dans la neurotoxicité et la neuroprotection dans une lignée cellulaire neuronale

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

Le Neuron-astrocyte indirecte coculture Assay: Un<em&gt; In vitro</em&gt; Mise en place pour l&#39;investigation détaillée des interactions Neuron-glie

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

Déclenchement Stress cellulaire et la mort Utilisation conventionnelle UV Laser Microscopie confocale

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Développement de dosage pour une Quantification contenue élevée de Formation globale de protéine mutante Sod1 dans les cellules vivantes

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major immune cells and only phagocytes in the brain, recent studies have shown that astrocytes also participate in various phagocytic processes, such as developmental synapse elimination and clearance of amyloid beta plaques in Alzheimer&#39;s disease (AD). Despite these findings, the efficiency of astrocyte engulfment and degradation of their targets is unclear compared with that of microglia. This lack of information is mostly due to the lack of an assay system in which the kinetics of astrocyte- and microglia-mediated phagocytosis are easily comparable. To achieve this goal, we have developed a long-term live-imaging in vitro phagocytosis assay to evaluate the phagocytic capacity of purified astrocytes and microglia. In this assay, real-time detection of engulfment and degradation is possible using pH indicator-conjugated synaptosomes, which emit bright red fluorescence in acidic organelles, such as lysosomes. Our novel assay provides simple and effective detection of phagocytosis through live-imaging. In addition, this in vitro phagocytosis assay can be used as a screening platform to identify chemicals and compounds that can enhance or inhibit the phagocytic capacity of astrocytes. As synaptic pruning malfunction and pathogenic protein accumulation have been shown to cause mental disorders or neurodegenerative diseases, chemicals and compounds that modulate the phagocytic capacity of glial cells should be helpful in treating various neurological disorders.

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

This protocol presents an in vitro live-imaging phagocytosis assay to measure the phagocytic capacity of astrocytes. Purified rat astrocytes and microglia are used along with pH indicator-conjugated synaptosomes. This method can detect real-time engulfment and degradation kinetics and provides a suitable screening platform to identify factors modulating astrocyte phagocytosis.

Un pH de Novel In Vitro Live-imagerie phagocytose médiée par dosage de l’Astrocyte utilisant Synaptosomes indicateur conjugué

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other proteins associated with neurodegenerative diseases, the reported in vitro aggregation kinetics for tau protein are less consistent presenting a relatively high variability. Here we describe the development of an in vitro aggregation assay that mimics the expected steps associated with tau misfolding and aggregation in vivo. The assay uses the longest tau isoform (huTau441) which contains both N-terminal acidic inserts as well as four microtubule binding domains (MBD). The in vitro aggregation is triggered by addition of heparin and followed continuously by thioflavin T fluorescence in a 96 well microplate format. The tau aggregation assay is highly reproducible between different wells, experimental runs and batches of the protein. The aggregation leads to tau PHF-like morphology which is very efficient in seeding the formation of de novo fibrillar structures. In addition to its application in studying the mechanism of tau misfolding and aggregation, the current assay is a robust tool for screening drugs that could interfere with the pathogenesis of tau.

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

The tau aggregation assay described in this manuscript mimics the anticipated features of in vivo tau misfolding and aggregation.

In Vitro Dosage pour l’étude de l’agrégation de la protéine Tau et de dépistage des drogues

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

En imagerie calcique Vivo des cellules ciliées ligne latérale chez le poisson zèbre larvaire

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

Transplantation sous-rétinienne de tissu rétinien dérivé de cellules souches embryonnaires humaines dans un modèle félin de grand animal

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...