Damos las gracias a los productos ReadiSorb por su generoso apoyo de estos estudios.

1. De cultivo celular de astrocitos y felino, el tratamiento con H 2 O 2 <ol><li> La línea de cultivo celular G355-5 en un frasco de 75 cm 2 con los medios de comunicación DMEM más nutrientes a 37 ° C y 5% de CO 2 hasta el 60% de confluencia (~ 5 días). Vuelva a colocar los medios de comunicación cada 1-2 días o según sea necesario para mantener una cultura saludable. </li><li> Retire el medio y agregar 2 ml de tripsina 0,25%. Levante las células mediante pipeteo durante 45 s - 1 min a temperatura ambiente (TA). </li><li> Rápidamente la transferencia de la suspensión a un tubo cónico de 15 ml que contienen 10 ml de los medios de comunicación. Mezclar suavemente. </li><li> Centrifugar a 300 xg, 23 ° C durante 3 minutos para obtener un pellet. Eliminar el sobrenadante sin perturbar el precipitado. </li><li> Agregar 6-7 ml de medio fresco y volver a suspender las células. </li><li> Añadir aproximadamente 1 ml de células en cada pocillo de una placa de 6 pocillos. Agregar 3.2 ml de los medios de comunicación a cada pocillo e incubar hasta el 80-90% confluente. </li><li> Prepare una solución de peróxido de 100μM (H 2 O 2) en los medios de comunicación. </li><li> Retire el medio de cada pocillo y reemplazar con 2 ml de medio fresco (control), 2 ml de H 2 O 2 (tratamiento) o 2 ml de DPBS. Se incuba a 37 ° C CO, 5% 2 de 3 h. </li><li> Quitar las soluciones y agregar 1 ml de tripsina para levantar las células. La transferencia de células a un tubo de 1,7 ml Eppendorf que contiene 0,5 ml de DPBS. Este paso se debe realizar en cada pocillo de forma individual con el fin de prevenir la muerte celular de incubación prolongado tripsina. </li><li> Giran a 300 xg durante 3 minutos, desechar el. Sobrenadante y resuspender en 1 ml de DPBS </li></ol> 2. La tinción de ROS <ol><li> Todos los pasos para la tinción debería ser realizada con una exposición mínima de luz para evitar decoloración. </li><li> Añadir 1 l de CCCP 50 mm para el control positivo. Se incuba a 37 ° C, 5% de CO 2 durante 5 min. </li><li> Añadir 5 l de una 10 mM CM-H 2 DCFDA solución a la H 2 O 2 grupos. Se incuba a 37 ° C, 5% de CO 2 durante 15-30 min. No mancha, se añade a la muestra de suero salino. </li><li> Lavar las muestras con 2 ml de DPBS para eliminar la mancha residual. </li><li> Giran a 300 xg durante 3 minutos para obtener un pellet. </li><li> Resuspender las células en 0,5 ml de DPBS. Añadir un PI l de cada muestra, con excepción de la solución salina. </li><li> Transferencia de muestras a un tubo de citómetro de flujo de 5 ml. </li><li> Ejecutar en el citómetro de flujo. </li></ol> 3. Citometría de flujo <ol><li> Corra las muestras en un FC500 Beckman Coulter (o equivalente) equipado con un láser de 488 nm de argón y los pasos siguientes bandas: 525, 775 y 620 nm, todos ± 20 nm. </li><li> Prepare los controles adecuados: las células no teñidas, CM-H 2 DCFDA manchada sólo, único IP, doble manchadas (control negativo). </li><li> Realizar un protocolo que tiene la histoplots siguiente: FS vs SS, PI vs CM-H DCFDA 2. También debe contar con los histogramas siguientes: el número de células frente a FL1, FL2 y FL3, que se corresponde con las manchas. </li><li> Antes de ejecutar los ejemplos, revise el equipo, los líquidos y contenedores de residuos. Caliente el equipo por lo menos 20 min. </li><li> Limpiar el equipo y realizar una calibración con las cuentas adecuadas de acuerdo a los estándares de los fabricantes. </li><li> Seleccione el protocolo y ejecutar los ejemplos. </li><li> Limpiar el equipo como se hizo anteriormente. </li><li> Limpie la línea de vacío de acuerdo con las normas de los fabricantes. </li><li> Apague la corriente y limpia la línea de la cabeza / de vacío. </li><li> Desactivar el software y el ordenador. </li></ol> 4. Los resultados representativos: Resultados de citometría de flujo se analizaron mediante FlowJo 7.6 y los controles de la mancha se utiliza para establecer objetivamente la compuerta. Sobre esta base, las células sanas a aparecer en la parte izquierda del gráfico y ROS (estrés oxidativo) se ha detectado como un cambio de celdas a la derecha. La figura 1 muestra los resultados de los efectos del peróxido de hidrógeno en astrocitos sanos felino. Los datos de FL1 se utilizó para medir la intensidad de la CM-H 2 DCFDA, lo que indica la presencia de ROS. Como era de esperar, una mayor cantidad de ROS se detectó en la muestra tratada con H 2 O 2. Esto se muestra en el histograma como un cambio en la intensidad de fluorescencia de izquierda a derecha. Al comparar las células sanas tratadas con DMEM contra DPBS, no hubo ningún cambio significativo en la cantidad de ROS (Fig. 2). <img src="/files/ftp_upload/2841/2841fig1.jpg" alt="Figura 1"/> Figura 1. Los resultados de citometría de flujo de los niveles de estrés oxidativo en los astrocitos sanos felino y el efecto de H 2 O 2 en las células sanas. Los resultados indican que el H 2 O 2 aumenta los niveles de ROS en las células sanas en comparación con las células sin tratamiento (DMEM). <img src="/files/ftp_upload/2841/2841fig2.jpg" alt="Figura 2"/> Figura 2. Resultados de citometría de flujo de los niveles de ROS detectado en las células sanas se incubaron durante 3 horas con DPBS.

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A menudo se sugiere un mecanismo de daño neuronal inducido por el virus es el estrés oxidativo 1, 4, 7, 9, 13, 15, 16, 18-22, 27, 31. Básicamente, se propone que a través de la exposición al virus de la glía (astrocitos y microglia) liberar los radicales de oxígeno reactivo, tal como hidróxido, el anión superóxido, óxido nítrico y / o peróxido de hidrógeno, que son tóxicos para las neuronas. Del mismo modo, el estrés oxidativo también ha sido sugerido como un importante mecanismo patológico ...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> DMEM </td><td> Cellgro </td><td> 15 a 013 CV </td><td></td></tr><tr><td> MEM Vitaminas </td><td> Cellgro </td><td> 25-020-cl </td><td> 1% (v / v) </td></tr><tr><td> L-glutamina </td><td> Hyclone </td><td> 17-605E </td><td> 1% (v / v) </td></tr><tr><td> No aminoácidos esenciales </td><td> Hyclone </td><td> 25-025-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> Aminoácidos Esenciales </td><td> Cellgro </td><td> 25-030-cl </td><td> 0,2% (v / v) </td></tr><tr><td> Vacío de 500 ml sistema de filtración </td><td> VWR </td><td> 87006-076 </td><td></td></tr><tr><td> 15 ml tubos cónicos </td><td> Halcón </td><td> 352097 </td><td></td></tr><tr><td> 75 cm 2 frascos de cultivo de tejidos </td><td> Halcón </td><td> 353136 </td><td></td></tr><tr><td> 6 y placa de cultivo de tejidos </td><td> Halcón </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> Yoduro de propidio (PI) </td><td> Sigma </td><td> P4170-10mg </td><td></td></tr><tr><td> Tripsina </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> HyQ antibióticos </td><td> Hyclone </td><td> SV30079.01 </td><td> 0,1% (v / v) </td></tr><tr><td> G355-5 células </td><td> ATCC </td><td> CRL-2033 </td><td> Cerebro felino normal </td></tr></tbody></table>

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

Un mecanismo frecuentemente sugerido inducida por el virus de daño neuronal es el estrés oxidativo. Los astrocitos tienen un papel importante en el control de estrés oxidativo del Sistema Nervioso Central (SNC). Los astrocitos ayudan a mantener un medio homeostático de las neuronas, así como la protección de las neuronas a partir de especies reactivas del oxígeno (ROS). CM-H2DCFDA es un indicador penetran en las células de la presencia de ROS. CM-H 2 DCFDA entra en la célula como un compuesto no fluorescente, y se convierte en fluorescente después de esterasas celulares eliminar los grupos de acetato, y el compuesto se oxida. El número de células, medido por citometría de flujo, que se encuentran para ser fluorescente verde es una indicación de la cantidad de células que se encuentran en un estado de oxidación. CM-H 2 DCFDA es susceptible a la oxidación por un gran número de diferentes ROS. Esta falta de especificidad, respecto de los cuales ROS puede oxidar CM-H 2 DCFDA, hace que este compuesto un regente valiosa para su uso en las primeras etapas de una investigación de la patogénesis, ya que este ensayo puede ser utilizado para la detección de un ambiente oxidativo celular, independientemente de que el oxígeno radical o una combinación de ROS son los responsables de las condiciones celulares. Una vez que se ha establecido que las ROS se presentan por la oxidación de la CM-H 2 DCFDA, entonces experimentos adicionales se puede realizar para determinar qué combinación de ROS o ROS están implicados en el proceso de patogénesis particular. Los resultados de este estudio demuestran que con la adición de peróxido de hidrógeno un aumento de la CM-H 2 DCFDA fluorescencia se detectó en relación con los controles de solución salina, lo que indica que este ensayo es una prueba muy útil para la detección de un ambiente oxidativo en las células G355-5, un la línea celular de astrocitos felino.

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

Un método de cribado para detectar la oxidación celular entornos es medir la oxidación de la CM-H2DCFDA. Una vez oxidado dentro de una célula, CM-H2DCFDA cambios no fluorescentes en un compuesto fluorescente. Este cambio en la fluorescencia se mide mediante citometría de flujo e indica el número de células en un ambiente oxidativo.

Ensayo de detección de estrés oxidativo en una línea celular astrocitos felina, G355-5

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

screening-assay-for-oxidative-stress-feline-astrocyte-cell-line-g355

Research

JoVE Journal

Neuroscience

14.3K vistas.  Western University of Health Sciences. Un método de cribado para detectar la oxidación celular entornos es medir la oxidación de la CM-H2DCFDA. Una vez oxidado dentro de una célula, CM-H2DCFDA cambios no fluorescentes en un compuesto fluorescente. Este cambio en la fluorescencia se mide mediante citometría de flujo e indica el número de células en un ambiente oxidativo.

Video: 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.

El ensayo neuroblasto: un ensayo para la generación y enriquecimiento de las células progenitoras neuronales de la diferenciación de la progenie de células madre neurales Usando citometría de flujo

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 ...

Investigación

Neurociencias

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 ensayo de migración celular cuantitativa para murinos entéricas Neuronales Progenitores

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.

Cell Analyzer basados ​​en impedancia Real-Time como una herramienta para delinear vías moleculares implicadas en la neurotoxicidad y neuroprotección en una línea celular neuronal

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.

La indirecta neurona-astrocito Coculture Ensayo: Un<em&gt; In Vitro</em&gt; Puesta en marcha de la investigación detallada de las interacciones neurona-glía

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.

Desencadenar el estrés celular y la muerte Uso convencional UV láser confocal Microscopía

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.

Desarrollo de ensayos para cuantificación de contenido alto de Sod1 mutante de la proteína total formación en células vivas

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 novela In Vitro Live-proyección de imagen de ensayo de astrositos mediado fagocitosis usando sinaptosomas conjugado con indicador

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 Análisis para el estudio de la agregación de la proteína Tau y detección de drogas

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 proyección de imagen del calcio Vivo de las células de pelo de la línea Lateral en el pez cebra larvas

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.

Trasplante subretiniano de tejido retiniano derivado de células madre embrionarias humanas en un modelo felino de animales grandes

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