Name: measuring near plasma membrane and global intracellular calcium dynamics in astrocytes
Uploaded: 2009-04-26T23:27:00
Description: Watch this Scientific Journal Video about Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes at JoVE.com

Ce travail a été soutenu par la Fondation Uehara Memorial du Japon (ES) ainsi que la Fondation de Whitehall, l&#39;Institut national des troubles neurologiques et des maladies et un prix de Stein-Oppenheimer de dotation (au BSK).

Procédures expérimentales La procédure expérimentale se compose de deux parties principales qui sont décrits d&#39;une façon par étapes ci-dessous. Partie 1: PRÉPARATION DES CULTURES hippocampique astrocyte Brièvement, mélangé hippocampique astrocyte-neurone cultures ont été préparés en utilisant un protocole bien établi 1,2,3. Nous avons optimisé la procédure de céder saine astrocytes mis en culture. Toutes ...

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Il est bien établi que les astrocytes affichage des élévations de calcium intracellulaire. Ils se produisent spontanément, peut être déclenchée par l&#39;activité neuronale ou par application d&#39;agonistes à activer les récepteurs à la surface des astrocytes 11. Une question importante et controversée est de savoir si astrocytes élévations de calcium intracellulaire peut déclencher la libération de molécules de signalisation qui activent les récepteurs des neurones 11, 12. Ceci est controversée, ca...

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		<th>Comment</th>
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<td>VWR&#174; Micro Cover Slips, Round, No. 1</td>
<td>Tool</td>
<td>VWR</td>
<td>48380-068</td>
<td>&nbsp;</td>
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<td>Poly-D-lysine hydrobromide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P0899</td>
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<td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td>
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<td>Sigma</td>
<td>L2020</td>
<td>&nbsp;</td>
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<td>Earle&#x2019;s Balanced Salt Solution (EBSS) (1X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
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<td>Minimum Essential Medium (MEM) (1X), liquid Contains Earle's salts, but no L-glutamine or phenol red</td>
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<td>Invitrogen</td>
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<td>Penicillin-Streptomycin liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15140-122</td>
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<td>Sodium pyruvate solution</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S8636</td>
<td>&nbsp;</td>
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<td>HEPES solution 1 M</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H0887</td>
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<td>N-2 Supplement (100X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17502-048</td>
<td>&nbsp;</td>
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<td>Horse Serum, Heat-Inactivated</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>26050-088</td>
<td>&nbsp;</td>
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<td>PAPAIN-022</td>
<td>Reagent</td>
<td>Worthington</td>
<td>LK003178</td>
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<td>Neurobasal&#x2122; Medium (1X) Liquid without Phenol Red</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>12348-017</td>
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<td>B-27 Serum-Free Supplement (50X), liquid</td>
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<td>Invitrogen</td>
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<td>L-Glutamine-200 mM (100X), liquid</td>
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<td>Invitrogen</td>
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<td>BD Falcon Multiwell Flat-Bottom Plates with Lids, Sterile</td>
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<td>BD Biosciences</td>
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<td>Sigma</td>
<td>S7653</td>
<td>&nbsp;</td>
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<td>KCl</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P3911</td>
<td>&nbsp;</td>
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<td>CaCl2 hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>21108</td>
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<td>MgCl2 hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M2670</td>
<td>&nbsp;</td>
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<td>HEPES free acid</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
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<td>D-(+)-glucose</td>
<td>Reagent</td>
<td>Sigma</td>
<td>G7528</td>
<td>&nbsp;</td>
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<td>Fluo-4, AM 1 mM solution in DMSO</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>F-14217</td>
<td>&nbsp;</td>
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<td>Pluronic&#174; F-127 20% solution in DMSO</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>P-3000MP</td>
<td>&nbsp;</td>
<tr>
<td>Immersion Oil TYPE DF</td>
<td>Microscope</td>
<td>Cargille</td>
<td>16242</td>
<td>&nbsp;</td>
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<td>Open chamber for 25 mm round coverslips, 100 &mu;l volume</td>
<td>Tool</td>
<td>WARNER Instruments</td>
<td>64-0362 (RC-21BDW)</td>
<td>&nbsp;</td>
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<td>P-2 platform for Series 20 chambers, non-heater</td>
<td>Tool</td>
<td>WARNER Instruments</td>
<td>64-0278 (P-2)</td>
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<td>FluoSpheres carboxylate-modified microspheres, 0.1 &mu;m, yellow-green fluorescent (505/515) 2% solids</td>
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measuring near plasma membrane and global intracellular calcium dynamics in astrocytes

Le cerveau contient des cellules gliales. Les astrocytes, un type de cellules gliales, ont longtemps été connues pour fournir un rôle passif de soutien pour les neurones. Cependant, plus de preuves suggèrent que les astrocytes peuvent aussi participer activement à la fonction cérébrale par des interactions fonctionnelles avec les neurones. Toutefois, de nombreux aspects fondamentaux de la biologie des astrocytes restent controversées, clairs et / ou expérimentalement inexploré. Une question importante est la dynamique des transitoires du calcium intracellulaire dans les astrocytes. Cette question est pertinente parce que le calcium est bien établi comme un messager important secondes et parce qu&#39;il a été proposé que les élévations de calcium astrocytaire peut déclencher la libération de transmetteurs d&#39;astrocytes. Cependant, il n&#39;a pas été toute description détaillée ou satisfaisante du calcium à proximité de la membrane plasmique de signalisation dans les astrocytes. Fluorescence totale de réflexion interne (FRBR) microscopie est un outil puissant pour analyser physiologiquement pertinents événements de signalisation dans environ 100 nm de la membrane plasmique des cellules vivantes. Ici, nous utilisons la microscopie TIRF et décrire la façon de surveiller à proximité de la membrane plasmique et la dynamique globale du calcium intracellulaire presque simultanément. Le raffinement et l&#39;application systématique de cette approche a le potentiel pour informer sur les détails précis de la signalisation calcique astrocytaire. Une compréhension détaillée de la dynamique du calcium dans les astrocytes pourrait fournir une base pour comprendre si, comment, quand et pourquoi les astrocytes et les neurones subissent calcium-dépendante des interactions fonctionnelles.

Watch this Scientific Journal Video about Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes at JoVE.com

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

Nous décrivons comment mesurer près de la membrane et la dynamique globale du calcium intracellulaire dans les astrocytes cultivés en utilisant la réflexion interne totale et de la microscopie à épifluorescence.

Mesure Proche membrane plasmique et mondiale dynamique du calcium intracellulaire dans les astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

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