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

Dit werk werd ondersteund door de Uehara Memorial Foundation van Japan (tot ES), alsmede de Whitehall Stichting, het Nationaal Instituut voor Neurologische Aandoeningen en Stroke en een Stein-Oppenheimer Endowment Award (voor BSK).

Experimentele procedures De experimentele procedure bestaat uit twee belangrijke onderdelen die hieronder worden beschreven in een stapsgewijze manier. Deel 1: VOORBEREIDING hippocampus astrocyten CULTUREN In het kort werden gemengd hippocampal astrocyten-neuron culturen bereid met behulp van een gevestigde protocol 1,2,3. We geoptimaliseerd de procedure om een ​​gezonde gekweekte astrocyten opleveren. Alle procedures hiero...

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Het is goed vast te staan ​​dat astrocyten intracellulair calcium verhogingen weer te geven. Deze zich voordoen spontaan, kan worden veroorzaakt door neuronale activiteit of door toepassing van agonisten aan receptoren activeren op de astrocyt oppervlak 11. Een belangrijke en controversiële kwestie is of de astrocyten intracellulair calcium hoogtes kan leiden tot het vrijkomen van signaalmoleculen die receptoren te activeren op neuronen 11, 12. Dit is controversieel omdat er bewijzen voor en tegen deze opvatting, ...

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<td>VWR&#174; Micro Cover Slips, Round, No. 1</td>
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<td>48380-068</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>
<td>Reagent</td>
<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>Invitrogen</td>
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<td>Sodium pyruvate solution</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S8636</td>
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<td>HEPES solution 1 M</td>
<td>Reagent</td>
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<td>H0887</td>
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<td>N-2 Supplement (100X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17502-048</td>
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<td>Horse Serum, Heat-Inactivated</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>26050-088</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>
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<td>B-27 Serum-Free Supplement (50X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17504-044</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>Cell Strainers</td>
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<td>352350</td>
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<td>BD Falcon Multiwell Flat-Bottom Plates with Lids, Sterile</td>
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<td>BD Biosciences</td>
<td>353046</td>
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<td>NaCl</td>
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<td>Sigma</td>
<td>S7653</td>
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<td>KCl</td>
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<td>P3911</td>
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<td>CaCl2 hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>21108</td>
<td>&nbsp;</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>
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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

De hersenen bevatten gliacellen. Astrocyten, een soort van gliale cel, zijn al lange tijd bekend dat een passieve ondersteunende rol te verstrekken aan neuronen. Echter, steeds meer bewijs suggereert dat astrocyten kunnen ook actief in de hersenfunctie deel te nemen door middel van functionele interactie met neuronen. Echter, veel fundamentele aspecten van de biologie astrocyte blijven controversieel, onduidelijk en / of experimenteel onontgonnen. Een belangrijk punt is de dynamiek van de intracellulaire calcium transiënten in astrocyten. Dit is relevant omdat calcium is goed ingeburgerd als een belangrijke tweede boodschapper en omdat is voorgesteld dat astrocyten calcium verhogingen kan de afgifte van zenders van astrocyten te activeren. Toch is er nog geen gedetailleerd of bevredigende beschrijving van de buurt plasmamembraan calcium signalisatie in astrocyten. Totale interne reflectie fluorescentie (TIRF) microscopie is een krachtig hulpmiddel om fysiologisch relevante signalering gebeurtenissen te analyseren binnen ongeveer 100 nm van het plasmamembraan van levende cellen. Hier gebruiken we TIRF microscopie en hoe dat monitor in de buurt plasmamembraan en de wereldwijde intracellulair calcium dynamiek vrijwel gelijktijdig. De verdere verfijning en systematische toepassing van deze aanpak heeft de potentie om te informeren over de precieze details van astrocyten calcium signalering. Een gedetailleerde kennis van astrocyten calcium dynamiek kan een basis bieden om te begrijpen of, hoe, wanneer en waarom astrocyten en neuronen ondergaan calcium-afhankelijke functionele interacties.

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

We beschrijven hoe te meten in de buurt van membraan en de wereldwijde intracellulair calcium dynamiek in gekweekte astrocyten met behulp van totale interne reflectie en epifluorescentie microscopie.

Het meten van de buurt plasmamembraan en Global intracellulair calcium Dynamics in Astrocyten

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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