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

Questo lavoro è stato sostenuto dalla Fondazione Memorial Uehara del Giappone (ES) e la Fondazione Whitehall, del National Institute of Neurological Disorder and Stroke e un Stein-Oppenheimer Premio Endowment (a BSK).

Procedure sperimentali La procedura sperimentale consiste di due parti principali che sono descritti in maniera saggia passo sotto. Parte 1: PREPARAZIONE CULTURE astrociti dell&#39;ippocampo In breve, misto astrociti-neuroni dell&#39;ippocampo culture sono state preparate utilizzando un protocollo ben definito 1,2,3. Abbiamo ottimizzato il procedimento per produrre sano astrociti in coltura. Tutte le procedure di seguito elenca...

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	<li>Richler, E., Chaumont, S., Shigetomi, E., Sagasti, A., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+approach+to+image+activation+of+transmitter-gated+P2X+receptors+in+vitro+and+in+vivo.">An approach to image activation of transmitter-gated P2X receptors in vitro and in vivo.</a> Nature Methods. 5, 87-93 (2008).</li><li>Granseth, B., Odermatt, B., Royle, S. 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A., Almers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+real-time+view+of+life+within+100+nm+of+the+plasma+membrane.">A real-time view of life within 100 nm of the plasma membrane.</a> Nat Rev Mol Cell Biol. 2, 268-275 (2001).</li><li>Jaiswal, J. K., Fix, M., Takano, T., Nedergaard, M., Simon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+vesicle+fusion+from+lysis+to+monitor+calcium-triggered+lysosomal+exocytosis+in+astrocytes.">Resolving vesicle fusion from lysis to monitor calcium-triggered lysosomal exocytosis in astrocytes.</a> Proc Natl Acad Sci U S A. 104, 14151-14156 (2007).</li><li>Araque, A., Carmignoto, G., Haydon, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+signalling+between+astrocytes+and+neurons.">Dynamic signalling between astrocytes and neurons.</a> Annu Rev Physiol. 63, 795-813 (2001).</li><li>Haydon, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GLIA:+listening+and+talking+to+the+synapse.">GLIA: listening and talking to the synapse.</a> Nat Rev Neurosci. 2, 185-193 (2001).</li><li>Bowser, D. N., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+excites+interneurons+and+astrocytes+to+increase+synaptic+inhibition+in+neuronal+networks.">ATP excites interneurons and astrocytes to increase synaptic inhibition in neuronal networks.</a> J Neurosci. 24, 8606-8620 (2004).</li><li>Bowser, D. 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E &#39;ben dimostrato che gli astrociti visualizzare prospetti del calcio intracellulare. Questi si verificano spontaneamente, può essere attivato da un&#39;attività neuronale o mediante l&#39;applicazione di agonisti per attivare i recettori sulla superficie astrociti 11. Un tema importante e controverso è se astrociti prospetti del calcio intracellulare può innescare il rilascio di molecole di segnalazione che attivano i recettori sui neuroni 11, 12. Questo è controversa perché c&#39;è stata la prova a favore ...

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		<th>Catalogue Number</th>
		<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>
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<td>Poly-D-lysine hydrobromide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P0899</td>
<td>&nbsp;</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>
<tr>
<td>Earle&#x2019;s Balanced Salt Solution (EBSS) (1X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>14155-063</td>
<td>&nbsp;</td>
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<td>Minimum Essential Medium (MEM) (1X), liquid Contains Earle's salts, but no L-glutamine or phenol red</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>51200-038</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>
<td>&nbsp;</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>
<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>BD Biosciences</td>
<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>S7653</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>
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<td>MgCl2 hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M2670</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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<td>Invitrogen</td>
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measuring near plasma membrane and global intracellular calcium dynamics in astrocytes

Il cervello contiene cellule gliali. Astrociti, un tipo di cellule gliali, sono da tempo noti per fornire un ruolo passivo di supporto ai neuroni. Tuttavia, una crescente evidenza suggerisce che gli astrociti possono anche partecipare attivamente nella funzione del cervello attraverso le interazioni funzionali con i neuroni. Tuttavia, molti aspetti fondamentali della biologia astrociti rimangono controversi, chiari e / o sperimentale inesplorati. Una questione importante è la dinamica dei transitori calcio intracellulare negli astrociti. Questo è importante perché il calcio è così affermato come un importante messaggero secondo e perché è stato proposto che elevazioni di calcio astrociti possono innescare il rilascio di trasmettitori da astrociti. Tuttavia, non vi è stata alcuna descrizione dettagliata e soddisfacente di calcio vicino a membrana plasmatica di segnalazione negli astrociti. Fluorescenza di riflessione interna totale (TIRF) microscopia è un potente strumento per analizzare gli eventi di segnalazione fisiologicamente rilevanti all&#39;interno di circa 100 nm della membrana plasmatica delle cellule vive. Qui, usiamo microscopia TIRF e descrivono come monitor vicino membrana plasmatica e globale dinamiche di calcio intracellulare quasi simultaneamente. L&#39;ulteriore perfezionamento e l&#39;applicazione sistematica di questo approccio ha il potenziale per informare sui dettagli precisi della segnalazione calcio astrociti. Una comprensione dettagliata delle dinamiche di calcio astrociti possono fornire una base per capire se, come, quando e perché astrociti e neuroni subiscono interazioni funzionali calcio-dipendente.

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

Noi descriviamo come misurare vicino a membrana e globale dinamiche di calcio intracellulare negli astrociti in coltura utilizzando riflessione interna totale e epifluorescenza.

Misura Vicino membrana plasmatica e Global Dynamics calcio intracellulare negli astrociti

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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