Name: whole-cell recording of calcium release-activated calcium (crac) currents in human t lymphocytes
Uploaded: 2010-12-21T15:00:00
Description: Watch this Scientific Journal Video about Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes at JoVE.com

Siamo grati al Dipartimento di Fisiologia e membrana Biologia, Università di California Davis per averci fornito servizi e un ambiente eccellente per lo studio dei canali ionici.

1. Preparazione di riposo linfociti T umani <ol><li> Utilizzando Cocktail RosetteSep umani arricchimento cellule T e Medium Density RosetteSep, purificare le cellule T da campioni di sangue umano secondo le istruzioni del produttore. La popolazione cellulare risultante dovrebbe contenere 95% di cellule T CD3 + riposo. Noi purificare i linfociti T umani da campioni di sangue periferico raccolti da volontari sani in conformità al protocollo approvato dal Consiglio di UC Davis interno Review. </li><li> Dopo l...

<ol>
	<li>Parekh, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Store-operated+CRAC+channels:+function+in+health+and+disease.">Store-operated CRAC channels: function in health and disease.</a> Nat Rev Drug Discov. 9, 399-410 (2010).</li><li>Feske, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRAC+channelopathies.">CRAC channelopathies.</a> Pflugers Arch. , (2010).</li><li>Lewis, R. S., Cahalan, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogen-induced+oscillations+of+cytosolic+Ca2++and+transmembrane+Ca2++current+in+human+leukemic+T+cells.">Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells.</a> Cell Regul. 1, 99-112 (1989).</li><li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+inactivation+of+depletion-activated+calcium+current+(ICRAC)+due+to+local+calcium+feedback.">Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback.</a> J Gen Physiol. 105, 209-226 (1995).</li><li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+calcium-dependent+inactivation+of+depletion-activated+calcium+current.+Store-dependent+and+-independent+mechanisms.">Slow calcium-dependent inactivation of depletion-activated calcium current. Store-dependent and -independent mechanisms.</a> J Biol Chem. 270, 14445-1451 (1995).</li><li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-dependent+potentiation+of+store-operated+calcium+channels+in+T+lymphocytes.">Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes.</a> J Gen Physiol. 107, 597-610 (1996).</li><li>Prakriya, M., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+characterization+of+currents+through+store-operated+CRAC+channels+and+Mg2+-inhibited+cation+(MIC)+channels.">Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels.</a> J Gen Physiol. 119, 487-507 (2002).</li><li>Fomina, A. F., Fanger, C. M., Kozak, J. A., Cahalan, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+channel+properties+and+regulated+expression+of+Ca(2+)+release-activated+Ca(2+)+(CRAC)+channels+in+human+T+cells.">Single channel properties and regulated expression of Ca(2+) release-activated Ca(2+) (CRAC) channels in human T cells.</a> J Cell Biol. 150, 1435-1444 (2000).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-Channel Recording. , (1995).</li><li>Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+for+liquid+junction+potentials+in+patch+clamp+experiments.">Correction for liquid junction potentials in patch clamp experiments.</a> Methods Enzymol. 207, 123-1231 (1992).</li></ol>

Le indagini elettrofisiologiche delle correnti CRAC a riposo cellule T è un compito impegnativo perché la endogeno ampiezza CRAC corrente in queste cellule è di piccole dimensioni a causa delle dimensioni piccole cellule (il riposo umano diametro delle cellule T è nel range di 5-8 micron). Qui vi presentiamo uno step-by-step procedura affidabile per registrare le correnti CRAC a riposo linfociti T umane isolate da cellule mononucleari del sangue periferico. Questa tecnologia ci permette di studiare la fisiologia e l...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>RosetteSep Human T Cell Enrichment Cocktail</td>
<td>&nbsp;</td>
<td>StemCell Technologies, Vancouver, BC, Canada</td>
<td>15061</td>
<td>&nbsp;</td>
<tr>
<td>RosetteSep Density Medium</td>
<td>&nbsp;</td>
<td>StemCell Technologies </td>
<td>15705</td>
<td>&nbsp;</td>
<tr>
<td>RPMI-in 1640 medium w/glutamine/HEPES</td>
<td>&nbsp;</td>
<td>Fisher, Waltham, MA</td>
<td>SH3025501</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Calf Serum</td>
<td>&nbsp;</td>
<td>Omega Scientific, Tarzana, CA</td>
<td>FB-01</td>
<td>&nbsp;</td>
<tr>
<td>GlutaMAX-I (100X solution)</td>
<td>&nbsp;</td>
<td>Invitrogen, Carlsbad, CA</td>
<td>35050</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640 vitamin solution (100X)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>7256</td>
<td>&nbsp;</td>
<tr>
<td>1640 amino acids solution (50X)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>R7131</td>
<td>&nbsp;</td>
<tr>
<td>Sodium pyruvate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S8636</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-Mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M7522</td>
<td>&nbsp;</td>
<tr>
<td>Inositol trisphosphate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>19766</td>
<td>&nbsp;</td>
<tr>
<td>BAPTA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A4926</td>
<td>&nbsp;</td>
<tr>
<td>Poly-L-Lysine Hydrobromide</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P2636</td>
<td>&nbsp;</td>
<tr>
<td>Lanthanum Chloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>262072</td>
<td>&nbsp;</td>
<tr>
<td>Thapsigargin</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>586005</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 Silicon Elastomer Kit</td>
<td>&nbsp;</td>
<td>Dow Corning, Midland, MI</td>
<td>3097358-1004</td>
<td>&nbsp;</td>
<tr>
<td>HIPEC R6101 Semiconductor Protective Coating</td>
<td>&nbsp;</td>
<td>Dow Corning, Midland, MI</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>63-500 Series High-Performance Vibration Isolation Lab Table</td>
<td>&nbsp;</td>
<td>Technical Manufacturing, Peabody, MA</td>
<td>63-540</td>
<td>&nbsp;</td>
<tr>
<td>EPC 10 patch clamp amplifier with headstage</td>
<td>&nbsp;</td>
<td>HEKA Instruments, Bellmore, NY</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulator </td>
<td>&nbsp;</td>
<td>Sutter Instrument, Novato, CA </td>
<td>MP-285</td>
<td>&nbsp;</td>
<tr>
<td>Olympus 1X71 Inverted microscope with 40x oil immersion objective</td>
<td>&nbsp;</td>
<td>Olympus America, Center Valley, PA</td>
<td>1X71</td>
<td>&nbsp;</td>
<tr>
<td>Windows Computer</td>
<td>&nbsp;</td>
<td>Dell </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pulse software</td>
<td>&nbsp;</td>
<td>HEKA Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Origin Scientific Graphing and Analysis Software</td>
<td>&nbsp;</td>
<td>OriginLab, Northampton, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Patch pipette puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass with filament (O.D.: 1.5mm and I.D.: 1.10mm</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>BF150-110-7.5</td>
<td>&nbsp;</td>
<tr>
<td>Narashige's Microforge</td>
<td>&nbsp;</td>
<td>Tritech Research, Los Angeles, CA</td>
<td>MF-830</td>
<td>&nbsp;</td>
<tr>
<td>Silicon O-rings </td>
<td>&nbsp;</td>
<td>McMASTER-CARR, Santa Fe Springs, CA</td>
<td>111 S70</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips 25 mm </td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>12-545-102 25 mm 25CIR.-1</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

whole-cell recording of calcium release-activated calcium (crac) currents in human t lymphocytes

Nei linfociti T, l&#39;esaurimento di Ca 2 + dal intracellulare Ca 2 + negozio porta all&#39;attivazione di plasmalemmal Ca + 2 canali, chiamati Calcio Release-Activated di calcio (CRAC) canali. Canali CRAC un ruolo molto importante nella regolazione della proliferazione delle cellule T e l&#39;espressione genica. Anormale funzione del canale CRAC nelle cellule T è stato collegato a gravi malattie autoimmuni immunodeficienza combinata e 1, 2. Studiare la funzione CRAC canale in cellule T possono scoprire nuovi meccanismi molecolari che regolano le normali risposte immunitarie e svelare le cause di malattie umane correlate. Le registrazioni elettrofisiologiche delle correnti di membrana forniscono la valutazione più accurata delle proprietà del canale funzionale e la loro regolamentazione. Valutazione elettrofisiologica CRAC correnti di canale in cellule Jurkat T, un essere umano linea di leucemia a cellule T, è stata eseguita più di 20 anni fa 3, tuttavia, le misurazioni CRAC corrente in normali cellule T umano rimane un compito impegnativo. Le difficoltà di registrazione dei canali CRAC correnti in normali cellule T sono aggravate dal fatto che i derivati ​​dal sangue i linfociti T sono molto più piccoli nelle dimensioni di cellule Jurkat T e, di conseguenza, l&#39;intero endogeno delle cellule correnti CRAC sono molto bassi in ampiezza. Qui, diamo uno step-by-step procedura che abitualmente utilizzano per registrare il Ca 2 + o Na + correnti attraverso canali CRAC a riposo cellule T isolate dal sangue periferico di donatori sani. Il metodo qui descritto è stato adottato dalle procedure utilizzate per la registrazione delle correnti CRAC in cellule Jurkat T e cellule T attivate umane 4-8.

Watch this Scientific Journal Video about Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes at JoVE.com

Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

Forniamo un passo-passo protocollo per la cellula intera registrazione patch clamp di calcio Release-Activated di calcio (CRAC) correnti in cellule mononucleate del sangue periferico derivati ​​linfociti T umani.

Whole-Cell Registrazione del calcio Release-Activated di calcio (CRAC) Correnti in Human Linfociti T

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated ...

Research

JoVE Journal

Biology

Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes

Loading Drosophila Nerve Terminals with Calcium Indicators

Caricamento terminazioni nervose Drosophila con gli indicatori di calcio

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Ricerca

Biologia

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

In Vivo 2-Photon Imaging di calcio a livello 2 / 3 dei topi

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

A cellula intera Recordings of Light Evocati eccitatorio Correnti Synaptic nel Slice Retinal

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

Calcium Imaging of Cortical Neurons using Fura-2 AM

Imaging di calcio di neuroni corticali usando Fura-02:00

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium ...

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

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, ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Monitoraggio Cambiamenti dei livelli di calcio mitocondriale durante l&#39;apoptosi mediante un sensore di calcio geneticamente codificato

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Misurare flussi di calcio veloce in cardiomiociti

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Valutazione del calcio Sparks in intatte fibre muscolari scheletriche

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

Misure calcio citosolico nelle cellule epiteliali renali mediante citometria di flusso

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Registrazione in tempo reale In Vivo di Arabidopsis segnali di calcio durante l'alimentazione dell'insetto utilizzando un biosensore fluorescente

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...