Name: real-time impedance-based cell analyzer as a tool to delineate molecular pathways involved in neurotoxicity and neuroprotection in a neuronal cell line
Uploaded: 2014-08-09T15:00:00
Description: Watch this Scientific Journal Video about Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line at JoV

Financial support for the experiments presented in the study was provided by the Marie Heim-V&#246;gtlin program of the Swiss National Science Foundation.
We thank Ms. Johanna Nyffeler for developing a set of modified MATLAB programs for screening for statistically significant differences in real-time cell analyzer&#8217;s data and Dr. Yama Abassi for helpful discussion.

1. Fremstilling <ol><li> Placer realtid celle analysator station i en vævsdyrkningsinkubator ved 37 ° C og med 5% CO2. Gennemfør alle cellekultur håndtering og farmakologiske behandlinger i en vævskultur hætte under sterile forhold. </li><li> BEMÆRK: nervecellelinjer kræver forskellige temperatur indstillinger i forhold til standard betingelser for kultur, bør justeres i overensstemmelse hermed. For svagt klæbende cellelinjer, brug coatingmidler at lette samspillet mellem celler med guld mikroelek...

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Den nuværende protokol viser nytten af ​​en real-time celle analysator løbende og under etiket-fri betingelser for at vurdere de neurobeskyttende / neurotoksiske effekter af forbindelser i neuronale cellelinjer og for at få indsigt i de second messenger veje involveret i effekt. Selvom realtid celle analysator værktøj til at studere cytotoksicitet og virkningerne af lægemidler på celleproliferation er generelt anerkendt, har kun få studier brugt det i neuronale relaterede cellety...

Neuronal celledød spiller en kritisk rolle i patofysiologien af mange hjerne-relaterede lidelser 1. Tilgængeligheden af pålidelig og høj-throughput in vitro toksicitet analyser er afgørende for at få bedre indsigt i de mekanismer for neurotoksicitet og at hjælpe vælge neurobeskyttende molekyler som terapeutiske kandidater i medicin udvikling 2. Men der er mange begrænsninger for de mest anvendte in vitro-neurotoksicitet assays.They vurdere neurotoksicitet / neuroprotection på en enkelt gang-point ikke tillader kinetisk beslutning; bruger ofte etiket eller sonde, som kan forstyrre de signalveje og begrænse yderligere undersøgelser i den samme celle befolkning, og er ofte arbejdskrævende, og i mange tilfælde ikke giver mekanistisk indsigt. I den foreliggende undersøgelse demonstrere vi anvendeligheden af ​​en real-time impedans-baserede celle analysator til at bestemme neurotoksicitet og neurobeskyttelse i en neuronal cellelinje i realtid og under etiket-fribetingelser, og at give indsigt i downstream mekanismer gennem analyse af second messenger veje involveret i effekt. Tidligere undersøgelser har bekræftet gyldigheden af real-time celle analysator at bestemme cytotoksicitet samt effekter på celleproliferation i cellelinjer i sammenligning med standard teknikker 3,4,5,6. For eksempel blev en god korrelation mellem udlæsninger af standard cellelevedygtighed WST-1 analysen og Cell indeksværdier på flere tidspunkter under basale spredning betingelser og efter to forskellige toksiske paradigmer i HeLa-celler 3. I A549 og MDA-MB-231 celler proliferation og cytotoksicitet provokeret med mikrotubulus stabilisator paclitaxel viste meget ens værdier, når vurderes ved Cell Index målinger og standardmæssigt anvendes sulforhodamin B (SRB) analyse 4. I den neuronale cellelinie immortaliserede hippocampusneuroner HT-22 Cell Index målinger blev valideret for deres evne to detektere celleproliferation, glutamat cytotoksicitet og cytobeskyttelse mod udbredte 3 (4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium-bromid (MTT)-assay 5. I samme undersøgelse MTT analyseresultater og Cell Indeks målinger korrelerede også godt i målingen neuronale stamceller spredning, cytotoksicitet efter vækstfaktorer afsavn og redning af cytotoksicitet ved den pan-caspaseinhibitor QVD 5. Cytotoksicitet induceres i NIH 3T3 celler ved Vandetanib (vaskulær endotel vækstfaktor-receptoren og epidermal vækstfaktor-receptor-inhibitor) viste lignende resultater målt med Cell indeksværdier eller neutral rød optagelse assay 6. Vi har for nylig anvendt realtid celle analysator system til at vurdere neurobeskyttende virkninger af serotonin 2A (5-HT-2A) receptoragonist (±) -2,5-dimethoxy-4-iodoamphetamine hydrochlorid (DOI) i en neuronal cellelinje ( SK-N-SH-celler) og screenet for inddragelse af second messenger veje gennem overvåger virkningen af deres kemiske hæmning på den observerede neurobeskyttelse 7. Interessant, 5-HT 2A-receptoren har både hallucinogene og nonhallucinogenic agonister (som DOI og lisurid, henholdsvis), som kan aktivere både almindelige og distinkte second messenger veje 8. Fordelene ved den præsenterede teknik er, at det giver mulighed for at indsamle oplysninger i realtid om celle overlevelse i løbet af dage, for at afgrænse anden messenger veje er involveret, for at vurdere mulige bidrag spredning effekter til neurobeskyttelse, og for at vælge en optimal tid yderligere end-point studier på samme celle population. Et skematisk diagram af arbejdsgangen i den nuværende protokol er vist i figur 1.

<table border="0" cellpadding="0" cellspacing="0" width="1015">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Name of Material/ Equipment</td>
			<td style="width:161px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:457px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">xCELLigence&#160; RTCA SP system bundle</td>
			<td style="width:161px;">ACEA Biosciences</td>
			<td style="width:119px;">No: 00380601030</td>
			<td>consists of RTCA Analyzer, RTCA SP Station and RTCA Control Unit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">E-plate 96</td>
			<td style="width:161px;">ACEA Biosciences</td>
			<td style="width:119px;">No: 05232368001</td>
			<td>for culturing the cells, inserted in the RTCA SP Station</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">SK-N-SH cells</td>
			<td style="width:161px;">ATCC - (in partnership with LGC Standards)</td>
			<td style="width:119px;">HTB-11</td>
			<td>can be replaced by another adherent neuronal cell line of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DMEM/F12&#160;</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8437</td>
			<td>cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">fetal bovine serum</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">16140-063</td>
			<td>supplements proliferation, but not serum deprivation medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">tisue culture flask T175</td>
			<td style="width:161px;">Sarstedt</td>
			<td style="width:119px;">83.1812.302&#160;</td>
			<td>for culturing cells, which will be later plated on the E-Plate 96</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">0.05% Trypsin-EDTA</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">25300-054</td>
			<td>for trypsinization of cells cultured in tissue culture flasks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Scepter 2.0 cell counter</td>
			<td style="width:161px;">Merck Millipore</td>
			<td style="width:119px;">PHCC20060</td>
			<td>automated cell counter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">phosphate-buffered saline</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">10010-015</td>
			<td>for washing the cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">(&#177;)-DOI hydrochloride</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D101</td>
			<td>5-HT2A agonist for cell culture treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">LY-294,002 hydrochloride</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">L9908</td>
			<td>PI3-K inhibitor for cell culture treatment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">lisuride maleate</td>
			<td style="width:161px;">Tocris Bioscience</td>
			<td align="right" style="width:119px;">4052</td>
			<td>compound with 5-HT2A agonistic activity for cell culture treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">dimethyl sulfoxide</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D4540</td>
			<td>for dissolving LY-294,002 and lisuride maleate</td>
		</tr>
	</tbody>
</table>

real-time impedance-based cell analyzer as a tool to delineate molecular pathways involved in neurotoxicity and neuroprotection in a neuronal cell line

Mange hjerne-relaterede lidelser har neuroncelledød involveret i deres patofysiologi. Forbedret in vitro-modeller for at studere neurobeskyttende eller neurotoksiske effekter af lægemidler og downstream veje involverede ville hjælpe at få indsigt i de molekylære mekanismer i neuroprotektion / neurotoksicitet og kan potentielt lette lægemiddeludvikling. Men mange eksisterende in vitro-toksicitet analyser har store begrænsninger - de fleste vurdere neurotoksicitet og neurobeskyttelse på et enkelt tidspunkt, ikke gør det muligt at overholde de frister, kursus og kinetik for effekten. Desuden vil muligheden for at indsamle oplysninger om downstream signalveje involveret i neuroprotektion i realtid være af stor betydning. I den nuværende protokol beskriver vi anvendelsen af en tidstro impedans-baserede celle analysator for at bestemme neurobeskyttende virkninger af serotonin 2A (5-HT-2A)-receptoragonister i en neuronal cellelinje under etiket-fri og tidstro conditionioner ved hjælp af impedans målinger. Endvidere viser vi, at inhibitorer af second messenger-veje kan anvendes til at afgrænse downstream-molekyler, der er involveret i neurobeskyttende virkning. Vi beskriver også anvendeligheden af ​​denne teknik til at bestemme, om en virkning på celleproliferation bidrager til en observeret neurobeskyttende virkning. Systemet anvender særlige mikroelektroniske plader benævnt E-plader, som indeholder alternerende guld mikroelektrode-arrays på bundfladen af ​​brøndene, der tjener som celle sensorer. Impedansen udlæsning modificeres ved antallet af adhærente celler, cellelevedygtighed, morfologi og adhæsion. En dimensionsløs parameter kaldet Cell indeks er afledt af den elektriske impedans målinger, og bruges til at repræsentere cellen status. Samlet set realtids-impedans-baserede celle analysator giver mulighed for real-time, etiket-fri vurdering af neurobeskyttelse og neurotoksicitet, og en evaluering af second messenger veje engagement, der bidrager til meredetaljeret og højkapacitetsidentifikation vurdering af potentielle neurobeskyttende forbindelser in vitro til udvælgelse af terapeutiske kandidater.

<html> Serum afsavn fører til fald i celle Index værdier, som kan overvåges kontinuerligt med real-time celle analysator Neurotoksiske stimuli til cellerne fører til et fald i Cell indeksværdier, som kan overvåges i realtid med den præsenterede teknik og dynamik, som er afhængig af det specifikke neurotoksiske stimulus og celletypen undersøgt. Figur 2 viser stigningen i celle Indeks da SK-N-SH-celler dyrkes i proliferationsmedium indtil den når et p...

Watch this Scientific Journal Video about Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line at JoV

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

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.

Real-Time impedans-baserede Cell Analyzer som et værktøj til at afgrænse Molekylær baner for neurotoksicitet og Neurobeskyttelse i en neuronal cellelinje

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

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