Name: real-time cytotoxicity assays in human whole blood
Uploaded: 2014-11-07T15:00:00
Description: Watch this Scientific Journal Video about Real-time Cytotoxicity Assays in Human Whole Blood at JoVE.com

Vi takker National Cancer Institute IMAT program fra NIH for at finansiere dette arbejde [R33 CA174616-01A1].

1. Target Cell Fremstilling <ol><li> Opretholde målceller (f.eks. Raji-lymfomceller) i vækstmedier (RPMI 1640 dyrkningsmedium, med 10% varmeinaktiveret føtalt bovint serum (FBS), 4 nM L-glutamin og 500 IU / ml penicillin / streptomycin) ved 37 ° C i en 5% CO 2-inkubator. </li><li> Centrifuger prøven for at pelletere målcellerne i et 15 ml rør. Centrifugering tid varierer med forskellige celletyper; centrifugeres i 3 minutter ved 468 xg i Raji celler. </li><li> Første aspirere eve...

<ol>
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WCA er et kritisk in vitro-anti-cancer screening værktøj med enkelt celle resolution 12-16, ideelt udnyttet efter de traditionelle målgrupper screeninger, såsom CDC og ADCC assays 9-11, og før prækliniske dyreforsøg. I øjeblikket er det primære target screening assays, såsom CDC eller ADCC assays alle udført i en forenklet medier eller et puffersystem. Men lægemiddelkandidater, der viser effekten på disse forenklede puffersystem er ikke altid effektive i mere komplekse fuldblo...

Nylige fremskridt inden for den farmaceutiske industri har ført til en øget interesse for at realisere de specifikke identifikationer af tumor celleantistoffer og personlige kræftbehandling; dog flere hindringer i processen. Kun 5% af stoffer, der har anticancer aktivitet i præklinisk udvikling er licenseret efter viser tilstrækkelig effekt i fase II-III-test 1,2. De prækliniske strategier (både in vitro og in vivo) er suboptimal så mange eksempler har vist, at antitumorlægemidler opfører sig anderledes i mennesker og i forsøgsdyr hovedsageligt på grund af deres forskellige blodkomponenter 3-5. At afhjælpe behovet for en antitumor drug screening platform og til at tilvejebringe en check-punkt før bekostelig dyreforsøg og kliniske forsøg, foreslås et humant fuldblod cytotoksicitetsassay (WCA) til evaluering antitumorlægemiddel effekt i en mere relevant biologisk miljø. Denfuldblod cytotoksicitet assay kan anvendes til at vurdere virkningen af ​​de enkelte celler til antistoffer og andre lægemiddelkandidater i humant fuldblod. WCA er udviklet ved at inkorporere high-throughput celle positionering teknologi med høj kapacitet og høj indhold imaging 6. Ved anvendelse af et automatiseret billedbehandlingssystem, kan antallet af både levende og døde celler bestemmes med en høj grad af præcision. På grund af det faktum, at målceller immobiliseres på samme fokale plan, WCA er i stand til at give kvantitative cytotoksicitet analyse i realtid uden fjernelse af røde blodlegemer. Desuden automatiske imaging system giver flere fordele, såsom at kun målceller, der har nået de angivne kriterier (f.eks., Fluorescens-mærkede celler og celle morfologi) er indhegnet og forarbejdes. Det giver også mulighed for produktion af 144 plader pr dag. Derfor er dette imaging kapacitet og overførselshastighed muliggør drift af high-cIndholdsproduktion og højt gennemløb eksperimenter samtidigt. Ved at kombinere WCA og automatiseret billedbehandlingssystem kan opnås high throughput kvantitativ cellecytotoksicitet analyse inden for et mere biologisk relevant miljø.

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		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Name of Material/ Equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:135px;">Name/Discription</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:336px;">Cell attachment 96 well plate kits&#160;</td>
			<td style="width:172px;">Adheren</td>
			<td style="width:119px;">AP9601</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:336px;">Suspension Single cell array 8 well chamber slide</td>
			<td style="width:172px;">Adheren</td>
			<td style="width:119px;">SS0801</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:336px;">Adherent Single cell array 8 well chamber slide</td>
			<td style="width:172px;">Adheren</td>
			<td style="width:119px;">SS0802</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Lymphoma cell line CD20+</td>
			<td style="width:172px;">ATCC</td>
			<td style="width:119px;">CCL-86</td>
			<td style="width:135px;">Raji cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Lymphoma cell line CD20-</td>
			<td style="width:172px;">ATCC</td>
			<td style="width:119px;">CRL-8083</td>
			<td style="width:135px;">MC/CAR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">RPMI 1640 with L-glutamine</td>
			<td style="width:172px;">Life Technologies</td>
			<td style="width:119px;">11875-119</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Fetal Bovine Serum</td>
			<td style="width:172px;">Thermo</td>
			<td style="width:119px;">SH30070.01HI</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Peni/Strep</td>
			<td style="width:172px;">Life Technologies</td>
			<td align="right" style="width:119px;">15070063</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Cytosolic dye</td>
			<td style="width:172px;">Life Technologies</td>
			<td style="width:119px;">C7025</td>
			<td style="width:135px;">Cell Tracker Green</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Rituxan (Biosimilar)&#160;</td>
			<td style="width:172px;">Eureka Therapeutics</td>
			<td style="width:119px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Human whole blood</td>
			<td style="width:172px;">Allcells</td>
			<td style="width:119px;">WB001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Propidium Iodide</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:119px;">P4170-10MG</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Automatic imaging system</td>
			<td style="width:172px;">Molecular Devices</td>
			<td style="width:119px;">Contact Vendor</td>
			<td style="width:135px;">Cell Reporter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Cell counting program</td>
			<td style="width:172px;">Molecular Devices</td>
			<td style="width:119px;">Contact Vendor</td>
			<td style="width:135px;">Cell Reporter</td>
		</tr>
	</tbody>
</table>

real-time cytotoxicity assays in human whole blood

En levende celle-baserede fuldblod cytotoksicitetsassay (WCA), der giver adgang til tidsmæssig information af den samlede celle cytotoksicitet er udviklet med high-throughput celle positionering teknologi. De målrettede tumor cellepopulationer første forprogrammeret til immobilisering i et array format, og mærket med grønne fluorescerende cytosoliske farvestoffer. Efter Cellearray formation, er antistoflægemidler tilsat i kombination med humant fuldblod. Propidiumiodid (PI) tilsættes derefter til at vurdere celledød. Cellearray analyseres med en automatisk imaging system. Mens cytosolisk farvestof etiketter de målrettede tumorcellepopulationer, PI etiketter de døde tumorcellepopulationer. Således kan andelen af ​​target cancercelledrab kvantificeres ved at beregne antallet af overlevende målrettede celler til antallet af døde målceller. Med denne metode, forskerne har adgang til tidsafhængig og dosisafhængig cellecytotoksicitet information. Bemærkelsesværdigt, ingen farlige radiokemikalier anvendes. Den WCA præsenteres her er blevet testet med lymfom, leukæmi, og cellelinjer fra solide tumorer. Derfor WCA giver forskerne til at vurdere drug effekt i et yderst relevant ex vivo tilstand.

Anti-CD20-antistoffer og lymfom celler (Raji celler og MC / CAR) blev valgt som en model for at demonstrere den fuldblod cytotoksicitetsassay (WCA) 7,8. Raji-celler havde højt kopiantal af CD20 på celleoverfladen, mens MC / CAR-celler havde lavt kopiantal af CD20 på deres membran. Målrettede celler blev først farvet grønne med grønne fluorescens cytosoliske farvestoffer og ordnet på 96-brønds plade. 10.000 - 50.000 target lymfomceller blev immobiliseret i hver brønd. 180 pi frisk udtaget humant helb...

Watch this Scientific Journal Video about Real-time Cytotoxicity Assays in Human Whole Blood at JoVE.com

Real-time Cytotoxicity Assays in Human Whole Blood

Helblodet cytotoksicitetsassay (WCA) er en cytotoksicitetsanalyse udviklet ved at inkorporere high-throughput celle positionering teknologi med fluorescensmikroskopi og automatiseret billedbehandling. Her beskriver vi, hvordan lymfom celler behandlet med et anti-CD20-antistof kan analyseres i realtid i humant fuldblod give kvantitative cellulær cytotoksicitet analyse.

Real-time cytotoksicitetsassays i humant fuldblod

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

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