Description:
NFkBLuciferaseReporterJurkatStableCellLineisderivedfromhumanTlymphocytecells,andstablyexpressfireflyluciferasereportergeneunderthecontroloftheNF-kBresponseenhancerelement. ThiscelllineisanidealcellularmodelformonitoringtheactivationofNFkBpathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
NF-κB isacriticalregulatorofinflammatoryresponses,proliferationanddifferentiationofT-cells.TheaberrantactivationofNFkB cancontributetothedevelopmentofautoimmunity,chronicinflammation,orlymphoidcancer. JurkatcellsarehumanTlymphocytecellswidelyusedtostudyTcellsignaling. SignosisdevelopedastableJurkatNFkB-luciferasereporterstablecellline,whichcanbeusedforeasilymonitoringtheactivationofNFkBactivationinTcellsthroughsensitiveluciferaseanalysis.ThiscelllinewasestablishedbytransfectionusingapTA-NFkB-luciferasereportervector,alongwithhygromycinexpressionvectorfollowedbyhygromycinselection. ThehygromycinresistantclonesweresubsequentlyscreenedforluciferaseactivityinducedbyTNFaphatreatment.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
AnalysisofSL-0050NFKBLuciferaseReporterJurkatTStableCellLine. 0.1x106Cellswereplatedona96-wellplateandincubatein100μlmedia+0.1%FBS.Additionstowellswereasfollows:(Control)Noaddition; (PHA)50μg/mlincubatedfor20hours;(PHA+TNFα)PHA50μg/mlincubatedfor4hoursthenTNFα20ng/mlwasaddedandcontinueincubatingfor16hours,(TNFα)TNFα20ng/mlincubatedfor16hours.
