ThePD-1/PD-L2InhibitorScreeningCell-BasedAssayisabioluminescentcell-basedassaythatcanbeusedtoscreenandprofileinhibitorsofthePD-1:PD-L2interaction.Theassayconsistsoftwomaincomponents:
•Growth-ArrestedPD-1Effectorcells(PD-1/NFATreporter-Jurkatcells):ReporterJurkatTcellsexpressingfireflyluciferasegeneunderthecontrolofNFATresponseelementsandalsoconstitutivelyexpressingHumanPD-1.Thesecryopreservedcellsareprovidedinathaw-anduseformatthatdoesnotrequirecellpropagation.Thesecellsaremodifiednottobeexpandedandareintendedtobeusedinasingleexperiment.
•ExpressionvectorsforTCRactivatorandHumanPD-L2:Transfection-readyvectorsareusedtotransfectcellstocreatethetargetcellsthatoverexpressPD-L2andanengineeredcellsurfaceTcellreceptor(TCR)activator.
•HEK293cellanditsgrowthmedium(Thawmedium1,#60187)orothercelllines
•Transfectionreagentformammaliancellline[WeuseLipofectamine™2000(Lifetechnologies#11668027).However,othertransfectionreagentsworkequallywell.]
•Opti-MEMIReducedSerumMedium(Lifetechnologies#31985-062)
•96-welltissueculture-treatedwhiteclear-bottomassayplate
•Luminometer
COMPONENTS:
•ScreenforactivatorsorinhibitorsofPD-1signalinginacellularcontext
•CharacterizetheBIOLOGicalactivityofPD-1anditsinteractionswithligands