1 Culture the cell line in complete culture medium (F12 medium cotining 10% FBS.1X penicillin 1 mM GlutaMax.10 ug/ml blasticidin 200 ug/ml zeocin) at 37c, 5%(v)CO²,overnight.On the follwing day. induce cells with 1 ug/ml of doxycycline for 24 hrs.On the day of the assay. dilute 5X Stim B buffer with ddH2O to make 1X Stim B.2 Prepare the compound as described above.Detach cells by removing media and adding 5 ml Accutase/T-225 flask and incubate at 37℃ for 3 minutes4 Add 10 ml complete culture medium to stop the reaction and pellet cells by centrifuging at 300 x g for 5 minutes.5 Resuspend cells in 2 ml of 1x stim B and then pass through a 100 micron cell strainer (to break up bigger chumps).6 Count cels density and viability by using cell counter. Only cels with >85% viability are used for the assay.7 Dilute cll to 3* 10 cll with the IX Stim B Seed cel at a density of 30,000 cll/ll in 384 well plates.8 Centrifuge the cell plate at 10000 rpm for I min and then agitate at 600 rpm for 2 min Incubate the plate at 37"C for 60 min.9 Prepare the IP1-d2 working solution and ati-lPl-rptate working solution ollowing the table bclow.10 Mix equal amount of IP1-d2 and at-lPlcryptate working solution to make dettion mix.11 Add 10 ulwell of detection mix to plates, Centrifuge the plate at 1000 rpm for I min and then agitate at 600 rpm for 2 min.12 Incubate for 1 hour at room temperature in the dark and read the plate by using an EnVison microplate reader (lex 320 nm, Àem *615 nm and 665 nm).