Victor x luminometer

The effect of the BsAb on blockade of PD-1 signaling was performed per manufacturer’s instructions with a cell-based method in which blocking PD-1 signaling allows T cell receptor (TCR) activation and induces luminescence via the NFAT pathway (Promega). Luminescence was detected by the addition of Bio-Glo reagent and quantitated on a PerkinElmer Victor X luminometer.

MM001 and MM047 were seeded in 24-well plates and transfected with 400 ng of pGL4.23-enhancer vector + 40 ng of pRL-TK Renilla vector (Promega) with Lipofectamine 2000 (Thermo Fisher Scientific). As positive controls, the previously published enhancers MLANA_5-I, IRF4_4-I and TYR_−9-D or ABCC3_11-I and GPR39_23-I were used for MM001 and MM047, respectively^{77 (link)}. One day after transfection, luciferase activity was measured through the Dual-Luciferase Reporter Assay System (Promega) by following the manufacturer’s protocol. Briefly, cells were lysed with 100 µl of passive lysis buffer for 15 min at 500 rpm. A total 20 µl of the lysate was transferred in duplicate in a well of an OptiPlate-96 HB (PerkinElmer) and 100 µl of luciferase assay reagent II was added in each well. Luciferase-generated luminescence was measured on a Victor X luminometer (PerkinElmer). A total of 100 µl of the Stop & Glo Reagent was added to each well and the luminescence was measured again to record Renilla activity. Luciferase activity was estimated by calculating the ratio luciferase/Renilla; this value was normalized by the ratio calculated on blank wells containing only reagents. Three biological replicates were done per condition for MM001 and two biological replicates for MM047.

The proliferation of Ba/F3 expressing WT or mutants TpoR mutants was assessed by Cell-Titer-Glo luminescent cell viability assay (Promega). Prior to the experiment, cells were washed three times in PBS. 96-wells plate were used to plate the cells at 10,000 cells/well in RPMI +10% FBS supplemented or not by different concentrations of recombinant Tpo (R&D Systems). After 72 hr, ATP-reacting substrate was added following the manufacturer’s instructions and luminescence readings were performed using a Perkin Elmer Victor X luminometer.