Celltiter glo atp detection system

The effect of UNC3866 and UNC4219 on cell viability was determined using a CellTiter-Glo™ ATP detection system (Promega #7573). Ten point, 1:3 dilution curves of compounds starting at 100 μM final concentration were diluted to 5× final concentration in PBS (vehicle control) and then 5 μL were added to 384-well white, clear bottom tissue culture plates (Corning #3707) with a Multimek automated liquid handling device (Nanoscreen, Charleston, SC). Twenty microliters of low passage, subconfluent HEK293T/PC3 cells grown in Dulbecco's Modified Eagle's Medium without phenol red (GIBCO® #31053) and supplemented with 10% Fetal Bovine Serum (GIBCO® #26140) were immediately added at a density of 5,000 cells per well using a Multidrop 384 (Titertek). Cell plates were incubated for 48 hours at 37°C and 5% C0_2, and then lysed with 25 microliters of CellTiter-Glo™ reagent. Luminescence was read on an Envision platereader (Perkin Elmer) after 15 minutes at room temperature in dim light.

The effect of SW2_110A and SW2_104A on cell viability was determined using a CellTiter-Glo ATP detection system (#G7573, Promega). THP1 cells were seeded in 0.1 × 10⁶ cells/mL density in 96-well clear bottom white microplate (#655098, Greiner Bio-One). Cells were treated with compounds SW2_110A and SW2_104A for 12 days, with fresh compounds replenishment at day 3, 6, 9. For dose-response studies, IC₅₀ was derived from an eight-point 2-fold titration ranging from 100 μM to 1.56 μM of SW2_110A or SW2_104A. CellTiter-Glo reagent was added to cells, and incubated with gentle shake for 15 minutes in dim light at R.T. Luminescence was read on a GloMax® microplate reader. Luminescence was normalized to DMSO-treated groups. The IC₅₀ was calculated using the “log[inhibitor] vs. the normalized response-variable slope” equation in GraphPad Prism 7.