Agilent bravo

To generate a focused compound library, compounds were purchased from Selleck Chemicals, Tocris and Sigma, either dissolved in dimethyl sulfoxide (DMSO, 472301 Sigma) at 10 mM or as dry powder that was dissolved at 10 mM in DMSO. Experimental compounds were excluded from the outer two rows and columns of each 384-well library plate to avoid the potential impact of edge effects. Each compound screen plate contained 24 compounds arrayed across a 10-point dose curve ranging from 20 μM to 3 nM (24 compound plates in total). To generate library plates, 75 μL of compound were pipetted into the wells of column 3 (C3-N3) and 13 (C13-N13) of an AB0781 plate (Thermo Fisher Scientific). Using an Agilent Bravo, 50 μl of DMSO were pipetted into the remaining wells. Three-fold serial dilutions were then created by aspirating 25 μl of compound (starting in columns 3 and 13) and dispensing into 50 μl of DMSO in the adjacent column, pipetting up and down five times and then transferring 25 μL to the next column. The resultant master compound plates contained at least 50 μl of compound at the desired range of concentrations (10 mM to 0.5 μM; corresponding to 20 μM to 3 nM final concentration in cells). Library screening plates were created by aspirating 11 μL of compound from the AB0781 master plates to AB1056 plates (Thermo Fisher Scientific).