Cell viability assay for drug synergy

Cells were seeded in 24-well plates and treated with no drug, single drug or in combination at the indicated concentrations for 72 hours. For the drug synergy tests, drug pairs were tested in wild-type K562 cells without Cas9, and thus represent effects independent of possible Cas9-induced DNA damage. Following drugs were used for the assay: A-1155463 (Chemietek CT-A115), A-1210477 (Chemietek CT-A121), CRT0044876 (Selleckchem S7449), KU-60019 (Selleckchem S1570), UMI-77 (Sigma SML1492), NU 7441 (Selleckchem S2638), PK-11195 (Enzo BML-CM118), PX-12 (Cayman 14192), KPT-330 (Selleckchem S7252), 1-benzyl-3,5-bis-(3-bromo-4-hydroxybenzylidene)piperidin-4-one (Millipore 217531), FK866 (Selleckchem S2799). K562 cells were seeded at 100,000 cells/ml, MV4;11 cells were seeded at 200,000 cells/ml, and GM12892 cells and CD34+ HSPCs were seeded at 300,000 cells/ml. After 72 hour incubation, the number of viable cells was counted by flow cytometry (FSC/SSC) using a BD Accuri C6 flow cytometer. Drug synergy was evaluated using the Bliss independence model (Bliss CI, 1939). Briefly, the predicted fractional growth inhibition of the drug combination is calculated using the equation F_A + F_B − (F_A × F_B), where F_A and F_B are the fractional growth inhibitions of the individual drugs A and B at a given dose. Bliss excess is the difference between the expected growth inhibition and the observed inhibition from the drug combination. Bliss scores greater than zero, close to zero, and less than zero denote synergy, additivity, and antagonism, respectively. Bliss sum is the sum of individual bliss scores in the 3 by 3 matrix of drug doses. P values where indicated were determined by one-tailed Student’s t-test between the observed effect of the drug combination and the expected effect calculated from the individual drug effects.