1100 plate reader

Cells were seeded at the densities grown as stated above. Drug combinations used included enzalutamide plus saracatinib and docetaxel plus saracatinib at their respective IC50 dosages for each cell line. Therapy was given, beginning at 2x the IC50 dose, then serial diluted by 2 till 9 dilution groups were established. Cell viability was measured using WST-1 at 1:10 dilution with CSS-FBS media at absorbance of 450 nm (Tecan 1100 Plate Reader). Measured absorbance was converted in percentile and inputted in the Bliss Independence equation (24 (link)) as well as CompuSyn 1.1, a computer software that determines synergy via Combination Index (25 (link)) between drugs based on individual dose response. Bliss independence is calculated as Fab=Fa x Fb, where Fa/b is the fraction of cells affected by drug A/B and Fab is the product of the two fractions, representing the predicted additivity of the two drugs. Bliss independence value is portrayed with Fab and the experimental values of each combination. Combinations with values greater than Fab are considered synergistic, while combination with values less than Fab are considered antagonistic. Each data point was conducted in technical triplicate.

Cells were seeded at the following densities: DU-145 (500 cells/0.1 mL), AD1 (2,000 cells/0.1 mL), R1D567 (1,000 cells/0.1 mL), LNCaP95 (4,000 cells/0.1 mL), 22Rv1 (2,000 cells/0.1 mL) in 96 well plates in RPMI media (Sigma-Aldrich) with 10% FBS, 1% penicillin-streptomycin, and 1% Glutamax (Corning). After an overnight incubation at 37°C and 5% CO2, media in the wells was replaced with fresh RPMI media with 5% charcoal-stripped (CSS)-FBS, 1% penicillin-streptomycin, and 1% Glutamax (Corning). Cells were then grown in the media for 3 days. One of the following three drug groups is then administered: enzalutamide, saracatinib, ranging from 0.39-100μM, and docetaxel 0.04-10nM. All drugs were obtained from Selleckchem. Treatment lasted for 6 days with replenishment of the media and drug after 3 days. Cell viability was measured using WST-1 at 1:10 dilution with CSS-FBS media at absorbance of 450 nm (Tecan 1100 Plate Reader). IC50 dosage was calculated using GraphPad Prism. Each data point was conducted in technical and biological triplicate.