ALDEFLUOR assay

Cells were grown in RPMI 1640 media (Corning) containing 2 mM glutamine, 10% FBS (Sigma), and 1× Pen/Strep (Gibco). When the cells reached 80% confluency, they were harvested by trypsinization and assayed for ALDH activity using the Aldefluor assay (STEMCELL Technologies) as previously described ²². Briefly, the cells were washed with PBS, resuspended in Aldefluor buffer and Aldefluor reagent was added. The cells were quickly mixed and evenly distributed into 1.5 mL Eppendorf tubes containing inhibitor or vehicle and incubated for 30 min at 37 ° C. Cells were washed, resuspended in fresh Aldefluor buffer that had been kept on ice until they were analyzed on a CytoFLEX S flow cytometer (Beckman Coulter). The percent of control values were calculated using the percentage of Aldefluor positive cells for a particular sample and the percentage of Aldefluor positive cells in the control sample (vehicle-treated). The percentage of Aldefluor positive cells was graphed in Prism 7 (GraphPad) and is displayed as mean ± SD. The Two-way ANOVA with Tukeys multiple comparison test within Prism 7 (GraphPad) was used to determine statistical significance between samples treated with compound or vehicle.