Evaluating ALDH Activity in Osteosarcoma Cells

SaOS-2 and SaOS-LM2 cell lines (10⁶) were cultured as described above and plated in 60 mm dishes (Fisher Scientific, Cat.# 08772B) for 24 h before any drug intervention to ensure adhesion of cells. Plated cells were then treated with enzalutamide, at IC₅₀ concentrations (SaOS-2: 109.7 µM and SaOS-LM2: 98.46 µM) for 48 and 72 h. Each group included three technical triplicates for analyses. At the end of the treatment periods, cells were harvested with Tryple Express (Fisher Scientific, Cat.# 12604021). The ALDEFLUOR™ kit (Stemcell Technologies, Cambridge, MA, USA, Cat.# 01700) was used to analyze the cells with ALDH enzymatic activity according to the manufacturer’s protocol as described previously [38 (link)]. Briefly, cells were incubated in the ALDEFLUOR™ assay buffer containing the ALDH substrate BAAA at 37 °C for 45 min. 7- Aminoactinomycin D (7-AAD) dye (Thermo Fisher Scientific, Cat.# A1310) was added to stain dead cells. Stained cells were analyzed using BD LSR Fortessa (BD Biosciences, San Jose, CA, USA) with Flowjo software (Version 10.5.2+, Flowjo LLC). Three separate ALDEFLUOR™ assays were performed.