Flow Cytometry Analysis of Apoptosis, Stemness, and Cell Cycle

An Annexin V-APC/7-AAD Apoptosis Kit (Abnova, #KA3808) was used for the apoptosis analysis as described previously [18 (link)]. ALDH assays were performed as described previously [22 (link)]. Cell cycle assays were performed as described, and propidium iodide(30 μg/ml) was used as DNA content dye [16 (link), 32 (link)]. For CD117 staining, 1 × 10⁶ primary culture tumor cells were stained based on a protocol recommended by the manufacturer. The anti-mouse CD117-PE(Miltenyi Biotec, #REA791) antibody was used. The REA control-PE (Miltenyi Biotec, # REA293) was used as a background control. A BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA) was used for the analysis. Results were analyzed by the Flow Jo software (Version 10.1, OR, USA). All experiments were performed in triplicates.