4.6. Flow Cytometry Analysis and Aldehyde Dehydrogenase (ALDH1) Activity Assay

MDA-MB-231 cells (1 × 10⁶ cells) were seeded in a 6-well plate for 24 h and treated with DMSO as the control or 20 µM physalin A for 24 h. After treatment with/without 20 µM physalin A for 1 day, cancer cells were detached by using 1× trypsin/EDTA. The detached cells were washed with 1× activated cell sorting (FACS) buffer. A total of 1 × 10⁶ cells were suspended with 100 µL of 1× FACS buffer and 10 µL of FITC-conjugated anti-human CD44 and phycoerythrin (PE)-conjugated anti-human CD24 (BD, San Jose, CA, USA) were added to each sample. After incubating on ice for 20 min, the samples were washed twice with 1× FACS buffer, and then analyzed using an Accuri C6 flow cytometer (BD, San Jose, CA, USA). The ALDH1 assay was performed using an ALDEFLUOR^TM assay kit (STEMCELL Technologies). The active reagent boron-dipyrromethene (BODIPY)-aminoacetaldehyde was added to each sample, which converts the reagent to fluorescent BODIPY-aminoacetate via ALDH. The ALDH inhibitor diethylaminobenzaldehyde (DEAB) was used as a negative control. MDA-MB-231 cells were treated with/without 20 µM physalin A for 24 h, and then trypsinized for detachment from the plate. After washing with ALDEFLUOR^TM assay buffer as per the manufacturer’s instructions, the proportion of ALDH1-positive cells was assayed using an Accuri C6 flow cytometer [59].