Identification of Cancer Stem Cells

To detect the CD44⁺CD117⁺ cell and ALDH-positive cell subsets, the CD44 and CD117 antibodies (eBioscience, USA) and the ALDH enzyme assay kit (Invitrogen, Carlsbad, CA, USA) were applied on a flow cytometer (FCM, BD Biosciences, USA) according to the manufacturer's instructions and our previous reports [16 (link), 19 (link)]. Briefly, a total of 2 × 10⁵ tumor cells from tumor tissues harvested from the mice immunized with different vaccines were suspended in PBS and labeled with anti-human/mouse CD44 fluorescein isothiocyanate (FITC) 1 : 100 (eBioscience, CA, USA) and anti-human CD117 phycoerythrin (PE) 1 : 20 (eBioscience, CA, USA) antibodies for immunofluorescence detection of the percentage of CSCs by a flow cytometer (FCM).