Six well ultralow attachment plates

The cells were plated in six-well ultra-low attachment plates (20 000 cells per well) (Corning, Corning, NY, USA) in serum-free DMEM/F12 medium, then added with 20 ng/ml human EGF, 10 ng/ml human bFGF, and 2% B27 (Invitrogen, Carlsbad, CA, USA). Cells were incubated at 37 °C with 5% CO₂. After 2 weeks, plates were analyzed for tumorsphere formation and counted by microscope.

For sphere-forming cultures, cells were plated at a density of 2 × 10⁴ cells per well in six-well ultra-low attachment plates (Corning Inc., Corning, NY) in serum free Bronchial Epithelial Cell Basal Medium (BEBM), supplemented with the following growth supplements: BPE, Hydrocortisone, hEGF, Epinephrine, Transferrin, Insulin, Retinoic Acid, Triiodothyronine, and GA-1000, provided as BEGM prepackaged SingleQuots (Lonza, Walkersville, MD) plus rhEGF (Sigma, St Louis, MO), bFGF (Sigma). Fresh aliquots of EGF and bFGF were added every three days. Floating spheres were subjected to mechanical dissociation, followed by replating of single cells for propagation into a second, and subsequently into a third batch of suspension culture as described in our previous reports [5 (link)-7 (link)].

After completing the designated intervention, HCC cells were plated at a density of 5000 cells per well in six-well ultra-low attachment plates (Corning, Corning, NY, USA) and then cultured with serum-free Dulbecco’s Modified Eagle Medium/F12 medium (Gibco) supplemented with 20 ng/mL human EGF, 1% B27 (Invitrogen, Carlsbad, CA, USA) and 20 ng/mL fibroblast growth factor. Subsequently, HCC cells were incubated at 37 °C with 5% CO₂ for 14 days. A microscope (Nikon Instruments Inc.) was used to count the number and determine the diameter of the tumourspheres at a magnification of ×200.

Single cells prepared by 0.25% trypsin-EDTA were seeded at a density of 2,000 cells/well in six-well ultralow attachment plates (Corning Inc.) and then cultured with serum-free DMEM/F12 medium (Thermo Fisher Scientific, Inc.) containing 20 ng/ml human EGF, 1% B27 and 20 ng/ml fibroblast growth factor for 7 days. The number of mammospheres (>20 μm) was counted under an inverted microscope (Olympus).

The validated CDR2L/ALDH6A1 and their mutant plasmids were co‐transfected with pMD2.G and psPAX2 into 293FT cells, and the supernatant was filtered after 2 days of culture. Then, A549 cells were infected with the supernatant and screened with puromycin. Stably expressed cells were selected for subsequent tumor sphere formation assay. For assessing the sphere‐forming ability, 1 × 10³ cells were seeded in six‐well ultra‐low attachment plates (Corning) in serum‐free medium containing DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 20 ng/mL basic fibroblast growth factor (bFGF; HARVEYBIO), 20 ng/mL epidermal growth factor (EGF; Invitrogen), and B27 supplement (Invitrogen). Sphere size and number were measured after 7 days of seeding. Images and numbers of tumor spheres were taken and counted with the use of KEYENCE BZ‐X800LE microscope (KEYENCE, Osaka, Japan). Tumor spheres greater than 75 μm were counted.

Cells were seeded at 1 × 10³ cells/well in six-well ultralow attachment plates (Corning Inc., Corning, NY, USA) in DMEM/F12 (Invitrogen, Carlsbad, CA) supplemented with N2 medium (Invitrogen), human EGF (10 ng/ml, Peprotech, USA), and human bFGF (10 ng/ml, Peprotech). After culturing for 14 days, the total number of spheres was counted.

Cells were plated in six-well ultralow attachment plates (Corning) at a density of 5000 cells/well in RPMI-1640 supplemented with B27 Supplement (Invitrogen), 10 ng/mL human EGF (Sigma-Aldrich), and 10 ng/mL human bFGF (Sigma-Aldrich). Cells were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. Fresh aliquots of EGF and bFGF were added every other day. After culture for 2 weeks, colonies larger than 50 μm in size were regarded as sarcospheres and quantitated by inverted phase contrast microscopy.