Pe labeled control antibody

Prior to harvesting, the cell lines were grown until an 80% confluency. For FACS (FACSAria; BD Biosciences, San Jose, CA, USA), the cells were detached using non-enzymatic cell dissociation solution (Sigma-Aldrich) and resuspended in Dulbecco's phosphate-buffered saline (DPBS, Invitrogen,USA). Approximately 5x10⁴ cells were incubated with an antibody (diluted 1:100 in FACS wash with 0.5% bovine serum albumin; 2 mM NaN3 and 5 mM EDTA) for 15 min at 4°C. An isotype and concentration-matched phycoerythrin (PE)-labeled control antibody (Miltenyi Biotec Ltd., Woking, Surrey, UK) was used and the samples were labeled with PE-labeled CD133/1 (clone AC133/1; Miltenyi Biotec Ltd.) and FITC-labeled CD44 (clone G44-26; BD Biosciences). After 3–5 minutes, the cells were washed and subsequently re-suspended. The cells were sorted to be CD 133^high/ CD44^high population (sorting cells) and non-sorting counterparts using a FACSAria flow cytometer, with post-sort analysis performed to confirm population purity. Sorted cell populations were cultured in two different settings, monolayer 2D culture or 3D multicellular tumor spheroid.

Three GC cell lines (KATO-III, SNU216, and SNU601) were purchased from the Korean Cell Line Bank and maintained in RPMI1640 medium (Hyclone, Logan UT, USA) supplemented with 10% (v/v) calf serum (Hyclone) at 37 °C in a 5% (v/v) CO₂ humidified atmosphere. The cells were harvested at 300×g for 5 min, incubated in cell-staining buffer containing phycoerythrin (PE)-labeled anti-CD133/1(AC133) antibody (1:10; Miltenyi Biotec, Bisley, UK) for 10 min in a dark refrigerator, and washed with 0.5% (w/v) bovine serum albumin in phosphate-buffered saline, pH 7.2, with 2 mM EDTA. An isotype-matched PE-labeled control antibody (Miltenyi Biotec) was used to label the samples and set gating levels. MoFlo XDP flow cytometry (Beckman Coulter, Brea, CA, USA) was also used to sort cell lines into CD133+ and CD133- populations. The data were analyzed using Summit software, version 5.2 (Beckman Coulter).

For FACS (Facs Aria, BD Biosciences, San Jose, CA, USA), cells were detached using non-enzymatic cell dissociation solution (Sigma-Aldrich) and ~5×10⁴ cells were incubated with antibody [dilution of 1:100 in FACS wash (0.5% bovine serum albumin, 2 mM NaN₃ and 5 mM EDTA)] for 15 min at 4°C. An isotype and concentration-matched PE-labeled control antibody (Miltenyi Biotech, Bisley, UK) was used and the samples were labeled with PE-labeled CD133/1 (clone AC133/1; Miltenyi Biotech) and FITC-labeled CD44 (clone G44-26; BD Pharmingen, San Diego, USA). After 3–5 min, the cells were washed with FACS wash and resuspended. The cells were sorted into CD133^high/CD44^high (CSC) and non-CSC subpopulations. The two subpopulations were cultured in two different settings, monolayer 2D culture or 3D multicellular tumor spheroid. Briefly, the experimental groups comprised of monolayer CSC (M⁺) and non-CSC (M⁻) and spheroid CSC (S⁺) and non-CSC (S⁻) subpopulations.