Fitc labeled cd44 clone g44 26

For FACS (FACSAria; BD Biosciences, San Jose, CA, USA), the cells were detached using non-enzymatic cell dissociation solution (Sigma-Aldrich) and ~5×10⁴ 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 min, the cells were washed with the FACS wash, and subsequently the cells were re-suspended. The cells were organized into a CD133^high/CD44^high subpopulation to become CSCs.

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.

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.