Mouse anti human cd44 conjugated with fitc

The spherical cells isolated from tumor spheroids of HCT-116 and HT-29 cell cultures without any treatment were firstly verified by their surface markers as we reported before (Wu et al., 2017 (link)). This was to ensure the colon CSCs have been enriched after the sphere culture. For the tumor spheroid culture after each treatment, 1 × 10⁵ spherical cells were used for staining with rabbit anti-human CD133 (prominin-1) antibody (Sigma-Aldrich); mouse anti-human CD44 conjugated with FITC (Invitrogen, Australia); and mouse anti-human CD24 antibody conjugated with RPE (Invitrogen, Australia). For CD133 staining, mouse anti-rabbit IgG-FITC (Sigma-Aldrich) was used as the secondary antibody. After 3 washes with 2% FCS/PBS, the cells were fixed in 2% paraformaldehyde (PFA)/PBS and analyzed by flow-cytometry (Accuri, BD) and CFlow Sampler software. Three biological repeats were performed.

The spherical cells obtained from sphere culture of HT-29 cells were firstly verified by their surface markers before subsequent experiments. This is to ensure the cancer stem cells have been enriched after sphere culture. The tumour spheres were treated with 1:1 diluted trypsin (2.5% Trypsin-EDTA, Invitrogen, Australia) for 5 min at 37 °C and washed. Spherical cells were dissociated by repeated pipetting and passing through a cell strainer (40 μM, Falcon, USA). After dissociation, 1 × 10⁵ cells were used for staining with rabbit anti-human CD133 (prominin-1) antibody (Sigma-Aldrich); mouse anti-human CD44 conjugated with FITC (Invitrogen, Australia); and mouse anti-human CD24 antibody conjugated with RPE (Invitrogen, Australia). For CD133 staining, mouse anti-rabbit IgG-FITC (Sigma-Aldrich) was used as the secondary antibody. After 3 washes with 2% FCS/PBS, the cells were fixed in 2% paraformaldehyde/PBS and analysed by flow-cytometry (Accuri, BD) and CFlow Sampler software.