Cyan adp instrument

Pacific Blue-conjugated anti-mouse CD24 or PerCP/Cy5.5-conjugated anti-human CD24 (Biolegend, San Diego, CA, USA) were used for cell surface staining of the cells. 7-amino actinomycin D (7-AAD, Biolegend) was used to gate live cells. Cells were stained at a concentration of 5 × 10⁶ cells/ml of fluorescence-activated cell sorting (FACS) buffer (PBS containing 0.1 % bovine serum albumin [BSA]) for 20 minutes on ice in the dark, after which, the cells were washed twice and resuspended in FACS buffer containing 7-AAD. Stained cells were analyzed using the CyAn ADP Instrument (Dako-Cytomation, Glostrup, Denmark) and the FlowJo 7.25 analysis software (Tree Star, Ashland, OR, USA). Flow cytometry-based cell sorting for CD24^- IGF1R-KD and CD24⁺ IGF1R-KD cells was performed using FACSAria (BD Biosciences, San Jose, CA, USA).

The following antibodies were used for cell surface staining of the Mvt-1 cell line, Pacific-Blue-conjugated anti-CD24, PE-conjugated anti-CD29, AF647 (Alexa Fluor 647)-conjugated anti-CD61/β3 and AF647 (Alexa Fluor 647)-conjugated anti-CD49F (Biolegend, San Diego, CA, USA): 7-Amino actinomycin D (7-AAD, Biolegend) was used to gate live cells. Cells were stained at a concentration of 5 × 10⁶ cells/ml of FACS buffer (PBS containing 0.1 % BSA) for 20 minutes on ice in the dark, after which the cells were washed twice and resuspended in FACS buffer containing 7-AAD. Stained cells were analyzed using the CyAn ADP Instrument (Dako-Cytomation, Glostrup, Denmark) and the FlowJo 7.25 analysis software. Intracellular staining was performed with the CytoWx/Cytoperm kit (BD PharMingen, San Diego, CA, USA), after CD24 cell surface staining. Cells were then washed with the Cytofix solution, and permeabilized with perm wash for 10 minutes on ice. To detect intracellular CD24, the antibody was added in combination with perm wash for 15 minutes on ice. Cells were washed twice and resuspended in FACS buffer until analysis. Flow cytometry-based cell sorting for CD24⁻ and CD24⁺ cells was performed using FACSAria (BD Biosciences, San Jose, CA, USA).