CD133/CD44 immunomagnetic double screening were improved and performed according to the methods reported in previous literature [17 (link)]. A single-cell suspension of Saos-2 and U2OS cells was incubated with magnetic microbeads-conjugated with the mouse anti-human CD133 monoclonal antibody (Miltenyi Biotec, USA) for 30 min. After washing, the CD133+ cells were separated using the magnetic cell sorting system (autoMACS; Miltenyi Biotec, USA). The purified CD133+ cells were expanded for 14 days by culturing and then harvested as a single-cell suspension to be incubated with magnetic microbeads-conjugated mouse anti-human CD44 monoclonal antibody (Miltenyi Biotec, USA) for 30 min. After washing, the CD44+ cells were separated using the magnetic cell sorting system as described above. This two-step isolation enabled us to obtain sufficient number of CD133+/CD44+ CSCs for the following experiment.
To verify the purity of the isolated CD133+/CD44+ CSCs, cells were stained according to the supplied antibody protocols. Mouse anti-Human CD133/1 (Clone: AC133)-PE and mouse anti-human CD44 (Clone: DB105)-FITC (Miltenyi Biotec, USA) were used. Then flow cytometry analysis was performed using a FACSCalibur instrument (Becton Dickinson).
To verify the purity of the isolated CD133+/CD44+ CSCs, cells were stained according to the supplied antibody protocols. Mouse anti-Human CD133/1 (Clone: AC133)-PE and mouse anti-human CD44 (Clone: DB105)-FITC (Miltenyi Biotec, USA) were used. Then flow cytometry analysis was performed using a FACSCalibur instrument (Becton Dickinson).