Caltag fix perm kit

For intracellular staining of Ki-67, at least 500,000 cells were processed using the Caltag Fix & Perm kit (Invitrogen) following the manufacturer’s guidelines. The antibodies used were FITC-conjugated anti-human/mouse Ki-67 and FITC-conjugated mouse IgG1k isotype control (BD Pharmingen, Buccinasco, Milan, Italy). Flow cytometry analysis was performed as reported above. The green fluorescence was measured as described in the above ‘Vector construction for miR-29b-1 expression and stable transfection’ paragraph. At least 1×10⁴ cells per sample were analyzed and data were stored in list mode files. Expression of cell marker was determined by comparison with isotype control.

Treated and untreated cells were detached by trypsin, counted and washed in 0.1% BSA in PBS. At least 500,000 cells were incubated with fluorescent-labeled monoclonal antibodies or respective isotype controls (1/10 diluted 4 °C for 30 min in the dark). After washing steps, the labeled cells were analyzed by flow cytometry using a BD FACS Aria III (Becton & Dickinson, Mountain View, CA, USA). The antibodies used were mouse anti-human CD133/2 PE (Miltenyi Biotec S.r.l. Calderara di Reno, Bologna, Italy), mouse anti-human CD29 PerCP-Cy5.5 (BD Pharmingen, Buccinasco, Milan, Italy), mouse anti-human CD44 FITC (BD Pharmingen, Buccinasco, Milan, Italy), and mouse anti-human CD324 PE (BD Pharmingen).
For intracellular and nuclear staining of vimentin, osteocalcin, OCT4 PE, Sox2 FITC and Nanog PerCP-Cy5.5, cells were processed using the Caltag Fix & Perm Kit (Invitrogen, Milan, Italy) following the manufacturer’s guidelines. Isotypes were used as controls. All data were analyzed using a Diva software 6.6.