Fitc conjugated antihuman cd24 antibody

SUM159 tumor masses were collected and digested as previously described. Cells were washed in PBS 1% BSA and pellets were resuspended in blocking buffer (PBS 10% BSA) and incubated for 30 min at 4°C light protected. After a wash in PBS 1% BSA, cells were incubated with antibodies solution (200 μl/1×10⁶ cells) for 45min at 4°C. In particular, cells were stained with FITC-conjugated anti-human CD24 antibody (Miltenyi Cat. #: 130–112-844, RRID: AB 2656554) and vioblue-conjugated anti-human CD44 antibody (Miltenyi Cat. #: 130–113-899, RRID: AB 2726390). Finally, cells were washed with PBS 1% BSA and sorted with MoFlo Astrios Cell Sorter (Beckman Coulter). To select a population highly enriched in CSCs, after sorting, CD44⁺CD24⁺ cells, CD44⁺CD24^– cells and CD44⁺CD24⁻ cells were plated to perform mammosphere forming assay. CD44⁺CD24^– population was selected to study CSCs.