Pe mouse anti human cd24

PE mouse anti-human CD24, APC mouse anti-human CD44, PE mouse IgG2a κ Isotypecontrol and APC mouse IgG2b κ Isotype Control were purchased from BD Pharmingen (San Diego, CA). Cells were resuspended at 1×10⁶cells per 100 μl of sorting buffer (1×PBS containing 0.5% bovine serum albumin) and incubated with pre-conjugated CD44-APC and CD24-PE primary antibodies for 10 min at 4°C. Three control groups were established for the first sorting: (1) cells labeled with the isotype antibodies of the above two antibodies, (2) cells labeled with the anti-CD44-APC antibody and the isotype control antibody for CD24, and (3) cells labeled with the anti-CD24-PE antibody and the isotype control antibody for CD44. The cells were washed in 1×PBS and centrifuged at 800g for 2 min. For flow cytometric analysis, cells were resuspended in sorting buffer after incubation with the primary antibodies.

The growing cells cultured in 100 mm dishes were washed with 1× PBS, trypsinized, centrifuged, and resuspended in 2% FBS in Hank’s balanced salt solution (HBSS). The viable cell number was counted using an automatic cell counter (trypan blue exclusion), and the cell density was adjusted to 1 × 10⁶ cells/100 μL. For CD24 and CD44 staining, 20 μL of antibodies (anti-CD24, anti-CD44 and PE-mouse IgG isotype control and APC-mouse IgG isotype control) were added to 100 μL of cell suspension and incubated at 4 °C for 30 min per the manufacturer’s protocol. However, for ABCG2 staining, 5 μL of APC Mouse Anti-Human CD338 and APC-mouse-IgG isotype control were added to 100 μL cell suspension and incubated at 4 °C for 30 min, according to the manufacturer’s instructions. After incubation, the cells were washed three times with HBSS and centrifuged at 2000 rpm for 3 min. Then, the cells were resuspended in 300 μL 1× PBS containing 1 μg/mL DAPI and were passed through tube filters to avoid cell aggregation. Finally, the cells were analyzed using flow cytometry. PE mouse anti-human CD24, APC mouse anti-human CD44, PE mouse IgG2α, κ isotype control (555574), APC mouse IgG2α, and κ isotype control antibodies were purchased from BD Bioscience (San Jose, CA, USA), and anti-mouse IgG₁ isotype control (MAB002) was purchased from R & D Systems, Inc. (Minneapolis, MN, USA).

For flow cytometry analyses and cell sorting tumor spheres were dissociated as single cells, washed and incubated with the appropriate dilution of control or specific antibody. Antibodies used were V450 Mouse Anti-Human CD44 (561292), PE-Cy7 Mouse Anti-Human CD45 (557748), PE Mouse Anti- Human CD146 (550315), PE-Mouse Anti-Human CD24 (555428), FITC Mouse Anti-Human CD90 (561969), FITC-Mouse Anti-Human EpCAM (347197), APC-Mouse Anti-Human CD10 (332777), (all from BD Bioscience Bedford, Franklin Lakes, NJ) and Mouse monoclonal antibody [5D3] to Cytokeratin 8 + 18 (Abcam Cambridge,UK, 17139). After 45 min incubation, cells were washed. Analysis was performed using a FACS Canto flow cytometer (BD Biosciences). Cell Sorting was performed by FACS ARIA cytometer equipped with three lasers (488, 633, 407 nm) (BD Biosciences). All the cytofluorimetric acquisitions were analyzed by BD FACSDiva Software version 6.1.3 (BD Biosciences). For the sorting, cells were incubated with antibodies for 1 h then washed in PBS. Antibodies were used following manufacturing protocol indication for sorting experiment. TO-PRO3 (Thermo Fisher) dye was used for viability evaluation and used following manufacturing protocol indication. All the cytofluorimetric acquisitions were analyzed by BD FACSDiva Software version 6.1.3 (BD Biosciences).