Pe conjugated anti cd24

2x10⁶ cells were collected and stained with 20 μ of phycoerythrin (PE)-conjugated anti-CD24, anti-Sox2, anti-Oct3/4, and anti-Nanog antibodies, or fluorescein isothiocyanate (FITC)-conjugated anti-CD44 (BD Biosciences), or with PE-conjugated anti-CD133 (Miltenyi Biotech), or co-stained with FITC-conjugated anti-CD44 antibodies and PE-conjugated anti-CD24 or PE-conjugated anti-CD133. In the process of staining for Sox 2, Oct3/4 and Nanog, BD Perm/Wash buffer (BD biosciences) was used according to the manufacturer's instructions. PE- or FITC-positive cells were quantified on a LSRII flow cytometer (BD Biosciences), and up to 5x10⁴ cells were counted per run.
For the bromodeoxyuridine (BrdU) incorporation assay, 10 μM BrdU was added to the cell suspension 2 hours before collection. Cells were then fixed with cold 70% ethanol, and labeled with a FITC-conjugated anti-BrdU monoclonal antibody according to the manufacturer's instructions (BD Biosciences). Propidium iodide was added before flow cytometric analysis. Detection of BrdU incorporation in DNA synthesizing cells was conducted by flow cytometry.

Tumor cells with different stem cell markers (ALDH⁺ cells and CD44^highCD24^low cells) were isolated using flow cytometry sorting (BD FACSAria III, USA). MDA-MB-231 cells and MDA-MB-468 cells were resuspended in PBS and stained with phycoerythrin (PE)-conjugated anti-CD24 at a dilution of 1:100 (Cat: 130-112-656, Miltenyi Biotec) and allophycocyanin (APC)-conjugated anti-CD44 at a dilution of 1:500 (Cat: 12211-MM02-A, SinoBiological) for 30 min at 4 °C. An ALDEFLUOR kit (Cat: # 01700, STEMCELL Technologies) was used to isolate cells with high ALDH enzymatic activity, each test tube (500 μl system) was added with 2.5 μl ALDEFLUOR reagent and each DEAB (Diethylaminoazobenzene) tube was added with 2.5 μl ALDEFLUOR reagent as well as 5 μl DEAB reagent and the ALDH⁺ population was determined according to the manufacturer’s instructions. The purity of the sorted populations was verified. Data were analyzed using Flowjo software (10.8.1) (BD biosciences, USA).

Flow cytometric analyses and sorting were performed on single-cell suspensions derived from each cell line. ALDH1 enzymatic activity was detected using the ALDEFLUOR assay kit (StemCell Tehnologies, Durham, NC). The basis for this assay is that uncharged ALDH substrate (BODIPY-aminoacetaldehyde [BAAA]) is taken up by living cells via passive diffusion. Once inside the cell, BAAA is converted into negatively charged BODIPY-aminoacetate (BAA) by intracellular ALDH. The negatively charged BAA is then retained inside the cell, causing the cell to become highly fluorescent. Diethylaminobenzaldehyde (DEAB) was used to inhibit the ALDEFLUOR reagent, providing a negative control. Antibodies (FITC-conjugated anti-CD44s, PE-conjugated anti-CD24, and anti-EpCAM were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). PE-conjugated-CD44s, -CD44v3, and -CD44v6 were purchased from R&D Systems (Minneapolis, MN). Fluorescence activated cell analyses and sorting were done by FACS Caliber and FACS Aria III (BD Bioscience, San Jose, CA). Hoechst 33342 was used for specifically staining the nuclei of the cells.