Pe conjugated cd133 antibody

Cells with different treatments were resuspended at 1 × 10⁶ cells per mL in PBS containing 2% FBS and then subsequently stained with PE‐conjugated CD133 antibody (BioLegend) for 30 minutes on ice in dark. After washing with ice‐cold PBS, each sample was analysed by flow cytometry (BD Bioscience). The positive CD133 subpopulations of Capan‐2 cells were sorted from using MACS separation (Miltenyi Biotec) according to the manufacturer’s protocol as described previously.²⁵

For PCSCs population analysis, prostate cancer cells were incubated with FITC-conjugated CD44 antibody (11-0441-82, BD Biosciences, San Diego, CA, USA) and PE-conjugated CD133 antibody (372804, Biolegend, San Diego, CA, USA) for 30 min at 37 °C. The CD44⁺/CD133⁺ subpopulation was quantified by flow cytometry and defined as PCSCs. Hyperactive aldehyde dehydrogenase (ALDH) activity is closely related to the physiological properties of CSCs^{50 (link)}. PCSCs population analysis was also conducted by flow cytometry using the ALDEFLUOR Stem Cell Identification Kit (01700, STEMCELL Technologies, Vancouver, Canada) according to the manufacturer’s instructions. The ALDH⁺ subpopulation was quantified and defined as PCSCs. As a specific inhibitor of ALDH activity, diethylaminobenzaldehyde (DEAB), was used to control for background fluorescence in the ALDH staining assay. For PCSCs sorting, the CD133⁺ subpopulation was isolated by the magnetic activated cell sorting (MACS) method using the CD133 MicroBead Kit (130-100-857, Miltenyi, North Rhine-Westphalia, Germany) in accordance with the manufacturer’s instructions.

The stable transfected cells were digested with trypsin and then collected. After being washed with PBS twice and fixed with 75% ethyl alcohol, the cells were stored at −20 °C overnight. The cells were then washed with PBS, incubated with RNase and stained with a Cell Cycle Staining Kit (CCS012, Multi Sciences, China) for 15 min in the dark. The cell-cycle was analyzed with a FACScan flow cytometer (BD, USA). To evaluate apoptosis of stable transfected cells, the cells were collected and stained with a PE Annexin V Apoptosis Detection Kit I (559763, BD) based on the manufacturer’s protocol. The ratio of apoptotic cells was determined by a FACScan flow cytometer (BD, USA). The collected stable transfected cells were incubated with PE-conjugated CD133 antibody (372804, Biolegend, USA) at RT for 90 min. Then the cells were placed on ice for 10 min and washed with precooled PBS before flow cytometric analysis for CD133(+) cells detection.