Pe labeled anti cd133 antibody

To silence PRDX2 expression in CD133⁺CD44⁺ CCSCs, we first generated HCT116 and HT29 cells exhibiting stable PRDX2 knockdown (shPRDX2) via lentiviral vector-mediated specific shRNA delivery, and a nontarget negative control lentivirus vector was also transduced into these cells to control for the impact of the lentiviral vector. The lentiviral cloning vectors Ubi-shPRDX2-EGFP-puromycin (LV-shPRDX2) (Sequence: TCTTTATCATCGATGGCAA) and Ubi-NC-EGFP-puromycin (LV-control) (Sequence: TTCTCCGAACGTGTCACGT) were purchased from Genechem Co., Ltd. (Shanghai, China). The cells were subcultured in medium containing puromycin (10 μg/ml, Sigma-Aldrich, USA), and antibiotic-resistant clones were picked and passaged as stable cells. Next, these stably transfected cells (shPRDX2-HCT116, shPRDX2-HT29, control-HCT116, and control-HT29) were used to isolate shPRDX2-HCT116-CD133⁺CD44⁺ CCSCs, shPRDX2-HT29-CD133⁺CD44⁺ CCSCs, control-HCT116-CD133⁺CD44⁺ CCSCs, and control-HT29-CD133⁺CD44⁺ CCSCs by magnetic bead sorting. The use of this approach resulted in a considerable enrichment of CD133⁺CD44⁺ CCSCs (regular purity > 90%), as identified via flow cytometry analysis with a PE-labeled anti-CD133 antibody (Miltenyi Biotech, Germany) and a PE-cy5-labeled anti-CD44 antibody (eBiosciences, USA). Three independent experiments were performed.

Cells (1 × 10⁶) detached from the dishes by TrypLE™ Select Enzyme (Thermo Fisher Scientific) were incubated simultaneously with both the PE‐labeled anti‐CD133 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany; 293C3) and the FITC‐labeled anti‐CD44 antibody (Beckman Coulter, Brea, CA, USA; IM1219U) in complete medium for 60 min on ice. After centrifugation, cells were washed several times with PBS and then resuspended in 1 mL PBS. Flow cytometry (BD FACSCanto™; Becton Dickinson, Franklin Lakes, NJ, USA) was used to analyze the percentage of CD133⁺/CD44⁺ subpopulations in different clones.

Cells detached from dishes by TrypLE (Invitrogen, Carlsbad, CA, USA) treatment were resuspended at a density of 1 × 10⁶ cells/ml in complete media containing FITC-labeled anti-CD44 antibody alone or with PE-labeled anti-CD133 antibody (Miltenyi Biotec, Auburn, CA, USA, 130-080-801). After 1-h incubation on ice, flow cytometry was carried out to analyze and isolate the CD44⁺ and/or CD44⁺CD133⁺ (double positive, DP) subpopulations in HCT-15, HT-29, Snail OE, and r29 cells. Thiostrepton-triggered apoptosis of HT-29 and r29 cells was analyzed after drug (5 or 10 μM) treatment for 48 h. Cells were harvested and incubated with 1 μg/ml FITC-labeled Annexin V (Beckman Coulter Inc.) and 10 μg/ml 7-amino-actinomycin D (7-AAD, AnaSpec, Fremont, CA, USA) in RPMI medium for 30 min on ice. Flow cytometry was performed to measure the percentage of apoptotic cells. To analyze the sub-G1 population induced by thiostrepton, drug-treated cells were fixed in cold 70% ethanol for 30 min on ice before being washed twice in PBS at room temperature. After RNase A (10 mg/ml, Sigma, St. Louis, MO, USA) treatment, cellular DNA was stained with 7-AAD and samples were then subjected to flow cytometry.