Facsverse laser flow cytometry analysis system

The cell cycles of 1 × 10⁶ HCT-116 and RKO cells in 6-well plates were observed by flow cytometry using a cell cycle analysis kit (PI/RNase Staining Buffer Solution, BD, USA). Briefly, after SCU administration, cells were digested with 0.25% EDTA-free trypsin, followed by centrifugation for 5–10 min at 1000 rpm. After cells collection, cells were re-suspended once with 1× PBS and centrifuged at 1000 rpm for 5–10 min. For cell cycle assay, cells were fixed by pre-chilled 70% ethanol at 4 °C for 18 h. After centrifugation and washes with ethanol to remove ethanol, 500 μl 1× staining solution was added followed by incubating at RT in dark for 15 min. Analysis was performed using a FACSVerse laser flow cytometry analysis system (Becton–Dickinson USA).

The cell cycle and apoptosis of HCT-116 and RKO cells were observed by flow cytometry using a cell apoptosis analysis kit (APC Annexin V Apoptosis Detection Kit with PI) as described before [11 (link)]. In short, after AUCAN administration, digestion of cells was followed with 0.25% EDTA-free trypsin, which was then centrifuged for 10 min at 1000 rpm (n = 5 wells per group). Cells were collected and resuspended once with 1× PBS (4°C), and centrifuged again for 10 min at 1000 rpm. Cells were then fixed and stained with propidium iodide (PI) for cell cycle assay, while for cell apoptosis assay, cells were resuspended with addition of 500 μl 1× binding buffer containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 140 mmol/l NaCl, and 5 mmol/l CaCl₂ at pH 7.4, and then 5 μl Annexin V-FITC and 5 μl PI were added into the 100 μl cell suspension, followed by incubation in dark at room temperature for 15 min. FACSVerse laser flow cytometry analysis system (Becton Dickinson, USA) was used for analysis.