Dna content quantification assay

The cell cycle was detected using the DNA content quantification assay (Solarbio, CA1510, Beijing, China). Briefly, cells were fixed with 75% alcohol overnight at 4 °C after digestion with trypsin (Gibco, 25200072, Waltham, MA, USA). RNase A was used the next day to remove RNA. Then PI staining solution was added, and the cells were incubated for 30 min in the dark at 4 °C. The cell cycle was finally detected using Beckman Coulter Cytoflex (BeckmanCoulter, Brea, CA, USA).
Apoptosis was detected using an Annexin V-FITC apoptosis detection kit (Solarbio, CA1020, Beijing, China). Briefly, cells were digested with an EDTA-free trypsin (Gibco, 15050057, Waltham, MA, USA) and resuspended in a 1× binding buffer. FITC-labeled anti-Annexin V was added for 5 min at room temperature in the dark. Finally, the PI staining solution was added, and cell apoptosis was detected using Beckman Coulter Cytoflex (BeckmanCoulter, Brea, CA, USA).

For the cell apoptosis assay, HCT116, DLD1, and RKO cells were stimulated with DHA (10 and 20 μM) for 48 h and then harvested for flow cytometric analysis (Beckman Coulter Cytoflex, Beckman, USA) using an APC Annexin V Apoptosis Detection Kit with 7-AAD (Cat No. 640930, BD Biosciences, USA) according to the manufacturer’s instructions.
For the cell cycle analysis, HCT116, DLD1, and RKO cells were treated with different DHA concentrations for 48 h and detached with trypsin after washing with cold PBS. Then, 70% ice-cold ethanol was used for cell fixation at 4°C overnight. After that, the cells were used for cell cycle analysis by a DNA Content Quantification Assay (Cell Cycle, Solarbio, China). Briefly, the cells were incubated with 100 μl of RNase at 37°C for 30 min, stained with 400 μl of propidium iodide (PI) for 30 min at 4°C in the dark, and then tested by flow cytometry. The data were analyzed by FlowJo 10.4 software (Becton, Dickinson & Company, NJ, USA).