4.6. Cell Cycle Analysis

The effect of the DMCH sample on the cell cycle of HT29 and SW620 cells was investigated using flow cytometry cell cycle analysis. Cell cycle assay was carried out using the CycleTEST Plus DNA Reagent Kit according to the instructions (BD Pharmingen, San Diego, CA, USA). The cells were seeded at a density of 2.0 × 10⁵ cells/well overnight. Then, cells were treated with different dosages of DMCH based on the concentration results of the MTT assay for 48 h and 72 h. After the treatment, cells were collected, and the pellet was resuspended in 200 μL of trypsin (solution A) and RNAse (solution B) for 10 min each followed by 250 μL of propidium iodide (solution C) for 15 min. After that, flow cytometric analysis was performed within 3 h using a Novocytes flow cytometer (ACEA Biosciences Inc., San Diego, CA, USA).