Flow cytometry to detect apoptosis and cell cycle changes

Caco-2 and HT-29 cells were grown to a density of 1 × 10⁵ per well on 6-wells plates, and transfected with plasmid expressing lncRNACNN3-206 (OE-lncRNACNN3-206), interfering RNA (si-lncRNACNN3-206), or scramble RNA as a control. Transfection was performed in triplicates for each group. At 48 h post-transfection, cells were collected and fixed with 200 µL of fixation solution (70% ethanol, 5% fetal bovine serum) for 1 h. To determine cell apoptosis, 400 µL of buffer-suspended cells were incubated with 5 µA of Annexin V-FITC at 2-8 °C in the dark for 15 min. Ten microlitre of propidium iodide (PI) was added for nuclei staining. All cells were analyzed 1 h after staining.