2.8. Fluorescence microscopic examination
The morphological characterisation of HT-29 cells was performed by using propidium iodide (PI) and acridine orange (AO) double staining according to the method described by Ng et al. (2013) and observed under a fluorescence microscope. Briefly,HT-29 cells were plated at a density of 1 _ 106 cells/ml in a 25 ml culture flask and treated with the IC50 concentration of WTE determined from MTT assay at varying time points of incubation. The cells were incubated in an atmosphere of 5% CO2 at 37 _C for 8, 12 and 24 h. Then the cells were harvested and washed twice using PBS after centrifuging at 1800 rpm for 5 min to remove the remaining media. An equal volume of fluorescent dye (AO/PI) containing AO (10 lg/ml) and PI (10 lg/ml) were added to the cellular pellet and freshly stained cells were observed under a UV fluorescence microscope within 30 min before the fluorescence colour started to fade.