Cytopathic Effect Measurement of Stevioside-Treated Trophozoites

The measurement of the cytopathic effect was performed according to [11 (link)]. Briefly, monolayers of the CaCo2 cell line were cultured, and after the end of the incubation time these cells were interacted with trophozoites previously treated with 9.53 mM stevioside for 24 h and untreated trophozoites for 15, 30, and 60 min. After the interaction times, as many trophozoites were removed as possible, and the cells were washed with cold 1× PBS. Subsequently, the cells were fixed with 2.5% paraformaldehyde. The remains of the monolayers were stained with methylene blue to later be subjected to chemical lysis of the cells via dye extraction and quantified by spectrophotometry at a 660 nm wavelength. Also, CaCo2 cells were attached in silanized coverslips, interacted with trophozoites as mentioned above, fixed with 2.5% paraformaldehyde, and washed with 1× PBS, then stained with Hematoxylin & Eosin (H&E) to be analyzed with a light microscope.