Permeation of the <i>Acanthamoeba</i> Neff to the vital dye SYTOX^® green caused by addition of IC₉₀ of the bioactive molecules.

<p>Molecules were added to cells (10⁵ cells/ml) in the presence of 1 μM SYTOX^® green in PBS + 20 mM d-glucose and the increase in fluorescence (<i>λ</i>_exc = 485 nm, <i>λ</i>_em = 520 nm) monitored. Negative Control cells: cells labeled with the Sytox green in presence of 0.5% Methanol. Positive control contained 2.5% Triton X-100 (A). Confocal microscopy of <i>Acanthamoeba castellanii</i> Neff labeled with SYTOX^® Green. Amoeba were plated as above and incubated for 3 h with IC₉₀ of the vanillin (B), vanillic acid (C) syringic acid (D) and ursolic acid (E), Negative control (F). Cells were observed in a Leica TSC SPE- confocal microscope equipped with inverted optics (<i>λ</i>_exc = 482 nm and <i>λ</i>_em = 519 nm).</p

Screening of 24 molecules against <i>Acanthamoeba spp</i> (IC₅₀ and IC₉₀ expressed in μg/ml).

<p>Screening of 24 molecules against <i>Acanthamoeba spp</i> (IC₅₀ and IC₉₀ expressed in μg/ml).</p

The effect of ursolic acid on the mitochondrial potential, JC-1 dye accumulates in the mitochondria of healthy cells as aggregates (red fluorescing) (Negative controle A); in cells treated with the IC₉₀ of the ursolic acid for 24 h, due to collapse of mitochondrial potential, the JC-1 dye remained in the cytoplasm in its monomeric form, which fluoresced green.

<p>The effect of ursolic acid on the mitochondrial potential, JC-1 dye accumulates in the mitochondria of healthy cells as aggregates (red fluorescing) (Negative controle A); in cells treated with the IC₉₀ of the ursolic acid for 24 h, due to collapse of mitochondrial potential, the JC-1 dye remained in the cytoplasm in its monomeric form, which fluoresced green.</p

Effects of the bioactive molecules against cysts were evaluated by incubating 10⁴ cysts of <i>A</i>. <i>castellanii</i> Neff with the previously calculated IC₉₀ values of the selected drugs in PYG medium, and cells were counted with a Neubauer chamber at between 96 and 168 h.

<p>Troph: Number of Trophozoite emerged from the cyst in the PYC medium.</p