Evos m5000 fluorescence inverted microscope

A selection of the products of relevance, those with the highest selectivity index, was used to study programmed cell death (PCD) in N. fowleri (ATCC® 30,808™). Therefore, the trophozoites were incubated in a 96-well plate at a concentration of 5 × 10⁵ cells/mL making use of a Countess 3 FL Automated Cell Counter (Thermo Fisher Scientific, Spain). Once attached to the wells, they were treated with IC₉₀ of the chemicals during 24 h. In order to expose the different metabolic events characteristic of a PCD, the reagents were added according to manufacturer's instructions under dark conditions to avoid their degradation. The results were revealed by fluorescence obtained using EVOS™ M5000 fluorescence inverted microscope (Invitrogen, Thermo Fisher Scientific, Madrid, Spain). For each objective lens thickness (40 × and 100 × ), five images were taken. The percentage of stained cells after the incubation of the cells with the dyes was also calculated. Moreover, a one-way analysis of variance (ANOVA) was used for data analysis on order to evaluate the differences between negative control and treated cells. The results are shown as the mean value of three different images ± standard deviation (SD).