Evos m5000 cell imaging system

For migration assays, cells were seeded in a 24-well plate until 80% confluency was reached. Cell monolayers were scratched with a sterile p200 tip, washed with PBS, and treated with 25, 50, and 100 µM PRIMA-1 containing mitomycin C (0.5 μg/mL) for the inhibition of cell proliferation. Images were obtained on the day of treatment and after 48 h with an EVOS brightfield microscope (EVOS M5000 Cell Imaging System, Life Technologies), and measurements were performed using ImageJ software (version 1.43r, National Institutes of Health).

hADSCs were cultured in osteogenic differentiation medium for 20 days, and then mineralization was assayed by Alizarin Red S (A5533 Sigma-Aldrich, Milan, Italy) staining. Cells were fixed in 10% formaldehyde solution for 20 min at room temperature. After washing twice with PBS, cells were treated with Alizarin Red Solution (2%, pH 4.2) for 40 min and then washed three times with distilled deionized water. Calcium deposits were imaged using an Evos microscope (EVOS M5000 Cell Imaging System, Life Technologies, Monza, Italy).

Cells were washed twice with PBS and fixed with precooled 10% formaldehyde for 15 min at −20 °C. After fixation, the cells were washed twice with PBS, stained for 30 min at room temperature in freshly diluted Oil Red O Solution 0.5% in isopropanol diluted at 3:2 with distilled water, filtered with a 0.45 µm filter (O1391-Sigma), and then washed twice with distilled water. Oil Red O is a lysochrome (fat-soluble dye) diazo dye used for the selective staining and detection of neutral triglycerides and lipids contained in lipid droplets of cultured cells. Images were acquired using a bright-field microscope (EVOS M5000 Cell Imaging System, Life Technologies) [29 (link)].

The aim of these assays was to evaluate the presence of different metabolic events that are shown in programmed cell death like process (PCD-like) undergoing cells. In these assays the inhibitory concentration 50 (IC₅₀) and the inhibitory concentration 90 (IC₉₀) of the yucatecone were used (28.53 ± 5.04 μM and 63.29 ± 0,12 μM respectively) incubating the N. fowleri ATCC® 30808™ trophozoites (5 × 10⁵ cells/mL) for 24 h. After the treatment of the cells with these concentrations of the molecules, a 50% and a 10%, respectively, of the amoebae in the well remain as viable. Finally, the protocol for each kit was performed following manufacturers’ instructions. Moreover, the percentage of stained cells after the incubation of the treated and non-treated cells with each kit and the ratio of fluorescence between the aggregate and monomer forms of the JC-1 was evaluated. For this, the EVOS M5000 Cell Imaging System (Life Technologies, Madrid, Spain) was used and each experiment was performed in triplicate. In each experiment five different images were evaluated with a minimum number of cells of 80.

The SYTOX Green kit (Life Technologies, Madrid, Spain) was used for the evaluation of the cellular plasmatic membrane damage. This stain binds to the DNA of those cells who have suffered alterations in the permeability of the plasmatic membrane and emits a high green fluorescence. Hence, no green fluorescence can be shown in healthy cells. This assay was performed following manufacturer's instruction and modified by Sifaoui et al. (2018) (link). An EVOS M5000 Cell Imaging System (Life Technologies, Madrid, Spain) was used to obtain the images.

The growth assays in saline tap water were performed following a very similar protocol to the one mentioned above. Firstly, the tap water was filtered with a 0.45 μm filter. After, the marine salt was diluted at 1, 5, 15 and 34 g/L. The growth media of the trophozoites was changed in order to add the different saline water dilutions. Finally, a concentration of 100 cells/mL was seeded (1 mL) in a 24 well plate. The same temperatures as in the previous experiments were used. The number of cells were determined at 24, 30, 48, 54 and 72 h after the incubation of the trophozoites in the EVOS M5000 Cell Imaging System (Life Technologies, Madrid, Spain). The experiments were performed in triplicate and the results are expressed as the mean value of each assay.

For the growth assays in saline bactocasitone (N. fowleri) and PYG medium (A. griffini), four different salt concentrations were used: 1, 5, 15 and 34 g/L (sea water salt concentration) (Chubarenko et al., 2023 (link); U.S. Geological Survey, U.S. Department of the Interior, 2024 ). The experiment started with the media change from fresh bactocasitone/PYG medium to the same growth media with the different NaCl concentrations and incubation of the trophozoites (1 mL) in a 24 well plate at a starting concentration of 10 cells/mL for N. fowleri and 100 cells/mL for A. griffini. The incubation was also done at three different temperatures: 20°C, 28°C and 37°C. In addition, N. fowleri cells were also incubated at 26°C. The number of cells that were present in each well was measured at 24, 30, 48, 54 and 72 h post incubation using the EVOS M5000 Cell Imaging System (Life Technologies, Madrid, Spain). The experiments were performed in triplicate and the results are expressed as the mean value of both assays.