Q fluoro software

To evaluate the effect of MPE on mitochondrial membrane potential (Δψm), HT29, Caco-2, and HCT116 cells (8 × 10³) were seeded in a 96-well plate. After exposure to MPE, cells were incubated for 15 min with JC-1 staining solution [48 (link)], and Δψm was assessed according to the protocol provided by the Cayman Chemical Company (Ann Arbor, MI, USA). JC-1 is a specific potentiometric dye that enters the mitochondria, producing a different staining of healthy and unhealthy cells. The compound forms aggregates with intense red–orange fluorescence, detectable with a rhodamine filter (excitation wavelength of 596 nm and emission wavelength of 620 nm) in healthy cells with polarized mitochondria and high Δψm. Conversely, JC-1 can remain in a monomeric form, showing green fluorescence detectable with an FITC filter (excitation wavelength of 485 nm and emission wavelength of 530 nm) in unhealthy cells that exhibit low Δψm. All images were visualized using a Leica fluorescence microscope (Leica Microsystems) equipped with a DC300F camera. The merging of images taken with the two filters (rhodamine vs. FITC) was performed using the Leica Q Fluoro Software (Wetzlar, Germany). We selected a minimum of three fields for each condition in order to calculate the JC-1 aggregates/JC-1 monomers ratio. The reduction of this ratio was considered to signify loss of Δψm.

HeLa cells were propagated on culture microscope slides under the conditions described above. After incubation for 24 h, the cultures were washed three times with phosphate buffered saline (PBS), fixed with acetone for 20 min at −20°C, washed with the same buffer and incubated overnight at 4°C with appropriate dilutions of the primary antibodies (Table 1). The slides were then washed for 30 min with PBS, placed in the dark and incubated with the secondary antibodies (Table 1) for 90 min in a humid chamber. The samples were washed three times with PBS and incubated successively with 1 μg/ml phalloidin-TRITC conjugate (Sigma-Aldrich) for 90 min and 10 ng/ml DAPI (Southern Biotech; Birmingham, USA). The preparations were visually examined and photographed in a Leica DMR-XA fluorescence microscope coupled to Leica Qfluoro software in the Image Processing facility of the University of Oviedo. The quantification of fluorescence for the subsequent statistical analysis was carried out using ImageJ analysis software (28 ).

The generation of autophagic vacuoles was detected using monodansylcadaverine (MDC) staining according to Munafò et al.’s procedure [38 (link)]. Briefly, following to the treatment in the presence of MG, the medium was replaced and 50 mM MDC in PBS was added to cells. Then, cells were washed in PBS and the fluorescence was analyzed with a Leica fluorescence microscope (Leica Microsystems, Wetzlar, Germany) using a 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) filter (excitation wavelength of 372 nm and emission wavelength of 456 nm). The analysis of autophagic vacuoles was also performed using acridine orange (AO) staining that specifically detects acidic vesicular organelles (AVOs) producing a bright red fluorescence, whereas it generates a bright green fluorescence for cytoplasm and nucleus [39 (link)]. For these analyses, cells were incubated for 15 min with 1 μg/mL AO prepared in PBS. Then, cells were analyzed under a Leica fluorescence microscope equipped with an image system (Leica Microsystems, Wetzlar, Germany) using Rhodamine (excitation wavelength of 596 nm and emission wavelength of 620 nm) and FITC (excitation wavelength of 485 nm and emission wavelength of 530 nm) filters. Merged images were obtained by combining pictures of both channels using Leica Q Fluoro software (Leica Microsystems, Wetzlar, Germany).

To assess the intracellular generation of reactive oxygen species (ROS), cells were plated in 96-well plates and allowed to adhere overnight. Cells were treated with MG and incubation with 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorochrome (Molecular Probe; Thermo Fisher Scientific, Inc., Life Technologies Italia, Monza, Italy) was performed as previously reported [37 (link)]. For this purpose, stock solutions of H2DCFDA were dissolved in DMSO and aliquots were stored at −20 °C until use. H2DCFDA working solution was prepared in a PBS solution containing 5 mM glucose and added to cells to the final concentration of 20 μM. Then, incubation was protracted for 30 min in the dark and ROS-positive cells were visualized using a fluorescein isothiocyanate (FITC) filter (excitation wavelength of 485 nm and emission wavelength of 530 nm) in a Leica inverted fluorescence microscope (Leica Microsystems S.r.l, Wetzlar, Germany) equipped with a DC300F camera. All pictures were captured using Leica Q Fluoro software (Leica Microsystems S.r.l, Wetzlar, Germany).

Apoptotic cell death was assessed by vital Hoechst 33342 staining [56 (link)] (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to vendor’s specifications. Morphological analysis of condensed or fragmented chromatin was estimated by a fluorescence microscope and pictures were taken with Leica Q Fluoro software (Leica Microsystems, Wetzlar, Germany; https://www.leica-microsystems.com/it, (accessed on 21 March 2024)).

Cell death was assessed by staining the cells with the vital dye Hoechst 33342, which evidences nuclei and allows for the detecting of chromatin condensation and fragmentation. For these experiments, 7 × 10³ cells/well were seeded in a 96-well plate, incubated with the compounds for the established times and then stained with Hoechst (2.5 µg/mL medium) for 30 min. After washings with PBS, cells were visualised using an inverted Leica fluorescent microscope (Leica Microsystems, Wetzlar, Germany) endowed with a 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) filter. Leica Q Fluoro Software (Leica Microsystems, Wetzlar, Germany) was used for image acquisition. Annexin V apoptosis detection assay was used to evidence early apoptotic cells. Briefly, 3 × 10⁵ cells were seeded in 6 cm diameter Petri dishes, allowed to adhere overnight and then treated with the compounds for 24 h. Cells were then harvested, washed twice in PBS, and 10⁵ were incubated for 15 min with 5 µL annexinV/PI in a 100 µL binding buffer. Following dilution (500 µL binding buffer final volume) analysis was performed by flow cytometry using FacsCanto BD. The percentage of annexin V positive cells was evaluated by Flowjo BD Software, Milan, Italy.