Evos imaging system

To determine the apoptosis and/or necrosis of the top compound on Snb19 and LN229 cell lines, the Dead Cell Apoptosis Kit with Annexin V FITC and PI (Thermo Fisher Scientific) was used. The apoptosis determination was performed followed by the standard protocol from the manufacture. Briefly, the cells were cultured in 6 well-plate with the initial density of 5 × 10⁵ cells/ well. The cells were treated with IC₅₀ concentration of the top compound, TMZ and negative control (DMSO) were harvested and washed in cold PBS. The cell pellets were then resuspended in 1× annexin-binding buffer provided in the kit. Then, 5 μL of FITC conjugated Annexin V and 1 μL of the 100 μg/mL PI working solutions were added to the 100 μL of cell suspension. The cells were incubated at room temperature for 15 min prior to the fluorescence measurements. The image acquisition was done by using EVOS imaging system (Thermo Fisher Scientific) with 20 × objective magnification.

Apoptosis induction assay was performed using Dead Cell Apoptosis Kit with Annexin V–fluorescein isothiocyanate (FITC) and propidium iodide (PI) (ThermoFisher Scientific, Waltham, MA, USA) followed by the manufacturer’s protocol. Briefly, GSC-LN229, NSCC-LN229, GSC-SNB19 and NSSC-SNB19 cells were seeded in 6 well-plate with the initial density of 5 × 10⁵ cells/well. The cells were treated with the IC₅₀ concentration of THTMP for 24 h, and then harvested and washed in ice-cold PBS. The cell pellets were then resuspended in 1X annexin-binding buffer provided along with the kit. Then, 5 μL of FITC conjugated Annexin V and 1 μL of the 100 μg/mL PI were added to 100 μL of the cell suspension. The cells were incubated at RT for 15 min prior to the fluorescence measurements. The image acquisition was done by using EVOS imaging system (ThermoFisher Scientific, Waltham, MA, USA) with 20× objective magnification. The positive control (TMZ) and negative control (DMSO) were also included in the study.

To study the effect of the lead compound on cell migration, an initial density of 1 × 10⁵ cells/well was plated on 12-well plates. The scratch assay was performed as previously described [2 (link)] and the scratched area was cleaned by washing the cells with 1 mL of phosphate-buffered saline (PBS). Then, 1 mL of culture medium was supplemented with 2% FBS and an IC₅₀ concentration of the lead compound and the DMSO control. The cells cultured with medium containing 2% FBS alone were used as a control. The scratched area was visualized by a phase contrast microscope every 2 h for a period of 8 h. To track the cell migration, the image acquisition was performed by using the EVOS imaging system (ThermoFisher Scientific, Waltham, MA, USA) with 10X objective magnification.

Matrigel (Trevigen, Gaithersburg, MD) was added to a 96-well plate (50 μL/well) and hardened in a humidified, 5% CO₂ incubator at 37 °C for 1 h. Cells (20,000 per well) were then incubated for seven hours in EGM-2 media (Lonza Allendale, NJ) before being stained with Calcein AM (Fisher Scientific, Pittsburg, PA) for 30 min. Their ability to form capillary-like networks was measured using an EVOS imaging system (ThermoFisher, Waltham, MA) and quantified with ImageJ (v1.47f, NIH, Bethesda, MD).

Quantitative assessment of apoptosis and necrosis for GA-T0 against SNB19 and LN229 cells was measured using a Dead Cell Apoptosis Kit using Annexin-V/fluorescein isothiocyanate (FITC) and propidium iodide (PI) (ThermoFisher Scientific, Waltham, MA, USA). Briefly, cells were seeded in 6-well plates at an initial density of 5 × 10⁵ cells/well. Cells were treated then with an IC₅₀ concentration of GA-T0 for 24 h. Positive control (TMZ), negative control (DMSO), and untreated samples were also included in the experiment. The cells were collected, washed in ice cold PBS, and the cell pellets were resuspended in 1× annexin-binding buffer. To 100 μL of cell suspension, 5 μL of FITC conjugated annexin-V and 1 μL of the 100 μg/mL PI was added and incubated at RT for 15 min. Fluorescent images of the viable, apoptotic, or necrotic cells with differences in plasma membrane integrity and permeability were captured using an EVOS imaging system (ThermoFisher Scientific, Waltham, MA, USA). All the experiments were performed with n = 6 in all the experimental conditions.

Cells were grown and differentiated onto sterile glass coverslips placed in 24-well plates. After differentiation, myotubes were challenged with either BSA, PA, or LA followed by fixation with 4% formaldehyde for 20 min at room temperature. Cells were then washed with PBS and permeabilised using a 0.5% triton X PBS solution for 15 min followed by incubation with 2% BSA dissolved in PBS containing 0.25% Triton X (blocking solution) in order to block non-specific bindings. After permeabilisation and blocking, cells were incubated for 2 h at room temperature with anti-TOMM20 diluted 1:1,000 in permeabilisation solution and then washed with PBS before being incubated with rabbit anti–mouse IgG secondary antibody (Alexa Fluor 488) for 1 h at room temperature in the dark. Incubation with secondary antibody was followed by three washes with PBS. Finally, coverslips were mounted on slides using fluoroshield with DAPI to counterstain the nuclei. Cells were imaged using the EVOS imaging system (Thermo Fisher Scientific, Bothell, WA, USA). Mitochondria morphology was evaluated using the ImageJ macro developed by Dagda et al. (42 (link)), as described previously (43 (link)). The circularity score was used to define mitochondria morphology, with an increase in the score indicating a higher degree of fragmentation.