Annexin 5 propidium iodide double staining uptake

To evaluate the levels of apoptosis following 18e derivative treatment, a flow cytometric analysis, using the cell apoptosis kit Annexin V/Propidium Iodide (PI) double staining uptake (Invitrogen, Life Technologies, Italy), was used. Vero-76 cells, at the density of 3 × 10⁵ cells/mL, were seeded in 12-well plates (Corning, New York, NY, USA) with complete medium (described in the cell culture section). After CV-B5 viral adsorption, the cells were incubated in the absence or presence of different concentrations of 18e for 48 h, until the cytopathic effect CPE of the virus control reached 70–80%. Cells were then washed once with PBS 1 X and re-suspended in 100 μL of Annexin binding buffer plus 1 μL of Annexin V and 1 μL of PI. Then, the reaction was performed in the dark for 15 minutes at room temperature. Stained cells were then analyzed by flow cytometry, measuring the fluorescence emission at 530 and 620 nm using a 488 nm excitation laser (MoFloAstrios EQ, Beckman Coulter, Pasadena, CA). Cell apoptosis was analyzed using the software Summit Version 6.3.1.1, Beckman Coulter.

To assess levels of apoptosis following 2b derivative treatment, a flow cytometric analysis, using the cell apoptosis kit Annexin V/Propidium Iodide (PI) double staining uptake (Invitrogen, Life Technologies, Italy), was used. Vero-76 cells, at the density of 2 × 10⁵ cells/mL, were seeded in 12-well plates (Corning, New York, NY, USA) with complete medium (described in cell culture section). After EV-A71 viral adsorption, the cells were incubated in the absence or presence of different concentrations of 2b for 96 h, until the cytopatic effect CPE of the virus control reached 70–80%. Cells were then washed once with PBS 1 X and re-suspended in 100 μL of Annexin binding buffer plus 1 μL of Annexin V and 1 μL of PI. Then, the reaction was performed in the dark for 15 minutes at room temperature. Stained cells were then analyzed by flow cytometry, measuring the fluorescence emission at 530 and 620 nm using a 488 nm excitation laser (MoFloAstrios EQ, Beckman Coulter, Pasadena, CA, USA). Cell apoptosis was analyzed using the software Summit Version 6.3.1.1, Beckman Coulter.

To assess which death mechanisms our compounds induced, cell apoptosis kit Annexin V/Propidium iodide (PI) double staining uptake (Invitrogen, Life Technologies, Italy) was used. 3b compound was selected as lead and employed in our assay. Human lung cancer cells (SK-MES 1) were seeded at the density of 8 × 10⁵ cells/ml in 6-well plates (Corning, United States) with a complete medium (described in cell culture section). After overnight incubations, the cells were treated with or without different concentrations of 3b for 96 h. Cells were then labeled with Annexin V and PI as previously described (Ibba et al., 2021 (link)). Stained cells were then analyzed by flow cytometry, measuring the fluorescence emission at 530 and 620 nm using 488 nm excitation laser (MoFloAstrios EQ, Beckman Coulter). Cell apoptosis was analyzed using Software Summit Version 6.3.1.1, Beckman Coulter.