*2.12. Apoptosis Induction Studies*

0.5 <sup>×</sup> 10<sup>6</sup> 1301 cells were cultivated in total volume 250 <sup>μ</sup>L for 72 h with amphiphilic dendron (5 μM), doxorubicin (3 μM) or doxorubicin-loaded dendrimersomes (doxorubicin content 3 μM). Non-treated cells were cultivated in parallel as a control. After cultivation, cells were collected, washed twice in cold Cell Staining Buffer (Biolegend, San Diego, CA, USA) and resuspended in Annexin V Binding Buffer (Biolegend, CA, USA). Ice-cold 70% ethanol solution was used for the necrosis induction control; and doxorubicin-treated cells were taken as the apoptosis induction control. For cell staining, the solutions of FITC Annexin V (5 <sup>μ</sup>L per probe with 0.25–1.0 <sup>×</sup> 107 cells) and 7-AAD (5 <sup>μ</sup>L per probe with 0.25–1.0 <sup>×</sup> 10<sup>7</sup> cells) were added. Cells were gently pipetted and incubated for 15 min at room temperature in the dark. Then, 400 μL of Annexin V Binding Buffer were added to each tube. Flow cytometric analysis was performed on a FACSCanto II cytometer (BD Biosciences, San Jose, CA, USA). 40,000 to 100,000 events per sample were acquired and analyzed using FACSDiva software. All experiments were run in triplicates.
