Nunc glass base dishes

U2OS cells transiently transfected with GFP- and AmCyan-tagged expression constructs or treated with commercial Baculovirus reagents were fixed on coverslips with 3% PFA for 20 min, washed with 0.1 M glycine in PBS and stained with Hoechst solution or propidium iodide for 5 min at room temperature. For immunofluorescence, after fixation with PFA, the cells were permeabilized with 0.1% Triton-X100 in PBS for 5 min, and blocked in PBS containing 5% BSA for 30 min at room temperature; then the slides were incubated with the appropriate antibodies (1:100) for 2 h at 37°C. Following 3 washings with PBS, samples were stained with secondary antibodies for 1 h at room temperature. Images were acquired with LSM510 confocal microscope (Zeiss), and processed using ImageJ software, freely available on the net. Imaging of live U2OS cells, grown on Nunc Glass Base dishes (Thermo Fisher Scientific) was performed on LSM510 confocal microscope (Zeiss), equipped with a cells incubator. Images were acquired every 2 min.

DU145 cells (10 × 10⁵) were inoculated on Nunc™ glass base dishes (ThermoFisher Scientific, Rochester, NY, USA) and left to grow overnight in 2 mL of DMEM complete media in the same conditions as elaborated earlier in the cell culture section. Cells were subsequently incubated with DOX, P-TPP-DOX and a combination of P-TPP-DOX with P-TPP-CQ for 1 h or 2 h in Opti-MEM. Specimens were examined on a multiphoton confocal microscope Leica TCS SP8 MP (Wetzlar, Germany) equipped with an Argon laser (excitation lines at 458, 476, 488, 496 and 514 nm), a DPSS 561 laser (excitation line at 561 nm) and an IR MaiTai DeepSee Ti:Sapphire laser (Spectra-Physics, Santa Clara, CA, USA) for multiphoton applications. Images were acquired with the spectral detector of the microscope using appropriate emission wavelength ranges; DOX (red) was excited at 561 nm with the DPSS laser, and emission was recorded between 570 and 650 nm, while MitoTracker^® Green FM was excited at 488 nm with the argon laser, and emission was recorded between 500 and 550 nm. Images were acquired with the LAS X software (Leica Microsystems CMS GmbH, Wetzlar, Germany) and are presented without any post-processing.