Nunc lab tek 2 chambered

HEK cells were seeded onto 8-well Nunc Lab-Tek II chambered (Thermo Fisher, cat. 155409) at 5 × 10⁴ cells/well density and grown for 48 h after transfection. Stably expressing HeLa cells were seeded onto ibiTreat µ-Slide 8 Well (Ibidi, cat. 80826) at 2 × 10⁴ cells/well density and grown for 24 h. The samples were gently washed with Dulbecco’s modified phosphate-buffered saline (DPBS) and then fixed with 4% paraformaldehyde in DPBS for 10 min at room temperature. After several washing steps, the cells were blocked for 1 h at room temperature in DPBS containing 2% bovine serum albumin, 1% fish gelatin, 0.1% Triton-X 100, and 5% goat serum (blocking buffer). The samples were then incubated for overnight at 4 °C with the primary antibody (Bxp-21, 1:200, Abcam, cat. ab3380) diluted in blocking buffer. After washing with DPBS, the cells were incubated for 1 h at room temperature with the secondary antibody (Alexa Fluor 647-conjugated goat anti-mouse, Thermo Fisher, cat. A-21235) diluted 1:250 in blocking buffer. Nuclei were stained with DAPI (1 µM final concentration). Cells were examined with Zeiss LSM 5710 laser scanning fluorescence confocal microscope (40 ×, oil immersion objective), and images were processed with ZEN 2012 software.

EMT6/AR-1 cells were seeded at a density of 20,000 cells per well in a 8-well Nunc Lab-Tek II chambered cover glass (Thermo Fisher Scientific, MA, USA) and allowed to adhere for 24 hrs at 37 °C in a 5% CO2 and 95% air humidified incubator. The concentration of doxorubicin or pDox was kept at 5 µM for doxorubicin control, DNPSs and VDNPSs. After incubation times of 2, 5 and 24 hours, cells were washed 3x with PBS, and Hoescht 33342 nuclei acid dye (Molecular Probes, Inc., Eugene, OR, USA) in PBS was added. Live cell imaging was performed using an Olympus FV1000 confocal microscope equipped with an oil immersion 60x lens. Excitation and emission wavelengths are as follows: for Hoescht 33342, excitation at 405 nm, and emission at 460 nm, and for doxorubicin, excitation at 488 nm, and emission at 550 nm.