Axioskop 40 microscopy

U-373 cells were seeded onto poly-l-lysin-coated coverslips for 24 h before exposure to NR-loaded SLN and c-SLN for 6 h. Briefly, cells were fixed in ethanol 70% at −20 °C, washed with phosphate-buffered saline (PBS). Then, cells were rinsed in PBS and incubated for 10 min with Hoechst solution to counterstain the nuclei. After rinsing with PBS, cells were mounted upside-down on a glass slide in a drop of Mowiol mounting medium (Merck KGaA, Darmstadt, Germany). Cells were photographed with AxioCam MRc5 mounted on Zeiss Axioskop 40 microscopy (Oberkochen, Germany).

ARPE-19 cells were seeded onto poly-L-lysin-coated coverslips for 24 h before exposure to either solvent, NIH or DMF, for 3 h. Immunocytochemistry was performed as previously described, with minor modifications (Marchesi et al., 2018 (link)). Briefly, cells were fixed in ethanol 70% at −20 °C, washed with phosphate-buffered saline (PBS), and permeabilized for 15 min with 0.01% Triton X-100 in PBS. Nonspecific binding sites were blocked at room temperature by incubation for 30 min with PBS containing 1% bovine serum albumin (BSA). Cells were then incubated for 1 h with a polyclonal antibody recognizing Nrf2 (NBP1-32822; Novus Biologicals, Centennial, CO, USA) diluted 1:50 in PBS/1% BSA solution. After a brief rinse with PBS solution, cells were incubated for 1 h with the Alexa Fluor 488-conjugated anti-rabbit secondary antibody (A27034; Invitrogen) diluted at 1:200 in PBS/1% BSA. Cells were rinsed in PBS, then incubated for 10 min with Hoechst solution to counterstain the nucleus. After rinse with PBS and distilled water, the cells were finally mounted up-side-down on a glass slide in a drop of Mowiol mounting medium (Merck KGaA). Cells were photographed with AxioCam MRc5 mounted on Zeiss Axioskop 40 microscopy.