Lsm710 confocal laser fluorescence microscope

Fibroblasts (6 × 10⁴ cells) were seeded on collagen-coated coverslips (12 mm in diameter) in a 4-well culture plate (Nunc 176740) and cultured for 24 h in a CO₂ incubator. After the treatment with mild PAM (500 μl) for 6 h in a CO₂ incubator, cells were washed with PBS followed by fixation with 3% paraformaldehyde, permeabilization with 0.1% Triton X-100, and blocking with 3% BSA solution. Cells were then incubated for 1 h with an anti-Nrf2 antibody (1:50) diluted with Can Get Signal Immunostain solution (Toyobo), followed by an incubation for 1 h with Alexa Fluor 488 goat anti-rabbit IgG (1:400). After the labeling of cell nuclei with Hoechst 33342 (1:1,000, Dojindo, Kumamoto, Japan), cells were washed and visualized under the LSM710 confocal laser fluorescence microscope (Carl Zeiss, Gottingen, Germany).

RhoNox-1, a turn-on fluorescent probe for the selective detection of ferrous iron, Fe(II), was prepared as described previously16 and preserved at −80 °C. A549 cells in a 96-well microplate (seeded at 2 × 10⁴ cells/well) were cultured for 24 h in a CO₂ incubator and then used in experiments. After the treatment of cells with PAM (80 μL) for the indicated hours in a CO₂ incubator, cells were washed once with PBS and then incubated with 5 μM RhoNox-1 in DMEM #1145 (Sigma-Aldrich) for 1 h in the CO₂ incubator. Fluorescence intensity was assayed (excitation, 525 nm; emission, 580 nm) after cells were washed once with PBS. The intensity of fluorescence was normalized relative to the cellular protein level in each sample.
A549 cells in a 4-well culture plate (seeded at 1 × 10⁵ cells/well) were cultured for 24 h in a CO₂ incubator and then used in experiments. Cells were treated with PAM (400 μL), as described above, and RhoNox-1 fluorescence-positive cells were visualized under an LSM 710 confocal laser fluorescence microscope (Carl Zeiss).