Triton x

Fluorescence
microscopy was done with the same microscope. For ROS determination
in HCT116 cells, the cells were seeded and treated with TiO₂ bulk and TiO₂ nanoparticles on coverslips in a 24-well
plate. Then, the cells were washed with chilled PBS and stained with
DCFDA with incubation in the dark for 20 min. The images were observed
and captured in the green channel of the microscope.
For the
neutral lipid metabolism analysis in HCT116 cells, the treated cells
were processed with a slight modification in the protocol, as described
by Nioi et al.^{29 (link)} Briefly, the cells were
stained after fixation with 2% paraformaldehyde and permeabilization
with 0.1% Triton X (Himedia, India). LipidTOX Deep Red neutral (Invitrogen)
was used to stain the neutral lipid present inside the cells. The
nuclei were stained with the Hoechst 33258 dye (blue).
Apoptosis
assay in HCT116 cells was performed by fluorescent microscopy
using the standard protocol of acridine orange (AO) staining.^{30 (link)} TiO₂ bulk- and TiO₂ nanoparticle-treated
cells were washed two times with PBS buffer and stained with 2 μg/mL
AO/EtBr dissolved in PBS for 20 min. After staining, they were again
washed two times to remove the extra stain. The images of the stained
cells were then taken in the green and red channels of the microscope.
The images were merged and presented.