Fluorsave mounting media

The internalization
and
localization of Co(OH)₂ NS into the cells were evaluated
by fluorescence probes. Typically, 150 000 cells were seeded
into a transparent microplate containing a glass coverslip. To label
Co(OH)₂ NS, 30 μg/mL rhodamine B was mixed with Co(OH)₂ NS under continuous rotation at room temperature for 1 h.
Although this was passive loading, it may also induce the intercalation
of small rhodamine B molecules inside the layer or cavities of layered
double hydroxides isostructural Co(OH)₂ NS. Then, the cell
lines were treated with 50 μg/mL Co(OH)₂ NS for different
time points (1, 6, and 24 h). After washing with PBS two times, the
cells were incubated according to the manufacturer’s instructions
with 200 nM LysoTracker Green DND-26 (Thermo Fisher Scientific) and
200 ng/mL Hoechst 33342 for lysosomes and nuclear staining, respectively.
After that, the cells were fixed with paraformaldehyde 4% for 20 min
and the coverslip was mounted using Fluorsave mounting media (Merck
Millipore, Burlington, MA) for cellular imaging. Hoechst, LysoTracker
Green DND-26, and Rhodamine B were detected under a fluorescence microscope
with appropriate filters. Images were analyzed with ImageJ and JacoP
plugins.

Briefly, A2780 and SK-OV-3 cells were seeded at a density 2x10⁵ cells/mL in a 6 well plate containing a glass coverslip for 24h. Next day, 10 µg/ml of AgNP labelled with 30 µg/ml of rhodamine B were added for 1 and 6h to the cells. After two wahes with PBS the cells were incubated with 200 nM of Lysotracker^TMGreen DND-26 (ThermoFisher Scientific, Waltham, MA, USA) to mark lysosomes and 200 ng/ml Hoechst 33,342 for nuclear staining. Next, 4% paraformaldehyde were used to fixed cells for 20 min and then coverslip was mounted using Fluorsave mounting media (Merck Millipore, Burlington, MA, USA) for cellular imaging. The cellular uptake of AgNP was detected by fluorescence microscope with appropriate filters. Image was analysed with ImageJ and JacoP plugins.