Highcontent screening microscope

The irreversible
bleaching of trimethine dyes fluorescence was monitored under constant
irradiation as a function of time. HeLa and HHF Hs27 cells were seeded
at concentration 2 × 10⁴ cells per well in 8 Well
Chambered Cover Glass with #1.5 high-performance cover glass (Cellvis)
24 h prior to the experiment. Before the experiment, the growth medium
was removed, and the cells were washed twice with preheated PBS. After
that, the cells were incubated with Hoechst 33342 in DMEM culture
medium (Gibco, USA) for 10 min at 37 °C in 5% CO₂.
Then cells were rewashed with PBS, and a solution of the cyanine dyes
in DMEM culture medium was added at their working concentrations (given
in Figure 6) for 20
min. After cells were rewashed twice with PBS and FluoroBrite DMEM
was added. Fluorescence was excited with 380 nm laser for Hoechst33342,
550 nm for T-1–T-3, and 480 nm for T-4–T-5 using the commercial Nikon High
Content Screening microscope equipped with an Andor Zyla 4.2 sCMOS
camera and heating and atmospheric control chamber, using a 40×
1.30NA Oil objective. The fluorescence intensity was measured using
a special mode in NIS-Elements AR 4.60 software for 8 min. The software
recorded values every 0.05 or 0.1 s. Fluorescence intensity was normalized
as I/I₀, where I₀ is the fluorescence intensity at zero time
point (0 min 00 s) and I is the fluorescence intensity
at a given time point (up to 8 min).