Bacmam 2

HepG2 cells and U251 cells were seeded
onto a single-well glass bottom dish at 2.0 × 10⁴ cells/well
in a final volume of 100 μL and incubated for ∼24 h at
37 °C, 5% CO₂. CellLight tubulin–GFP, BacMam
2.0 (Thermo Fisher Scientific) was added to the cells (1 μL
per well) and incubated for further 24 h, at 37 °C, 5% CO₂. After removal of the medium, 10 μM TP–TMR in the medium was added to the cells and incubated for a designed
period. For evaluation of the effects of MT-binding drugs, 10 μM
taxol or 10 μM colchicine was coincubated with 10 μM TP–TMR at the same time. For evaluation of the endosomal
escape, 0.1 mg/mL FITC–transferrin was coincubated with 10
μM TP–TMR at the same time. After washing
with PBS, the cells were incubated with 5 μg/mL Hoechst 33342
for 10 min at 37 °C, 5% CO₂. After washing with PBS,
the medium was added to the cells and fluorescence images were acquired. TP–TMR was excited with 550 nm and observed through
a 574 nm emission band-pass filter (red). Tubulin–GFP was excited
with 489 nm and observed through a 510 nm emission band-pass filter
(green). FITC–transferrin was excited with 495 nm and observed
through a 519 nm emission band-pass filter (green). Hoechst 33342
was excited with 352 nm and observed through a 455 nm emission band-pass
filter (cyan).