Ix81 fv3000

SUN-449, 7402 and Raw 264.7 cells were seeded in glass-bottom culture dishes (Φ 20 mm) at a density of 8 × 10⁴ cells per well and cultured overnight. After incubation with ONs (25 µg/mL) at 37 °C for 4 h, cells were rinsed with PBS three times, followed by staining with LysoTracker Red DND-99 (100 nM, 30 min), MitoTracker Red CMXRos (200 nM, 50 min), ER-Tracker Red (1 µM, 50 min) and Hoechst 33,324 (10 µg/mL, 20 min). Upon complete washes, cells were observed on Confocal laser scanning microscopy (CLSM, (Olympus IX81-FV3000, Japan) with a 60 oil-immersion objective.

The photostability of ONs was compared with that of the commercial lysosomal tracker probe LysoTracker Red DND-99 after long-term irradiation. SUN-449 cells were seeded in glass-bottom culture dishes (Φ 20 mm) at a density of 8 × 10⁴ cells per well and cultured overnight. Subsequently, cells were contained with ONs (25 µg/mL, 3 h) and LysoTracker Red DND-99 (100 nM, 30 min). Upon complete washes, cells were scanned on CLSM (Olympus IX81-FV3000, Japan) every 15.4 s for 463 s. For ONs, excitation: 488 nm; emission collected: 500–540 nm. For LysoTracker Red DND-99, excitation: 561 nm; emission collected: 570–670 nm.