Eclipse ts2 fl fluorescence microscope

Cells were seeded at 40,000 cells/well in 24-wellplates and treated with SI and/or DMOG the next day for 16 h under 3% O₂ in a hypoxia chamber. After treatment, cells were washed with warm culture medium three times and incubated for 30 min under 3% O₂ with 10 µM Image-iT lipid peroxidation sensor (C10445, Thermo Fisher Scientific). After incubation, cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min at 3% O₂. The Image-iT lipid peroxidation sensor is based on BODIPY 581/591 C11 which has a fluorescence emission peak at approximately 590 nm in its native form. Upon oxidation, the emission shifts to approximately 510 nm. Fluorescence signal was detected using an Eclipse Ts2-FL fluorescence microscope (Nikon, Amsterdam, Netherlands) and quantified with IMAGEJ.

Fluorescence microscopy was performed with a Nikon ECLIPSE Ts2-FL fluorescence microscope (Nikon Instruments Inc., Melville, NY, USA) equipped with a C-mount camera, F-mount camera, a 20× objective, an excitation and emission filter set (EGFP: 488/509 nm; mCherry: 587/610 nm) and OPLENIC software (version x64, 10.1.14643.20190511).

Oxidative stress measurements were evaluated using CellROX Green Reagent (Thermo Fischer). In all, 6 × 10⁴ PANC1 cells or 4 × 10⁴ A549 cells have been spread out and subsequently KD was induced. After 3 days under normoxic or hypoxic conditions (1%O₂), cells were incubated with 5 µM CellROX for 2 h. The CellROX itself is non-fluorescent, but if it gets oxidized in cells, the excitation at 488 nm leads to the emission of intense green fluorescence with wavelengths around 520 nm. The fluorescence was detected using an Eclipse Ts2-FL fluorescence microscope with a 20x CFI LWD objective (Nikon, Amsterdam, Netherlands) and quantified with the image processing and analysis software IMAGEJ.