Intracellular H2O2 Measurement Protocol

Intracellular H₂O₂ production was measured using 5- and 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCFDA; Molecular Probes, Eugene, OR, USA) as previously described^{48 (link)}. Briefly, the cells were grown in serum-starved McCoy’s 5A medium supplemented with 1% FBS for 24 h. The cells were then switched to serum-free DMEM without phenol red and exposed to LCA for 30 min. The cells were treated with 10 mM metformin or 1 mM NAC 1 h prior to the LCA treatment to assess their effects on ROS production activated by LCA. The cells were incubated with 5 ng/ml DCFDA for 15 min and immediately observed using an LSM 510 laser-scanning confocal microscope (Carl Zeiss, Germany). The DCF fluorescence was excited at 488 nm using an argon laser, and the emission evoked was filtered with a 515 nm longpass filter.
All obtained fluorescence images taken with the LSM 510 confocal microscope were analyzed using the LSM 5 Image Browser software.