Fluorescence detecting microplate reader

Ten milligrams of mice liver tissue was homogenized in 1 mL of PBS and DCFH levels were determined as an index of the peroxide production as previously reported [26 (link)]. Briefly, 50 µL of freshly prepared liver homogenate was mixed with 4.85 mL of 100 mM potassium phosphate buffer (pH 7.4) and incubated with DCFH-DA at a final concentration of 5 μM for 15 min at 37 °C. After centrifuging at 10,000× g for 10 min at 4 °C, the pellet was suspended in 5 mL of potassium phosphate buffer (pH 7.4) on ice and incubated for 60 min at 37 °C as described previously [26 (link)]. In order to investigate the effect of PN3 extract on AA + iron-induced ROS production, HepG2 cells were incubated with AA (15 h) + iron (1 h) and/or 50–100 µg/mL PN3. Cells were stained with 10 µM DCFH-DA for 30 min at 37 °C. The collected cells were then washed twice with a wash buffer, and H₂O₂ generation was determined using a fluorescence-detecting microplate reader (Jemini, Molecular Device, Sunnyvale, CA, USA) at excitation/emission wavelengths of 485/530 nm. The production of ROS was normalized as per the protein concentration of each treated sample and was defined relative to the vehicle-treated control.

Cellular ROS levels were quantified by measuring the DCF fluorescence intensity [16 (link)]. ROS generation was evaluated in HT22 cells treated with RSL3 and/or GBH for 1 h. HT22 cells were stained with DCFH-DA (10 μM) in an incubator maintained at 37 °C for 30 min. The collected HT22 cells were then washed with PBS, and ROS levels were measured using a fluorescence-detecting microplate reader (Molecular Devices) with excitation/emission wavelengths of 485/530 nm. ROS generation was normalized to the protein concentration of each treated sample and was defined relative to the vehicle-treated control.