Amplex red

Free fatty acids and glycerol released in the media were measured on a BioTek Synergy H1 microplate reader as previously detailed^{49 (link)}. Free fatty acid release was determined using a NEFA-HR kit (Fujifilm) adapted to fluorescence detection with Amplex Red (10-Acetyl-3,7-dihydroxyphenoxazine, Cayman Chemical) and the fluorescence was read for Resorufin with excitation at 554 nm and emission at 593 nm. Glycerol release was determined using Free Glycerol Reagent (Sigma) and the absorbance was measured at 540 nm.

H₂O₂ release was measured through the conversion of Amplex red reagent by peroxidase to produce the red-fluorescent oxidation product, resorufin [22] (link). Following treatment, IPF fibroblasts were washed twice, and incubated with a reaction mixture (100 µM Amplex red [Cayman Chemical, Ann Arbor, MI], 5 U/ml horseradish peroxidase, 1 mM HEPES in Hank’s Balanced Salt Solution without phenol red). After a 90 minute incubation, signals were measured with excitation and emission wavelengths at 544 and 590 nm, respectively. H₂O₂ concentrations were calculated by plotting against a standard curve.

Cells were seeded on black 96-well plates (2 × 10⁴ Neuro2a cells/ well, 3 × 10⁴ SH-SY5Y cells/ well) and treated as described above.
For determination of H₂O₂ accumulating in the medium during the treatment period, 50 µl conditioned cell culture supernatant of each well were transferred to a new 96-well plate and supplemented with 5 µM Amplex Red (Cayman Chemical, Ann Arbor, USA) and 0.01 U/ml HRP (Thermo Fischer Scientific, Schwerte, Germany). A potential interference between the added agents and the HRP-catalyzed reaction between Amplex Red and H₂O₂ was analyzed by repeating this experiment in a cell-free system utilizing freshly prepared DMEM/0.1% FCS + phospholipids (10 µM) or solvent in the presence of supplemented H₂O₂ (0.000007%).
For measuring the H₂O₂ freshly released by pretreated cells, the supernatant was removed and 50 µl of Amplex Red reaction mixture (5 μM Amplex Red and 0.01 U/ml HRP in phenol red-free DMEM/0.1% FCS) was added to each well.
The fluorescence signal of resorufin was determined at an excitation wavelength of 530 ± 17 nm and an emission wavelength of 590 ± 17 nm for 16 min (120 s (seconds) intervals) at 37 °C under light exclusion in a Safire² Fluorometer (Tecan, Crailsheim, Germany). The increase of fluorescence over time was calculated for each well and used for further data analysis.