Softmax pro microplate data acquisition analysis software

Hydroxyproline content in tissues was determined as previously described^{67 (link)}. Trans-4-hydroxy-L-proline (0–300 µg/mL) was used to generate a standard graph. Absorbance was measured at 550 nm using a SpectraMax M2e Multi-Mode Microplate Reader and SoftMax Pro Microplate Data Acquisition & Analysis Software (Molecular Devices, USA). The value of unknown hydroxyproline was determined from the standard graph. The weight of each respective wound biopsy was used for normalization.

CHO-mTRPA1 cells were seeded into flat-bottom 96-well microtiter plates (Greiner Bio-One) and grown to confluency. Cells were treated with APs or H₂O₂ (300 μM) in PBS for 10 and 30 min at 37 °C in a cell culture incubator. The compounds were removed and BODIPY 581/591 C11 (Thermo Fisher Scientific, Eugene, OR, USA, 10 μm final concentration) was added. After 30 min in culture, cells were washed and resuspended in PBS and the fluorescence of C11 BODIPY was monitored with excitation/emission of 581/591 nm for the reduced dye, and excitation/emission of 488/510 nm for the oxidized dye using a Flexstation III Benchtop Multi-Mode Microplate Reader and the SoftMax Pro Microplate Data Acquisition & Analysis Software (Molecular Devices). The ratio of the emission fluorescence intensities at 591 nm to 510 nm gives the read-out for lipid peroxidation in cells.