Spectramax i3x microplate spectrophotometer

Free radical scavenging activity was assessed using a modified version of the method proposed by Cheng, et al. [54 (link)] (n = 4–6 per group). In brief, 100 μL of sample extracts (4, 20, 100, 500, 2500 μg/mL) or isolated compounds (10, 25, 50, 100, 200 μM) dissolved in 50% ethanol were added to 100 μL of 0.2 mM DPPH (Fisher Scientific, Leicestershire, UK) solution, reaching a final concentration of 0.1 mM DPPH. Each reaction mixture was shaken for five seconds and left to stand for 30 min in the dark at 25 °C. The absorbance of each mixture was measured at 515 nm against a blank (50% ethanol) using a SpectraMax i3x Microplate spectrophotometer (Molecular devices). The inhibition rate of DPPH radicals was calculated as follows:

The production of superoxide was determined by measuring the superoxide dismutase (SOD)-inhibitable reduction of tetrazolium salt, WST-1, as described previously with modifications [14 (link)]. C57BL/6 J mixed glia cultures in 96-well plates were treated with vehicle or iC3b in 100 µl of 1 × HBSS without phenol red. After the treatment, 50 µl of WST-1 (1 mM) in HBSS with and without 800 U/ml SOD was added immediately. The absorbance at 450 nm was read with a Spectra Max i3X microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA), and the data at 30–40 min post-treatment were analyzed. The difference in absorbance observed in the presence and absence of SOD was considered to be the amount of superoxide produced, and results were expressed as the percentage of vehicle-treated control cultures.
The levels of tumor necrosis factor α (TNF-α) in the culture medium were measured with commercial ELISA kits from R&D Systems following the manufacturer’s instructions. The results were quantified using a Spectra Max i3X microplate spectrophotometer.