Fluostar optima 96 well plate reader system

Fluorescence experiments were performed on a multi-plate reader FLUOstar Optima 96-well plate reader system (BMG Labtech) at 25 °C. Two automatic injectors were used to add ascorbate and hydrogen peroxide solutions into the wells during experiments. Coumarin-3-carboxylic acid (CCA) was used as a probe to detect HO^•, as it reacts with CCA to form 7-hydroxycoumarin-3-carboxylic acid (7-OH-CCA), which is fluorescent at 452 nm upon excitation at 395 nm. The intensity of the fluorescence signal is proportional to the number of 7-OH-CCA molecules formed, which in turn is proportional to the HO^• radicals released.26 (link) A fixed volume of 2 μL of ascorbate (2 mM) and 2 μL of hydrogen peroxide (5 mM) were added every 15 min for 2 hours to 200 μL of phosphate buffered (pH 7.4, 50 mM) solutions containing CCA (0.5 mM) and Cu(ii) (20 μM) with or without the Aβ peptide (Aβ₁₆, Aβ₇, AcAβ₁₆, D7H–Aβ₁₆, Aβ₂₈, AcAβ₂₈, or Aβ₄₀) (25 μM). The fluorescence was monitored every 30 s.