Ferrozine-iron (II) chelation assay

The iron (II)-chelating activity of PAC-1 was assessed as previously described¹⁷, under nitrogen. Briefly, 19.6 mL double-distilled water (DDW) and 400 μL of 0.2 mM FeCl₂ (final concentration 4 μM) were premixed, and an aliquot of 198 μL was added into each well of a 96-well plate (Corning 3599). Next, 2 μL/well of 100× drugs in DMSO were added. The plate was shaken on a microplate shaker for 5 min and incubated for 30 min to allow for equilibration. Next, ferrozine (final concentration 200 μM) was added, and the plate was shaken on a microplate shaker for 10 min. Finally, the absorbance at 550 nm was scanned using a SpectraMax M5 instrument (Molecular Devices, Eugene, OR). The signal for control group 1, which contained only DDW and ferrozine, was set as 100% iron chelation, whereas the signal for control group 2, which contained DDW, FeCl₂, and ferrozine, was set as 0% chelation. The chelation rate for the treatment groups was calculated as follows:

CR = (OD_treatment - OD_control2) ∕ (OD_control1 - OD_control2) × 100%