C11-BODIPY581/591, an oxidation-sensitive fluorescent lipid peroxidation probe: (micro)spectroscopic characterization and validation of methodology

C11-BODIPY581/591 is a fluorescent ratio-probe for indexing lipid peroxidation and antioxidant efficacy in model membrane systems and living cells, with excellent characteristics: (i) emission in the visible range of the electromagnetic spectrum, with good spectral separation of the nonoxidized (595 nm) and oxidized (520 nm) forms; (ii) has a high quantum yield and because of this, low labeling concentrations can be used, ensuring minimal perturbation of the membrane whilst retaining favorable signal to noise ratios; (iii) has a good photo-stability and displays very few fluorescence artifacts; (iv) is virtually insensitive to environmental changes, i.e., pH or solvent polarity; (v) is lipophilic and as such easily enters membranes; (vi) once oxidized, C11-BODIPY581/591 remains lipophilic and does not spontaneously leave the lipid bilayer; (vii) C11-BODIPY581/591 localizes in two distinct pools within the lipid bilayer, a shallow pool at 18 Å and a deep pool at < 7.5 Å from the center of the bilayer; (viii) is not cytotoxic to rat-1 fibroblasts up to 50 μM; (ix) is sensitive to a variety of oxy-radicals and peroxynitrite, but not to superoxide, nitric oxide, transition metal ions, and hydroperoxides per se; (x) its sensitivity to oxidation is comparable to that of endogenous fatty acyl moieties.