Fig.1.

Spontaneous oxidation of DHR and effect of added HDL. In a 96-well flat-bottom plate 50 µM DHR was added to each well alone or with 5 µg (cholesterol) of FPLC-purified HDL from a donor with anti-inflammatory HDL (aHDL) and from a donor with proinflammatory HDL (pHDL) in a total volume of 175 µl (saline with 150 mM NaCl and HEPES 20 mM, pH 7.4), each in quadruplicate. Spontaneous air-oxidation of DHR at 37°C was then followed in 2 min intervals using a fluorescence microplate reader set to detect 485/538 nm excitation/emission. A: The means and standard deviations of the quadruplicate fluorescence measurements are plotted over time. B: The rates of change in fluorescence between 20 and 50 min (calculated by linear regression) are plotted for the quadruplicates, as well as means/standard deviations. C: FPLC-purified HDL was added in varying concentrations (cholesterol) to 50 µM DHR in 175 µl in a 96-well flat-bottom plate, and the rate of change in fluorescence was measured. The rates of change in fluorescence (means and standard deviations) are plotted against the amounts of added HDL. DHR with no added HDL demonstrated a rate of 25,342 ± 2,619 fluorescence units/minute (not plotted).