Quantifying Plasma CPP Levels

Plasma levels of CPPs were measured in accordance with Miura et al.^{30 (link)}. Plasma (5 μl) was added to 45 μl of Dulbecco’s modified Eagle’s medium (DMEM) containing 100 mM HEPES (pH 8.0) supplemented with 0.5 μM OsteoSense 680EX (PerkinElmer) and incubated at 25 °C for 60 min. The mixture was then applied to a gel-filtration spin column (Bio-rad, molecular weight cut-off 40 kDa) and centrifuged at 1000 × g for 2 min. The flow-through fraction was diluted with the same volume of 2% sodium dodecyl sulfate and 100 mM EDTA. The fluorescence intensity of OsteoSense was quantified using an infrared fluorescence scanner.