Gel filtration spin column

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.

Serum CPP levels were measured by the gel filtration assay as we reported previously^{8 (link)}. Briefly, serum and OsteoSense 680EX (PerkinElmer), a near-infrared fluorescent probe that binds to crystalline CaPi, was added to Dulbecco’s Modified Eagle Medium (DMEM) containing 100 mM HEPES (pH 8.0). After incubation at 25 °C for 60 min, the mixture was applied to a gel filtration spin column (Bio-rad, molecular weight cut-off 40 kDa) and centrifuged at 1000g for 2 min. The flow-through was diluted with the same volume of 2% SDS and 100 mM EDTA and subjected to quantification of the OsteoSense fluorescence using a scanner (Odyssey CLx, LI-COR, excitation at 685 nm, emission at 700 nm).