Concentrations of CoQ10 and free cholesterol (FC) in cells were determined by HPLC as reported previously.(4 (link)) Briefly, cells were seeded in 24-well plates and incubated for various times. The cell media were removed and the cells were washed twice with ice cold PBS. Then, 400 µl of HPLC grade 2-propanol (Fisher Chemicals, Fairlawn, NJ) was added. Samples were collected and centrifuged. Extracts thus obtained were injected into the HPLC-ECD system. Mobile phase: 50 mM NaClO4 in methanol/2-propanol (7/3, v/v); flow rate: 1.0 ml/min; analytical column: KANTO RP-18 (L) GP, 5 µm × 150 mm × 4.6 mm (Kanto Chemical, Tokyo, Japan); post-reduction column: RC-10, 15 mm × 4 mm (IRICA, Kyoto, Japan). CoQ10 and FC concentrations were determined by an electrochemical detector (600 mV; NANOSPACE SI-1, Shiseido, Tokyo, Japan) and a UV detector (210 nm; SPD-10A, Shimadzu, Kyoto, Japan), respectively.
CoQ10 in sucrose gradient fractions was extracted by using a 5-volume quantity of methanol and a 10-volume quantity of hexane. Triolein was used as an internal standard. Hexane fractions thus obtained were dried under a nitrogen gas stream and redissolved in 2-propanol. The samples were directly injected into the HPLC system described above.
CoQ10 in sucrose gradient fractions was extracted by using a 5-volume quantity of methanol and a 10-volume quantity of hexane. Triolein was used as an internal standard. Hexane fractions thus obtained were dried under a nitrogen gas stream and redissolved in 2-propanol. The samples were directly injected into the HPLC system described above.
