Total cholesterol assay kit

The lens and whole brain tissues of control and Nestin-CreER^T2;Qk^L/L mice at P19 were homogenized in the lipid extraction buffer (chloroform: isopropanol: NP-40 = 7: 11: 0.1) (200 μl of the lipid extraction buffer per 10 mg of tissue). After centrifugation at 13,000 × g at 4 °C for 10 min, the supernatant was collected and air-dried to remove the organic solvent. Dried lipid was used to measure cholesterol levels by Total Cholesterol Assay Kits (Cell Biolabs) according to the manufacturer’s instructions. For plasma cholesterol measurement, blood was collected retro-orbitally from control and Nestin-CreER^T2;Qk^L/L mice at P19, immediately followed by treatment of 0.5 M EDTA as an anticoagulant. Then, the blood samples are centrifuged at 2000 × g at 4 °C for 10 min, and the top layer was used with 1:50 dilution for cholesterol measurement as shown above.

The corpus callosum tissues in Qk-Nestin-iCKO mice (n = 6) and control mice (n = 5 mice) were homogenized in a lipid extraction buffer (chloroform:isopropanol:NP-40, 7.0:11.0:0.1). After centrifugation at 13,000 g at 4°C for 10 min, the supernatant was collected and air-dried to remove the organic solvent. Dried lipid was used to measure cholesterol levels using Total Cholesterol Assay Kits (Cell Biolabs).

Cells were FBS starved for 6 hours with DMEM containing 0.1% BSA and incubated with KRH buffer (4 mM KH₂PO₄, 1 mM MgSO₄, 1 mM CaCl₂, 120 mM NaCl, 10 mM NaHCO₃, 30 mM HEPES, pH 7.4) for 20 minutes. Cells were then treated with insulin at final concentrations of 0, 1, 10, or 100 nM for 20 minutes. Glucose uptake was assessed using 2-NBDG (Thermo Fisher Scientific) at a final concentration of 100 μM for 1 hour. After incubation, cells were washed twice with PBS and the signal read using a fluorescence plate reader at excitation of approximately 465 nm and emission of approximately 540 nm. Plasma and liver cholesterol levels were measured using Total Cholesterol Assay Kits (STA-384, Cell Biolabs Inc.). Plasma and liver triglyceride levels were measured using the Triglyceride Colorimetric Assay Kit (10010303, Cayman).