4.10. Hepatic Triglyceride Analysis

Triglycerides from mouse liver were extracted and quantified as proposed by Adhyatmika et al. [49], and Smith et al. [50]. Briefly, hepatic tissue was homogenized for 40 s in 1 mL of extraction buffer that contained 25 mM Tris, 10 mM sodium phosphate, 150 mM NaCl, 0.1% SDS, 1% Triton-X100, and protease inhibitor. The tissue sample was then diluted with 5 mL of a chloroform/methanol (2:1, v/v) solution vortex briefly and incubated under an ice bath for two hours. The solution was centrifuged at 1650× g for 10 min at 4 °C, and three phases were identified. The upper phase was collected and subject to a second extraction step by adding 600 µL of 4 mM MgCl₂ and vortexed vigorously and incubated under an ice bath for 30 min, followed by centrifugation 1650× g for 10 min at 4 °C. The bottom phase from the first extraction was combined with the bottom phase of the second extraction. The chloroform was removed by evaporation under nitrogen gas in a 37 °C water bath. The triglycerides were suspended with 200 µL of distillate water and vortexed vigorously to create the final emulsion. Finally, the triglyceride content was quantified using the Infinity Triglyceride Reagent (Thermo Scientific, Waltham, MA, USA) following manufacturer’s instructions and normalized to protein content.