Infinity triglyceride reagent

Triglyceride assay was performed as previously described [9 (link)]. Briefly, adult flies Flies (12 per genotype, 1:1 ratio of males:females) were weighed and homogenized in PBS containing 0.1% Triton-X100 in an amount (μl) that is 8 X the total weight of flies (μg). The homogenates were centrifuged and the supernatants were removed and incubated with Infinity Triglyceride Reagent (Thermo Electron) for 30 min at 37°C. The absorbance at 540 nm was then measured and TG content was calculated from a standard curve constructed with solutions of known TG concentrations (Thermo Electron). The results were normalized to the protein concentration (μg/μl) of each sample with the Bradford assay (5000002, Bio-Rad).

Liver tissue and cell samples (20 mg) were homogenized in 400-μl HPLC-grade acetone. After overnight incubation with agitation at room temperature, 50 μl aliquots of acetone-extracted lipid suspension were used to determine triglyceride concentrations, employing the Infinity triglyceride reagent (Thermo Electron). Hepatic lipid content was defined as mg of triglyceride per g of total liver or cellular proteins, as described earlier.

Triglyceride assay was performed as previously described [43 (link)]. Flies (10–15 per genotype) or third-instar larvae (10–15 per genotype) were weighed and homogenized in PBS containing 0.1% Triton-X100 in an amount (μl) that is 8 X the total weight of flies (μg). The homogenates were centrifuged and the supernatants were removed and incubated with Infinity Triglyceride Reagent (Thermo Electron) for 30 min at 37°C. The absorbance at 540 nm was then measured and TG content was calculated from a standard curve constructed with solutions of known TG concentrations (Thermo Electron). The results were normalized to the protein concentration (μg/μl) of each sample (Bradford assay).

HFD mice treated with either vehicle or SN-401 were anesthetized with 1–4% isoflurane followed by cervical dislocation. Gross liver weights were measured and identical sections from the right medial lobe of the liver were dissected for further examinations. Total triglyceride content was determined by homogenization of 10–50 mg of tissue in 1.5 ml of chloroform:methanol (2:1 v/v) followed by centrifugation at 16,260×g for 10 min at 4 °C. A 20 µl of the supernatant was evaporated in a 1.5 ml microcentrifuge tube for 30 min. Triglyceride content was determined by adding 100 µl of Infinity Triglyceride Reagent (Fisher Scientific) to the dried sample followed by 30 min incubation at RT. The samples were then transferred to a 96-well plate along with standards (0–2000 mg/dl) and absorbance was measured at 540 nm and the final concentration was determined by normalizing to tissue weight. For histological examination, liver sections were fixed in 10% zinc formalin and paraffin-embedded for sectioning. Hematoxylin and eosin (H&E) stained sections were then assessed for steatosis grade, lobular inflammation, and hepatocyte ballooning for nonalcoholic fatty liver disease (NAFLD) scoring^{46 (link),95 (link),96 (link)}.