Triglyceride assay kit

TAG levels were measured using a triglyceride assay kit (Applygen Technologies Inc.) following the manufacturer's protocols (Ding et al, 2018 (link)). Fat bodies were homogenized in 100 μl of lysis buffer and heated at 70°C for 10 min to inactivate the endogenous enzymes. The samples were incubated with triglyceride reagent for 30 min at 37°C and read with a SpectraMax Plus 384 instrument at a wavelength of 540 nm. The protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Fisher), and the TAG content was calculated on the basis of a standard curve for TAG, with the standard samples being run in parallel with the experimental samples.
Glycogen levels were determined using the Glycogen Colorimetric Assay Kit II (BioVision Inc.). In brief, fat bodies were homogenized in 200 μl of ddH₂O on ice and boiled for 10 min to inactivate enzymes. The samples were centrifuged at 13,000 g for 15 min. The supernatant was used for the glycogen assay. The measured values were normalized to lysate protein levels.

A dry chemistry analyzer was used to measure the concentrations of aspartate transaminase (AST) and alanine aminotransferase (ALT). The liver tissue samples were homogenized, the supernatant was collected, and hepatic triglyceride (TG) concentrations were measured with a triglyceride assay kit (Applygen Technologies Inc., China). Endotoxin was quantified indirectly with lipopolysaccharide-binding protein (LBP) due to the inaccurate results of direct measurements, and serum LBP concentrations were determined using an LBP ELISA Kit (Guduo, Shanghai, China) according to the manufacturer’s protocols.