Triglyceride colorimetric assay kit

3T3L1 cells were plated in 12-well plates and kept for differentiation. On Day 1 of differentiation, cells were treated with gymnemagenin at different concentrations (50, 25, 12.5, 6.25, 3.125, 1.56 μM). Triglyceride was measured on Day 4 and Day 8 of differentiation using a triglyceride colorimetric assay kit following the manufacturer’s protocol (Elabscience).

Fat bodies were collected at 24 h post-L. plantarum, -PGN (Lysine-type), or -PBS injection to determine TAG and cholesterol levels, as described [62 (link)]. Fat bodies were homogenized in isopropanol and centrifuged at 10,000 × g for 10 min. The supernatants were used for TAG and cholesterol quantification using a triglyceride colorimetric assay kit (Elabscience, Wuhan, China) and total cholesterol colorimetric assay kit (Elabscience), respectively. For Drosophila, larvae were collected at 3 h post-PGN challenge, homogenized, and centrifuged, following which the supernatants were used for assays.

Plasma levels of insulin, glucose, triglycerides, cholesterol, and non-esterified fatty acid (NEFA) were measured with the following commercial kits: ultrasensitive insulin ELISA kit (Mercodia, Uppsala, Sweden), glucose liquid (Química Analítica Aplicada SA, Tarragona, Spain), triglyceride colorimetric assay kit (Elabscience, Houston, TX, USA), cholesterol liquid kit (Química Analítica Aplicada SA, Spain), and NEFA colorimetric assay kit (Elabscience, USA), respectively. The HOMA-IR index was calculated as fasting plasma insulin (mU/L) × fasting plasma glucose (mmol/L)/22.5.
The citrate synthase activity was measured in the BAT using a colorimetric assay kit (BioVision, Milpitas, CA, USA). Hepatic glycogen was quantified according to the manufacturer’s instruction of a commercial kit (Sigma-Aldrich, USA). In addition, the lipid content was quantified in the liver after extraction with chloroform, as previously described [23 (link)]. Briefly, the tissues were homogenized in chloroform/methanol (2:1) solution. After 3 h of shaking, Milli-Q water was added, and the organic layer was separated by centrifugation (16,000× g, 20 min) and dried overnight. Triglyceride concentration was measured as described above.