Triglyceride quantification kit

Oil red O staining was used to determine the accumulation of cellular lipid droplets. After the differentiation process, the cells (2 × 10⁴ cells/well in 24-well plate) were washed with PBS (pH 7.4) and fixed with 10% w/v formalin for 15 min at room temperature. Then, oil red O solution was added to stain cellular lipid droplets for 1 h. After removal of excessive staining solution, the cells were rinsed three times with deionized water and 60% isopropanol. The stained adipocytes were observed under a Nikon Ts2 inverted microscope (Tokyo, Japan). The dye retained in the cells was extracted with 100% isopropanol, and the optical density (OD) was measured at 570 nm with a microplate reader (Anthros, Durham, NC, USA). The OD at 570 nm was calculated as a relative value compared to the total protein content (as determined by BCA assay kit) and presented as % oil red O staining.
In addition, the level of released glycerol in differentiated 3T3-L1 adipocytes was measured with a glycerol assay kit, and the amount of cellular triglyceride was determined with a triglyceride quantification kit, following manufacturer’s instructions (Sigma Aldrich, St. Louis, MO, USA). The triglyceride content was normalized with the total cellular protein content.

Triglycerides were quantified using a Triglyceride Quantification Kit (Sigma-Aldrich) according to the manufacturer’s recommendations. Briefly, triglycerides are broken down into free fatty acids and glycerol, which is then oxidized to generate a fluorescent product (λ_ex/em = 535/587 nm). Fluorescence was monitored using the Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific). Prior to the extraction of the triglycerides, the cells were trypsinized. An aliquot was stained with 0.4% trypan blue solution and were counted using a Luna Automated Cell Counter (Logos Biosystems, Anyang-si, Gyeonggi-do, Korea). The fluorescent results were normalized on the cell number. The results are the mean of three independent experiments that were performed in triplicate ± SD.

The triglyceride content of fetal hearts was measured using a Triglyceride Quantification Kit (Sigma) according to the manufacturer’s protocol.

The liver tissues from d21 male mice were ground in liquid nitrogen. One portion of the ground liver was weighed and used for TG isolation and quantification. TG isolation and quantification were performed using the Triglyceride Quantification Kit (MAK266, Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s manual. The hepatic TG levels were expressed as mg/gram of liver tissue.

Liver tissues were homogenized in 5% NP 40 solution. The concentration of triglyceride (TG) was measured using triglyceride quantification kit (Sigma) according to the manufacturer’s protocol. TG contents were normalized by the protein concentration (BCA protein assay kit, Bio-Rad).

Liver samples were collected after perfusion with 0.1 M PBS. The liver samples were then immediately frozen on dry ice and stored at −80 °C until the analysis. Liver TG concentration was measured using a triglyceride quantification kit (#MAK266; Sigma-Aldrich, St. Louis, MO, United States) following the manufacturer’s instructions. Briefly, Liver tissue samples (100 mg) were homogenized in 1 mL of 5% Nonidet™ P40 Substitute (NP40; #74385; Sigma-Aldrich, St. Louis, MO, United States) as described by Huang et al., 2020. Next, liver samples were heated to 80°C–100°C for 2–5 min and cooled to room temperature with this step repeated once, followed by adding lipase to convert TG to glycerol and fatty acids and adding the master reaction mix to each well to complete the reaction. Finally, colorimetric detection was performed by measuring the absorbance at 570 nm.

Triglyceride in the liver was measured as previously described (Le Marchand et al., 1973 (link); Fujii et al., 2008 (link); Komazaki et al., 2017 (link); Sasaki et al., 2018 (link)). Briefly, the aliquots of liver lysates were added to a microcentrifuge tubes containing 37.5% KOH and heated at 70°C for 30 min. The tubes were placed in 55°C water bath overnight (n = 4). Subsequently, 50% ethanol was added, and the tubes were centrifuged. The supernatants were separated, treated with MgCl₂, left on ice for 10 min, and then centrifuged again. The supernatants and a triglyceride standard (Sigma Aldrich [St. Louis, MO, USA]) were placed in a 96 well black plate with a clear flat bottom, and triglyceride levels were measured using a commercially available kit (Triglyceride quantification kit, Sigma Aldrich [St. Louis, MO, USA]). Preparation was performed by following the manufacturer’s instruction. Fluorescence intensity (λex = 540 nm/λem = 590 nm) was measured with a plate reader (BMG Labtech FLUOstar OPTIMA-6).