Tg gpo pap kit

Lipids were extracted from the freeze-clamped liver tissue by the method of Bligh and Dyer [19 (link)]. TG levels were quantifiably determined by an enzymatic method specific for TG using a TG GPO-PAP kit (Roche Diagnostics, Australia). A representative image hepatic steatosis in H&E staining for each group was also supplemented.

The concentrations of triglyceride, phospholipid, free and total cholesterol, and protein in whole cervical lymph and its fractions were determined using the following commercial assay kits: TG GPO-PAP kit from Roche (New South Wales, Australia), Phospholipase C assay kit from Wako Diagnostics (Osaka, Japan), Amplex Red Cholesterol assay kit from Thermo Fisher Scientific, (Victoria, Australia) and bicinchoninic acid (BCA) assay kit from Thermo Fisher Scientific (Victoria, Australia), respectively. The manufacturers’ protocols were followed without modification. Cholesteryl ester concentration was determined by deducting free cholesterol concentration from total cholesterol concentration.
Lymph to plasma protein concentration ratio was calculated using the following equation: $\raisebox{1ex}{$\text{Lymph}$}\!\left/ \!\raisebox{-1ex}{$\text{plasma}\text{protein}\text{ratio}$}\right.=\raisebox{1ex}{${\mathrm{C}}_{\mathrm{L}}$}\!\left/ \!\raisebox{-1ex}{${\mathrm{C}}_{\mathrm{P}}$}\right.$
where C_L is the total protein concentration in the lymph and C_P is the total protein concentration in the plasma (obtained from data previously published by our lab) (Gracia et al., 2020 (link)).

Liver triglycerides (TG) were extracted by the method of Folch and determined by a TG GPO-PAP kit (Roche Diagnostic, Australia) as previously described [18 (link)]. Briefly, 30–40mg of each liver sample was homogenized in 4 ml of chloroform/methanol (2:1) using a glass pestle tissue grinder. After the homogenization, the samples were rotated at room temperature overnight to ensure the complete solubilisation of the liver TG. The next day, 2 ml of 0.6% NaCl was added to each sample and followed by centrifugation to separate the aqueous from the organic phases. The lower chloroform layer contained liver TG were carefully transferred into a glass vial and dried completely under the nitrogen or air at 45°C. The extract was reconstituted in absolute ethanol for the determination TG using a POLARstar microplate reader (BMG Labtech, Germany).