Phospholipase c assay kit

BAL isolated using PBS 2 mM EDTA was centrifuged and the supernatant and cell pellet collected. Hyaluronan Quantikine ELISA Kit (R&D Systems) was used to determine the HA concentration in the BAL supernatant. Total protein was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Total PC concentration was determined using the Phospholipase C assay kit (Wako Diagnostics).

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)).