A simple and high-throughput in-cell Western assay using HepG2 cell line for investigating the potential hypocholesterolemic effects of food components and nutraceutics.

Since saving time and money are critical issues while developing innovative functional foods and nutraceutics, the use of specific and high-throughput assays for the fast screening of potentially bioactive ingredients is crucial. In this context, the aim of the present investigation was the development of an in-cell Western (ICW) assay, a quantitative colorimetric cell-based technique, at the HepG2 cell line for screening and evaluating the effects of potentially bioactive compounds on the low density lipoprotein (LDL) receptor (LDLR). It is known that LDLR plays a pivotal role in the binding and endocytosis of circulating LDL, increasing its plasma clearance. The ICW was optimised and validated using monacolin K, the main hypocholesterolemic component of red yeast rice. This provided a robust and reproducible assay useful for characterising the cholesterol-lowering properties of bioactive food components. To our knowledge, this is the first application of the ICW technique in the field of functional foods and nutraceutics.