Cholesterol Efflux Assay

Summary

The cholesterol assay is designed to quantitate the rate of cholesterol efflux from cultured cells and the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of labelling cells with cholesterol, equilibration of cholesterol among intracellular pools and release of cholesterol to an extracellular acceptor.

Abstract

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis ¹. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare ². Excessive cholesterol is more common problem ³. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I.

The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required.

The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.

Protocol

1. Preparation of [³H]-cholesterol

1. In the fume hood, dispense the required amount of [³H]cholesterol into a 1.5 ml microfuge tube (0.5 μCi (19 kBq) per well is required for a typical assay).
2. If [³H]-Cholesterol is suspended in toluene, dry it down completely with N2 gas and resuspend with 100% ethanol to a final concentration of 1 μCi (37 kBq/μl. Vortex and mix well.

2. Plating Cells and Labelling Cellular Cholesterol

This protocol has been tested using the following cell types: human monocytes ^4,5,THP-1 human monocyte-macrophages ^6,7,8

Discussion

The described method to measure cholesterol efflux is designed to measure movement of cholesterol from cells to an extracellular cholesterol acceptor. There are several critical considerations in understanding this methodology.

Labelling and equilibration

In the described methodology labelled cholesterol is added to serum-containing serum. Although never investigated in detail, it is assumed that cholesterol is incorporated into serum lipoproteins and they are taken up.......