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, RAW 264.7 murine macrophages ^5,9,10,11, HeLa cells ¹², human umbilical vein endothelial cells (HUVEC), BHK-21 cells ⁹, human and mouse fibroblasts ^13,14, HepG2 human hepatocarcinoma cells ¹⁵ and, in modified form, from platelets ¹⁶ .

1. Resuspend cells and count them. Plate cells into 12-well plates at the final density of 0.2×10⁶ cells per well in 0.9 ml complete medium. Cells will continue growing and will reach 0.8×10⁶ cells per well by the time of the efflux experiment. For cells that do not divide, such as differentiated THP-1 cells, the seeding density should be 0.8×10⁶ cells per well. If using 6- or 24-well plates, double or half the number of cells per well respectively. The number of wells should be sufficient for quadruplicate determinations for each experimental condition.
2. If differentiation of THP-1 cells is required, add PMA (final concentration 0.1 μg/ml) to the media for 48-72 h.
3. Add a small aliquot of media into the microfuge tube containing the [³H]cholesterol.
4. Wash sides of tube with media before adding [³H]cholesterol into a separate aliquot of complete media (100 μl/well).
5. Add the media containing [³H]cholesterol to the wells with cells (final volume per well is 1 ml). If no treatment of cells is required before labelling, cells may be plated in [³H]cholesterol-containing medium.
6. Incubate cells for 48 hours in cell culture incubator (37 °C, 5% CO₂).

3. Equilibration Incubation

1. After 48 hour incubation, check cells under microscope. Ensure they are healthy and are at approximately 80% confluence.
2. Remove media containing [³H]cholesterol. Wash cells gently with PBS. Repeat 3 washing times.
3. Prepare serum-free media. If required, activate cells by adding LXR agonist (such as TO-901317 at 1-4 μMol/L) or cAMP (0.3 mMol/L; for cells of mouse origin only) to serum free-media.
4. Add 500 μl serum free media to each well.
5. Incubate for 18 hours in the cell culture incubator (37 °C, 5% CO₂).

4. Cholesterol Efflux Incubation

1. After 18 hour incubation in serum-free medium, check cells under microscope. Ensure they are healthy and confluent (minimum 80% confluency).
2. Prepare solution of cholesterol efflux acceptors in serum-free medium. Examples of acceptors include :
   Apolipoprotein A-I (apoA-I) ( final concentration 10 μg/ml)
   High density lipoprotein (HDL)( final concentration 20 μg/ml)
   Cyclodextrin (final concentration 200 μg/ml)
   Plasma (final concentration 1-2%)
3. Wash cells gently with PBS.
4. Add 250 μl of serum-free media with acceptors to each well.
5. Leave one set of wells to determine background efflux (efflux to serum free media with NO acceptor)
6. Incubate cells for 2 hours in cell culture incubator (37 °C, 5% CO₂). Duration of the efflux incubation may vary from 30 min to 8 h if required.

5. Processing Samples

1. After 2 hours incubation check cells under microscope.
2. Collect media into 1.5 ml microfuge tubes. Spin at 14,000 rpm for 1-10 min at room temperature to remove cellular debris.
3. Transfer 100 μl media into 7 ml scintillation vial. Add 5 mls of Insta-gel Plus (PerkinElmer) and vortex mixture. Store remaining samples at 4 °C.
4. Place plates in freezer for 30 mins. Add 500 μl dH₂O to each well. Check cells under microscope to ensure that all cells have lifted from the bottom of wells. If still adherent, either leave plates with dH₂O at 4 °C overnight or scrape wells.
5. Once all cells have lifted off, pipette up and down to break up cell clumps. Transfer 100 μl into 7 ml scintillation vials. Add 5 mls of Insta-gel Plus (PerkinElmer) and vortex mixture. Store remaining plates at 4 °C.
6. Place scintillation vials on scintillation counter. Configure counter to count for [³H] in dpm units.

6. Analysing the Results

1. The rate of cholesterol efflux is usually expressed as a proportion of cholesterol moved from cells to the acceptor. The following formula is used:

2. The specific efflux is calculated as a difference between the efflux in the presence or absence of the acceptor (blank).

7. Timeline

8. Representative Results

An example of an outcome of a cholesterol efflux experiment is shown in Fig. 1. In this experiment THP-1 human monocytes were differentiated into macrophages and cholesterol efflux to different acceptors was tested. Cholesterol efflux to medium with no acceptors (“blank”) was 0.79% and this value was regarded as a non-specific efflux and was subtracted from other values. Cholesterol efflux to human apoA-I (final concentration 30 μg/ml) was 4.75%. A reference plasma sample was included into this experiment to monitor inter-experimental variability. A reference sample can be used for normalization of data across a large number of experiments, but we found it prudent to repeat the assay if variability is high. A patient plasma (final concentration 2%) was tested before and after the patient was treated with medication. It was concluded that in this patient medication had a negative impact on the capacity of plasma to support cholesterol efflux.

Another example of an outcome of the efflux experiment is shown in Fig. 2. In this experiment RAW 264.7 macrophages were activated or not activated by overnight incubation with LXR agonist TO-901317 (final concentration 1 μmol/L) and cholesterol efflux to the same sample of human plasma (2%) was tested. It was concluded that activation of cellular expression of ABC transporters with LXR agonist increases the capacity of cells to release cholesterol to extracellular acceptor.

Figure 1. Cholesterol efflux from THP-1 cells to various acceptors. Percentage of cholesterol efflux (i.e. the proportion of labelled cholesterol moved from cells to the specified acceptor) is shown after subtraction of blank values. Mean ± SD of quadruplicate determinations, *p<0.05.

Figure 2. Cholesterol efflux from RAW 264.7 activated or not activated with LXR agonist to human plasma. Percentage of cholesterol efflux (i.e. the proportion of labelled cholesterol moved from cells to the specified acceptor) is shown after subtraction of blank values. Mean ± SD of quadruplicate determinations, *p<0.001.