Cholesterol efflux assay

The more widely accepted test is the cholesterol efflux assay on cultivated skin fibroblasts. After equilibration with radiolabeled cholesterol, fibroblasts are incubated with albumin in the presence or absence of lipid-free apoA-I. ApoA-I substantially increases cholesterol efflux from normal cells but not from Tangier cells [11, 30]. 

Human skin fibroblasts are cultured from skin biopsy samples. The dermis is cut into small pieces (0.5 mm on each side) and placed into a dish in DMEM containing 10% (v/v) FCS and 1% (v/v) antibiotic-antimycotic solution. When these primary cultures are confluent they are split and cells between passage three and six are used for experiments. For the cholesterol efflux assay, cells are grown in 24-well plates to 60-80% confluence and are labeled with [1,2-3H]-cholesterol (1 pCi/well) for 24 h. Cells are then washed with DMEM and incubated for 4 h at 37°C with DMEM containing BSA (0.2%, v/v) and either 0 (negative control) or 5-30 pg/ml apoA-I. The efflux medium is collected and centrifuged to remove cell debris. Cells are solubilized in 0.1 mol/1 NaOH and the radioactivity in the efflux media and in the cell lysates is determined by scintillation counting [11, 30, 75]. 

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