Beckman ls 6000sc scintillation counter

Six-well plates were treated with 1 ml Poly-L-lysine for 1 h at RT and washed three times in 1 ml Dulbecco’s phosphate buffered saline (DPBS). HEK293T cells (4.5 × 10⁵) were seeded on the pre-treated plates 24 h prior to transfection. The cells were always triple transfected to co-express ATP8B1, CDC50, and ABCB4 wild-type or variant (as described above). Twenty-four hours post-transfection, the cells were fed 2 μCi [methyl-³H]choline (PerkinElmer, Waltham, MA, USA) and cultured for 24 h. The medium was removed and cells were washed three times in 1 ml fresh medium pre-warmed to 37 °C, and then incubated in 2 ml medium supplemented with 2 mM sodium taurocholate hydrate (TC) (Sigma, St Luis, MO, USA). After 24 h incubation, 50 μl of culture media from each well were analysed for radioactivity content in a Beckman LS 6000SC scintillation counter (Beckman Coulter, Fullerton, CA, USA). The cells attached to the dish were washed three times in 1 ml DPBS and lysed in 2 ml 0.5% (v/v) Triton-X 100. An aliquot (50 μl) from each lysate was analysed for radioactivity to determine the cellular radioactive content. PC efflux was calculated as PC detected in the media as a percentage of the total (media plus lysate), then normalised against an internal non-functional control; the Walker B mutant, ABCB4^E558Q.