Quantifying Lipid Flippase and Scramblase Activity

Flippase was measured as previously described [14 (link)]. Briefly, cells were resuspended in 3ml of flippase assay buffer and NBD-PS (Avanti Polar Lipids) was added to a final concentration of 3 μM. Cells with NBD-PS were divided into aliquots and incubated for 0, 1, 5, 15, 30 or 45 minutes. After each incubation time, half of the cell suspension was separated for non-extracted sample and kept on ice. The remaining half was spun down to remove non inserted NBD-PS and subjected to BSA extraction of NBD-PS from the outer leaflet by adding 3% fatty acid free BSA (MP Biomedicals) in flippase assay buffer. After incubation on ice for 20 minutes, freshly prepared sodium dithionite (Sigma) was added to a final concentration of 10 mM and incubated a further 10 minutes, to reduce the NBD lipids in the outer cell surface. The cells were spun down and resuspended in flippase assay buffer containing 0.25% BSA and 2 μg/ml PI. NBD-PS signal from unextracted and extracted samples was measured by flow cytometry from living cells after exclusion of PI-positive dead cells, using a BD Fortessa. Non-extractable NBD-PS in the BSA and sodium dithionite treated sample was presented as the percentage of total amount in the control unextracted sample. Scramblase was measured in the same way but with NBD-PC (Avanti Polar Lipids).