Fig. 2.

Efficiency and kinetics of lipid exchange between lipid-loaded MαCD and A549 cells at 37 °C. (A) Kinetics of lipid exchange using 1.5 mM bSM (or 1:9 mol:mol NBD-DPPE/bSM) and 40 mM MαCD. Removal of endogenous SM was measured by ³H SM remaining in ³H labeled A549 cells after lipid exchange; 10% of the samples were analyzed. Delivery of NBD-labeled fluorescent lipid was assayed from the level of cell-associated NBD fluorescence. Fluorescence units are arbitrary. (B) Effect of MαCD concentration on the residual percentage ³H SM = [(cpm SM after exchange)/(cpm SM before exchange)] × 100% remaining in A549 cells after lipid exchange. Percentage exchanged endogenous SM = 100% − percentage residual ³H SM. The exogenous lipid was 1.5 mM bSM. (C) Effect of the concentration of lipid mixed with MαCD on the residual percentage ³H SM remaining in A549 cells after lipid exchange. MαCD concentration was 40 mM. Some cells rounded up when exogenous bSM concentration was 0.2 mM or less. (D) Different lipid combinations resulted in similar levels of percentage ³H SM exchange. The lipids were composed of 1.5 mM bSM, 3 mM POPC, 3 mM 1:1 (mol:mol) bSM/POPC, or bSM/1,2-dioleoyl-sn-glycero-3-phosphocholine. MαCD concentration was 40 mM. For B–D, data were normalized to the level of radioactive PS+PI in the same sample, using the equation percentage ³H SM = (cpm SM after exchange/cpm SM before exchange) × (cpm PS+PI before exchange/cpm PS+PI after exchange) × 100%. PS and PI, which comigrate on TLC, are inner leaflet lipids inaccessible to exchange (as confirmed in Fig. 3A). For A–D, average values and SDs from three experiments are shown. cpm, counts per minute.