The choice of lipid is crucial, given that commercially available lipids possess different compositions and properties that can affect transport activity. In this regard, it is advisable to use both the same batch and type of lipid if transport results are to be compared. The chosen lipid should (a) be easy to handle in the laboratory, (b) provide repeatable results, (c) be affordable, (d) be as similar as possible to the biological membranes it mimics (this is especially important when transport assays are performed to explore the activity of novel therapeutic agents). A literature search shows that egg yolk phosphatidylcholine (EYPC) and 2-oleoyl-1-palmitoyl-sn-glycero-3phosphocholine (POPC, Figure ) are the most commonly used lipids; however, 1palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2oleoyl-sn-glycero-3-phosphoglycerol (POPG) have also been employed. For instance, Matile's, Talukdar's, and Ballester's research groups have used EYPC from Avanti Polar Lipids, while Chen and Manna have also used EYPC but purchased from Sigma-Aldrich. On the other hand, the Gale, and Quesada 45, As indicated before, when it comes to choosing the appropriate lipid to conduct transport assays, the decision should be made by considering, among other factors, the biological membranes to be mimicked. In this respect, EYPC is usually selected because it can emulate mammalian phospholipid composition. EYPC is a natural lipid mixture in which POPC is the main ingredient. This mixture is made of lipids with various tail lengths and a number of unsaturated bonds, which may result in slightly different compositions and average molecular weights for different batches; however, this disparity between batches depends on the purity of the purchased product. In contradistinction, POPC is a synthetic lipid product with a certified purity, which ensures a higher consistency is maintained between batches and, in turn, affords reliable data. Like EYPC, POPC also very closely mimics a mammalian phospholipid composition due to the positions of the (un)saturations on the chains while also having a known distribution of fatty acids. However, it should be noted that the free fatty acid content (not the type) may differ depending on the supplier. While this does not affect the chloride/nitrate transmembrane exchange monitored by a chloride-selective electrode, it may have some impact on the results of HPTS-based assays, as fatty acids enhance proton transport across the vesicles' membrane (see the Experiments section). Finally, it is important to note that sometimes a mixture of lipid and cholesterol has been prepared to carry out transport experiments due to the contribution of the latter to the stabilisation of the vesicles' membrane. Their proportions may vary, but Quesada's group uses a 7:3 POPC:cholesterol mixture with good results.