Pyranine

The ratiometric pH-sensitive fluorophore pyranine (from Molecular Probes, Eugene, OR) was prepared at a concentration of 10 mM in milli-Q water. pyranine (final concentration of 300 μM) was mixed with the stocked liposomes (4 mg of lipid) and 100 mM KPi (pH 7.0) in a total volume of 1 mL. pyranine was encapsulated in the liposomes by three freeze-and-thaw cycles at 30°C (65°C for mixtures containing DPPE, DPPC, and DPPG lipids). The osmolality of the liposome lumen is ∼190 mosmol/kg. This value equals the osmolality of the assay buffer (100 mM KPi (pH 7.0)). After extrusion through a 200 nm polycarbonate filter at room temperature (65°C for mixtures containing DPPE, DPPC, and DPPG lipids) to homogenize the vesicles, the liposomes were eluted through a 22-cm-long Sephadex-G75 (Sigma-Aldrich) column pre-equilibrated with the assay buffer to remove the external pyranine. For blank correction, empty liposomes were prepared using the same procedure without the addition of pyranine. The collected 1 mL fractions containing the liposomes were identified using either an ultraviolet lamp (for liposomes filled with pyranine) or a NanoDrop spectrophotometer (for empty liposomes) and diluted in a total volume of 12 mL of the assay buffer.

Sph, POPC and SM from Egg, Chicken were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Chol and TX-100 were obtained from Sigma-Aldrich (St. Louis, MO, USA). trans-parinaric acid (t-PnA) and pyranine were purchased from Molecular Probes/Invitrogen (Eugene, OR, USA). 8-Aminonaphthalene-1,3,6-Trisulfonic Acid, Disodium Salt (ANTS) and p-xylene-bis-pyridinium bromide (DPX) were supplied by Life Technologies (Carlsbad, CA, USA). The organic solvents were obtained from Fluka (St. Louis, MO, USA).
The concentration of the lipid and of the probes stock solutions were determined as previously described^{26 (link)}.