Liposomes were generated by lipid film hydration as described (Kallert et al., 2015 (link)). Briefly, dimethyldioctadecylammonium (DDA; 0.3 mg/ml; Avanti Polar Lipids), D-(+)-trehalose 6,6′-dibehenate (TDB; 0.25 mg/ml; Avanti Polar Lipids, Sigma Aldrich) and L-α-phosphatidylcholine (PC; 0.9 mg/ml; Avanti Polar Lipids) were mixed in chloroform (VWR): methanol (Sigma-Aldrich; 9:1, v/v). The organic solvents were evaporated under nitrogen flow. Liposomes were formed by hydrating the lipidfilm in 500 μl 10 mM Tris-buffer (pH 7.4; Sigma-Aldrich) for 25 min at 57°C within between vortexing, as previously described (Kennerknecht et al., 2020 (link)). Angie1 or Angiogenin were added in a 1:1 mixture to the liposomes at a final concentration of 2.7 mM. The size of liposomes was determined by Nanoparticle Tracking Analysis (NTA) using a ZetaView TWIN (Particle Metrix, Inning, Germany). Samples were diluted in TRIS-buffer and videos of the light-refracting particles were recorded with the following settings: 25°C fixed temperature, 11 positions, 1 cycle, sensitivity 85, shutter 100, 15 fps, 2 s videos/position, and six measurements. The number and size distribution were evaluated by ZetaView Analyze (Version 08.05.05 SP2), as previously described (Conzelmann et al., 2020 (link)).
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