Nuclepore track etched membrane

Liposome generation and flotation was performed as described in Vollmer et al., 2015 (link). In short, E. coli polar lipids (Avanti Polar Lipids) dissolved in chloroform and supplemented with 0.2 mol% 18:1 Liss Rhodamine PE (Avanti Polar Lipids) were vacuum-dried on a rotary evaporator, dissolved as liposomes in PBS by freeze/thawing cycles and extruded by passages through Nuclepore Track-Etched Membranes (Whatman) with defined pore sizes using an Avanti Mini-Extruder to generate small unilamellar liposomes of defined sizes. For liposome flotations, proteins (6 μM) were mixed 1:1 with liposomes (6 mg/mL) and floated for 2 hr at 55,000 rpm in a TLS-55 rotor (Beckman) at 25°C through a sucrose gradient. Binding efficiency was determined by Western blot analysis using an EGFP antibody (Roche, 11814460001, 1:2000). As secondary antibody, an anti-mouse, horseradish peroxidase conjugated antibody (Calbiochem, 401215, 1:5000) was used. The ImageQuant LAS-4000 system (Fuji) and the AIDA software were used to compare band intensities of start materials with floated liposome fraction.

Bottled water (still, low mineralization) marketed in 1.5 L single use PET bottles from the five top-selling five brands, was purchased from local retailers by the consumers’ organization OCU (Organization of Consumers and Users of Spain). Still water in 1.5 L PET bottles is the most common format sold in the country and the five brands selected represented about 40% or the market share for that type of water in Spain. We sampled 45 L for each brand (225 L for all brands). In all cases, water was bottled in origin at their respective springs as indicated in their labels.
Water was stored in a dark place protected from extreme temperatures and filtered as soon as possible using Whatman Nuclepore Track-Etched Membranes, 25 mm, pore size 0.8 μm, made of black polycarbonate. Black filters were used to make it easy the visual identification of anthropogenic particles, many of which were white or transparent. We conducted vacuum filtration using multiple filters for each brand, filtering a set of bottles with each filter. Each bottle underwent filtration in three 0.5 L aliquots, with the cap being recapped after each aliquot. This process required the caps to be removed three times per bottle (and repositioned twice), replicating the typical handling of bottled water by consumers.

An appropriate amount of chloroform was added to a vial containing lipid or lipid mixture with 1 mol% Rhod-PE for fluorescence observation to obtain 17 mM lipid solutions. The lipid we used is DOPC, SOPC, or POPE. The lipid mixture we used consisted of 50 mol% DOPC and 50 mol% DOPE or 50 mol% DOPC, 20 mol% DOPE, and 30 mol% DOPS. After the chloroform was evaporated with Ar, the vial was placed under vacuum for at least 120 min to obtain a dried lipid film. The dried lipid film was hydrated with DI water under gentle agitation. Suspensions of the hydrated lipid film that contained multilamellar, micrometer-sized vesicles were obtained, followed by extrusion 21 times through a 50 nm polycarbonate membrane filter (Nuclepore track-etched membranes, Whatman, Inc., UK) using an extruder (Avanti Polar Lipids, Inc., USA). The SUV dispersion was stored at 4 °C for future use. The prepared SUV dispersion was spin-coated on the SU-8 microwell templates, which were cleaned with IPA and DI water at a rate of 100 rpm for 30 s and 1000 rpm for 30 s, followed by drying in a freeze dryer for more than 6 h to remove any traces of the aqueous solution.