Isopore membrane filter

The phospholipids were dissolved at 0.67 mM with 0.04 mol % TexasRed-DHPE, in 300 μL of a mixed organic solvent (CHCl₃–MeOH, 2:1, v/v). The solution was poured into a 10 mL round-bottom flask. The organic solvent was removed for 5 min by a rotary evaporator (N-1110, EYELA, Tokyo, Japan), equipped with a vacuum pump. The speed of the rotation of the evaporator was 180 rpm, the exhaust rate was 1.2 L/min, and the temperature was set to 40 °C by a water bath. After a lipid film was formed at the bottom of the flask, the flask was placed in a desiccator to remove any residual solvent from the lipid film under a reduced pressure at room temperature, for 17 h.
Next, 2 mL of phosphate-buffered saline (PBS) was heated to 37 °C and was gently poured into the round-bottom flask containing the lipid film. The flask was then sealed and incubated at 37 °C for 2 h. The flask was shaken by a vortex mixer, hourly, and for further dispersing, the sample in the flask was ultrasonicated for 1 h. The room temperature was kept at 22 °C. In order to remove undesired tubular vesicles and giant or large vesicles, which are occasionally formed during lipid film hydration, 2 mL of the sample was taken from the vesicle dispersion and passed through a 0.1-μm IsoporeTM Membrane Filter (Merck Millipore, Darmstadt, Germany), twice. This filtering treatment led to small vesicles of phospholipids in PBS.

3 µL of diluted HAS nanoparticle suspension (0.25 mg/mL) was applied on a 0.1 µm membrane filter (Isopore^TM membrane filter, Merck Millipore, Darmstadt, Germany) and dried overnight in a desiccator. Afterwards, the membrane filter was sputtered with gold (Sputter SCD 040, BALTEC, Liechtenstein) under argon atmosphere. SEM was performed on a CamScan CS4 microscope (Cambridge Scanning Company, Cambridge, United Kingdom) and the sample was visualised with an accelerating voltage of 10 kV, a working distance of 10 mm, and 10,000-fold magnification.

Mid- to late-log phase precultures of Sj were filtrated gently using an Isopore^TM membrane filter (pore size 1.2 μm, Merck Millipore) to minimize bacterial contamination and possible bacterial sucrose synthesis, and then resuspended at 5 ml Sj cell suspension to 5.5 ml fresh MA medium with or without 10 g l⁻¹ NaCl in 20-ml test tubes. Tubes were incubated in the chamber described above for 24 h. Before sucrose extraction, cultures were again filtrated using the Isopore™ membrane filter (pore size 1.2 μm) to minimize bacterial contamination. Cells on the filter were resuspended in MA medium. A 300-μl aliquot of the suspension was used for chlorophyll extraction and measurement, and the remaining suspension was filtered using a Durapore® membrane filter (pore size 0.65 μm, Merck Millipore). The filter with Sj cells was soaked in 7 ml of 80% MeOH and the supernatant subjected to sucrose extraction (Ehira et al., 2014 (link)). Sucrose concentration was measured by high-performance liquid chromatography using a chromatograph equipped with a SUGAR SP0810 column (8 × 300 mm; Shodex, Tokyo, Japan) maintained at 80°C and a refractive index detector, with deionized water as the eluent at a flow rate of 1 ml min⁻¹ and a sample volume of 20 μl. Sucrose content was normalized to chlorophyll a concentration as in previous study (Ehira et al., 2014 (link)).

In the laboratory, sample cups were gently shaken to suspend cells in a homogeneous solution before a subsample (10 mL) was taken. Large swimmers were removed using a 1-mm mesh nylon sieve. The subsample from every cup was filtered through a 3 µm pore size polycarbonate filter using a <50 mmHg vacuum. The filtrate was then collected onto a 0.2 μm Millipore Isopore membrane filter (Millipore, Schwalbach, Germany). Filters were washed with sterile Kara Sea water (~50 mL) as recommended by Metfies et al. [19 (link)]. Total DNA from the filters and from the Sterivex unit was extracted with a NucleoSpin Plant Kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s instructions.
A subsequent analysis of DNA was carried out according to Belevich et al. [28 (link)]. A fragment of the 18S rDNA containing the hypervariable V4 region was amplified with the primers EuF-V4(f) (5′-CCAGCASCCGCGGTAATWCC-3′) and picoR2(r) (5′-AKCCCCYAACTTTCGTTCTTGAT-3′) [32 (link)]. The library preparation and sequencing of the DNA fragments were carried out with TruSeq Nano DNA Kit according to the manufacturer’s protocol by using the Illumina MiSeq system (Illumina, San Diego, CA, USA). The read length was 250 bp; reading was performed from both sides of the fragments. The sequencing was conducted by BioSpark (Moscow, Russia).