Millex gp syringe filter unit

For the analysis of phenylpropanoid glycosides, the methanolic extracts were prepared using 300 mg of plant material (lyophilized biomass of each in vitro culture and dried herb of a soil-grown plants). Extraction was carried out in ultrasonic bath (Polsonic 3, Warsaw, Poland) with methanol. Each sample was extracted five times with 3-mL portions of solvent (30 min per extraction). After extraction, five extracts of each sample were pooled together and centrifuged. The resulting supernatant was filtered using a syringe filter (0.22 µm; Millex-GP Syringe Filter Unit, Millipore, Burlington, MA, USA).
Analysis of phenolic acids was carried out in samples prepared from 10-mL portions of each extract dried in crystallizers to a constant weight and dissolved in 2 mL of methanol. The extracts were filtered through a syringe filter (0.22 µm, Millex-GP Filter Syringe Unit, Millipore).
In addition, culture media (20 mL) collected after each experiment were lyophilized, dissolved in 1 mL of methanol, and filtered using syringe filters (0.22 µm, Millex-GP Filter Syringe Unit, Millipore).

Synchronized worms were grown in liquid culture with the density of 3 worms per microliter and fed with 25 mg OP50 per milliliter until day 1 of adulthood^{67 (link)}. Because a proportion of extracellular vesicles are secreted out of the worms^{68 (link)–70} , the synchronized worms were soaked in M9 to collect EVs. In brief, for each sample, 300,000 worms were collected and washed by M9 to remove excess bacteria. Worms were subsequently cultured in 25 ml M9 at 20 °C for 12 h with shaking at 180 rpm. Worms were sedimented by gravity, whereas the supernatant was collected and filtered by a 0.22 μm Millex-GP Syringe Filter Unit (Millipore). Extracellular vesicles (EVs) were isolated from the filtered medium using Total Exosome Isolation Reagent (Thermo Fisher Scientific) following the manufacturer’s instruction. For gene expression, the isolated EVs were suspended in 700 μl QIAzol Lysis Reagent (QIAGEN). EVs were suspended in 600 μl PBS for other experiments.

Bacteriophage MS2 (ATCC 15597-B1) was propagated using the double agar layer method. The bottom layer (of the tryptone soya agar (TSA) Escherichiacoli agar plates) consisted of TSA (Oxoid, Basingstoke, Hampshire, UK) and the top layer consisted of 4.5 mL of soft TSA mixed with 500 µL of overnight E. coli (ATCC 700891) culture (which had been incubated overnight at 37 °C in typtone soya broth (Oxoid)) and 200 µL of freeze thawed MS2 bacteriophage solution. The plates were then incubated overnight at 37 °C. The plaques were harvested in peptone water (Oxoid) and purified by centrifugation at 3000 rpm for 15 min to separate the host cell debris and the bacteriophage. The supernatant was filtered through a 0.22 μm Millex-GP Syringe Filter Unit (Millipore, catalog number SLGP033RS, Tullagreen, Cork Ireland) and used as a stock solution. This stock was serially diluted in sterile water and the concentration was determined by plating and counting plaques using the double agar layer method described above.