Millex gv

Saliva samples were thawed on ice and then, to inhibit enzymatic activity, 1 μL of RiboLock RNase Inhibitor (40 U/μL) (Thermo Fisher Scientific, Oslo, Norway), together with 5 μL of cOmpleteTM Mini Protease Inhibitor (Roche), were added per 1 mL of saliva and mixed well. Filtered phosphate-buffered saline (PBS) (0.1 μm filter, Millex^®-GV, Merck Millipore, Cork, Ireland) was subsequently added to each sample at a ratio of 1:1. Two centrifugation steps were performed: 300 g for 10 min, 4 °C, to remove cells, bacteria, and food residue, and 10,000× g for 20 min, 4 °C, to remove cellular debris and other particulate matter. Next, 500 μL of each sample was mixed with an equal volume of filtered PBS and then filtered with a 0.22 μm filter (Millex^®-GV, Merck Millipore, Cork, Ireland).

Cultures from different S. Typhimurium strains were normalized by their OD₆₀₀ readings. Next, cells were pelleted down at 5,000 rpm 4 C. In order to remove residual cells, the supernatant was filtered using a 0.45-µm-pore-size polyvinylidene difluoride (PVDF) membrane followed by a 0.2-µm PVDF membrane (Millex GV; Millipore). The filtrate was subjected to ultracentrifugation at 100,000 × g for 3 h to harvest OMV (Optima L-90K ultracentrifuge; Beckman Coulter). The supernatant was discarded, the vesicle pellet was washed with sterile PBS, and the ultracentrifugation step was repeated. The final vesicle pellet was resuspended in PBS.

To prepare C. difficile culture supernatants for cytotoxicity assay, C. difficile isolates were grown on BHI-T plates for 48h anaerobically and a single colony was transferred into TY broth [69 ] for an additional 4 days culturing in an anaerobic chamber at 35°C. Bacteria cultures were subsequently centrifuged at 4690 x g for 15min. Supernatants were filter sterilized with 0.22 μm low protein binding filters (Millex-GV, Cat# SLGV033RS, Millipore, Etobicoke, Canada) and aliquots were stored at 4°C for subsequent cytotoxicity test and toxin concentration assay by ELISA.

HEV positive swine fecal sample was from an experimentally infected pig with a genotype 4 HEV strain (GenBank accession no.: EF570133) with a titer of 10³-10⁴ pfu/mL [22 (link)]. This sample was proved to be negative for PEVs (including PTV and PEV1-10), haemagglutinating encephalomyelitis virus, Aujeszky’s disease virus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, classical swine fever virus, Japanese encephalitis virus, porcine transmissible gastroenteritis virus, porcine epidemic diarrhoea virus, porcine rotavirus, porcine sapovirus, cytomegalovirus, porcine Torque-Teno virus and porcine parvovirus by RT-PCR or PCR methods [22 (link)]. The fecal sample was converted to 10% (w/v) suspensions in PBS (pH7.4) and clarified by centrifugation at 15, 000 g for 30 min. Supernatants were purified by passage through 0.22 μm microfilters (Millex-GV, Millipore, Japan) before viral inoculation. The HEV negative swine fecal samples were treated in the same way as control.

Defrozen pollen material (0.25 g) was mixed with 10 mL of solution and 2.0 mg mL⁻¹ of protease. Samples were sonicated with an ultrasound probe for 2 min and stirred in a water bath at 37 °C for 4 h. Then, samples were cooled at room temperature and centrifuged for 10 min at 9000 rpm. The supernatant was filtered through 0.22 μm Millex GV filters (Millipore Corporation, Billerica, MA, USA). The standards solutions (1, 5, 10, and 20 μg L⁻¹) for inorganic (i.e., selenite, SeO₃⁻² and selenate, SeO₄⁻²) and organic (i.e., selenocystine, (SeCys2); Se-(methyl) selenocysteine, (SeMeSeCys); selenomethionine, (SeMet) were employed. Se forms were prepared with ultrapure (18.2 MΩ cm) water. Speciation of Se was performed by HPLC (Agilent 1100, Agilent Technologies, Santa Clara, CA, USA) using an anion exchange column (Hamilton, PRP-X100, 250 × 4.6 mm², 5 μm particle size). The mobile phase was made by ammonium acetate with gradient elution. The analytical method and instrumental conditions were previously described in [9 (link)].