Gibco dulbecco s phosphate buffered saline dpbs

Mice were sacrificed by cervical dislocation, and the middle colonic contents were collected and frozen immediately in liquid nitrogen and stored at −80 °C until use. The colonic contents (approximately 100 mg) were diluted 10-fold with GIBCO® Dulbecco’s phosphate buffered saline (D-PBS) (Thermo Fisher Scientific, Waltham, MA, USA) and extracted twice by intense mixing for 1 min and incubating for 5 min on an icebox without agitation. One minute after the extraction, the upper aqueous portion without precipitation at the bottom was collected and centrifuged (10,000 × g for 10 min at 4 °C), and 100 µL supernatant was centrifugally filtered using a 5-kDa cut-off filter Ultrafree-MC (Millipore). The filtrate was stored at −80 °C until further use.

Human feces samples containing HuNoV were acquired anonymously from the existing collection of samples of the University Hospital of Leuven (Belgium). From each stool sample, 100 mg was aliquoted and resuspended in 1 mL of sterile Gibco Dulbecco’s phosphate-buffered saline (DPBS; Thermo Fisher Scientific, Waltham, MA) followed by thorough mixing using a vortex mixer and subsequent centrifugation (5 min at 10,000 × g). Next, the supernatant was harvested, whereby ~500 μL was filtered through a sterile 0.22-μm membrane filter (Merck Millipore, Burlington, MA) using a 1-mL sterile syringe. The supernatants were stored at −80°C.
The virus-DPBS suspensions were used for DNA and RNA extractions, quantification by RT-qPCR, and zebrafish larvae injections. Additionally, 10 μL of the unfiltered and filtered HuNoV suspensions were aerobically cultured on tryptic soy agar (TSA) plates at 37°C to examine the presence of bacteria. HuNoV RNA was extracted from 50 μL virus suspension by adding 350 μL of TRIzol reagent (Thermo Fisher Scientific, Waltham, MA) to the sample, vortexing, and using the Direct-zol RNA miniprep (Zymo Research, Irvine, CA) according to the manufacturer’s protocol.

If not stated otherwise, all chemicals were purchased from Merck KGaA (Darmstadt, DE). 20 mM sodium phosphate buffer (SPB) or a 25 mM multi-component buffer (MCB) containing different urea concentrations (0–4 M) were prepared with ultrapure water (PURELAB Ultra, ELGA LabWater, Lane End, United Kingdom), while Gibco^® Dulbecco’s phosphate-buffered saline (DPBS, Life Technologies Corporation, Grand Island, US-NY) was used as purchased. The MCB had a global capacity of 25 mM in the range of pH 6 to pH 9 and consisted of 47 mM N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS), 11 mM 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO) and 38 mM sodium citrate. At a buffer temperature of 22°C, the buffer was pH-adjusted using 4 M sodium hydroxide solution and filtered through a 0.45 µm cellulose acetate membrane (Pall Corporation, New York, NY, United States).