Ultracell 100 membrane

Vero and Vero-furin cells were grown in 2- and 10-chambered cell stacks (Corning, New York) in DMEM as detailed above. Cells were infected at 80% confluence (~ 3 x 10⁹ cells) with DENV2 16681 at a multiplicity of infection (MOI) of 0.1. Cells were gently agitated at room temperature for 2 h after which they were overlaid with DMEM containing 2% FBS and incubated further. Culture supernatant was harvested and replaced with fresh media on days 3, 4, 5, and 6 post-infection. Virus containing supernatants were clarified by centrifugation at 6000 rpm (JA-10, Beckman Coulter) for 30 min at 4°C, overlaid atop a 24% sucrose cushion prepared in TN buffer (20 mM Tris, 100 mM NaCl, pH 8.0), and centrifuged at 32,000 rpm (Type 50.2 Ti, Beckman Coulter) for 2 h at 4°C. The virus-containing pellet was solubilized in a small volume of TN buffer and loaded on a 10–30% potassium tartrate and 7.5–26% glycerol discontinuous gradient prepared in TN buffer and centrifuged at 32,000 rpm (SW41, Beckman Coulter) for 2 h at 4°C. The virus band was recovered from the 20% density zone by puncturing the tube using a 22-gauge needle. The virus sample was subjected to buffer exchange and was concentrated to a volume of 50–100 μl with an Amicon Ultra-4 centrifugal filter unit with an ultracell-100 membrane (EMD Millipore).

Medium molecular weight chitosan (MMW-CHT; ref.448877; 75% degree of deacetylation calculated by ¹H NMR – Figure S1 - Bruker DRX 300 Avance at 300.13 MHz; CHT was dissolved in a solution containing 98% D₂O and 2% DCl (v/v)), alginic acid sodium salt (ref. A3249), glacial acetic acid and phosphate buffer saline (PBS) were purchased from Sigma-Aldrich, and used as received. Sodium chloride (NaCl) was purchased from LabChem, and sodium hydroxide (NaOH) from AkzoNobel. Deuterium oxide and deuterium chloride (20% in Deuterium oxide) were purchased from Acros. Amicon® Ultra 4 mL centrifuge tubes with Ultracell 100 membrane (100 kDa cutoff), were purchased from Merck Millipore. Mouse fibroblast cell lines L929 (ref. 85011425) were purchased from Sigma-Aldrich, and human adipose-derived mesenchymal stem cells (hASCs; ref. ATCC® PCS-500-011™) from ATCC. Cell culture media Dubelcco’s Modified Eagle Medium (DMEM) was purchased from Sigma-Aldrich (ref. D2902). Minimum Essential Medium Eagle-Alpha Modification (αMEM; ref. 12000014), fetal bovine serum (FBS; ref. 10270106), Alamar Blue reagent (ref. DAL1025), Calcein AM (ref. C3099) and propidium iodide (ref. P1304MP), TrypLe™ Express Enzyme (ref. 12605010) and penicillin/streptomycin solution (ref. 15140122) were acquired from ThermoScientific.