Optima l 100 ultracentrifuge

Post-nuclear supernatant prepared in PBS were incubated with 1% sarkosyl for 10 min on ice and centrifuged at 30,000 r.p.m. for 3 h at 4°C using Optima L-100 Ultracentrifuge and Sw55TI rotor (Beckman Coulter, Inc, USA). After collecting soluble fractions (supernatant), insoluble fractions (pellets) were rinsed with PBS and dissolved in 4x sample buffer (8% SDS, Tris HCl 250 mM, pH 6.8, 40% Glycerol, 0.08 mg/ml bromophenol blue and 1.4 M β-mercaptoetanol) with vigorous vortexing and sonication. One twentieth of the supernatant volume was used to dissolve the pellets.

The concentration and purification of the IDVs was carried out modifying a published protocol (Hutchinson et al., 2014 (link)). Briefly, after replication of the viruses on MDCK, the cellular supernatant was collected and centrifuged at 3,000 g for 30 min, at 4°C. The clarified supernatant was centrifuged at 112.000 g in SW28 (Beckman Coulter Optima L-100 Ultracentrifuge) for 2 h, at 4°C, and subsequently, the pellet was suspended in phosphate buffered saline (PBS) and loaded onto a 30–60% (w/v) sucrose gradient and centrifuged at 209,000 g, in SW21 for 2.5 h, at 4°C. The purified virus was collected and the sucrose removed by centrifugation at 112,000 g in SW28 for 2 h, at 4°C.
Inactivation and labeling of the purified influenza D virus were done according to van Riel et al. (2007) (link). Inactivation was carried out by dialysis against 0.1% formalin for 72 h. After inactivation, the virus was dialyzed against formalin in PBS. The inactivated virus was labeled through a 1 h incubation at room temperature (RT) with a 1:1 (v/v) 0.5 mol/L bicarbonate buffer solution (pH 9.5) containing 0.1 mg/ml of FITC (Sigma-Aldrich, United States). The virus was dialyzed overnight in PBS to release unbound FITC, using Pur-A-Lyzer tubes (Sigma-Aldrich, United States).