Amicon 100k centrifugal filter

It is well known that two forms of EV71 viral particles, full and empty particles, existed during propagation in cells [3] (link), [26] (link). Based on historical poliovirus studies, the full particles are infectious and immunogenic but the empty particles are not [27] (link). Therefore, we purified EV71 infectious (full) particle of the reference viruses for rabbit immunizations. The EV71 culture supernatant was concentrated 10-fold with a Amicon 100K centrifugal filter (Millipore). The crude virus concentrate was loaded onto a 15–65% continuous sucrose gradient and centrifuged at 28000 rpm for 4 hr. Fractions (2 mL per fraction) were collected and the viral titer and protein concentration of each fraction were determined by TCID₅₀ and BCA assays (Thermo Scientific), respectively. Fractions with high infectious virus titers in 32–38% sucrose concentration were merged and concentrated by diafiltration using Amicon 100K centrifugal filter and centrifugation at 3500 g. The purified EV71 viruses were further verified by using Western blot and electron microscopy analysis.

For viral production, HEK293FT cells were cultured in ten plates of 150 × 25‐mm cell culture dishes (SPL #20150) up to 80%~90% confluency. And then cells were transfected with pAAV‐densin shRNA vector, pHelper, and RC plasmids using PEI (Polysciences, Inc. #23966), while pAAV‐GFP was transfected to other ten plates to produce the control AAV virus. After 3 days of transfection, cells were collected, pelleted by centrifugation, re‐suspended in freezing buffer (0.15 M NaCl and 0.05 M Tris, pH 8.0) for 10 min, and then immediately thawed in 42°C for 10 min with shaking. After two cycles of freeze–thaw process for the proper lysis of cells, the mixtures being treated with benzonase (0.5 μl/ml; Sigma, #E1014) were incubated at 37°C for 1 hr and cell debris was removed by centrifuging at 3,580 g at 4°C. The supernatants were flown onto HiTrap Heparin columns (GE Healthcare), which were then washed with cold 5‐ml PBS‐MK (1‐mM MgCl₂, 2.5‐mM KCl in DPBS). Viruses caught in heparin column were eluted by flowing PBS‐MK NaCl (PBS‐MK plus 0.5 M NaCl). The virus solution was concentrated by using Amicon 100 K centrifugal filter (Millipore, # UFC910096) up to 150 μl and stored at −80℃.