Amicon centrifugation filter units

The cells were cultured in DMEM without FBS in a 10-cm culture dish and infected with EV71/2231 for 1 h. Next, they were washed with PBS to remove unbound virus and maintained in DMEM supplemented with 2% FBS for 2 h. The cells were then washed again with PBS and cultured in DMEM only for 6 h. After incubation, the cell culture medium and attached cells were processed separately as described below. The cell culture medium was carefully transferred to 10-kDa Amicon centrifugation filter units (Millipore) and centrifuged at 8000g for 45 min at 4 °C. The retentate was resuspended in lysis buffer (1% Triton X-100, 50 mM sodium chloride, 1 mM EDTA, 1 mM EGTA, 20 mM sodium fluoride, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate in 20 mM Tris-HCl, pH 7.4) containing a protease inhibitor cocktail (Roche) and then collected as the supernatant fraction. The attached cells were washed with PBS, resuspended in lysis buffer, and centrifuged at 17 900g for 10 min at 4 °C. The supernatant was collected as the whole-cell lysate.

Viruses (10⁵ PFU) were incubated with DMSO or RA (280 μM) in a total volume of 1 mL at 4°C for 1 h. The mixtures were transferred to 100-kDa Amicon centrifugation filter units (Millipore) and centrifuged at 8000 × g for 20 min at 4°C. After washing twice with DMEM, the concentrated viruses were resuspended in DMEM and then subjected to plaque assay for titre determination.