Heat shock protein 90ß in the Vero cell membrane binds Japanese encephalitis virus

Wang, Yuan; Li, Yan; Ding, Tianbing

Flow cytometry (FCM). FCM was performed using a routine procedure. Briefly, the Vero cells were treated with PBS containing 3.5 mM EDTA, washed 3 times with FCM dilution, and resuspended (adjusting concentration to 1x10 6 /ml). Vero cells (50 µl per tube) were consecutively incubated with rabbit anti-human HSP90β mAb (1:100 and 1:50), JEV (MOI=1), anti-JEV mAb 2H4 (1:10) and FITC-conjugated goat anti-mouse IgG (1:100) at 4˚C for 1 h with gentle shaking. Each incubation step was interspersed by washing 3 times with an FCM dilution solution and centrifugation at 1,000 rpm and 4˚C for 5 min. Finally, the cells were fixed in 300 µl 4% PBS-buffered formaldehyde, and immunofluorescence was detected using a flow cytometer (Epics Elite ESP; Beckman Coulter Inc., Indianapolis, IN, USA). All detection experiments were performed in quadruplicate.