Flow cytometry.

Cell surface and intracellular ORF3 protein localization was additionally examined by flow cytometric analysis using a CytoFLEX flow cytometer (Beckman Coulter; 405/488-nm lasers) and Cytexpert software (2.3 version). Vero cells grown in 6-well plates were infected with PEDVs. After the desired time of incubation, the cells were trypsinized and harvested by centrifugation, after which they were washed twice by gentle resuspension in PBS and centrifugation. Half of the cells were fixed with 4% PFA and permeabilized for 30 min with ice-cold methanol. Both permeabilized and nonpermeabilized cells were resuspended in fluorescence-activated cell sorting (FACS) buffer (PBS containing 0.1% BSA) and subsequently stained with antibodies against the ORF3 protein as well as, for comparison, with antibodies against the PEDV structural proteins S, M, and N and with antibodies against cell surface marker hsp90β. Permeabilized and nonpermeabilized cells were stained with primary antibodies for 1 h at room temperature, washed twice with FACS buffer, and finally resuspended in 100 μl of the same buffer. The primary signal was detected using Alexa Fluor 647-conjugated goat anti-rabbit IgG (1:100) or Alexa Fluor 488-conjugated goat anti-mouse IgG (1:200). At least 5,000 events were acquired and FACS data were computed using Cytexpert software (2.3 version).