Fitc conjugated goat anti mouse ab

Human ICAM‐1/CD54 Ab (0.5 µg; R&D Systems, Minneapolis, MA, USA) followed by a secondary FITC‐conjugated goat anti‐mouse Ab (1 : 50; Jackson ImmunoResearch Laboratories) was used for ICAM‐1 detection using flow cytometry as previously described [22]. Antigen expression was determined using Flow Cytometer S100EXi (Stratedigm; San Jose, CA, USA) with cellcapture software (Stratedigm, Inc., San Jose, CA, USA) and flowjo v10 (BD biosciences, Ashland, OR, USA). Dead cells were gated out from the analysis.

RD cells grown in 24-well dishes were fixed with 4% paraformaldehyde and penetrated using 0.2% Triton X-100. The following antibodies (Abs) were used to probe EV serotypes: mouse anti-EV71 Ab (1:1000; MAB979, Merck Inc., Kenilworth, NJ, USA) for EV71 and CVA16; mouse anti-coxsackie virus B blend Ab (1:1000; MAB9410, Merck Inc., Kenilworth, NJ, USA) for CVB1 and CVB2; and mouse anti-echovirus blend Ab (1:1000; MAB9670, Merck Inc., Kenilworth, NJ, USA) for Echo9 and Echo30. A fluorescein isothiocyanate (FITC)–conjugated goat antimouse Ab (1:100; Jackson) was used as a secondary Ab. The nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI; 0.1 µg/mL; Merck Inc., Kenilworth, NJ, USA). The cells were viewed under a fluorescent microscope (Leica DM6000B) equipped with both FITC and UV filters. EV antigen–positive cells and DAPI-positive cells from each field (at least four fields from each experiments) were counted and analyzed using the associated MetaMorph software (Nashville, TN, USA).