Visualizing EMCV Infection Dynamics in Macrophages

EMCV was labeled with Syto‐82 as described previously (Brandenburg et al, 2007 (link)). Briefly, Vero cells were infected with MOI 1 EMCV for 2 h and then incubated with 25 μM Syto‐82 (Invitrogen) at 37°C, 5% CO₂ for 4 h. At end of the labeling reaction, the cells were rinsed, harvested, and freeze‐thawed twice to release the labeled virus for following experiments. Cell debris was removed by filtration through 0.45 μm filter. The viral titer was determined by plaque assay. BMDMs were treated with Syto‐82‐labeled‐EMCV at MOI 1 for 10 min, rinsed and cultured at 37°C, 5% CO₂ for 30, 60, or 90 min, and subjected for live imaging analysis. To determine the co‐localization of EMCV with the endosome‐lysosome network, BMDMs were treated with Syto‐82‐labeled‐EMCV at MOI 1 for 15 min and then stained with 100 nM LysoTracker Green DND‐26 (Invitrogen) for 45 min. Syto‐82 signals were acquired on Zeiss Axio Observer 7 40X lens or 63X oil lens with ApoTome. The images were processed by using the Zeiss Zen 2012 software and quantified by ImageJ.