Preparation of Conditioned Media for Virus Studies

Conditioned media was prepared as described previously [7 (link)]. Briefly, cells were allowed to reach 80–90% confluence, and counted to ensure proper multiplicity of infection (MOI). Cells were infected with or without HSV1716 or UV-inactivated HSV1716 and incubated at 37 °C 5% CO₂ for 24 h. The conditioned media was harvested under sterile conditions and centrifuged at 4000× g for 10 min. Then, supernatants were filtered once using a 0.22 µm Steriflip filter (EMD Millipore, Darmstadt, Germany) followed by a final filtration using a 0.1 µm filter (Sartorius AG, Goettingen, Germany). Conditioned media from virus infected cell lines underwent plaque forming assay to ensure complete virus removal.