Astrocytes activated by BDV-infected neurons induce microglia activation in mixed cultures. A – Lectin staining of round microglia in mixed cultures left either uninfected and untreated (control), or BDV-infected and untreated, or uninfected but treated with conditioned media from AF5-activated astrocytes. Scale bar – 50 μm. The quantitative data for AF5-activated astrocytes are presented in C (column a). B – LPS-induced production of TNF-α and IL-1β by mixed cultures. TNF-α and IL-1β were measured by ELISA. Note a significant up-regulation of the cytokines in mixed cultures activated by conditioned media from astrocytes pre-treated with media collected from BDV-infected AF5 neuronal cells. Values are given as ratio compared to concentrations measured in mixed cultures exposed to conditioned media from astrocytes pretreated with media collected from mock infected AF5 neuronal cells, n = 3–5. C – Activation of microglia in mixed cultures following exposure to medium from astrocytes conditioned with medium from different types of infected neuronal cells. Astrocytes can be activated by persistently BDV-infected neuronal AF5 cells (a) or CA77 cells (b). Medium from persistently BDV-infected astrocytes (c) as well as LPS treatment of mixed cultures (d) were used as positive controls. Conditioned media from different pre-treated astrocyte cultures were added to mixed cultures to activate microglia. The results are numbers of round microglia cells in BDV-treatments normalized to respective mock-treated sister cultures, n = 3–6. The results of the paired t-test on two populations (mock vs. mean): *p < 0.05.