Figure 8
IL-10 contributes to neuroprotection by ΔRR-infected microglia. (A). EOC20 cells were mock infected with PBS or infected with ΔRR or ΔPK (moi = 5) and CM were collected at 48 hrs p.i. The CM were UV-treated, as described in Materials and Methods, incubated (1 hr; 37°C) with IL-10 neutralizing antibody (20 μg/ml) and assayed for IL-10 by ELISA. (B). Primary hippocampal cultures treated (3 hrs) with NMDA (50 μM) or PBS were extensively washed with MEM and re-incubated with a mixture (1:1) of Neurobasal medium containing B27 supplement and CM from infected microglia that had been treated or not with 20 μg/ml of IL-10 neutralizing antibody. They were fixed 24 h later and co-stained with AlexaFluor-546 conjugated antibody to active caspase-3 (caspase-3p20) and FITC-conjugated antibody to βIIITubulin (neuronal marker). Each experiment was done in triplicate and the % staining cells was determined by counting 5 randomly selected fields (at least 250 cells each, in a 3 mm2 area). Results are expressed as % caspase-3p20+ cells/total number of cells determined by DAPI staining (C). ***p < 0.001, **p < 0.01 as compared to NMDA + Mock CM + IL-10 antibody.