Figure 3

Conditioned media from high dose of CD300f-IgG-treated cocultures is neurotoxic. (A) Hippocampal enriched neuronal cultures were incubated with rCD300f-IgG2a or control IgG2a and neuronal viability was defined by MTT (two independent experiments were performed). (B) Mixed hippocampal glial cultures were incubated with rCD300f-IgG2a or control IgG2a and cellular viability was determinate by MTT (two independent experiments were performed). (C) Hippocampal enriched neuronal cultures were incubated with the conditioned media from cocultures treated with rCD300f-IgG1 (1.0 μg/ml) or control IgG1 (1.0 μg/ml), as additional controls we incubated neurons with control conditioned medium and fresh rCD300f-IgG1 (1.0 μg/ml), or fresh rCD300f-IgG1 (1.0 μg/ml) (three independent experiments were performed). (D) Hippocampal enriched neuronal cultures were incubated with mixed-glia conditioned media (one experiment is shown here where the conditioned media was neuron media, an additional experiment with glial media is shown in Suppl. Figure 3). (E) Cytokine levels were measured in conditioned media from glia cultures treated with rCD300f-IgG1 and from neuron-glia cocultures treated with IgG1 and rCD300f-IgG1. (F) Hippocampal enriched neuronal cultures were incubated with conditioned media from cocultures treated with rCD300f-IgG1 (1.0 μg/ml) or control IgG1 (1.0 μg/ml), and co-treated with glial-derived neurotrophic factor (GDNF, 1.0 ng/ml); NMDA glutamate receptor inhibitor 3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP, 100 μM), or GAP junction and connexin hemichannel inhibitor carbenoxolone (CBX, 100 μM) (two independent experiments were performed). Data show mean ± SEM; p corresponds to one way ANOVA followed by Tukey’s test (A–F).