Fig 1: Effects of vitronectin-regulated astrocyte secretions on neuronal cell death. (A) Experimental timeline for primary astrocyte and cortical neuron cultures. After the isolation of astrocyte, these were treated with LPS and VN for 6 h, followed by washing out of LPS. The astrocytes were incubated with VN for additional 12 h and ACM was collected. Primary cortical neurons were exposed to ACM and CD59 for 8 h and fixed, for neuronal cell death analysis. (B) Immunofluorescence staining images of primary cortical neurons treated with ACM, which was collected from astrocytes with or without LPS and VN, using anti-cleaved caspase 3 (green) and anti-MAP2 (magenta) antibodies, as well as DAPI (white). Arrowheads indicate cleaved caspase 3+ primary cortical neurons. (C) Ratio of cleaved caspase 3+:MAP2+ primary cortical neurons treated with ACM collected from astrocytes with or without LPS and VN. At least 12 images were captured from each coverslip from n = 3 independent experiments, and each image contained > 30 neurons. Statistics used one-way ANOVA with multiple comparisons. (D) Quantification of C3 concentration in ACM collected from astrocytes treated with or without LPS and VN. Data represent mean ± SEM of at least n = 5 independent experiments. Statistics used one-way ANOVA with multiple comparisons. (E) Ratio of cleaved caspase 3+:MAP2+ primary cortical neurons treated with CD59 and ACM collected from astrocytes treated with or without LPS and VN. At least 12 images were captured from each coverslip from n = 3 independent experiments, and each image contained > 30 neurons. Data represent mean ± SEM. Statistics used two-way ANOVA with multiple comparisons.