Fig. 4

Control studies supporting a complement bystander mechanism for neuron injury. a Astrocyte-neuron cocultures were incubated for 2 h with 20 μg/ml AQP4-IgG and 2% C1q-depleted serum (i) or with 20 μg/ml AQP4-IgG and 2% C6-depleted serum (ii); cocultures were incubated for 2 h with 20 μg/ml AQP4-IgG and 2% human complement that was pre-exposed for 1 h to 1 μg/ml Fc hexamer (complement inhibitor) (iii); AQP4^−/− astrocyte-neuron cocultures were incubated for 2 h with 20 μg/ml AQP4-IgG and 2% human complement (iv). GFAP, MAP2, and C5b-9 immunofluorescence as indicated, with dead cells stained red. b Direct astrocyte injury caused by α-aminoadipic acid. Cocultures were exposed to 2 mM α-aminoadipic acid for 75 min, with AQP4 and MAP2 immunofluorescence and dead cell stain shown. Filled arrowheads show dead astrocytes. Micrographs representative of three sets of studies done on different cocultures