Specified concentrations of AQP4-IgG (or control human IgG, Thermo Fisher Scientific, Rockford, IL) and human complement (Innovative Research, Novi, MI) were added in Hank’s buffer, and cells were incubated at 37 °C for specified times. In some experiments, cells were exposed to serum of an AQP4-IgG seropositive NMO patient who met the revised diagnostic criteria for clinical disease. A fixable dead-cell stain (amine-reactive dye, Invitrogen, Eugene, OR) at 1:1000 dilution was added 30 min prior to cell fixation. In some experiments, C1q- or C6-deficient human complement (Innovative Research, Novi, MI) was used instead of normal complement. In some experiments, the astrocyte toxin α-aminoadipic acid (Santa Cruz Biotechnology, Dallas, TX) at 2 mM was added to astrocyte-neuron cocultures for 75 min.
For live-cell real-time imaging, astrocyte-neuron cocultures were grown on 6-well plates and imaged by phase-contrast optics using a 20×, 0.45 NA objective lens on a Nikon Eclipse Ti microscope equipped with an environmental chamber at 37 °C and 5% CO2. Ethidium homodimer-1 (1 μM, Invitrogen, Eugene, OR) was added to the culture medium prior to image acquisition. Transmitted light (phase-contrast) and red fluorescence images were obtained sequentially every 2 min for a 30-min baseline period and then for 2 h following addition of 20 μg/ml AQP4-IgG and 2% complement.
For live-cell real-time imaging, astrocyte-neuron cocultures were grown on 6-well plates and imaged by phase-contrast optics using a 20×, 0.45 NA objective lens on a Nikon Eclipse Ti microscope equipped with an environmental chamber at 37 °C and 5% CO2. Ethidium homodimer-1 (1 μM, Invitrogen, Eugene, OR) was added to the culture medium prior to image acquisition. Transmitted light (phase-contrast) and red fluorescence images were obtained sequentially every 2 min for a 30-min baseline period and then for 2 h following addition of 20 μg/ml AQP4-IgG and 2% complement.