To test modulating activity of the patient septin antibodies, live
neurons were exposed to patient CSF (1:2) or serum (1:10) diluted in Neural
Basal Plus media with B27 supplement (Gibco, Gaithersburg, MD). Cells were
incubated 6.5 hours at 37°C in a humidified atmosphere of 5%
CO2. Cells were placed on ice, washed with cold PBS and exposed
for 30 minutes to FITC-conjugated goat anti-human IgG (1:200; Southern Biotech).
After washing with PBS, cells were fixed in 4% PFA for 15 minutes at ambient
temperature, exposed for 5 minutes to 0.2% Triton X-100 and blocked in 10% NGS
for 30 minutes. Cells were incubated overnight at 4oC with primary
commercial antibodies, washed in PBS and probed after 1 hour at ambient
temperature with secondary antibodies.
neurons were exposed to patient CSF (1:2) or serum (1:10) diluted in Neural
Basal Plus media with B27 supplement (Gibco, Gaithersburg, MD). Cells were
incubated 6.5 hours at 37°C in a humidified atmosphere of 5%
CO2. Cells were placed on ice, washed with cold PBS and exposed
for 30 minutes to FITC-conjugated goat anti-human IgG (1:200; Southern Biotech).
After washing with PBS, cells were fixed in 4% PFA for 15 minutes at ambient
temperature, exposed for 5 minutes to 0.2% Triton X-100 and blocked in 10% NGS
for 30 minutes. Cells were incubated overnight at 4oC with primary
commercial antibodies, washed in PBS and probed after 1 hour at ambient
temperature with secondary antibodies.