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Neurons incubated with conditioned media
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In order to avoid the direct effects of serum IgG on neurons [6], instead of co-culture of microglias and neurons and treating both kinds of cells with IgG at the same time, here we cultured microglias or neurons separately. IgG from children was added into the culture media of microglias to collect conditioned media, and then, conditioned media were filtered with protein G agarose to get the IgG-free fraction. Finally, the culture media of neurons were replaced with IgG-free conditioned media from microglias. This method has been used before [29].
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