In principle, it should be absolutely fine to nucleofect pure neuron cultures (e.g. after purification by Ficoll). Glial cells are not necessary for transfection. What one would have to consider for example, is to get the required number of neurons, if we recommend using 2-5x106cells per sample, this would mean using 2-5x106neurons. If mixed glial cultures are nucleofected, you not only get transfected neurons, but some glial cells as well. The majority of transfected cells should be neurons.