The following method was used to calculate the efficiency of neuronal induction. The total number of Tuj1 positive cells with a neuronal morphology, defined as cells having a circular, three-dimensional appearance that extend a thin process at least three times longer than their cell body, were quantified at indicated time points. We determined this number in at least 15 randomly selected 20× visual fields on a Zeiss Axio Observer microscope. The average and standard deviation per field was determined and then used to extrapolate the total number of iN cells present in the entire dish based the known surface areas of a 20× visual field and the respective culture dish. We then divided this number by the number of cells plated before infection to get the percentage of the starting population of cells that adopted neuron-like characteristics. In multiple independent experiments we verified that this extrapolation method yields cell numbers very similar to those measured by a hemocytometer at the time of plating. Data are presented as Mean ± SD.
