Dissociated cell culture for testing effects of carbon nanotubes on neuronal growth.

Cell culture has emerged as an important research method for studying the effects of carbon nanotubes (CNTs) on primary neurons. We describe the procedure for preparation of dissociated mixed cell culture from postnatal rat hippocampi. Based on morphological criteria and specific neuronal cell markers, neurons can be selected within this mixed cell culture and studied. We present the procedure for the assessment of neuronal cell morphology based on intracellular fluorescence of the vital dye calcein that accumulates in live neurons. This procedure encompasses fluorescence imaging and measurement of the following parameters: neurite number, total neurite length, mean neurite length, number of growth cones, number of branches, and number of branches per neurite. These combined cell culture and fluorescence microscopy approaches can be successfully used for assessment of the effects that CNTs, as water-soluble agents, have on neuronal cell growth and neurite outgrowth.