Simplified protocol for isolation of multipotential NG2 cells from postnatal mouse.

Background: The functions of NG2 cells have attracted much attention since they were identified. At present, our understanding of their properties and functions is still limited due to the lack of an easy protocol for isolating them from mice.

New Method: In the present study, in postnatal mouse cortical tissue cultures, cell confluence was achieved at DIV 6-8 by frequently changing the medium in the absence of viable neurons, and abundant NG2 cells grew on top of the astrocyte layer before microglia started to thrive. Thus, we developed a simple protocol to separate mouse NG2 cells by shaking the cultures on an orbital shaker at 37 °C for only 3-4h.

Results: The yield and purity of NG2 cells were sufficiently high, and the cells displayed immunological and electrophysiological phenotypes typical of NG2 cells. They expressed a large delayed-rectifier K+ current (ID) and a transient A-type K+ current (IA) that were electrophysiologically different from astrocytes and neurons. They showed significantly enhanced chemo-attractive migration after application of GABA. They also showed properties of multipotential neuronal precursor cells and were capable of generating oligodendrocytes (54.2±8.1%), neurons (up to 13.3±6.8%), and astrocytes (93.9±4.3%) under defined conditions. Comparison with Existing Method(s): When compared to other methods available for the isolation of mouse NG2 cells, the procedure we present is simple, relatively fast, and economical.

Conclusions: Overall, we present evidence that this new method for isolating NG2 cells from postnatal mice is simple, economical, and effective.