An ex vivo Approach to Assess Mitochondrial ROS by Flow Cytometry in AAV-tagged Astrocytes in Adult Mice.

Mitochondrial reactive oxygen species (mROS) are naturally produced signalling molecules extremely relevant for understanding both health- and disease-associated biological processes. The study of mROS in the brain is currently underway to decipher their physiopathological roles and contributions in neurological diseases. Recent advances in this field have highlighted the importance of studying mROS signalling and redox biology at the cellular level. Neurons are especially sensitive to the harmful effects of excess mROS while astrocytic mROS have been shown to play a relevant physiological role in cerebral homeostasis and behaviour. However, given the complexity of the brain, investigating mROS formation in a specific cell-type in adult animals is methodologically challenging. Here we propose an approach to specifically assess mROS abundance in astrocytes that combines i) a targeting strategy based on the use of adeno-associated virus (AAV) vectors expressing the green fluorescent protein (GFP) under an astrocyte (glial fibrillary acidic protein or GFAP) promoter, along with, ii) a robust and widely extended protocol for the measurement of mROS by flow cytometry using commercial probes. The significance of this work is that it allows the selective study of astrocytic mROS abundance by means of easily accessible technology.