DAF-FM detection of nitric oxide in embryonic cortical neurons

In situ imaging of nitric oxide (NO) in cells can be performed using 4-amino-5-methylamino-2’,7’-difluororescein diacetate (DAF-FM) (1). The ability to visualise NO production upon activation of endogenous NOS isoforms in a culture system is becoming increasingly important for studying NO signalling. We have detected NO accumulation in embryonic cortical neurons upon activation of endogenous nNOS following stimulation with the neurotrophin brain-derived neurotrophin factor (BDNF) (2). Importantly, we were able to detect NO accumulation within both the cytoplasm and the nucleus. This was achieved through culturing cortical neurons in low serum and the use of a DAF-FM-DA probe in combination with a CellTracker Red CMTPX probe.