Testing the nitric oxide dissociation hypothesis for photobiomodulation in cultured cell models

A widely accepted hypothesis for the mechanism underlying photobiomodulation therapy (PBMT) is the light-induced release of nitric oxide (NO) from the active site of cytochrome c oxidase, or complex IV (C-IV) of the electron transport chain (ETC). We used cultured cell models to test the NO photodissociation hypothesis using the fluorescent dye DAF-FM (Thermofisher) for detecting NO release and Cell Titer Glo 2.0 (Promega) for measuring cellular ATP concentration. After optimum dosimetry for cellular NO release at 447 nm was determined, a time course of ATP production was performed to determine if the NO dissociation led to a detectable increase in cellular energy. Additionally, the fluorescent dye indicator, JC-1, was used to distinguish between mitochondria with low and high membrane potential in response to blue light using confocal microscopy.