Cell experiment:

The HL60 cells are incubated at 6×106 cells/mL in Krebs-Ringer buffer at 37℃ containing 10 μM Dihydrorhodamine 123 (DHR). The generation of O2- is initiated by the addition of 50 nM phorbol 12-myristate 13-acetat (PMA) and the progress of the generation of rhodamine 123 is monitored in 50-μL aliquots (3×105 cells) diluted tenfold before analysis. The uninduced HL60 cells are loaded with 5 μM carboxy SNARF-1 AM acetate (SNARF-AM) in the Na+ medium for 10 min at 37℃ and washed by centrifugation and resuspension to remove unhydrolysed SNARF ester[1].

Dihydrorhodamine 123 is a fluorescent probe (λex=488 nm, λem=525 nm).

In the presence of 10 μM Dihydrorhodamine 123 (DHR) the stimulation of the neutrophil NADPH oxidase by the addition of 50 nM phorbol 12-myristate 13-acetat (PMA) resultes in an increase in the rate of rhodamine generation. The fluorescent intensity of the cells, in the presence of 10 μM Dihydrorhodamine 123, increases with time following the addition of 50 nM PMA. In the presence of 10 μM Dihydrorhodamine 123, induced HL60 cells show a sustained increase in fluorescence following the addition of 50 nM PMA[1].

[1]. Lydia M. Henderson et al. Dihydrorhodamine 123: a fluorescent probe for superoxide generation? Eur.J.Biochem. 217, 973-980.