Cell experiment:

Neutrophils (2.5×106/mL) are preequilibrated in 0.25 μM 3,3'-Dihexyloxacarbocyanine iodide (DiOCg(3)) for 5 min at 37℃ in a 1 cm path length cuvette. Final volume is 2 mL. Fluorescence measurements are made in a spectrofluorimeter (emission wavelength: 510 nm, excitation wavelength: 470 nm). Stimuli are injected by a syringe through an injection port on the spectrofluorimeter, and their volumes never exceed 5% (v/v)[1].

3,3'-Dihexyloxacarbocyanine iodide is a carbocyanine dye which can be used to monitor changes in mitochondrial membrane potential.

Addition of cells to the cuvette containing 0.25 μM 3,3'-Dihexyloxacarbocyanine iodide (DiOCg(3)) leads to an increase in fluorescence, equilibration is rapid and is complete by 4 min. When f-met-leu-phe (10-7 M) is added to neutrophils preequilibrated with 3,3'-Dihexyloxacarbocyanine iodide, there is a short lag period of 5 seconds. The lag period is followed by a rapid loss of fluorescence. Examination of 3,3'-Dihexyloxacarbocyanine iodide loaded neutrophils using fluorescence microscopy demonstrates that in resting neutrophils the brightest fluorescence is associated with long thin organelles which corresponds to the distribution of mitochondria[1].

[1]. Korchak HM, et al. A carbocyanine dye, DiOC6(3), acts as a mitochondrial probe in human neutrophils. Biochem Biophys Res Commun. 1982 Oct 29;108(4):1495-501.