Phagosomal pH determination by dual fluorescence flow cytometry

Several methods have been developed to measure the pH of phagosomes, using fluorescein derivatives as reporter of pH, and spectrofluorimetry, fluorescence microscopy, or flow cytometry as quantification technique. All have major disadvantages, including either a slow or inaccurate response. In the present study, pH determination was achieved on J774-cell phagosomes containing dual-labeled zymozan particles using dual fluorescence flow cytometry with an argonion laser excitation wavelength at 488 nm. This allowed zymozan-containing macrophages to be distinguished from other cells and their fluorescence to be measured rapidly. The use of a new probe, namely Oregon Green 488 which has a pKa lower than carboxyfluorescein with the same maximum excitation and emission wavelengths, allowed investigation of pH value below 5. The dual labeling with Oregon Green 488 and carboxytetramethylrhodamine as pH-sensitive and pH-insensitive probes, respectively, overcame the absence of an isobestic point in the Oregon Green 488 spectrum. The phagosomal pH was determined using a calibration curve of phagosomal pH established by adding ionophores in phagocyte suspension and measuring the fluorescence intensity ratio (535 nm/585 nm) for different pHs. A phagosomal pH of 4.5 +/- 0.1 can be accurately determined. This method permits pH measurements down to 4, even in the presence of nonengulfed reporter particles.