PHAGOSOMAL PH DETERMINATION BY DUAL FLUORESCENCE FLOW-CYTOMETRY

Several methods have been developed to measure the pH of phagosomes, u sing fluorescein derivatives as reporter of pH, and spectrofluorimetry , fluorescence microscopy, or how cytometry as quantification techniqu e. 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 argon-ion laser excitation wavelen gth at 488 nm. This allowed zymozan-containing macrophages to be disti nguished from other cells and their fluorescence to be measured rapidl y. The use of a new probe, namely Oregon Green 488 which has a pK(alph a) lower than carboxyfluorescein with the same maximum excitation and emission wavelengths, allowed investigation of pH value below 5. The d ual labeling with Oregon Green 488 and carboxytetramethylrhodamine as pH-sensitive and pH-insensitive probes, respectively, overcame the abs ence of an isobestic point in the Oregon Green 488 spectrum. The phago somal pH was determined using a calibration curve of phagosomal pH est ablished by adding ionophores in phagocyte suspension and measuring th e fluorescence intensity ratio (535 nm/585 nm) for different pHs. A ph agosomal pH of 4.5 +/- 0.1 can be accurately determined. This method p ermits pH measurements down to 4, even in the presence of nonengulfed reporter particles. (C) 1998 Academic Press.