Neutrophil NADPH oxidase does not assemble on macropinocytic vacuole membranes

NADPH oxidase assembly is considered to occur mainly on the plasma membrane and phagolysosome membranes and to a lesser extent in intracellular granules. Stimulation of neutrophils with phorbol 12-myristate 13-acetate (PMA) in conjunction with chemiluminescent substrates has been widely used as a model to understand the sub-cellular locations of NADPH-oxidase activity. Interpretation of data from such studies is complicated by observations that PMA does not trigger phagocytosis but results in formation of large macropinocytic vacuoles. Here we show by laser-scanning confocal microscopy that PMA triggers uptake of lucigenin into macropinocytic vacuoles and that no chemiluminescence (CL) can be detected at this location when NADPH oxidase assembly on the plasma membrane still occurs. This shows that the macropinocytic vacuole membrane is distinct from the phagocytic vacuole membrane although both compartments are derived from the plasma membrane.