IL-4 activation does not modify rate or extent of lysosomal contribution to the phagosome. (A-B) Rates of modification of the phagosomal membrane by fusion with lysosomes were assessed by following loss of the early endosomal marker EEA1 and gain of the lysosomal marker LAMP1 on maturing phagosomes in untreated and IL-4–activated BMMØs. After the phagocytosis of IgG-coupled 3-μm experimental particles, BMMØs were fixed with formalin at the times indicated. Phagosomes were assessed for association with the maturation markers EEA1 and LAMP1 by Immunofluorescent microscopy in a blinded fashion. (A) Averaged percentage of phagosomes deemed positive for EEA1 and LAMP1 from 3 independent experiments. Error bars denote SEM. (B) Representative images of untreated and IL-4–activated BMMØs stained for EEA1 (green) and LAMP1 (red) at 10 and 30 minutes. (C) Real-time averaged traces of the phagosomal activity of the lysosomally derived β-galactosidase in untreated and IL-4–activated BMMØs. Phagosomal β-galactosidase activity was measured through the hydrolysis of the β-galactosidase substrate 5-dodecanoylaminofluorescein di-β-d-galactopyranoside conjugated to IgG-opsonized experimental particles relative to calibration fluorescence. RFU values are proportional to the degree of substrate hydrolysis. (D) Averaged phagosomal β-galactosidase activities relative to untreated controls from 4 independent experiments. Activities were determined by calculation of the slope of the linear portion of the real-time trace (as described by y = mx + c, where y = relative fluorescence, m = slope, and x = time) and expressed relative to untreated controls. Error bars denote SEM.