ATP induces translocation of A-SMase to the plasma membrane in macrophages. (A) MDMs were treated with a control vehicle or BzATP (200 µM) for 10 minutes. The cells were fixed, permeabilized, and stained for A-SMase using A-SMase–specific antibodies followed by secondary antibodies conjugated with AF567 fluorescent dye. During the secondary antibody labeling, the cells were also labeled with a general cell membrane marker PHK67 green fluorescent cell linker. The immunofluorescence staining was analyzed by confocal microscopy. Arrowheads in the middle panel indicate immunostaining of A-SMase in the plasma membrane following ATP treatment. Yellow color in the right panel indicates colocalization of A-SMase with the membrane marker, illustrating mobilization of A-SMase from intracellular compartments to vesicular membranes for translocation to the other leaflet. (B) Quantification of A-SMase translocation to the plasma membrane. The A-SMase fluorescence intensity on the plasma membrane (30 cells or more) was measured using FIJI software (ImageJ2, Wayne Rasband, National Institute of Mental Health). Mann-Whitney U test was used to determine the significance between control and BzATP treatments. **P < .01.