Figure 7.

Localization of C5aR–GFP relative to the plasma membrane of fixed PLB-985 cells. C5aR-GFP–expressing cells were differentiated with 1.3% DMSO, labeled with DiD, and plated on glass coverslips. Cells were then stimulated with a uniform concentration (20 nM) of C5a for 3 min at room temperature and fixed. The GFP and DiD signals were acquired alternatively in the FITC and Texas Red channels, respectively, as described in MATERIALS AND METHODS in successive 0.25-μm focal planes through the sample; out-of-focus light was removed with a constrained iterative deconvolution algorithm (Agard et al., 1989). (A) Maximum intensity projections of three-dimensional data stacks from a polarized DiD-stained, C5aR-GFP–expressing-cell. (B) A single focal plane of the cell in panel A. The arrow and arrowhead point to the front and the back of the cell, respectively. Bar, 10 μm.