Fluorescence microscopy study of polymorphonuclear leukocyte substrate attached materials

We describe a technique to visualize substrate-attached materials (SAM) of polymorphonuclear leukocytes (PMN) using the fluorescent lipid analog 1,1'-dioctadecyl-3,3,3',3',-tetramethylindocarbocyanine-perchlorate (DiC18Icc). DiC18Icc was incorporated into the membranes of living cells or SAMs. Since cell preparation does not require fixation, SAMs can be rapidly visualized by fluorescence microscopy. SAMs are generated by subjecting attached cells to a shearing force by rinsing with phosphate-buffered saline (PBS). The SAM-labeling protocol identified a membrane compartment as shown by detergent extraction. The SAMs of PMN leukocytes observed with this technique display complex patterns of interconnecting filaments, foci with radiating filaments, and smooth membranous areas with interconnecting filaments. The sensitivity and nondestructive nature of the DiC18Icc-labeling procedure have allowed us to observe filopodia of motile cells. The results are consistent with the hypothesis that locomotion involves a series of attachment and detachment steps. After 60 minutes of locomotion, these trailing filopodia have been measured at lengths up to 100 micron. The amount of membrane associated with these filopodia accounts for roughly 10% of the total membrane area of resting cells. These data set limits for models of membrane flow during chemotaxis.