Fluorescence microscopy study of polymorphonuclear leukocyte substrate attached materials

Abstract

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.

Sequential expression of cell surface C3bi receptors during neutrophil locomotion

“…As Figure lb,d shows, fluorescence is restricted to uropods and trailing filopodia. The neutrophil's trailing filopodia are not observed by bright-field photomicroscopy; these structures have been previously detected by fluorescence microscopy (Francis et al, 1988). The data shown in Figure 1 and Table 1 indicate that CR3 redistributed into clusters during spontaneous polarization of neutrophils.…”

“…An accumulation of neutrophils at sites of infection is required for the efficient elimination of microbial pathogens (Miller, 1975). A sequential series of adherence events involving the plasma membrane and extracellular matrix in vivo (Atherton and Born, 1971) or an artificial substrate in vitro (Francis et al, 1988;Marino et al, 1985) are thought to be involved in neutrophil locomotion. However, the detailed biochemical mechanism of neutrophil locomotion on substrates is unknown.…”

Superoxide‐mediated lysis of erythrocytes: The role of colloid‐osmotic forces

“…Scanning electron microscopy Scanning electron microscopy was performed as we have previously described (Francis et al, 1988b). Washed RBCs were suspended in Tris-sucrose buffer or PBS at a concentration of 3 x lo6 celldml.…”

Cell surface distribution of Fc receptors II and III on living human neutrophils before and during antibody dependent cellular cytotoxicity

“…The specificity of the neutrophil's plasma membrane response to bound IgG-opsonized erythrocytes was examined. Neutrophils were labeled with the fluorescent lipid analog diC181cc as described (Petty and Francis, 1986;Francis et al, 1988b). The cell surface distribution of diC181cc was not affected by the bound IgG- coated targets (Fig.…”