Fig. 2.

MT6-MMP is localized in lipid rafts of resting and stimulated human PMNs. Freshly isolated PMNs were left untreated or were stimulated with IL-1α (100 U ml⁻¹) for 20 min at 37°C before processing for sucrose gradient fractionation (A) or confocal microscopy (B). In (A), PMN lysates (4 × 10⁷ cells) were overlaid by a sucrose gradient and ultracentrifuged. Nine fractions were collected from the top of the gradient and boiled in reducing or non-reducing 2× sample buffer, as indicated, before MT6-MMP or flotillin-1 detection. (B) PMNs (1 × 10⁶ cells) were fixed by PFA and permeabilized by 0.1% Triton X-100. The cells were then stained for lipid rafts with cholera toxin (CT-488) and the MT6-MMP hinge antibody, as described in Methods. Images were acquired by confocal microscopy, and co-localization between CT-488 and MT6-MMP is shown in the overlay as intense yellow pixels. These results are representative three independent blood donors.