Fig. 4.
MT6-MMP faces inward in human living PMNs. Freshly isolated PMNs (2.5 × 105 cells) were left untreated (non-permeabilized) (A) or were permeabilized by 0.1% Triton X-100 (B) and then stained with PI and antibodies against MT6-MMP hinge region or CD16 or with an appropriate isotype control. Resulting fluorescence was read by flow cytometry gating on PI-negative cells (non-permeabilized cells) or PI-positive cells (permeabilized cells). In (C), surface proteins of freshly isolated PMNs were biotinylated as described under Methods. Biotinylated proteins were then captured by avidin beads and subjected to reducing SDS–PAGE followed by MT6-MMP detection. Lane 1, non-biotinylated PMNs; lane 2, biotinylated PMNs and lane 3, recombinant MT6-MMP. Shown here are representative results that were replicated in three different blood donors.