Plasmin Activity Assay in Cells

Cells were seeded at 1 × 10⁵ cells per well on a 96-well plate and adhered overnight. Cells were serum-starved overnight and then incubated with 1 µM Glu- or Lys-Plg, as specified in figure legends; Plg concentration was determined experimentally after testing doses from 1–10 µM (data not shown), and the lowest dose was chosen to mimic normal serum Plg concentrations [36 (link)]. A plasmin activity assay was then run according to the manufacturer’s specification (Plasmin Activity Assay Kit, Abcam, Cambridge, UK). Briefly, after cell incubation with test samples for 1–12 h, as indicated in figure legends, 50 µL of plasmin substrate was added to each well. Substrate cleavage was measured by fluorescence detection at Ex/Em 360/450 nm in kinetic mode (read every 2.5 min for 22 min) on a Synergy HTX Multi-Mode Microplate Reader. To measure activity in the supernatant, experiments were set up as above, then supernatants collected and moved to clean assay wells following a 1 h incubation with Plg. Supernatants were replaced on cells with serum-free media. Pla substrate was then added to cells and supernatants separately, and activity assessed as above.