ε aminocaproic acid εaca

The kinetics of plasminogen activation by tPA with rGAPDH (or not) was further measured using an enzyme-linked immunosorbent assay (ELISA). rGAPDH (20 μg/mL) and plasminogen (20 μg/mL) were mixed and incubated at 37°C for 1 h. The mixture was added to a flat-bottom 96-well plate. Next, 500 ng/mL tPA was added to the wells, the mixture was incubated for 15 min, 0.4 mM substrate was added, and the plates were incubated at 37°C. The OD₄₀₅ was measured every 15 min from 15 min to 120 min. Wells containing only plasminogen with tPA, rGAPDH with plasminogen, or rGAPDH with tPA served as the controls. The experiments were performed in triplicate.
Meanwhile, an ELISA was also applied to determine the role of C-terminal lysine residues in the binding of GAPDH to plasminogen. The methods were as same as the ones described above except that the plates were incubated with 100 μL of plasminogen (2.5 μg/mL or 12.5 μg/mL) for 3 h at 37°C in the presence or absence of 200 mM ε-aminocaproic acid (ε-ACA) (Sigma-Aldrich) (a lysine analog). After 48 h, the activity was estimated from the OD values measured at 405 nm.

TE plaques without HA particles were created following the methodology described previously.²⁷ (link) To summarize, human vena saphena cells (HVSCs) (1.5 × 10⁶ cells/ml) were seeded in 1.0 × 1.5 cm-sized fibrin gels, a suspension of bovine fibrinogen (10 mg/ml, Sigma F8630), and bovine thrombin (10 U/ml, Sigma T4648), cast between two Velcro strips (1 cm long). After seeding, the samples were cultured in a growth medium consisting of advanced DMEM (ThermoFisher) supplemented with 10% Fetal Bovine Serum (Life Technologies), 1% Glutamax (Gibco), 0.1% gentamycin (Invitrogen), 1:167 Fungizone (Invitrogen), and L-ascorbic acid 2-phosphate (vitamin C, 0.25 mg/ml, Sigma A8960) for 21 days under static conditions (37 °C, 5% CO₂). For the first 7 days of culture, ε-Amino Caproic Acid (ε-ACA, 1 mg/ml, Sigma) was added to prevent fibrin break-down.²⁸ (link)

Serous ovarian cancer cells (OAW28, OV-90 & OVCAR-3) were plated in 96 well plates (5000 cells/well) and treated with ATRA (1-5 μM) for 6 days. Cells were washed with 0.1 M phosphate buffered saline (PBS), pH 7.4 and treated for 10 min ± 0.5 μM plasminogen (P7999, Sigma Aldrich) and in the presence or absence of plasmin inhibitor (ε-aminocaproic acid; ε-ACA, 100 mM, Sigma Aldrich) prior to the addition of the plasmin substrate, chromozyme PL (3 mM in 100 mM glycine, Roche Diagnostics, Mannhein, Germany). Plasmin activity was measured at 405 nm using the Triad series multimode detector (Dynex Technologies, VA, USA) over a 2 h period as described previously [6 (link)]. The colorimetric change resulting in the formation of yellow p-nitroaniline is a direct measure of the plasmin activity. Plasmin (0.1 U/ml, P1867, Sigma Aldrich) was used as a positive control and negative controls included parallel wells containing no chromozyme PL substrate and growth media alone, which served as the reagent blank.