Fluorometric Assay for MMP Activity

Leukemia cell lines were lysed with RIPA buffer (Sigma) following the manufacturer’s instructions. Protein concentrations of the lysates were measured with the BCA protein reagent (Pierce Chemical). 20 µl of lysate was added to 80 µl MMP buffer (10 mM Tris pH 7.5, 150 mM NaCl, 10 mMCaCl2, 0.05% Triton X-100). Fluorogenic peptide substrate IX (R&D Systems) was added to a final concentration of 10 µM. Samples were read in a fluorescent plate reader (excitation 320 nm, emission 405 nm) every 10 min for 70 minutes. Relative fluorescence units were normalized to protein concentration of the lysates [20] (link).