Effects of Blood Collection Methods on Gelatin Zymography of Matrix Metalloproteinases

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Abstract
We simultaneously collected blood samples from 20 healthy volunteers (median age, 36 years; range, 23–55 years) into plastic tubes with no additive, plastic tubes with a silica gel-coated surface as coagulation accelerator, lithium heparin-coated plastic tubes, and potassium EDTA-coated plastic tubes (Monovette Systems; Sarstedt). The tubes were kept at room temperature and centrifuged immediately after venipuncture (1600g for 15 min at 4 °C), and the supernatants were stored at −80 °C until analysis. Human gelatinases were prepared as described previously (7). Gelatin zymography was performed under nonreducing conditions on 7.5% polyacrylamide mini slab gels (Bio-Rad), copolymerized with 1.5 g/L 90 Bloom gelatin (Sigma) (8). Aliquots containing 50 μg of total protein (Bio-Rad) were used for each zymographic test. To assay gelatin lysis, scaled aliquots of proteins were run in triplicate and submitted to computer-assisted densitometric scans using Image Pro-Plus software (Cybernetics); the semiquantitative results were expressed as a percentage vs control or calibrator.