Notes & Tips Application of gelatin zymography for evaluating low levels of contaminating neutrophils in red blood cell samples Cesare Achilli a , Annarita Ciana a , Cesare Balduini a , Angela Risso b , Giampaolo Minetti a,⇑ a Department of Biochemistry, University of Pavia, 27100 Pavia, Italy b Department of Biology and Plant Protection, University of Udine, 33100 Udine, Italy article info Article history: Received 30 September 2010 Received in revised form 15 October 2010 Accepted 15 October 2010 Available online 2 November 2010 abstract Supposedly ‘‘homogeneous’’ red blood cell (RBC) samples are commonly obtained by ‘‘washing’’ whole blood free of plasma, platelets, and white cells with physiological solutions, a procedure that does not result, however, in sufficient removal of polymorphonuclear neutrophils (PMNs), leading to possible arti- factual results. Pure RBC samples can be obtained only by leukodepletion procedures. Proposed here is a version of gelatin zymography adapted to detect matrix metalloproteinase 9 (MMP-9), selectively expressed by PMNs, in heterogeneous mixtures of RBCs and PMNs that can reveal contamination at levels as low as 1 PMN/10 6 RBCs. Ó 2010 Elsevier Inc. All rights reserved. In red blood cell (RBC) 1 studies, it is common practice to obtain ‘‘homogeneous’’ RBC samples by simply ‘‘washing’’ the whole blood free of its plasma, platelets, and white blood cells (WBCs) with phys- iological solutions. However, what is eliminated with the wash is mainly mononuclear cells (lymphocytes and monocytes), whereas granulocytes, of which polymorphonuclear neutrophils (PMNs) are the major subset, remain mixed with the RBCs. The consequences of this contamination can be deleterious when certain types of stud- ies are carried out in bulk RBC suspensions [1]. Suitable leukodeple- tion procedures should be adopted to obtain pure RBC samples, and a sensitive method for counting low levels of contaminating PMNs is often required. In this work, we propose a novel application of the gelatin zymographic technique showing that whole cell samples can be separated in the electrophoretic gel and that detection of trace amounts of PMNs can be reproducibly obtained even in the presence of large amounts of RBCs at ratios of RBCs to PMNs in the original sample as high as 10 6 :1. Depending on the experimental system, contaminating PMNs may release the content of their cytoplasmic granules (where cathepsin G and neutrophil elastase, the most abundant PMN pro- teases, are stored in fully active form and at the high concentration of 1 lg/10 6 PMNs each) because of accidental cell rupture, treat- ment with detergents, or cell activation by various factors (e.g., contact with glass). Particularly at risk is the common procedure of ‘‘ghost’’ membrane preparation from RBCs by hypotonic lysis, which is prone to proteolytic artifacts if traces of PMNs are present and antiproteases are not used efficiently. Moreover, when study- ing WBC-contaminated RBC suspensions, a certain function and/or component can be erroneously attributed to RBCs when in fact it belongs to leukocytes. Various techniques other than washing have been devised to suitably purify RBCs from blood, starting from filtration through cotton [2] to immunomagnetic cell sorting [3]. At the lab level, we consistently and effectively use a filtration through cellulose method [4]. Having complied with the essential requirement of leukodepletion, researchers would also want to evaluate the purity of the RBC population. Unfortunately, standard techniques cannot be used because the residual WBC content is often below the sen- sitivity threshold for detection by standard automated hematology analyzers and other more sophisticated techniques are not com- monly available at the lab level. A valid method to test contamination by PMNs is the analysis of specific markers of this cell type. Matrix metalloproteinase 9 (MMP-9, gelatinase B) is expressed only in PMNs among human blood cells. Its inactive form, pro-MMP-9, is stored inside the ‘‘spe- cific’’ and ‘‘gelatinase’’ types of PMN granules, and it undergoes activation by proteolytic cleavage only following PMN activation and degranulation. MMP-9 could be revealed by Western blotting and chemiluminescence detection [5,6], but a much more sensitive procedure is that of gelatin zymography [6,7]. This technique is based on the separation of proteins by nonreducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) in a special polyacrylamide gel made by inducing acrylamide to polymerize in the presence of gelatin as the selective substrate of MMP-9. During electrophoresis, pro-MMP-9 is activated in a nonproteolytic manner by SDS [8]. Following electrophoretic separation and a ‘‘renaturation’’ step in the presence of Triton X-100, the gel is incubated at 37 °C in a Ca 2+ -containing buffer 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.10.019 ⇑ Corresponding author. Fax: +39 0382 987240. E-mail address: minetti@unipv.it (G. Minetti). 1 Abbreviations used: RBC, red blood cell; WBC, white blood cell; PMN, polymor- phonuclear neutrophil; MMP-9, matrix metalloproteinase 9; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Analytical Biochemistry 409 (2011) 296–297 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio