REPORT Cell in situ zymography: an in vitro cytotechnology for localization of enzyme activity in cell culture Aastha Chhabra & Astha Jaiswal & Umang Malhotra & Shrey Kohli & Vibha Rani Received: 13 March 2012 /Accepted: 14 June 2012 /Published online: 21 July 2012 / Editor: T. Okamoto # The Society for In Vitro Biology 2012 Abstract In situ zymography is a unique technique for de- tection and localization of enzyme–substrate interactions ma- jorly in histological sections. Substrate with quenched fluorogenic molecule is incorporated in gel over which tissue sections are mounted and then incubated in buffer. The enzy- matic activity is observed in the form of fluorescent signal. With the advancements in the field of biological research, use of in vitro cell culture has become very popular and holds great significance in multiple fields including inflammation, cancer, stem cell biology and the still emerging 3-D cell cultures. The information on analysis of enzymatic activity in cell lines is inadequate presently. We propose a single-step methodology that is simple, sensitive, cost-effective, and func- tional to perform and study the ‘in position’ activity of enzyme on substrate for in vitro cell cultures. Quantification of enzy- matic activity to carry out comparative studies on cells has also been illustrated. This technique can be applied to a variety of enzyme classes including proteases, amylases, xylanases, and cellulases in cell cultures. Keywords Cell culture . Fluorescent assay . in situ zymography . MMPs . Proteases Introduction A variety of molecular techniques are available with researchers for characterization of enzymes and determining their biological activity. Substrate zymography has long been used as an electrophoretic technique for detection of substrate specific enzymatic activity in polyacrylamide gels (Hawkes et al. 2001). Certain limitations pertaining to ho- mogenization of tissue samples, obstruction in localization owing to protein extraction, and incorporation of complex substrates in gel, have compelled the scientists to look for alternative techniques which have the ability to detect, lo- calize, and assess the functional activity of enzyme on site/ in position within the tissue sections or cell cultures (Pucci- Minafra et al. 2001; Stephens et al. 2001; Troeberg and Nagase 2003). Thus, an upsurge to devise methodologies which are more superior and focused for the identification of a wide range of clinically relevant enzymes like proteases shall be of great significance (Sternlicht and Werb 2001; Lopez-Otin and Overall 2002; Turk 2006). In situ zymography (ISZ), an adaptation and modification of substrate zymography, is being carried out by researchers primarily for detection, localization, and estimation of substrate-specific enzymatic activity in tissue sections (Nemori and Tachikawa 1999; Yan and Blomme 2003). The basic principle of this technique involves digestion of specific substrate by activated enzymes present at their native location, followed by detection of the liberated signal by light or fluorescent microscopy. Based on the nature of substrate, it has been classified into two methodologies, wherein one method utilizes a substrate containing photo- graphic emulsion overlay while the other uses a fluorescen- tally labeled substrate (Galis et al. 1995; Mungall et al. 1998). An improved version of the latter has also been developed that uses substrate protein conjugated to quenched fluorescein, which emits higher fluorescence as the protease cleaves the substrate, thereby liberating the quenched fluorescein molecule (Frederiks and Mook 2004). To our knowledge, ISZ is mostly being used for study of protease activity in tissue sections (George and The authors Aastha Chhabra, Astha Jaiswal, Umang Malhotra, Shrey Kohli, and Vibha Rani have contributed equally to this paper. A. Chhabra : A. Jaiswal : U. Malhotra : S. Kohli : V. Rani (*) Department of Biotechnology, Jaypee Institute of Information Technology, A-10, Sector-62, Noida 201307 Uttar Pradesh, India e-mail: vibha.rani@jiit.ac.in In Vitro Cell.Dev.Biol.—Animal (2012) 48:463–468 DOI 10.1007/s11626-012-9529-5