EnSens® Fluorescent 3D Live Cell Imaging Kit for MMP-13 Activity Tracking
Pricing (select from Available Options, above):
Trial size (25 assays) $159
96 assays $599
The EnSens® Fluorescent 3D Live Cell Imaging Kit for MMP-13 Activity Tracking is a fluorescence based protease activity detection kit for use with live three-dimensional (3D) cell culture or co-culture. The kit provides non-toxic, protease-selective substrate and far-red fluorescent dye to be mixed directly into invasion matrix / scaffold / hydrogel (e.g. Matrigel®, or collagen gel), enabling real-time monitoring of specific protease activity by fluorescent microscopy. Kit components are stable for at least six days in culture, and highly resistant to photobleaching. Custom substrate development also available. Excitation 625-635nm/ Emission 655-665nm. Cy5 or similar filters can also be used.
Imaging the activity of a specific protease in live cells: The micrographs below demonstrate fluorescent live-cell 3D imaging of protease activity (in this case using the EnSens® MMP-14 Activity Detection Substrate) in an invading MDA-MB-231/fibroblast spheroid. Spheroids were formed in the wells of a Corning® 96-well Spheroid Microplate, then surrounded with a layer of Matrigel® Matrix. Data were generated by BioTek® Instruments using their CytationTM 5 Cell Imaging Multi-mode Reader (brightfield and Cy5 channels) and Gen5TM data analysis software.
Selectivity of the EnSens® MMP-13 Activity Detection Substrate: Despite the highly-overlapping substrate specificities of the collagenase MMPs (MMP-1, MMP-8, and MMP-13), the EnSens® MMP-13 substrate is cleaved by MMP-13, but not by MMP-1 or MMP-8. The substrate is cleaved somewhat by MMP-2, -3, -10, and 12 in addition to MMP-13, but the MMP-13-selective (and commercially available) inhibitor CL-82198 can be included in the assay to optimize the selectivity of the substrate.
The graph on the left shows selective inhibition of MMP-13 by CL-82198 when 50nM of each enzyme was assayed separately.
In the graph on the right, mixtures of MMPs (4nM each of MMP-2, MMP-3, MMP-10, MMP-12, with or without 4nM MMP-13) were assayed in the absence or presence of CL-82198. The inhibitor makes little to no difference in total activity when MMP-13 is not included in the mixture (light-colored bars). When MMP-13 is included in the mixture, CL-82198 significantly reduces the total activity (dark-colored bars).
This demonstrates that, with the use of MMP-13 inhibitor CL-82198, the EnSens MMP-13 substrate can selectively measure MMP-13 enzyme activity in a mixture of MMPs, either qualitatively or quantitatively (using a standard curve built from purified enzyme). For reference to this inhibitor strategy, see Berger AB et al. 2006 Identification of early intermediates of caspase activation using selective inhibitors and activity-based probes. Mol Cell 23: 509-521; and Corasaniti MT et al. 2007 Cell signaling pathways in the mechanisms of neuroprotection afforded by bergamot essential oil against NMDA-induced cell death in vitro. Br J Pharmacol. 151: 518-29.
•EnSens® MMP-13 Activity Detection Substrate
•Far Red Fluorogen (FRF dye)
•Pre-activated substrate (EnSens®-PA, substrate positive control)
MMP-13 is one of the three collagenase matrix metalloproteases (MMP-1, MMP-8, and MMP-13). By virtue of its substrate profile (extracellular matrix and connective tissue proteins such as collagen, gelatin, aggrecan, decorin, and fibronectin), it is involved in arthritis and cancer/metastasis. It also cleaves and activates TGF-beta.
EnSens Fluorescent 3D Live Cell Imaging Kit for MMP-13 Activity Tracking
- Product Code: 022315_11
- Availability: In Stock