EnSens® Fluorescent 3D Live Cell Imaging Kit for Factor Xa 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 Factor Xa 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® Factor Xa Activity Detection Substrate: Despite the highly-overlapping substrate specificities of Factor Xa and Thrombin, EnSens® substrates are able to differentiate between the two enzymes. In the graph below, the EnSens® Factor Xa activity detection substrate is cleaved extremely well by Factor Xa, but not at all by Thrombin.
EnSens Factor Xa kit was challenged with purified active Factor Xa protease or purified active Thrombin protease. The assay was run in 384-well plates in a reaction volume of 20 uL. Excitation 625-635nm and Emission 660-670nm settings were used on a Tecan Infinite M1000 fluorescent plate reader and readings were taken every 2 minutes over 2 hours.
•EnSens® Factor Xa Activity Detection Substrate
•Far Red Fluorogen (FRF dye)
•Pre-activated substrate (EnSens®-PA, substrate positive control)
(Coagulation factor Xa, FXa, prothrombinase, prothrombase, thrombokinase, thromboplastin, Stuart factor, Stuart-Prower factor)
A clotting cascade protease initially expressed as inactive proenzyme and activated by other coagulation factor proteases, factor Xa cleaves and activates prothrombin in the presence of factor Va. Factor Xa is inactivated by the serpin ZPI (protein Z-dependent protease inhibitor). It is the target of recent drugs rivaroxaban (XARELTO); apixaban (Eliquis); enoxaparin (Lovenox); edoxaban (Lixiana); betrixaban (LY517717); darexaban (YM150); TAK-442; and eribaxaban (PD0348292).
Together, factor Xa and thrombin proteases play prominent roles in the coagulation cascade and are major targets for emerging anti-coagulant therapies and associated diagnostics. EnSens fluorimetric reagents enable selective tracking and quantification of Factor Xa and Thrombin activation in purified protease, plasma, and whole blood samples.
EnSens Fluorescent 3D Live Cell Imaging Kit for Factor Xa Activity Tracking
- Product Code: 022315_07
- Availability: In Stock