FAM FLICA Caspase-1 Kit from GenWay Biotech

February 01, 2013

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Northwestern University
Microbiology-Immunology
PhD Student

Company:

GenWay Biotech, Inc.

Product Name:

FAM FLICA Caspase-1 Kit

Catalog Number:

GWB-Q00437

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The FAM FLICA Caspase-1 kit is a convenient method to assess caspase activity and cell death in vitro. Caspase-1 is a protease capable of processing several host proteins including IL-1β and IL-18. Its activity has been implicated in inflammatory autoimmune diseases as well as inflammasome activation during host responses to pathogens. This kit utilizes fluorescent probes that form irreversible covalent bonds specifically with active caspase-1 enzyme. Traditional methods have assessed active caspase abundance by Western blot, but these can be challenging since antibodies often recognize both the pro and mature forms of these proteases. The ability of FLICA reagents to quantify caspase activity as fluorescence intensity provides great advantages when studying eukaryotic cell death.

Experimental Design and Results Summary

Application

One of the biggest advantages of using FAM FLICA reagents for staining active caspases is the variety of ways in which fluorescence can be measured. We have measured fluorescence of stained cells using a plate reader, however caspase activity can also be assessed using fluorescence microscopy or flow cytometry.

Starting Material

We have used this kit to stain mouse lung homogenates. Another advantage of this system is its possible use with both primary cells and immortalized cell lines from either mouse or human origins. This kit can also be used to stain frozen tissue sections.

Protocol Overview

Overall, the staining protocol is very simple and straightforward. After performing desired treatments on your cells of interest, FAM FLICA stain is added to cells in a 96-well plate and incubated for 1 hour at 37°C. Cells are then washed twice with provided wash buffer (FLICA probes are cell permeable, so unbound probe is washed away). Fluorescence is measured using a plate reader with excitation wavelength of 488 nm and emission wavelength of 530 nm.

Tips

None

Results Summary

Results are always very consistent between replicates. Most tissues will have some basal level of caspase activity, which can easily be induced using positive control treatments.

Additional Notes

If performing analysis at a later time, washed cells may be fixed with provided fixative and stored at 4°C.

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Summary

The Good

The ease of use, variety of stainable cell types, and different possible fluorescence detection methods provide advantages over traditional methods.

The Bad

This is still fairly expensive technology.

The Bottom Line

This kit would be helpful for anyone studying caspase-1 activation during a variety of disease conditions.

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