Caspase activation is not only a hallmark of apoptosis but is also involved in several physiological, non-apoptotic cell responses. Therefore, there is a growing need for sensitive and reliable assays to monitor caspase activation in a wide variety of experimental conditions. Caspases are cysteine-proteases that hydrolize target proteins with specific amino acid sequences. The affinity of the catalytic site for these amino acid residues has been exploited to develop caspase inhibitors, which block activity.
The Biovision CaspGLOW Red Active Caspase Staining Kit is a complete set of reagents for providing an easy, fast and versatile assay to detect cells with activated caspases. The assay exploits the irreversible binding between activated caspase and a general caspase inhibitor (VAD-FMK) conjugated to sulf-rhodamine (Red-VAD-FMK) as a fluorescent marker of activity. The staining procedure is a very simple, two step protocol: 1) cells are incubated for 30 min with Red-VAD-FMK, which is non toxic and cell permeable thanks to the FMK group; 2) the excess reagent is then flushed out by washes and the caspase-bound reagent is specifically retained into cells with activated caspases. As a result, this procedure represents a specific, in situ labeling method. The kit contains Red-VAD-FMK, washing buffer and VAD-FMK (negative control).
One of the most interesting features of the Biovision CaspGLOW Red Active Caspase Staining Kit is its versatility. In fact, once the cells are labled, the signal can be visualized and/or quantified by a variety of means, with or without fixation. In addition to qualitative analysis of active caspase (performed with a fluorescence microscopy), quantitative analysis can be performed using either flow cytometry or a fluorescence plate reader. In my laboratory, we were able to develop two additional assays using this kit to evaluate caspase activation. First, we were able to set a fluorometric macro assay using a standard fluorometer equipped with 2 ml cuvettes: after incubation of the cells with Red-VAD-FMK, followed by washes, the cells are broken in lysis buffer and the released fluorescent signal is measured. Second, we applied the CaspGLOW red Active Caspase assay to fresh tissue cryosections with a histochemical approach: freshly sectioned tissue (skeletal muscle) is incubated with Red-VAD-FMK for cell staining, washed, and fixed. Caspase activity is preserved in frozen tissue and can be visualized in situ by fluorescence microscopy. Normally, we combine the caspase assay with immuno-fluorescence to higlight tissue architecture or localize specific factors.
Even though one can always call for improved products (e.g. caspase substrates conjugated to brighter, long lasting fluorochromes), I would say this kit is great as such. The main benefits include reasonable price, good size of the reagents, reliability, easiness of use. In conclusion, I find the Biovision CaspGLOW Red Active Caspase Staining Kit to be a powerful, very practical approach to analyze capase activity qualitatively and quantitatively. Thanks to the competitive price (the FMK-conjugated caspase inhibitors are quite expensive in general), I find this product a very good value and recommend it to other researchers in the field of caspase activity and function.
Dario Coletti, PhD
University of Rome La Spaienza