I am working in the Department of Biophysics, Panjab University, Chandigarh, India as a Senior Research Fellow. I had an opportunity to use and analyze the Caspase 3 Fluorescence Assay Kit from Cayman Chemical. As part of my work, I was in search of a convenient and efficient protocol for evaluation of Caspase 3 activity as I am working on phytochemicals in the area of cancer research. Caspase 3 is a cysteine-aspartic acid protease and plays a central role in the execution phase of cell apoptosis. Further, it is now well established that Caspase 3 is activated in the mitochondrial initiated apoptotic pathway. Also, regulation of caspase 3 activation is one of the focuses in discovery for anti-cancer drugs. I tested and evaluated various protocols. I rated them with respect to sensitivity and specificity as well as other aspects such as ease-of-use, hands-on time, the level of complexity of results interpretation and the required skill level of the user.
The chemistry utilized in the Caspase 3 Fluorescence Assay Kit involves a specific caspase 3 substrate: N-Ac-DEVD-N-MC-R110, which upon cleavage by active caspase 3, generates a highly fluorescent product that can be monitored using excitation and emission wavelengths of 485nm and 535nm, respectively. Further, the kit also contains caspase 3 inhibitors for verifying the specificity of the substrate.
I found the Cayman Caspase 3 Fluorescence Assay Kit instructions to be clear, concise and detailed enough so that anyone skilled in the sciences could perform the assay. Secondly, in the same kit, it is very easy to verify the specificity of the substrate with the help of a specific inhibitor which adds to authenticity to the observations. Also, the protocol is sensitive enough to allow estimation in a small sample size. The drawbacks of the Caspase 3 Fluorescence Assay Kit are its cost and that it cannot be stored at a temperature above -80°C. Also, a plate reader with flouresence filters must in the lab.
My research involves the use of tissue homogenates. So, prior to dissection, lung tissues were perfused with Tris Buffer, pH 7.4 to remove any red blood cell contamination. Tissue homogenates were then made, followed by centrifugation at 10.000 rpm for 15 minutes at 4°C. The resultant supernatant was used for the above assay. Then, as directed in the kit, different reagents: caspase 3 substrate solution, active caspase 3 standard, and caspase 3 inhibitor solution, to be used in the assay were prepared. The kit also includes directions for the use of wells on the plate. Accordingly, for the standard wells 100 ul of active caspase standard was pipetted into the standard wells. In the sample wells 90 ul of the samples were added followed by addition of 10 ul of the sample caspase assay buffer. Further, in the inhibitor wells, 10 ul of caspase 3 inhibitor solution was added instead of caspase 3 assay buffer. This was followed by the addition of 100 ul of substrate to each of the wells as directed in the kit. The fluorescent intensity of each well was read at excitation 485 nm and emission 535 nm using a multilabel plate reader.
I recommend the Caspase 3 Fluorescent Assay Kit for its technology, reproducibility, sensitivity, and specificity. The instructions were clear, precise and accurate. In addition, there was very little clean up and preparation time.
Department of Biophysics
Panjab University