Caspase (poly, 3/7, 8, 9, 1) & Mitochondria Membrane Potential Detection – MitoCasp

Caspase enzymes specifically recognize a 4 amino acid sequence (on their substrate) which necessarily includes an aspartic acid residue. This residue is the target for the cleavage reaction, which occurs at the carbonyl end of the aspartic acid residue(6). Caspases can be detected via immunoprecipitation, immuno-blotting techniques using caspase specific antibodies, or by employing fluorogenic substrates which become fluorescent upon cleavage by the caspase. MitoCasp uses a novel approach to detect active caspases (7-9). The methodology is based on carboxyfluorescein (FAM) labeled fluoromethyl ketone (FMK)-peptide inhibitors of caspases. These inhibitors are cell permeable and non-cytotoxic. Once inside the cell, the inhibitor binds covalently to the active caspase (10). Cells that contain bound inhibitor can be analyzed by flow cytometry or fluorescence microscopy.

Cell Technology utilizes a cationic dye to visualize mitochondrial membrane potential (15-17). The cationic dye is cell permeable and has a strong fluorescent signal in the red region and exhibits low membrane potential independent (non specific) binding and toxicity. In healthy cells the cationic dye is accumulated by the mitochondria in proportion to the DeltaPsi (membrane potential). In most cell lines, accumulation of the cationic dye in the mitochondria results in a higher fluorescence intensity. In apoptotic cells, where the mitochondrial membrane potential is compromised, the cationic dye does not accumulated in the mitochondria and these cells exhibit a lower fluorescence signal. Utilizing these two reagents in combination Caspase activity and mitochondrial membrane potential can be analyzed simultaneously. Citations Identification of single-domain, Bax-spec

Jurkat cells were stimulated with Staurosporine for 3 hours (B) or DMSO (A). The cells were then stained with the MitoCasp kit according to the protocol. The cells were then washed twice and analyzed by flow cytometry: Ex:488nm Em: FL1 and FL2.

Fig A. Healthy cells show a strong red fluorescence indicating intact mitochondria and no green fluorescence, indicating no active caspases.

Fig B. Apoptotic cells show a loss of red fluorescence (y axis) indicating loss of mitochondrial membrane potential and positive green fluorescence (x axis) indicating active caspases.