Caspase Apoptosis Assay

Caspases are the main executors of the apoptotic process, as caspases upon activation, mediate apoptosis by proteolysis of specific substrates. In this assay caspase activity is measured using a caspase specific inhibitor sequence linked to a fluorescent probe. This assay is known as Fluorochrome-Labelled Inhibitor of Caspases Assay (FLICA).

The non-cytotoxic caspase specific inhibitor is cell permeant and passes through the intact plasma membrane and covalently binds to the reactive cysteine residue on the large subunit of the active caspase heterodimer. Unbound caspase inhibitor diffuses out of the cell and is washed away, thus there is no interference from pro-caspases or inactive forms of the enzyme. The fluorescence measured thus gives a direct measure of the amount of active caspase in the whole living cell. Non-viable cells are identified using propidium iodide.

Figure: Jurkat cells were grown in the absence (upper row) or in the presence (lower row) of camptothecin (CPT). Cells were stained with Hoechst-33342, FLICA reagent (FAM) and Propidium Iodide (PI) and analysed using the Caspase Assay and a NucleoCounter^® NC-3000™. Scatter plots and histograms were obtained from the NucleoView™ NC-3000™ software. Polygons and markers in the displayed plots were used to demarcate the various cell populations. In this example camptothecin causes a dramatic increase of early apoptoctic cells (Caspase FAM positive and PI negative cells).