Caspase 3/7, 8, or 9 Assay

Detection of apoptosis using the FLICA Assay

  • Determine active caspase 3/7, caspase 8 or caspase 9
  • User-friendly protocol with predefined settings
  • Quantify specific caspase activity at a cellular level
  • Ascertain if cells are in early or late stage of apoptosis/necrosis
  • Fast, automated single cell analysis
  • Clear data presentation with Plot Manager feature
  • Automated PDF reports
  • Export data in FCS/ACS formats
In the Caspase 3/7, 8, or 9 Assay, cells are stained with FLICA, Solution 15, and Solution 16, loaded into a cell counting slide, and counted with the NucleoCounter® NC-3000™.

Overview of the caspase Assay

Caspases (cysteine-aspartic proteases, cysteine aspartases or cysteine-dependent aspartate-directed proteases) are a family of protease enzymes essential for the execution of programmed cell death. They are the main executors of the apoptotic process; upon activation, caspases mediate apoptosis by proteolysis of specific substrates.

Our Caspase 3/7, 8 or 9 Assay measures activity using a caspase-specific inhibitor sequence linked to a fluorescent probe. 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 (PI). This assay is known as Fluorochrome-Labeled Inhibitor of Caspases Assay (FLICA).

Assay Principle

When using our caspase apoptosis assay (FLICA), caspase activity is measured with a caspase-specific inhibitor sequence linked to a fluorescent probe.

The non-cytotoxic caspase-specific inhibitor is cell permeant and passes through the intact plasma membrane where it covalently binds to the reactive cysteine residue on the large sub-unit of the active caspase heterodimer. Unbound caspase inhibitor diffuses out of the cell and is washed away, leaving no interference from pro-caspases or inactive forms of the enzyme. Therefore, the fluorescence measured gives a direct quantification of the amount of active caspase in the entire living cell. Non-viable cells are identified using PI.

Data image from the Caspase 3/7, 8, or 9 Assay where the analyzed cells are shown in the NucleoView™ software.

Using fluorescence microscopy and image analysis, the NucleoCounter® NC-3000™ system automates the detection of apoptotic cells based on caspase activity. Cells are stained with Hoechst 33342, PI, and carboxyfluorescein-labeled FLICA reagent.

The total cell population is stained with Hoechst 33342 (blue), while early apoptotic cells and late apoptotic/necrotic cells are stained with carboxyfluorescein-labeled FLICA reagent (green) and PI (red), respectively.

Results presented in plot manager

The diagram demonstrates the effect of adding camptothecin (CPT) to Jurkat cells in culture. CPT is a commonly used topoisomerase poison, which induces apoptosis, thus activating caspases. Jurkat cells are an immortalized line of human T lymphocyte cells, commonly used for studying the effectiveness of potential anti-cancer compounds.

Jurkat cells were grown in the absence (upper row) or presence (lower row) of camptothecin (CPT).

Image of processed data from the Caspase 3/7, 8, or 9 Assay in the NucleoView™ software, in the form of histograms and scatter plots.

Cells were stained with Hoechst 33342, FLICA reagent (FAM) and PI and analyzed using the Caspase 3/7, 8, or 9 Assay with the NucleoCounter® NC-3000™. Scatter plots and histograms were obtained in NucleoView™ NC-3000™ software. Polygons and markers in the displayed plots were used to demarcate the various cell populations. In this example, CPT causes a significant increase of early apoptotic cells (marked as Caspase FAM positive and PI negative cells in the lower right quadrant of the second plot).