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

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.

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). 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).

Publications

  • Instrument: NucleoCounter® NC-3000™
  • Cell Type: Cancer Cells
  • Research area: Oncology​
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