NC-3000™ Caspase 3/7, 8 & 9 Assay
– For easy, fast and objective measurement of apoptosis
Obtain single caspase information on the cellular level
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.
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Principle:
Using fluorescence microscopy and image analysis; the NucleoCounter® NC-3000™ system automates 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, while early apoptotic and late apoptotic/necrotic cells are stained with carboxyfluorescein labeled FLICA reagent and PI, respectively.
Jurkat cells stained with Hoechst 33342 (appearing blue), FLICA reagens (appearing green) and PI (appearing red). Cells were grown in the absence (1A) or presence (1B) of camptothecin for 16 h at 37 °C and 5 % CO2. Cells were harvested, washed and stained according to the protocol and analysed on the NC-3000™ using the Caspase Assay protocol. In this example 1 % of the untreated Jurkat cells are early apopotic and 5 % late apoptotic, whereas 13 % of the CPT treated Jurkat cells are in early apoptosis and 23 % in late apoptosis. Lower panel shows the images acquired for the two samples.
Key benefits
- Measure caspase 3/7, caspase 8 or 9
- User friendly protocol with predefined settings
- Obtain single caspase information on the cellular level
- Information about both early apoptotic and late apoptotic/necrotic cells provided
- Standardized results – even with different users
- Fast automated single cell analysis