Question: I would like to quantify apoptosis in living cells and I am interested in your FLICA product. Could you tell me how FLICA works? Additionally, I would like to recover the cell after incubation and use DNA for analysis. Will the FLICA assay reduce apoptosis and DNA fragmentation?
Caspases play important roles in apoptosis and inflammation. ICT’s FLICA assay kits are used by researchers seeking to quantitate apoptosis via caspase activity in cultured cells. The FLICA reagent enters each cell and irreversibly binds to activated caspases. Because the FLICA reagent becomes covalently coupled to the active enzymes, it is retained within the cell, while any unbound FLICA reagent diffuses out of the cell and is washed away. The remaining fluorescent signal is a direct measure of the active caspase enzyme activity present in the cell at the time the reagent was added. Cells that contain the bound FLICA can be analyzed by fluorescence microscopy or flow cytometry. Cells labeled with the FLICA reagent may be read immediately or preserved for 16 hours using the fixative.
FLICA reagents contain caspase inhibitor sequences preferred by their target caspase, linked to a fluorescent reporter tag (e.g. FAM, SR, 660), and an FMK reactive group. Each fluorescent FLICA molecule covalently binds to an active caspase enzyme and this results in inhibition of the caspase – it can no longer cleave substrates. However, as FLICA reagents are typically used at low micromolar concentrations, the amount of inhibition has no discernable effect on the caspase cascade and the cell’s ability to undergo programmed cell death/apoptosis continues. Essentially, FLICA technology takes advantage of the caspase’s tendency to cleave at certain peptide sequences and harnesses it to tag those caspases for detection. Because the apoptosis cascade continues, the apoptotic cells will eventually reach late stage apoptosis and DNA fragmentation will occur as expected. Therefore, FLICA does not reduce apoptosis and DNA fragmentation.
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