Our FLICA® probes are non-cytotoxic Fluorescent Labeled Inhibitors of CAspases that covalently bind to active caspase enzymes. FLICA® measures the intracellular process of apoptosis instead of a side-effect, such as the turnover of phosphatidyl serine, and eliminates the incidence of false positives that often plagues methods like Annexin V and TUNEL staining. FLICA® can also be used to measure pyroptosis, a highly inflammatory form of programmed cell death.
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.
Cells labeled with FAM-FLICA can be counter-stained with reagents such as the red live/dead stains Propidium Iodide (included in FAM-FLICA kits) and 7-AAD (catalog 6163) to distinguish apoptosis from necrosis. Nuclear morphology can be concurrently observed using Hoechst 33342, a blue DNA binding dye (included in FLICA kits). Cells can be viewed directly through a fluorescence microscope or the fluorescence intensity can be quantified using a flow cytometer or fluorescence plate reader. FAM-FLICA optimally excites at 488-492 nm and has a peak emission at 515-535 nm.
FLICA® can be used to label suspension or adherent cells and thin tissue sections. After labeling with FAM-FLICA®, cells can be fixed or frozen. For tissues that will be paraffin-embedded after labeling, use our red sulforhodamine SR-FLICA® probes.
We have kits for the detection of: caspase-1 (YVAD or WEHD) (also recognizes caspases 4 and 5), -2 (VDVAD), -3/7 (DEVD), -6 (VEID), -8 (LETD), -9 (LEHD), and -10 (AEVD).
FLICA kits are available with a green, red, or far-red fluorescent label.