FLICA™ Poly Caspases Kits
Detect all caspases in vitro with FAM-VAD-FMK and SR-VAD-FMK
ICT's FLICA poly caspases detection assays are simple yet accurate methods to measure apoptosis via caspase activity in whole cells. Simply add the FLICA poly caspases reagent to the media and incubate. Cells containing active caspases will fluoresce green or red. FLICA is not an ELISA and does not involve the use of any antibodies.
ICT's FLICA (Fluorescent Labeled Inhibitors of Caspases) inhibitor probes are comprised of 3 segments:
- a green (FAM) or red (SR) fluorescent label
- an inhibitor peptide sequence targeted by active caspase enzymes (VAD)
- a fluoromethylketone group (FMK), which forms a covalent bond with the active enzyme.
ICT's FLICA Poly Caspases inhibitor probes are cell permeant:
No lysis or permeabilization steps are necessary. Just culture cells, add FLICA to the media, incubate, and wash. Active caspase enzymes will form a covalent bond with FLICA and retain the fluorescent signal within the cell. Because the caspase itself binds with FLICA, there is no interference from pro-caspases nor inactive forms of the enzyme. FLICA makes it easy to identify apoptotic cells.
For further analysis, add a necrotic stain, like PI (included in the green FAM kits) or 7-AAD to distinguish apoptotic cells from necrotic cells. To label DNA for visualization, Hoechst 33342 is included. Once labeled with FLICA, cells can be fixed, embedded, dually-stained with an antibody, or frozen for storage.
To analyze, read the fluorescent signal with a microscope, plate reader, or flow cytometer. The green FAM-FLICA probes excite at 490nm and emit at 530nm (FAM manual); the red SR-FLICA probes excite at 560nm and emit at 590nm (SR manual). We recommend reading the cells within 24 hours, as the fluorescent label may photobleach; however, samples have been frozen for 8 weeks and re-analyzed with equivalent results.
FLICA works with suspension cells, adherent cells, and thin tissue sections. FLICA will not work with previously fixed or paraffin embedded cells, as those treatments inactivate the caspase enzyme. However, cells may be fixed, embedded, or frozen after labeling with FLICA.
FLICA poly caspases detection kits are available in green or red fluorescence and in 2 sizes: 25 tests and 100 tests. 1 "test" is a 300mcL aliquot of cells grown at 1X10^6 cells/mL and analyzed on a fluorescence plate reader. Plate readers tend to require the most reagent, flow cytometers the least.
The green FAM-FLICA kit contains:
- ICT's FLICA poly caspases detection probe, FAM-VAD-FMK
- 10x apoptosis wash buffer
- Hoechst stain (a blue DNA stain)
- Propidium iodide (a red live/dead stain)
- Fixative (for use after labeling with FLICA)
Trial size, ~25 tests:
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Regular size, ~100 tests:
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The red SR-FLICA kit contains:
- ICT's FLICA poly caspases detection probe, SR-VAD-FMK
- 10x apoptosis wash buffer
- Hoechst stain (a blue DNA stain)
- Fixative (for use after labeling with FLICA)
Trial size, ~25 tests:
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Regular size, ~100 tests:
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Sample protocol:
- Culture your cells up to 1 x 10^6 cells/mL.
- Induce apoptosis following your protocol, and create positive and negative controls.
- Reconstitute the reagent with 50mcL DMSO to form the stock concentrate (which can be frozen for future use).
- Dilute the stock concentrate with 200mcL 1X PBS to form the working solution.
- Add ~10mcL of the working solution directly to a 300-500mcL aliquot of your cell culture for labeling.
- Incubate 1-4 hours.
- Wash and spin cells twice, or let incubate for 1 hour with fresh media or 1x apoptosis wash buffer.
- If desired, label cells with Hoechst stain.
- If desired, label cells with Propidium Iodide or 7AAD.
- If desired, fix cells.
- Analyze data using a fluorescence microscope, plate reader, or flow cytometer.