Detect caspase activity in whole living cells.
ICTs FLICA™ kits make it easy to measure active caspases in whole living cells: apoptotic cells fluoresce red or green. Use FLICA™ to quantitate apoptosis, to distinguish apoptosis from necrosis, and measure cytotoxicity. Detect all caspases at once using the poly caspases FLICA™ kits, or detect specific caspase 1, 2, 3&7, 6, 8, 9, 10, or 13 using ICT’s specialized FLICA™ kits (prices).
FLICA™ kits are not ELISAs and they do not use antibodies for detection. Instead, they use an inhibitor sequence of caspases (such as VAD, which reacts with all caspases) linked to a green (carboxyfluorescein, FAM) or red (sulforhodamine, SR) fluorescent probe. FLICA™= Fluorescent-Labeled Inhibitor of CAspases.
FLICA™ kits are fairly simple to use and experiments can be completed in 1 day. The reagents are cell-permeant, so you dont have to lyse the cells or permeabilize the membranes. Just add FLICA™ to your cell culture media, let it incubate 1-4 hours, wash the cells, and analyze with a fluorescence microscope, plate reader, or flow cytometer.
Once added to the media of your cell culture, FLICA™ will pass through the cell wall. If there is an active caspase enzyme inside the cell, it will form a covalent bond with FLICA™ and retain the green or red fluorescent signal within the cell. Because the caspase enzyme itself binds with FLICA™, only active caspases will be measured. There is no interference from pro-caspases nor inactive forms of the enzyme. FLICA™ probes constantly fluoresce, therefore, any unbound reagent must be washed back out of the cell to remove any background fluorescence. This is done by several quick rinse and spin steps, or further incubation with the wash buffer or media.
Add a necrotic stain, like PI (which is included in the green FAM kits) or 7-AAD to distinguish apoptotic cells from necrotic cells. To label DNA, Hoechst 33342 is included in all FLICA™ kits. Once labeled with FLICA™, cells can be fixed, embedded, or frozen for storage (a fixative is included).
Read the fluorescent signal with a microscope, plate reader, or flow cytometer. Green FAM-FLICA™ excites at ~490nm and emits at 530nm (FAM manual). Red SR FLICA™ excites at ~560 and emits at 590 (SR manual).
FLICA™ works with suspension cells, adherent cells, thin tissue sections, and thin frozen sections from human, monkey, chicken, mouse, rat, drosophila, yeast, and paramecia. FLICA™ kits have been used successfully with THP-1, HL60, MCF7, HeLa, Jurkat, epithelial cells, retinal cells, primary neurons, macrophages, lymphocytes, and fibroblasts, to name a few. To image apoptotic cells in live animals in vivo, use ICTs FLIVO™ kits.
FLICA™ will not work with fixed nor paraffin embedded cells, as the caspase enzymes have become inactivated. However, cells can be fixed, embedded, or frozen after labeling with FLICA™ (a fixative is included). We recommend reading within 24 hours, as the fluorescent label may photobleach, however samples have been frozen for 8 weeks and re-analyzed with equivalent results.
FLICA™ kits come in 2 sizes: 25 tests and 100 tests. For example, 1 test is a 300uL 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. Set up an initial titration experiment to determine of the amount of reagent and washing procedure that will work best for your experimental conditions. Create positive controls by inducing apoptosis with ICTs camptothecin or staurosporine.
FLICA™ makes it simple to identify apoptotic cells. To detect apoptosis in general, use the green or red poly caspases kits. These kits use a general peptide sequence (VAD) which reacts with all active caspases. To distinguish apoptotic from necrotic cells, use ICTs green FAM-FLICA™ kit to label apoptotic cells green, then add PI (included with the kit) or 7-AAD to stain necrotic cells red.
The best way to assess the total health of cultured cells via flow cytometry is to combine ICTs green fluorescent poly-caspases FAM-VAD-FMK FLICA reagent with 7-AAD, a red fluorescent necrotic stain. To use, just take a small aliquot of cells, add a low dose of FAM-FLICA, let that incubate 30”, add 7AAD for 5”, then read on a flow cytometer to quantitate living, dead, and apoptotic cells. This procedure requires no wash steps, as the sheathing fluid running through the flow cytometer acts as a wash step to remove any unbound probe.
To target a specific caspase, use ICT’s specialized FLICA™ kits for caspase 1, 2, 3&7, 6, 8, 9, 10, or 13. All kits are available with a green fluorescent probe. 3 kits are available with a red fluorescent probe: poly caspases, caspases 3&7, and caspase 9. Use the red SR-FLICA™ kits with GFP transfected cell lines and for dual-labeling studies with other green reagents. Add a colored secondary antibody to generate more data with 1 experiment.
Advantages over Alternative Methods
More reliable than annexin:
FLICA™ kits are more accurate and reliable than annexin. Annexin detects phosphatidyl serine which is exposed upon the turnover of the cell membrane. This is not an accurate indicator of apoptosis as this turnover can occur for other reasons. Annexin tends to bind to all thymus-derived cells, apoptotic or not.
Earlier and easier than TUNEL:
ICTs kits are easier and more sensitive than TUNEL. TUNEL is a long procedure based on DNA laddering. FLICA™ kits detect apoptosis based on caspase activity, which occurs before DNA laddering, so you get an earlier indicator of apoptosis.
1. Culture your cells up to 1 x 10^6 cells/mL.
2. Induce apoptosis following your protocol, and create positive and negative controls.
3. Reconstitute the reagent with 50 uL DMSO to form the stock concentrate (which can be frozen for future use).
4. Dilute the stock concentrate with 200uL 1X PBS to form the working solution.
5. Add ~10 uL of the working solution directly to a 300-500 uL aliquot of your cell culture for labeling.
6. Incubate 1-4 hours.
7. Wash and spin cells twice, or let incubate for 1 hour with fresh media or 1x apoptosis wash buffer.
8. If desired, label cells with Hoechst stain.
9. If desired, label cells with Propidium Iodide or 7AAD.
10. If desired, fix cells.
11. Analyze data using a fluorescence microscope, plate reader, or flow cytometer.
Green FLICA™ Kits
Red FLICA™ Kits
Two page FLICA flyer (pdf).
Frequently Asked Questions
Please refer to the section "FAQ: FLICA Caspase & Apoptosis Detection Kits".
FLICA kits have worked successfully with these cell lines:
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